摘要
目的:观察骨髓间充质干细胞移植治疗急性心肌梗死对血管内皮功能的影响。方法:实验于2005-09/2006-06在唐山工人医院中心实验室完成。①选用清洁级健康近交系SD大鼠40只,随机数字表法分为假手术组、模型对照组、干细胞移植组、细胞培养基组,10只/组。另取1只大鼠用于骨髓间充质干细胞的提取。②大鼠处死后无菌条件下分离股骨和胫骨,剔除肌肉、骨膜,剪一侧骨端,用10mL无血清DMEM低糖培养基冲出骨髓细胞后,收集冲洗液制成单细胞悬液,过滤离心,弃上清液,密度梯度法分离培养骨髓间充质干细胞。待细胞80%贴满培养瓶底时,用乙二胺四乙酸和胰蛋白酶混合消化传代。将4,6-二脒-2-苯基吲哚以50mg/L浓度加入第3代细胞培养基中,制成细胞悬液备用。③术前各组大鼠均行气管插管,建立急性心肌梗死模型,以远端供血区心肌组织颜色苍白、心电图检测Ⅱ导联ST段持续抬高为模型建立成功的标志。假手术组仅开胸予以前降支穿线但不结扎。④造模成功后1～3h,干细胞移植组用微量注射器吸取4,6-二脒-2-苯基吲哚标记好的第3代骨髓间充质干细胞悬液50μL,直接注入梗死区边缘的心肌组织。细胞培养基组按干细胞移植组的方法于相同部位注射等量无血清DMEM低糖培养基(pH值6.9)。⑤造模后4周处死各组大鼠,切取心脏组织,于梗死边缘区心肌组织制作切片,荧光显微镜下观察4,6-二脒-2-苯基吲哚标记的供体细胞。留取2mL全血,离心后抽取血清500μL,酶联免疫吸附法测定血清中细胞间黏附分子1与血管内皮细胞黏附分子1的含量。结果:40只大鼠均进入结果分析。①体外培养骨髓间充质干细胞的性状观察:从正常大鼠骨髓中分离培养的原代骨髓间充质干细胞,24～48h贴壁生长,短棒状;7～10d达到80%～90%融合;传至第3代骨髓间充质干细胞分布均匀,呈纺锤形。②各组心脏标本4,6-二脒-2-苯基吲哚标记移植细胞的存活状况:干细胞移植组可见成团及散在的4,6-二脒-2-苯基吲哚标记的阳性细胞,其余3组心脏标本中均未见荧光细胞。③各组大鼠血清中细胞间黏附分子1与血管内皮细胞黏附分子1的含量检测:与假手术组比较,模型对照组、细胞培养基组、干细胞移植组细胞间黏附分子1与血管内皮细胞黏附分子1的含量均明显升高(P<0.01或0.05);与模型对照组比较,干细胞移植组细胞间黏附分子1与血管内皮细胞黏附分子1的含量均有所降低[(3.37±0.14),(2.10±0.15)μg/L;(2.64±0.13),(1.74±0.06)μg/L;P均<0.05]。结论:骨髓间充质干细胞移植到实验性大鼠心肌梗死模型后成功存活,能降低血清中增高的细胞间黏附分子1与血管内皮细胞黏附分子1含量,提示骨髓间充质干细胞移植可能通过降低黏附分子的表达来改善血管内皮功能。
AIM： To explore the influence of mesenchymal stem cells （MSCs） transplantation for acute myocardial infarction （AMI） on the endothelial function. METHODS： The experiment was conducted in the Central Laboratory of Tangshan Worker＇s Hospital from September 2005 to June 2006. ①TotaUy 40 SD rats of clean grade of healthy inbred strain were selected, and were divided into sham operation group, model control group, MSCs transplantation group and medium group through the random digits table method, 10 rats in each group. Another rat was used to extract the MSCs. ②After the rats were killed sterilely, the fems and tibs were isolated clearly, and then mon-side extremities were cut off after removing muscle and periosteum. The MSCs were run-out by serum-free low carbohydratal DMEM medium （10 mL）, and unicell suspension was collected, filtered and efferenced, and the supematant fluid was refused, thus the MSCs were isolated, cultured by density gradient method. The pepsis and passage were executed by EDTA and pancreas protease after the MSCs were 80 percentage of culture flask. The MSCs of the third generation were labeled by 50 mg/L 4, 6-diaidino-2-phenylindole （DAPI）, and the cell supematant fluid was produced. ③The preoperative lifesaver were conducted in every group, then the AMI rats model were made. The successful sign： the cardiac muscular tissues were pale in the blood-supply region of distal end and lasting raise ST sect on lead Ⅱ of electrocardiogram detection. In addition, the rats in the sham operation group were braided only, not be obstructed the anterior descending branch. ④1-3 hours after the AMI rats model were made, the MSCs cells suspension （50 μL） labeled by DAPI in the third generation was imbibed with microinjector, were rejected in different myocardial infarction region of rats in the MSCs transplantation group. At the same time, in cell medium group, serum-free low carbohydratal DMEM medium （50μL, pH=6.9） were rejected in same region according to the method of the MSCs transplantation group. ⑤The rats were killed after 4 weeks, and the cardiac tissues were made into sections, then the DAPI labeling MSCs were observed by fluorescence microscope. The whole blood （2 mL） was remained and serum （500 μL） was obtained after centrifuge, then the contents of intercellular adhesion molecules-1 （ICAM-1） and vascular cell adhesion molecules-1 （VCAM-1） in serum were measured by enzyme-linked assay （ELISA）. RESULTS： Totally 40 rats were involved in the result analysis. ①characteristic observation of the MSCs cultured in vitro. the MSCs from bone marrow of normal rats grew adheredly after 24-48 hours, became short rod-shape; and became confluent （80%-90%） each other up to 7-10 days; Then became well-distributed and fusiform until the third era. ②survival conditions of the MSCs： Agglomerate DAPI labeled cells were seen in MSCs transplantation group, and were not seen in the other groups. ③amount detection of ICAM-1 and VCAM-1 in each group： The amount of ICAM-1 and VCAM-1 in model control group, cell media and MSCs transplantation group were significantly higher than that in sham operation group （P 〈 0.01 or P 〈 0.05 ）, while MSCs transplantation group could markedly reduce the amount of ICAM-1 and VCAM-1 as compared with the model control group [（3.37±0.14）, （2.10±0.15） μg/L; （2.64±0.13）, （1.74±0.06） μg/L;P〈 0.05]. CONCLUSION： The implanted MSCs labeled with DAPI survive. The MSCs transplantation treatment can reduce the amount of ICAM-1 and VCAM-1 in the serum of AMI rats. It indicates that the MSCs transplantation treatment can improve endothelial function according to decreasing the expression of intercellular adhesion molecules.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第11期2087-2090,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家高新技术发展计划(八六三计划)(2005AA205232)~~
