可注射水凝胶携带Bcl-2和VEGF双基因共表达MSC移植治疗大鼠心肌梗死及其机制研究

发布时间：2018-04-25 11:20

本文选题：骨髓间充质干细胞 + 可注射水凝胶　；参考：《南方医科大学》2017年博士论文

【摘要】：背景:骨髓间充质干细胞(Mesenchymal Stem Cell,MSC)移植治疗心肌梗死(MyocardialInfarction,MI)极有发展前景,但临床试验结果显示治疗效果微弱,移植细胞在缺血心肌极低的滞留率和存活率是主要原因之一。对MSC进行基因修饰或使用生物材料来承载后移植可改善这一问题。Bcl-2(B-cell lymphoma 2,Bcl-2)基因能抑制细胞凋亡。VEGF(Vascular Endothelial Growth Factor,VEGF)基因能促血管生成。可注射水凝胶(Injectable Hydrogel,IH)具有可注射性、生物降解性和组织细胞相容性,能承载细胞进行移植。目的:使用IH联合Bcl-2和VEGF双基因修饰拟提高MSC移植对MI的疗效,为临床使用提供实验基础及理论依据。方法和结果:1、PCR扩增获取Bcl-2和VEGF基因并使用T2A序列连接以实现双基因的共表达。构建能同时携带Bcl-2和VEGF基因的慢病毒载体并筛选构建能稳定过表达Bcl-2和VEGF基因的SD大鼠MSC。荧光定量PCR和Western blot结果证实Bcl-2和VEGF双基因共表达MSC对比Bcl-2或VEGF单基因表达MSC对Bcl-2和VEGF基因有更高的表达。CCK-8结果说明Bcl-2和VEGF双基因共表达MSC对比Bcl-2或VEGF单基因表达MSC增殖更快。2、体外糖氧剥夺(Oxygen and Glucose Deprivation,OGD)模型模拟体内缺血缺氧微环境,实验分为五组:MSC-GFP组、MSC-VEGF组、MSC-Bcl-2组、MSC-BV 组、MSC-GFP 正常培养组。AnnexinV-FITC/PI 流式细胞术和 Western blot检测凋亡相关蛋白Bax、Bcl-2、Cleaved-caspase-3,结果表明OGD诱发了MSC的凋亡,Bcl-2或VEGF单基因修饰的MSC凋亡减少,Bcl-2和VEGF双基因修饰的MSC-BV较Bcl-2或VEGF单基因修饰的MSC凋亡减少更显著。ELISA结果显示Bcl-2和VEGF双基因修饰能增强OGD环境下MSC旁分泌VEGF,bFGF,HGF 和 IGF-1。3、IH携带Bcl-2和VEGF双基因共表达MSC进行对MI大鼠的移植治疗,实验分为六组:SHAM组、PBS组、IH组、MSC组、MSC+BV组、MSC+BV+IH组。细胞移植治疗4周后心脏超声结果提示MSC+BV+IH组的心脏收缩功能改善更显著。HE和Masson染色结果提示MSC+BV+IH组的梗死面积显著缩小。cTnT免疫荧光结果提示MSC+BV+IH组心肌存活更多。TUNEL和Western blot结果提示MSC+BV+IH组心肌细胞凋亡减少更显著。vWF和a-SMA免疫荧光结果提示MSC+BV+IH组血管密度更高。CM-Dil标记MSC移植提示MSC+BV+IH组的细胞滞留改善更明显。4、原代培养SD乳鼠心肌细胞,与MSC使用Transwell共培养系统置于1%02缺氧处理24h,实验分为四组:心肌细胞正常培养组、心肌细胞缺氧组、心肌细胞+MSC+GFP缺氧组、心肌细胞+MSC+BV缺氧组。采用Annexin V-FITC/PI流式细胞术、Western blot检测凋亡蛋白Cleaved-capase-3、TUNEL分析心肌细胞凋亡,结果表明与Bcl-2和VEGF双基因共表达MSC共培养更能减少缺氧状态下心肌细胞的凋亡。结论:1、成功构建了 Bl-2和VEGF双基因共表达重组慢病毒载体;筛选构建了Bcl-2和VEGF双基因共表达MSC;对比Bcl-2或VEGF单基因表达MSC,其对目的基因Bcl-2和VEGF有更高的表达,更快的增殖速度。2、Bcl-2和VEGF双基因共表达MSC对比Bcl-2或VEGF单基因表达MSC在体外糖氧剥夺环境下发挥更好的自我保护和旁分泌作用。3、Bcl-2和VEGF双基因共表达MSC移植对心肌梗死有更好的治疗作用,可注射水凝胶的承载移植进一步增强了 MSC的治疗作用。4、Bcl-2和VEGF双基因共表达MSC体外通过旁分泌作用保护缺氧心肌细胞。
[Abstract]:Background: Mesenchymal Stem Cell (MSC) transplantation is very promising for the treatment of myocardial infarction (MyocardialInfarction, MI), but the results of clinical trials show that the treatment effect is weak. The very low retention rate and survival rate of the transplanted cells in the ischemic myocardium is one of the main reasons. Gene modification to MSC or the use of biological materials is used. Transplantation can improve this problem.Bcl-2 (B-cell Lymphoma 2, Bcl-2) gene can inhibit apoptosis.VEGF (Vascular Endothelial Growth Factor, VEGF) gene can promote angiogenesis. Injectable hydrogel (Injectable Hydrogel) has injectable, biodegradability and tissue cell compatibility, can carry cells for transplantation. IH combined with Bcl-2 and VEGF double gene modification to improve the effect of MSC transplantation on MI, provide experimental basis and theoretical basis for clinical use. Methods and results: 1, PCR amplification to obtain Bcl-2 and VEGF genes and the use of T2A sequence connection to realize the co expression of double genes. The results of MSC. fluorescence quantitative PCR and Western blot in SD rats that could stabilize the expression of Bcl-2 and VEGF genes confirmed that Bcl-2 and VEGF double genes co expressed MSC contrastive Bcl-2 or VEGF monogenic expression. .2, Oxygen and Glucose Deprivation (OGD) model was used to simulate the microenvironment of ischemic anoxia in vivo. The experiment was divided into five groups: MSC-GFP group, MSC-VEGF group, MSC-Bcl-2 group, MSC-BV group, MSC-GFP normal culture group,.AnnexinV-FITC/PI flow cytometry and apoptosis related protein The results showed that the apoptosis of MSC was induced by OGD, and the apoptosis of MSC modified by Bcl-2 or VEGF was reduced. The MSC-BV of Bcl-2 and VEGF modified MSC-BV was more significant than that of Bcl-2 or VEGF single gene modification. The experiment was divided into six groups: group SHAM, group PBS, group PBS, group IH, group IH, group MSC, MSC+BV group, MSC+BV+IH group. The cardiac ultrasound results suggested that the cardiac contractile function of the MSC+BV+IH group was improved more notably by the.HE and Masson staining results, suggesting that the infarct area of the MSC+BV+IH group was significantly reduced. The results of pestilence fluorescence suggested that the myocardium survived more.TUNEL and Western blot in the MSC+BV+IH group, suggesting that the cardiomyocyte apoptosis decreased more significantly in MSC+BV+IH group.VWF and a-SMA immunofluorescence results suggested that the blood vessel density of MSC+BV+IH group was higher and.CM-Dil marked MSC transplantation suggested that the cell retention of MSC+BV+IH group was more obvious. Cell, and MSC Transwell co culture system was placed in 1%02 hypoxia treatment 24h, and the experiment was divided into four groups: normal culture group of myocardial cells, hypoxia group of myocardial cells, +MSC+GFP anoxic group of myocardium, +MSC+BV anoxic group of myocardial cells, Annexin V-FITC/PI flow cytometry, Western blot detection of apoptotic protein Cleaved-capase-3, TUNEL analysis myocardium The results showed that co expression of Bcl-2 and VEGF double genes co expressed MSC co culture could reduce the apoptosis of cardiomyocytes in anoxic state. Conclusion: 1, Bl-2 and VEGF double genes were successfully constructed to co express recombinant lentivirus vector, and Bcl-2 and VEGF double genes were constructed to co express MSC, and Bcl-2 or VEGF single gene expression MSC was compared to the target gene. Bcl-2 and VEGF have higher expression, faster proliferation rate.2, Bcl-2 and VEGF double genes co express MSC to compare Bcl-2 or VEGF single gene expression MSC to play a better self protection and paracrine role in the environment of oxygen deprivation in vitro.3, Bcl-2 and VEGF double gene co expression transplantation has a better therapeutic effect on myocardial infarction, and can be injected hydrogel. The bearing transplantation further enhanced the therapeutic effect of MSC..4, Bcl-2 and VEGF coexpressed MSC in vitro to protect hypoxic cardiomyocytes through paracrine action.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R542.22

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