Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic ...Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). Methods:A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria.This recombinant adeno-associated virus type 2/serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR.The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigeninlabeled H1 probe.Expression of ND4 in mitochondria was evaluated by immunofluorescence. Results: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0×1011 vg/mL,confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. Conclusion:A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.展开更多
The study aimed to detect the expression of the Th1-specific cell surface protein T cell Ig and mucin domain-containing molecule-3(Tim-3)mRNA peripheral blood lymphocytes isolated from asthmaticin patients and to exami...The study aimed to detect the expression of the Th1-specific cell surface protein T cell Ig and mucin domain-containing molecule-3(Tim-3)mRNA peripheral blood lymphocytes isolated from asthmaticin patients and to examine the correlation among Tim-3 mRNA,interleukin-4(IL-4),interferon-γ(IFN-γ)level,and FEV1/FVC(force expiratory volume in one second/forced vital capacity)to explore the role of Tim-3 in the development and progression of asthmatic inflammation.Tim-3 mRNA expression was detected by reverse transcription polymerase chain reaction(RT-PCR).The IL-4 and IFN-γlevels were determined by using enzyme-linked immunosorbent assay(ELISA).The correlation among Tim-3 mRNA,IL-4,IFN-γlevel,and pulmonary ventilatory capacity was analyzed.The expres-sion of Tim-3 mRNA in patients with acute asthma exacerbation was 0.39�0.06,significantly higher than that in patients at the remission stage and controls(0.18�0.05 and 0.07�0.03,P<0.05).The level of IL-4 in patients with acute asthma exacerbation was 68.42�10.54,significantly higher than that in the patients at the remission stage and controls(41.83�9.37 and 32.75�8.16,P<0.05).The level of IL-4 in patients in remission was significantly higher than that in controls(P<0.05).The level of IFN-γin patients with acute asthma exacerbation was 65.74�7.85,significantly lower than that in patients in remission and the control group(120.84�11.62 and 139.65�13.47,P<0.05).The level of IFN-γin patients in asthma remission was significantly lower than that in controls(P<0.05).Tim-3 mRNA expression was positively correlated with the level of IL-4(r=0.68,P<0.05)and negatively with the level of IFN-γand pulmonary ventilatory capacity(r=–0.85,r=–0.76,both P<0.01).The increased expression of Tim-3 mRNA in peripheral blood lymphocytes might be involved in the development and progression of asthmatic inflammation.展开更多
The lacrimal endoscope is applied to the diagnosis and therapy of the disorders of the lacrimal system in combination with laser or micro drills under orthophoria.The changes of mucous membranes,the characteristics of...The lacrimal endoscope is applied to the diagnosis and therapy of the disorders of the lacrimal system in combination with laser or micro drills under orthophoria.The changes of mucous membranes,the characteristics of pathological changes and the predilection sites of lacrimal system diseases were initially approached.One hundred and forty six pairs of eyes of 128 patients with lacrimal system disease were observed by the lacrimal endoscope in the Ophthalmology Department of Tongji Hospital from June 2006 to March 2007.The dynamic changes in mucous membranes,lesion sites,secreted substances and formation of membrane could be observed under orthophoria.Combined with laser or micro drill,the endoscope was applied to the therapy of lacrimal system disorders and the difference before and after the treatment was observed.Results are as follows:(1)The examination and therapy using the lacrimal endoscope were completed under topical anesthesia in 122 patients,and 6 patients of neonatorum dacryocystitis were examined and treated under general anaesthesia.All patients reported painless.(2)Sharp images of the lacrimal system were obtained by the endoscope.Normal lacrimal mucosal membrane was smooth and light pink,expanded and unobstructed during irrigation.In chronic dacryocystitis patients,the inhomo-geneous colour of mucosal membranes was red and white,with different degrees offibrotic membranes at the superior,middle and inferior parts of the nasolacrimal canals and secreted substances at sac could be observed.The lacrimal ducts could not be expanded and obstructed during irrigation.The patients with lacrimal system obstruction had different extents of membrane formation,and stenosis or complete obstruction of the lacrimal duct could be observed,and the corresponding mucosal membrane was not smooth which could not be expanded and obstructed during irrigation.(3)After the treatment by the endoscope combined with laser or micro drill,the major proliferation of the membrane disappeared and the lacrimal duct was unobstructed during irrigation.The cure rate and effective rate were 80.1%and 93.1%,respectively.The lacrimal endoscope is a new method in the diagnosis of lacrimal system diseases.Through a combination with laser or micro drill to carry out the therapy under orthophoria,it will bring a great change to the diagnosis and therapy of lacrimal system diseases.展开更多
基金financially supported by National Natural Science Fund.Molecule mechanism of the resistance of HRD1 against diabetic retinopathy (NO.81271015)Molecule mechanism of the resistance of P58IPK against diabetic retinopathy (NO.30872823)
文摘Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). Methods:A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria.This recombinant adeno-associated virus type 2/serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR.The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigeninlabeled H1 probe.Expression of ND4 in mitochondria was evaluated by immunofluorescence. Results: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0×1011 vg/mL,confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. Conclusion:A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.
基金supported by the National Natural Science Foundation of China(Grant No.30801260).
文摘The study aimed to detect the expression of the Th1-specific cell surface protein T cell Ig and mucin domain-containing molecule-3(Tim-3)mRNA peripheral blood lymphocytes isolated from asthmaticin patients and to examine the correlation among Tim-3 mRNA,interleukin-4(IL-4),interferon-γ(IFN-γ)level,and FEV1/FVC(force expiratory volume in one second/forced vital capacity)to explore the role of Tim-3 in the development and progression of asthmatic inflammation.Tim-3 mRNA expression was detected by reverse transcription polymerase chain reaction(RT-PCR).The IL-4 and IFN-γlevels were determined by using enzyme-linked immunosorbent assay(ELISA).The correlation among Tim-3 mRNA,IL-4,IFN-γlevel,and pulmonary ventilatory capacity was analyzed.The expres-sion of Tim-3 mRNA in patients with acute asthma exacerbation was 0.39�0.06,significantly higher than that in patients at the remission stage and controls(0.18�0.05 and 0.07�0.03,P<0.05).The level of IL-4 in patients with acute asthma exacerbation was 68.42�10.54,significantly higher than that in the patients at the remission stage and controls(41.83�9.37 and 32.75�8.16,P<0.05).The level of IL-4 in patients in remission was significantly higher than that in controls(P<0.05).The level of IFN-γin patients with acute asthma exacerbation was 65.74�7.85,significantly lower than that in patients in remission and the control group(120.84�11.62 and 139.65�13.47,P<0.05).The level of IFN-γin patients in asthma remission was significantly lower than that in controls(P<0.05).Tim-3 mRNA expression was positively correlated with the level of IL-4(r=0.68,P<0.05)and negatively with the level of IFN-γand pulmonary ventilatory capacity(r=–0.85,r=–0.76,both P<0.01).The increased expression of Tim-3 mRNA in peripheral blood lymphocytes might be involved in the development and progression of asthmatic inflammation.
基金supported by the Fund of Tongji Hospital New Technique and Therapy in 2006.
文摘The lacrimal endoscope is applied to the diagnosis and therapy of the disorders of the lacrimal system in combination with laser or micro drills under orthophoria.The changes of mucous membranes,the characteristics of pathological changes and the predilection sites of lacrimal system diseases were initially approached.One hundred and forty six pairs of eyes of 128 patients with lacrimal system disease were observed by the lacrimal endoscope in the Ophthalmology Department of Tongji Hospital from June 2006 to March 2007.The dynamic changes in mucous membranes,lesion sites,secreted substances and formation of membrane could be observed under orthophoria.Combined with laser or micro drill,the endoscope was applied to the therapy of lacrimal system disorders and the difference before and after the treatment was observed.Results are as follows:(1)The examination and therapy using the lacrimal endoscope were completed under topical anesthesia in 122 patients,and 6 patients of neonatorum dacryocystitis were examined and treated under general anaesthesia.All patients reported painless.(2)Sharp images of the lacrimal system were obtained by the endoscope.Normal lacrimal mucosal membrane was smooth and light pink,expanded and unobstructed during irrigation.In chronic dacryocystitis patients,the inhomo-geneous colour of mucosal membranes was red and white,with different degrees offibrotic membranes at the superior,middle and inferior parts of the nasolacrimal canals and secreted substances at sac could be observed.The lacrimal ducts could not be expanded and obstructed during irrigation.The patients with lacrimal system obstruction had different extents of membrane formation,and stenosis or complete obstruction of the lacrimal duct could be observed,and the corresponding mucosal membrane was not smooth which could not be expanded and obstructed during irrigation.(3)After the treatment by the endoscope combined with laser or micro drill,the major proliferation of the membrane disappeared and the lacrimal duct was unobstructed during irrigation.The cure rate and effective rate were 80.1%and 93.1%,respectively.The lacrimal endoscope is a new method in the diagnosis of lacrimal system diseases.Through a combination with laser or micro drill to carry out the therapy under orthophoria,it will bring a great change to the diagnosis and therapy of lacrimal system diseases.
