BACKGROUND Massive intragastric clotting(MIC) makes endoscopic therapy difficult in patients with acute upper gastrointestinal bleeding. Literature data on how to address this problem are limited. Here, we report on a...BACKGROUND Massive intragastric clotting(MIC) makes endoscopic therapy difficult in patients with acute upper gastrointestinal bleeding. Literature data on how to address this problem are limited. Here, we report on a case of massive stomach bleeding with MIC that was successfully treated endoscopically using an overtube of singleballoon enteroscopy.CASE SUMMARY A 62-year-old gentleman with metastatic lung cancer was admitted to the intensive care unit due to tarry stools and hematemesis of 1500 mL of blood during hospitalization. Emergent esophagogastroduodenoscopy revealed massive blood clots and fresh blood in the stomach with evidence of active bleeding.Bleeding sites could not be observed even by changing the patient’s position and aggressive endoscope suction. The MIC was successfully removed using an overtube connected with a suction pipe, which was inserted into the stomach with an overtube of a single-balloon enteroscope. An ultrathin gastroscope was also introduced through the nose into the stomach to guide the suction. A massive blood clot was successfully removed, and an ulcer with oozing bleeding at the inferior lesser curvature of the upper gastric body was revealed, facilitating endoscopic hemostatic therapy.CONCLUSION This technique appears to be a previously unreported method to suction MIC out of the stomach in patients with acute upper gastrointestinal bleeding. This technique could be considered when other methods are not available or if they fail to remove massive blood clots in the stomach.展开更多
The clotting protein(CP) plays important and diverse roles in crustaceans,such as coagulation and lipid transportation.A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis(name...The clotting protein(CP) plays important and diverse roles in crustaceans,such as coagulation and lipid transportation.A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis(named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography.Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+,suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase in shrimp hemocytes.The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE.CP exists as disulfide-linked homodimers and oligomers.The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon,Farfantepenaeus paulensis and Litopenaeus vannamei;and similar to that of other decapods.The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight(MALDI-TOF/TOF) mass spectrometry.Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.展开更多
Objective To explore the influence of styptic fiber on clotting time in rabbits so as to provide experiment data for its development.Methods Onto 0.1 mL aliquots of citrated anti-coagulant rabbit blood placed in a sur...Objective To explore the influence of styptic fiber on clotting time in rabbits so as to provide experiment data for its development.Methods Onto 0.1 mL aliquots of citrated anti-coagulant rabbit blood placed in a surfacial plate 25 ul of 0.2 mol·L-1 CaCl2 solution was dropped,and mixed well with glass stirrer;the resulting mixture was immediately capped with a piece of styptic fiber(test product group)or absorptive gelatin sponge(positive control group)of 2 cm diameter.Then,the surficial plate was rinsed with 30ml of purified water at 5,10,20,30,40 and 50 min after capping;the rinsings were allowed to stand for 1 h and were subjected to OD determination at a wavelength of 541 nm.The above procedure was repeated twice,the average value of the twice experiments was taken for evaluation of the hemostatic effect of test product.For negative control group,all procedures except for capping were same as the test product group.The haemostatic effect was judged by percent OD relative to OD at 0 min in negative control group(OD 0 min)(OD 0 min was considered as 100%);if OD value at a time was less than 80% of OD 0 min,it should be designated as primary clotting time(PCT),less than 20% as complete clotting time(CCT).Results The measured PCT was 20min for both negative and positive control groups;CCT was 50,30 and 5 min for negative control,positive control and test product groups,respectively,showing the test styptic fiber had a CCT 8 times shorter than untreated blood,10 times shorter than negative control and 6 times shorter than positive control.Conclusions The test styptic fiber has powerful hemostatic effect.展开更多
Objective： To investigate the correlation of fibrinogen level and absorbance change in both PT and APTT clotting curves on BCSXP Analyzer. Methods：A serial of standard fibrinogen and 250 patient plasma samples with ...Objective： To investigate the correlation of fibrinogen level and absorbance change in both PT and APTT clotting curves on BCSXP Analyzer. Methods：A serial of standard fibrinogen and 250 patient plasma samples with different qualities（normal, hemolysis, icterus, and lipemia） were run on BCSXP for assays PT, APTT and Fibrinogen. The absorbance change（DeltaA） from baseline to plateau in clotting curve was retrieved and analyzed on its correlation with the Fibrinogen result. Influence of plasma quality and PT/ APTT result on this correlation was also studied respectively. Results：Both PT-DeltaA and APTT-DeltaA showed good linear regres- sion with fibrinogen level in the sample, with R2 close to 0.90 in both standard and patient samples. Hemolysis（H）, itcterus（I） and lipemia（L） of the sample with valid clotting curves were found to have no significant difference in this correlation from normal（N） sample（R2： 0.83H, 0.92I, 0.81L and 0.79N in PT; 0.89H, 0.95l, 0.91L and 0.89N in APTT） in either PT or APTT curve. PT or APTT result also has little impact on this correlation（0.71 in range 7 - 10 sec, 0.56 inl0 - 20 sec, and 0.70 in 20 sec-; R2 in APTT： 0.88 in 20-30 sec, 0.92 in 30-40 sec, and 0.95 in 40 sec-）. Conclusion：The absorbance change in either PT or APTT clotting curve correlates well with the fibrinogen level in plasma, which is independent of plasma quality PT or APTT results. The absorbance change can be used as an alternative way to roughly estimate fibrinogen level in either PT or APTT clotting curve when the result of clauss-based fibrinogen measurement is not available.展开更多
This paper presents a solution methodology for H<sub>∞</sub>-feedback control design problem of Heparin controlled blood clotting network under the presence of stochastic noise. The formulaic solution pro...This paper presents a solution methodology for H<sub>∞</sub>-feedback control design problem of Heparin controlled blood clotting network under the presence of stochastic noise. The formulaic solution procedure to solve nonlinear partial differential equation, the Hamilton-Jacobi-Isaacs equation with Successive Galrkin’s Approximation is sketched and validity is proved. According to Lyapunov’s theory, with solutions of the nonlinear PDEs, robust feedback control is designed. To confirm the performance and robustness of the designed controller, numerical and Monte-Carlo simulation results by Simulink software on MATLAB are provided.展开更多
We report the clinical case of a 40-year-old Italian woman, who soon after her booster vaccination with mRNA-1273 after the two previous vaccinations with BNT162b2, developed severe headache, high fever, and Musculo-s...We report the clinical case of a 40-year-old Italian woman, who soon after her booster vaccination with mRNA-1273 after the two previous vaccinations with BNT162b2, developed severe headache, high fever, and Musculo-skeletal pain. She went to the emergency department, where computerized tomography (CT) scans of chest and brain were performed, resulting in both negative for pathologic findings. On the contrary, white blood count was strongly lowered and D-dimer severely elevated. She improved after treatment with enoxaparin and the blood analyses returned in the normal range after ten days. This case supports the hypothesis that COVID-19 vaccines could increase blood clotting in some predisposed subjects. Therefore, we believe that robust and well-designed clinical trials, considering the evaluation of D-dimer levels, should be performed to eliminate any doubts on this issue.展开更多
In this study, texture analysis method was used for the determination of rennet flocculation time (tfloc) and rennet clotting time (tclot) of rennet-induced reconstitued milk samples with different CaCl2 concentration...In this study, texture analysis method was used for the determination of rennet flocculation time (tfloc) and rennet clotting time (tclot) of rennet-induced reconstitued milk samples with different CaCl2 concentrations. The rennet flocculation time (RFT) and rennet clotting time (RCT) were also determined by using the Berridge test and sensory evaluation. The hardness value versus renneting time curves derived from texture analysis gave a good modified exponential relationship for each CaCl2 concentration and the curves were used to calculate flocculation time and clotting time parameters. It was found that the parameters (tfloc and tclot) appeared strongly correlated with RFT and RCT, respectively. Texture analysis was proved as a suitable method to control the rennet-induced coagulation and determine the rennet clotting time. It was also determined that enrichment of milk with CaCl2 leaded to a decrease in flocculation and clotting times and an increase in rate of clotting and gel hardness.展开更多
Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hF...Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the poten-tial to be optimized as a safe and effective therapeutic modality for hemophilia B.展开更多
To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retrovira...To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of factor Ⅸ-deficie human primary skin fibroblasts, FDⅨ cells. The factor Ⅸ secretion rate of the infected FDⅨ cells was about 549 ng/10~6 cells/24h and over 75% of secreted factor Ⅸ was biologically active. We are convinced that this factor Ⅸ-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.展开更多
Background Due to lack of point-of-care testing, the use of low-molecular-weight heparin （LMWH） therapy in some special patients is restricted. This study was designed to explore the effects of LMWH on clot rate （C...Background Due to lack of point-of-care testing, the use of low-molecular-weight heparin （LMWH） therapy in some special patients is restricted. This study was designed to explore the effects of LMWH on clot rate （CR） and activated clotting time （ACT）, and to search for an appropriate method for bedside monitoring of anticoagulant activity of LMWH. Methods Thirty-two healthy volunteers were selected from the staff of Beijing Tongren Hospital. CR and ACT were measured with different reagents （glass beads, diatomite, kaolin and magnetic bar） on blood samples spiked with increasing concentrations of LMWH （dalteparin, 0.2-1.8 IU/ml）. Correlations between concentrations of LMWH and values of CR and ACT were analysed based on the data obtained and regression analysis was performed to establish a regression equation. Results With the increase in doses of dalteparin, CR values reduced gradually. The values of CR of four reagents （glass beads, diatomite, kaolin and magnetic bar） were 20.4-4.5 IU/min, 27.4-6.9 IU/min, 27.5-7.9 IU/min and 7.8-0.1 IU/min respectively and an linear relationship was observed between the CR values and dalteparin concentrations （P〈0.05）. The values of ACT were 173-615 seconds, 130-270 seconds, 123-226 seconds, 337-1411 seconds respectively, which showed a linear regression between the ACT values and dalteparin concentrations （P〈0.01）. Differences in slope of the regression curves of ACT were observed with all the reagents tested （glass beads 248.2 s/IU, diatomite 74.8 s/IU, kaolin 58.2 s/IU and magnetic bar 1112.2 s/IU, P〈0.01）. While the minimum concentration of dalteparin was 0.2 IU/ml, 0.4 IU/ml, 1.4 IU/ml and 0.2 IU/ml separately, the ACT values of the four coagulants （glass beads, diatomite, kaolin and magnetic bar） were beyond the normal limit and showed a noticeable increase respectively （P〈0.01）. Conclusions This study showed that there was an excellent linear relationship between the CR and ACT values and dalteparin concentrations for all the four reagents （glass beads, diatomite, kaolin and magnetic bar） in vitro. The sensitivity of different coagulation reagents to LMWH different. Choosing a suitable reagent, both CR and ACT were possible to be used as a convenient bedside test for LMWH.展开更多
DNA-DNA molecular hybridization is commonly used in the detection of transferred foreign gene in transgenic mice, which is an effective tool in the detection of transgenic mice due to its high sensitivity and reliabil...DNA-DNA molecular hybridization is commonly used in the detection of transferred foreign gene in transgenic mice, which is an effective tool in the detection of transgenic mice due to its high sensitivity and reliability. The method, however, is very laborious and time-consuming, and requires a large number of DNA samples展开更多
THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system...THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system is costly, laborious and tim-cosuming. In 1994,Johanna et al. reported that secretion of a foreign protein, human growth hormone (hGH),in milk had been achieved after direct introduction of the cDNA into the mammary gland ofgoats by replication-defective retroviral vector. In that work, the in vivo expression of展开更多
Culture of patient somatic cells, transfected with therapeutic genes into genomic DNA with retroviral vectors, followed by colony selection, amplification, and reimplantation of transformed cells into patient, has bee...Culture of patient somatic cells, transfected with therapeutic genes into genomic DNA with retroviral vectors, followed by colony selection, amplification, and reimplantation of transformed cells into patient, has been Widely used for clinical trial of gene therapy in the past years. But the disadvantages of this protocol are obvious. (i) To a great extent the expression level of the transfected cells depends on the different integration sites,which cause the various expression rates from different colonies; besides, the life span展开更多
Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein ...Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.展开更多
Clotting factor Ⅸ is an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder——hemophilia B, or Christmas disease. Factor Ⅸ is synthesized in...Clotting factor Ⅸ is an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder——hemophilia B, or Christmas disease. Factor Ⅸ is synthesized in liver hepatocytes where it undergoes complex vitamin K-dependent post-translational modifications which are essential for the activation of factor Ⅸ. Recently, several groups have cloned factor Ⅸ cDNA and reported that, in the presence of vitamin K, active factor Ⅸ could be secreted in human and mouse fibroblasts,展开更多
The murine model of subarachnoid hemorrhage(SAH)is a valuable experimental tool for investigating molecular and cellular mechanisms,and the endovascular filament perforation technique can be used to simulate prominent...The murine model of subarachnoid hemorrhage(SAH)is a valuable experimental tool for investigating molecular and cellular mechanisms,and the endovascular filament perforation technique can be used to simulate prominent pathophysiological features observed after human SAH;however,current validation methods for assessing an appropriate SAH model are limited.Here,we introduce a simple procedure for se-lecting a mouse model of diffuse SAH.SAH was induced in 24 mice using a standard filament perforation method.After confirming survival at 24 h,SAH was scored 0-1 based on T2*-weighted images on whole-brain magnetic resonance imaging(MRI)and visual surveillance of the cisterna magna(CM)through the dura mater.The CM-based SAH grading correlated well with a reference parameter defined by extracted brain(r^(2)=0.53,p<0.0001).The receiver operating characteristic curve revealed a sensi-tivity of 85%and a specificity of 91%for detecting diffuse SAH,with a similar area under the curve(0.89±0.06[standard error of the mean])as the MRI-based grading(0.72±0.10,p=0.12).Our data suggest that confirming an SAH clot in the CM is a valuable way to select a clinically relevant diffuse SAH model that can be used in future experimental studies.展开更多
Hemophilia B, a serious bleeding disorder, is an inherited X chromosome-linked diseasecaused by the deficiency or inactiveness of human clotting factor IX (FIX). The conven-tional clinical treatment of plasma infusion...Hemophilia B, a serious bleeding disorder, is an inherited X chromosome-linked diseasecaused by the deficiency or inactiveness of human clotting factor IX (FIX). The conven-tional clinical treatment of plasma infusion is expensive and associated with a high risk展开更多
Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has be...Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has been successfully introduced in gene diagnosis, mutation detection, polymorphism analysis and nucleotide sequencing. In the study of gene transfer and gene therapy, it is necessary to analyse DNA in the展开更多
Primary rabbit skin fibroblasts (RSF) containing lacZ reporter gene or human clotting factor IX (hFIX) cDNA could long termly exist and be expressed after being implanted in rabbit muscles. Among them, the beta glacto...Primary rabbit skin fibroblasts (RSF) containing lacZ reporter gene or human clotting factor IX (hFIX) cDNA could long termly exist and be expressed after being implanted in rabbit muscles. Among them, the beta glactosidase expression lasted at least 3 months, and hFIX expression lasted 1 month. These results will be helpful to organically combine the advantages of both skin fibroblasts and muscle, and provide a new way for somatic gene therapy of hemophilia B and other genetic diseases.展开更多
Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cD-NA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained w...Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cD-NA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained which covered the entire cFIX coding region. Furthermore, GINaCcIX (driven by hCMV promoter) and GlNaMBcLX (driven by MCK enhancer and β-actin promoter) were constructed using the retroviral vector backbone of GINa. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/106 cell/24 h (GINaCcIX) and 211 ng/106 cell/24 h (GINaMBcIX) respectively. Those data offered a promising result for further animal study.展开更多
基金Supported by Natural Youth Science Foundation of China,No.82104743Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Innovation Project,No KY2056.
文摘BACKGROUND Massive intragastric clotting(MIC) makes endoscopic therapy difficult in patients with acute upper gastrointestinal bleeding. Literature data on how to address this problem are limited. Here, we report on a case of massive stomach bleeding with MIC that was successfully treated endoscopically using an overtube of singleballoon enteroscopy.CASE SUMMARY A 62-year-old gentleman with metastatic lung cancer was admitted to the intensive care unit due to tarry stools and hematemesis of 1500 mL of blood during hospitalization. Emergent esophagogastroduodenoscopy revealed massive blood clots and fresh blood in the stomach with evidence of active bleeding.Bleeding sites could not be observed even by changing the patient’s position and aggressive endoscope suction. The MIC was successfully removed using an overtube connected with a suction pipe, which was inserted into the stomach with an overtube of a single-balloon enteroscope. An ultrathin gastroscope was also introduced through the nose into the stomach to guide the suction. A massive blood clot was successfully removed, and an ulcer with oozing bleeding at the inferior lesser curvature of the upper gastric body was revealed, facilitating endoscopic hemostatic therapy.CONCLUSION This technique appears to be a previously unreported method to suction MIC out of the stomach in patients with acute upper gastrointestinal bleeding. This technique could be considered when other methods are not available or if they fail to remove massive blood clots in the stomach.
基金supported by the National Natural Science Foundation of China(No.30600458)
文摘The clotting protein(CP) plays important and diverse roles in crustaceans,such as coagulation and lipid transportation.A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis(named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography.Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+,suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase in shrimp hemocytes.The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE.CP exists as disulfide-linked homodimers and oligomers.The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon,Farfantepenaeus paulensis and Litopenaeus vannamei;and similar to that of other decapods.The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight(MALDI-TOF/TOF) mass spectrometry.Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.
文摘Objective To explore the influence of styptic fiber on clotting time in rabbits so as to provide experiment data for its development.Methods Onto 0.1 mL aliquots of citrated anti-coagulant rabbit blood placed in a surfacial plate 25 ul of 0.2 mol·L-1 CaCl2 solution was dropped,and mixed well with glass stirrer;the resulting mixture was immediately capped with a piece of styptic fiber(test product group)or absorptive gelatin sponge(positive control group)of 2 cm diameter.Then,the surficial plate was rinsed with 30ml of purified water at 5,10,20,30,40 and 50 min after capping;the rinsings were allowed to stand for 1 h and were subjected to OD determination at a wavelength of 541 nm.The above procedure was repeated twice,the average value of the twice experiments was taken for evaluation of the hemostatic effect of test product.For negative control group,all procedures except for capping were same as the test product group.The haemostatic effect was judged by percent OD relative to OD at 0 min in negative control group(OD 0 min)(OD 0 min was considered as 100%);if OD value at a time was less than 80% of OD 0 min,it should be designated as primary clotting time(PCT),less than 20% as complete clotting time(CCT).Results The measured PCT was 20min for both negative and positive control groups;CCT was 50,30 and 5 min for negative control,positive control and test product groups,respectively,showing the test styptic fiber had a CCT 8 times shorter than untreated blood,10 times shorter than negative control and 6 times shorter than positive control.Conclusions The test styptic fiber has powerful hemostatic effect.
文摘Objective： To investigate the correlation of fibrinogen level and absorbance change in both PT and APTT clotting curves on BCSXP Analyzer. Methods：A serial of standard fibrinogen and 250 patient plasma samples with different qualities（normal, hemolysis, icterus, and lipemia） were run on BCSXP for assays PT, APTT and Fibrinogen. The absorbance change（DeltaA） from baseline to plateau in clotting curve was retrieved and analyzed on its correlation with the Fibrinogen result. Influence of plasma quality and PT/ APTT result on this correlation was also studied respectively. Results：Both PT-DeltaA and APTT-DeltaA showed good linear regres- sion with fibrinogen level in the sample, with R2 close to 0.90 in both standard and patient samples. Hemolysis（H）, itcterus（I） and lipemia（L） of the sample with valid clotting curves were found to have no significant difference in this correlation from normal（N） sample（R2： 0.83H, 0.92I, 0.81L and 0.79N in PT; 0.89H, 0.95l, 0.91L and 0.89N in APTT） in either PT or APTT curve. PT or APTT result also has little impact on this correlation（0.71 in range 7 - 10 sec, 0.56 inl0 - 20 sec, and 0.70 in 20 sec-; R2 in APTT： 0.88 in 20-30 sec, 0.92 in 30-40 sec, and 0.95 in 40 sec-）. Conclusion：The absorbance change in either PT or APTT clotting curve correlates well with the fibrinogen level in plasma, which is independent of plasma quality PT or APTT results. The absorbance change can be used as an alternative way to roughly estimate fibrinogen level in either PT or APTT clotting curve when the result of clauss-based fibrinogen measurement is not available.
文摘This paper presents a solution methodology for H<sub>∞</sub>-feedback control design problem of Heparin controlled blood clotting network under the presence of stochastic noise. The formulaic solution procedure to solve nonlinear partial differential equation, the Hamilton-Jacobi-Isaacs equation with Successive Galrkin’s Approximation is sketched and validity is proved. According to Lyapunov’s theory, with solutions of the nonlinear PDEs, robust feedback control is designed. To confirm the performance and robustness of the designed controller, numerical and Monte-Carlo simulation results by Simulink software on MATLAB are provided.
文摘We report the clinical case of a 40-year-old Italian woman, who soon after her booster vaccination with mRNA-1273 after the two previous vaccinations with BNT162b2, developed severe headache, high fever, and Musculo-skeletal pain. She went to the emergency department, where computerized tomography (CT) scans of chest and brain were performed, resulting in both negative for pathologic findings. On the contrary, white blood count was strongly lowered and D-dimer severely elevated. She improved after treatment with enoxaparin and the blood analyses returned in the normal range after ten days. This case supports the hypothesis that COVID-19 vaccines could increase blood clotting in some predisposed subjects. Therefore, we believe that robust and well-designed clinical trials, considering the evaluation of D-dimer levels, should be performed to eliminate any doubts on this issue.
文摘In this study, texture analysis method was used for the determination of rennet flocculation time (tfloc) and rennet clotting time (tclot) of rennet-induced reconstitued milk samples with different CaCl2 concentrations. The rennet flocculation time (RFT) and rennet clotting time (RCT) were also determined by using the Berridge test and sensory evaluation. The hardness value versus renneting time curves derived from texture analysis gave a good modified exponential relationship for each CaCl2 concentration and the curves were used to calculate flocculation time and clotting time parameters. It was found that the parameters (tfloc and tclot) appeared strongly correlated with RFT and RCT, respectively. Texture analysis was proved as a suitable method to control the rennet-induced coagulation and determine the rennet clotting time. It was also determined that enrichment of milk with CaCl2 leaded to a decrease in flocculation and clotting times and an increase in rate of clotting and gel hardness.
基金Project supported by the State High Technology Development Program and Technology and Development Program of Shanghai.
文摘Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the poten-tial to be optimized as a safe and effective therapeutic modality for hemophilia B.
基金This work was supported by a grant from the State High Technology Development Program 863-102-17-40
文摘To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of factor Ⅸ-deficie human primary skin fibroblasts, FDⅨ cells. The factor Ⅸ secretion rate of the infected FDⅨ cells was about 549 ng/10~6 cells/24h and over 75% of secreted factor Ⅸ was biologically active. We are convinced that this factor Ⅸ-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.
文摘Background Due to lack of point-of-care testing, the use of low-molecular-weight heparin （LMWH） therapy in some special patients is restricted. This study was designed to explore the effects of LMWH on clot rate （CR） and activated clotting time （ACT）, and to search for an appropriate method for bedside monitoring of anticoagulant activity of LMWH. Methods Thirty-two healthy volunteers were selected from the staff of Beijing Tongren Hospital. CR and ACT were measured with different reagents （glass beads, diatomite, kaolin and magnetic bar） on blood samples spiked with increasing concentrations of LMWH （dalteparin, 0.2-1.8 IU/ml）. Correlations between concentrations of LMWH and values of CR and ACT were analysed based on the data obtained and regression analysis was performed to establish a regression equation. Results With the increase in doses of dalteparin, CR values reduced gradually. The values of CR of four reagents （glass beads, diatomite, kaolin and magnetic bar） were 20.4-4.5 IU/min, 27.4-6.9 IU/min, 27.5-7.9 IU/min and 7.8-0.1 IU/min respectively and an linear relationship was observed between the CR values and dalteparin concentrations （P〈0.05）. The values of ACT were 173-615 seconds, 130-270 seconds, 123-226 seconds, 337-1411 seconds respectively, which showed a linear regression between the ACT values and dalteparin concentrations （P〈0.01）. Differences in slope of the regression curves of ACT were observed with all the reagents tested （glass beads 248.2 s/IU, diatomite 74.8 s/IU, kaolin 58.2 s/IU and magnetic bar 1112.2 s/IU, P〈0.01）. While the minimum concentration of dalteparin was 0.2 IU/ml, 0.4 IU/ml, 1.4 IU/ml and 0.2 IU/ml separately, the ACT values of the four coagulants （glass beads, diatomite, kaolin and magnetic bar） were beyond the normal limit and showed a noticeable increase respectively （P〈0.01）. Conclusions This study showed that there was an excellent linear relationship between the CR and ACT values and dalteparin concentrations for all the four reagents （glass beads, diatomite, kaolin and magnetic bar） in vitro. The sensitivity of different coagulation reagents to LMWH different. Choosing a suitable reagent, both CR and ACT were possible to be used as a convenient bedside test for LMWH.
基金State High-Technology Development ProgramState Commission of Education, PRC
文摘DNA-DNA molecular hybridization is commonly used in the detection of transferred foreign gene in transgenic mice, which is an effective tool in the detection of transgenic mice due to its high sensitivity and reliability. The method, however, is very laborious and time-consuming, and requires a large number of DNA samples
文摘THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system is costly, laborious and tim-cosuming. In 1994,Johanna et al. reported that secretion of a foreign protein, human growth hormone (hGH),in milk had been achieved after direct introduction of the cDNA into the mammary gland ofgoats by replication-defective retroviral vector. In that work, the in vivo expression of
基金the State High-Technology Development Program of China.
文摘Culture of patient somatic cells, transfected with therapeutic genes into genomic DNA with retroviral vectors, followed by colony selection, amplification, and reimplantation of transformed cells into patient, has been Widely used for clinical trial of gene therapy in the past years. But the disadvantages of this protocol are obvious. (i) To a great extent the expression level of the transfected cells depends on the different integration sites,which cause the various expression rates from different colonies; besides, the life span
基金supported by the State High Technology Development Program(863).
文摘Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.
基金Project supported by grant from the State High Technology Development Program 863-102-17-40 and grant from State Education Commission Doctoral Foundation 32780266.
文摘Clotting factor Ⅸ is an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder——hemophilia B, or Christmas disease. Factor Ⅸ is synthesized in liver hepatocytes where it undergoes complex vitamin K-dependent post-translational modifications which are essential for the activation of factor Ⅸ. Recently, several groups have cloned factor Ⅸ cDNA and reported that, in the presence of vitamin K, active factor Ⅸ could be secreted in human and mouse fibroblasts,
基金Japan Society for the Promotion of Science,Grant/Award Number:22K09110。
文摘The murine model of subarachnoid hemorrhage(SAH)is a valuable experimental tool for investigating molecular and cellular mechanisms,and the endovascular filament perforation technique can be used to simulate prominent pathophysiological features observed after human SAH;however,current validation methods for assessing an appropriate SAH model are limited.Here,we introduce a simple procedure for se-lecting a mouse model of diffuse SAH.SAH was induced in 24 mice using a standard filament perforation method.After confirming survival at 24 h,SAH was scored 0-1 based on T2*-weighted images on whole-brain magnetic resonance imaging(MRI)and visual surveillance of the cisterna magna(CM)through the dura mater.The CM-based SAH grading correlated well with a reference parameter defined by extracted brain(r^(2)=0.53,p<0.0001).The receiver operating characteristic curve revealed a sensi-tivity of 85%and a specificity of 91%for detecting diffuse SAH,with a similar area under the curve(0.89±0.06[standard error of the mean])as the MRI-based grading(0.72±0.10,p=0.12).Our data suggest that confirming an SAH clot in the CM is a valuable way to select a clinically relevant diffuse SAH model that can be used in future experimental studies.
基金Project supported by the "863" State High Technology Development Program.
文摘Hemophilia B, a serious bleeding disorder, is an inherited X chromosome-linked diseasecaused by the deficiency or inactiveness of human clotting factor IX (FIX). The conven-tional clinical treatment of plasma infusion is expensive and associated with a high risk
基金Project supported by High Technique Research of the State.
文摘Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has been successfully introduced in gene diagnosis, mutation detection, polymorphism analysis and nucleotide sequencing. In the study of gene transfer and gene therapy, it is necessary to analyse DNA in the
文摘Primary rabbit skin fibroblasts (RSF) containing lacZ reporter gene or human clotting factor IX (hFIX) cDNA could long termly exist and be expressed after being implanted in rabbit muscles. Among them, the beta glactosidase expression lasted at least 3 months, and hFIX expression lasted 1 month. These results will be helpful to organically combine the advantages of both skin fibroblasts and muscle, and provide a new way for somatic gene therapy of hemophilia B and other genetic diseases.
基金Project Supported by the state High Technology Development Program
文摘Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cD-NA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained which covered the entire cFIX coding region. Furthermore, GINaCcIX (driven by hCMV promoter) and GlNaMBcLX (driven by MCK enhancer and β-actin promoter) were constructed using the retroviral vector backbone of GINa. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/106 cell/24 h (GINaCcIX) and 211 ng/106 cell/24 h (GINaMBcIX) respectively. Those data offered a promising result for further animal study.
