经HIF-1α基因修饰的心脏干细胞移植对心肌梗死后心衰大鼠心功能的影响

发布时间：2018-04-16 02:01

本文选题：细胞培养 + 流式细胞术　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:经缺氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)基因修饰的心脏干细胞移植与单纯心脏干细胞移植相比,对心衰大鼠心功能、心脏干细胞存活率、梗死区边缘毛细血管、心肌细胞凋亡的影响。方法:1心脏干细胞(cardiac stem cells,CSCs)培养:采用1日龄SD雄性大鼠进行心脏干细胞分离鉴定与纯化,进行传代培养心脏干细胞。2基因重组腺病毒感染心脏干细胞;采用RT-qPCR法检测心脏干细胞HIF-1αmRNA表达。3模型制备及分组:将24只SD大鼠随机等分为3组:缺氧诱导因子1α修饰的心脏干细胞组、单纯心脏干细胞组和模型组,以开胸结扎冠脉前降支的方法制备心肌梗死后心衰模型,2周后分别注射经基因修饰的心脏干细胞、单纯心脏干细胞及PBS液。4观察指标:4周后进行结果采集:超声测定心功能;荧光显微镜下观察CSCs存活情况并计算CSCs密度;HE染色法评估梗死边缘区心肌病理学改变;TUNEL法检测梗死边缘区心肌细胞凋亡率;Western Blot法检测梗死边缘VEGF蛋白表达情况;免疫组化染色检测CD31的表达,计算梗死边缘区毛细血管密度;分析左心室射血分数差值与心脏干细胞存活密度相关性。结果:1原代培养的大鼠心脏组织块7~8天后,圆形、体积小、折光性强的心脏干细胞开始从组织块边缘爬出;2通过抗c-kit抗体和链亲和素-FITC标记,在波长490 nm可见光激发后,可清晰见到被FITC显影的c-kit+CSCs,经流式细胞仪,c-kit+CSCs可被系统识别并收回,培养的细胞中CSCs纯度为13%左右,继续培养纯化的CSCs至第3代;3重组腺病毒均可成功感染CSCs,感染后干细胞生长状态良好,感染效率高,无毒副作用;经测定MOI值为300时感染效率最佳;在HIF1α-EGFP+CSCs组(4.483±0.156)中HIF1α基因转录的mRNA量显著高于在EGFP+CSCs组(1.007±0.13)和CSCs组(1.18±0.03)(P0.05);4细胞移植4周后,各组间心功能指标表现出显著性差异:与模型组(31.51±4.34)相比,MI+CSCs+HIF1α组(43.99±4.95)和MI+CSCs组(39.41±3.72)的LVEF显著升高,其中以MI+CSCs+HIF1α组变化更为显著(P0.05);5移植4周后,MI+CSCs+HIF1α组心脏干细胞存活率(12.391±1.881)显著高于MI+CSCs组(6.756±1.181)(P0.05);移植前后左心室射血分数差值与梗死边缘区CSCs存活率呈正相关(r=0.867,P0.01);6移植4周后,3组均可见凋亡细胞,MI+CSCs+HIF1α组细胞凋亡率(47.40±3.06)%和MI+CSCs组(53±3.65)%比较显著降低(P0.05),而MI+CSCs组细胞凋亡率又明显低于MI组(61.20±3.91)%(P0.05);7术后4周CSC+HIF1α+MI组(4.747±0.969)比MI组(1.17±0.088)VEGF蛋白表达水平显著增加,具有统计学意义(P0.01);MI+CSCs+HIF1α组毛细血管密度(12.82±0.86)显著高于MI+CSCs组(8.86±1.08),具有统计学意义(P0.05)。结论:1 HIF1α基因修饰的CSCs移植和单纯CSCs移植均可减少心肌梗死大鼠心肌细胞凋亡、促进梗死周边区域血管再生,改善心功能,其中HIF1α基因修饰的CSCs效果更加显著。2移植前后LVEF差值与CSCs存活率呈正相关。3 HIF-1α腺病毒载体感染心脏干细胞后可以增加心脏干细胞中HIF1αmRNA的表达和蛋白含量。
[Abstract]:Objective: the hypoxia inducible factor 1 alpha (hypoxia inducible factor-1 HIF-1 alpha, alpha) gene modified cardiac stem cell transplantation and simple cardiac stem cell transplantation compared on cardiac function in rats with heart failure, cardiac stem cell survival rate, the edge of the infarct area capillaries, cardiomyocyte apoptosis. Methods: 1 cardiac stem cells (cardiac stem cells, CSCs) training: the 1 day old male SD rats cardiac stem cells and purified recombinant.2 gene adenovirus infected cells cultured cardiac stem stem cells in the heart; detection of cardiac stem cells HIF-1 mRNA expression and.3 model group prepared by RT-qPCR method: 24 SD rats were randomly divided into 3 groups: cardiac stem cells group, hypoxia inducible factor 1 alpha modified, simple heart stem cell group and model group, reduced system model of heart failure after myocardial infarction with coronary artery ligation method in open chest before and after 2 weeks were injected by radical Because of the modification of cardiac stem cells, simple cardiac stem cells and PBS liquid.4 were observed: 4 weeks after the acquisition of heart function were measured by ultrasound; fluorescence CSCs were observed under microscope and calculated the density of CSCs; HE staining method to evaluate myocardial infarct marginal zone pathological changes; apoptosis of myocardial cells in infarcted zone by TUNEL rate the expression of VEGF protein in myocardial infarction; Western edge Blot method; the expression of CD31 by immunohistochemical staining, the capillary density calculation of infarct marginal zone; analysis of left ventricular ejection fraction and cardiac stem cell survival difference density. Results: 1 primary cultured rat heart tissue block 7~8 days, round, small size the strong refraction of cardiac stem cells began to climb out from the edge of the tissue block; 2 by anti c-kit antibody and streptavidin labeled with -FITC, in the visible light excitation wavelength of 490 nm, can clearly see the developing c-kit+CSCs by FITC, By flow cytometry, c-kit+CSCs system can be identified and recovered, the purity of CSCs in cultured cells was about 13%, continue to cultivate the purified CSCs to the third generation; 3 recombinant adenovirus can be successfully infected with CSCs infection after stem cell growth in good condition, with high efficiency, non-toxic side effects; the determination of the MOI value is 300 when the infection efficiency of the best; in HIF1 -EGFP+CSCs group (4.483 + 0.156) mRNA in the amount of HIF1 alpha gene transcription was significantly higher than that in group EGFP+CSCs (1.007 + 0.13) and CSCs group (1.18 + 0.03) (P0.05; 4) 4 weeks after cell transplantation, heart function index between groups showed significant differences: with the model group (31.51 + 4.34) compared with MI+CSCs+HIF1 group, alpha (43.99 + 4.95) and MI+CSCs group (39.41 + 3.72) LVEF significantly increased the MI+CSCs+HIF1 alpha group changes more significant (P0.05); 4 weeks after the 5 transplantation, MI+CSCs+HIF1 group alpha cardiac stem cell survival rate (12.391 + 1.881) significantly (6.7 higher than that of MI+CSCs group 56 + 1.181) (P0.05) before and after transplantation; left ventricular ejection fraction and the difference of CSCs infarction survival rate was positively correlated (r=0.867, P0.01) 4; 6 weeks after transplantation, apoptotic cells were found in 3 groups, MI+CSCs+HIF1 group alpha cell apoptosis rate (47.40 + 3.06)% and (53 + 3.65) MI+CSCs group% were decreased (P0.05), and the apoptosis rate of MI+CSCs group was significantly lower than that of group MI (61.20 + 3.91)% (P0.05); 7 and 4 weeks after operation CSC+HIF1 +MI group (4.747 + 0.969) than the MI group (1.17 + 0.088) VEGF protein expression level increased significantly, with statistical significance (P0.01) a group of MI+CSCs+HIF1; capillary density (12.82 + 0.86) was significantly higher than that of group MI+CSCs (8.86 + 1.08), with statistical significance (P0.05). Conclusion: CSCs transplantation and simple transplantation of CSCs gene modified HIF1 alpha 1 can reduce myocardial cell apoptosis in rats with myocardial infarction, promote the surrounding area infarction angiogenesis, improve cardiac function. The HIF1 alpha gene modified The effect of CSCs was more significant. The difference of LVEF between.2 before and after transplantation was positively correlated with CSCs survival rate..3 HIF-1 alpha increased the expression of HIF1 alpha mRNA and protein content in cardiac stem cells after infection with adenovirus vector.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22;R541.6

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