小型猪成体心肌干细胞的体外培养及鉴定

发布时间：2018-05-17 10:58

本文选题：心肌干细胞 + 细胞培养　；参考：《天津医科大学》2015年硕士论文

【摘要】：目的:心肌干细胞再生疗法是心肌梗死的新型治疗方法,但是心肌干细胞数量少,扩增难度大,其体外培养方法并没有统一的标准,国内许多实验室获得的心肌干细胞未能实现其多向分化潜能的鉴定,从而尚不能作为心肌干细胞的有力佐证。本研究旨在探讨一种高效、稳定的小型猪心肌干细胞体外培养方法,并且对其进行全面系统的鉴定,为心肌梗死的细胞生物疗法奠定基础。方法:选取成年巴马小型猪,雌性,体重25-30kg。通过心脏外科手术经右侧第二肋间做小切口钳取右心耳组织,用组织块培养法获取原代心肌干细胞,以心肌干细胞培养液进行培养,视细胞生长情况进行传代。选取分选纯化后的第3代细胞行流式检测鉴定细胞表型:c-kit、CD34、CD45、CD31、CD90、Lineage;行Western Blot检测心肌早期特异性转录因子Nkx2.5和GATA-4的表达;经诱导剂诱导后行免疫荧光检测CX43、cTnT、α-SMA、vWF的表达以观察心肌干细胞的多向分化潜能。结果:1.从成年小型猪右心耳组织中分离的原代细胞于倒置显微镜下观察呈“小、圆、亮”的特征,生长缓慢,传代后细胞贴壁生长,形态逐渐变成梭形,较原代易于生长。2.纯化后细胞经流式细胞仪鉴定细胞表型为c-kit+CD34-CD45-CD31-CD90-Lineage-。3.Western Blot检测结果示心肌早期特异性转录因子Nkx2.5和GATA-4表达阳性。4.纯化后细胞经不同诱导剂诱导培养后,能够表达:心肌细胞特异性蛋白CX43和cTnT,平滑肌细胞特异性蛋白α-SMA,以及内皮细胞特异性蛋白v WF。结论:1.自成年小型猪右心耳组织成功分离获得细胞表型为c-kit+CD34-CD45-CD31-CD90-Lineage-的细胞,阳性表达干细胞表面标记物。2.本研究获得的细胞能够表达心肌早期特异性转录因子Nkx2.5和GATA-4,提示该细胞具备调控自身向心肌细胞早期分化的能力。3.经诱导剂诱导培养后细胞能够表达CX43、cTnT、α-SMA、v WF等特异性标记蛋白,提示该细胞具有向心肌细胞、平滑肌细胞及内皮细胞等多方向分化的潜能。4.本研究自原代组织块中分离、培养、纯化所得细胞经流式细胞仪、Western Blot测定、多向分化潜能检验等过程鉴定为心肌干细胞。本研究探索出一种高效、统一的小型猪心肌干细胞体外培养及鉴定方法,对于临床治疗具有指导意义。
[Abstract]:Objective: myocardial stem cell regeneration therapy is a new treatment for myocardial infarction, but the number of myocardial stem cells is small and the expansion is difficult. Myocardial stem cells obtained in many laboratories in China have not been able to realize the identification of their multidirectional differentiation potential, so they can not be used as evidence of myocardial stem cells. The purpose of this study was to explore an efficient and stable culture method for myocardial stem cells of miniature pigs in vitro and to identify them systematically and to lay a foundation for cell biotherapy of myocardial infarction. Methods: adult Bama miniature pig, female, weight 25-30 kg. Primary myocardial stem cells were obtained from right auricular tissue by small incision forceps between the right second ribs after cardiac surgery. Primary myocardial stem cells were obtained by tissue block culture and cultured in the medium of myocardial stem cells, and then subcultured according to the condition of cell growth. After the third generation of purified cells were selected, the phenotype of the cells was identified by flow cytometry, the expression of specific transcription factors (Nkx2.5 and GATA-4) and the expression of specific transcription factors (Nkx2.5 and GATA-4) in the myocardium were detected by Western Blot, and the phenotype of the cells was identified by flow cytometry (FCM), and the expression of specific transcription factors (Nkx2.5 and GATA-4) was detected by Western Blot. The expression of CX43 cTnT, 伪 -SMA-vWF was detected by immunofluorescence after induction to observe the multidirectional differentiation potential of myocardial stem cells. The result is 1: 1. The primary cells isolated from the right atrial appendage of adult miniature pigs were observed under inverted microscope as "small, round and bright". The cells grew slowly. After passage, the cells became fusiform and grew more easily than the primary cells. The phenotype of the purified cells was identified by flow cytometry as c-kit CD34-CD45-CD31-CD90-Lineage-.3.Western Blot. The results showed that the expression of Nkx2.5 and GATA-4 was positive. 4. The purified cells were induced by different inducers to express cardiomyocyte specific protein CX43 and cTnT, smooth muscle cell specific protein 伪 -SMAand endothelial cell specific protein v WF. Conclusion 1. The cell phenotype of c-kit CD34-CD45-CD31-CD90-Lineage- was successfully isolated from adult miniature pig's right atrial appendage, and the positive expression of stem cell surface marker. 2. The cells obtained in this study were able to express Nkx2.5 and GATA-4, suggesting that the cells have the ability to regulate their early differentiation into cardiomyocytes. The cells were induced to express CX43 cTnT, 伪 -SMAV WF and other specific proteins, which indicated that the cells had the potential of differentiating into cardiomyocytes, smooth muscle cells and endothelial cells in many directions. The cells isolated, cultured and purified from primary tissue mass were identified as myocardial stem cells by flow cytometry (FCM) Western Blot assay and multidirectional differentiation potential test. In this study, an efficient and uniform method for the in vitro culture and identification of miniature porcine myocardial stem cells was explored, which is of guiding significance for clinical treatment.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.22

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