本文選題：細胞培養(yǎng) + 流式細胞術(shù)�。� 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:經(jīng)缺氧誘導(dǎo)因子1α(hypoxia inducible factor-1α,HIF-1α)基因修飾的心臟干細胞移植與單純心臟干細胞移植相比,對心衰大鼠心功能、心臟干細胞存活率、梗死區(qū)邊緣毛細血管、心肌細胞凋亡的影響。方法:1心臟干細胞(cardiac stem cells,CSCs)培養(yǎng):采用1日齡SD雄性大鼠進行心臟干細胞分離鑒定與純化,進行傳代培養(yǎng)心臟干細胞。2基因重組腺病毒感染心臟干細胞;采用RT-qPCR法檢測心臟干細胞HIF-1αmRNA表達。3模型制備及分組:將24只SD大鼠隨機等分為3組:缺氧誘導(dǎo)因子1α修飾的心臟干細胞組、單純心臟干細胞組和模型組,以開胸結(jié)扎冠脈前降支的方法制備心肌梗死后心衰模型,2周后分別注射經(jīng)基因修飾的心臟干細胞、單純心臟干細胞及PBS液。4觀察指標:4周后進行結(jié)果采集:超聲測定心功能;熒光顯微鏡下觀察CSCs存活情況并計算CSCs密度;HE染色法評估梗死邊緣區(qū)心肌病理學改變;TUNEL法檢測梗死邊緣區(qū)心肌細胞凋亡率;Western Blot法檢測梗死邊緣VEGF蛋白表達情況;免疫組化染色檢測CD31的表達,計算梗死邊緣區(qū)毛細血管密度;分析左心室射血分數(shù)差值與心臟干細胞存活密度相關(guān)性。結(jié)果:1原代培養(yǎng)的大鼠心臟組織塊7~8天后,圓形、體積小、折光性強的心臟干細胞開始從組織塊邊緣爬出;2通過抗c-kit抗體和鏈親和素-FITC標記,在波長490 nm可見光激發(fā)后,可清晰見到被FITC顯影的c-kit+CSCs,經(jīng)流式細胞儀,c-kit+CSCs可被系統(tǒng)識別并收回,培養(yǎng)的細胞中CSCs純度為13%左右,繼續(xù)培養(yǎng)純化的CSCs至第3代;3重組腺病毒均可成功感染CSCs,感染后干細胞生長狀態(tài)良好,感染效率高,無毒副作用;經(jīng)測定MOI值為300時感染效率最佳;在HIF1α-EGFP+CSCs組(4.483±0.156)中HIF1α基因轉(zhuǎn)錄的mRNA量顯著高于在EGFP+CSCs組(1.007±0.13)和CSCs組(1.18±0.03)(P0.05);4細胞移植4周后,各組間心功能指標表現(xiàn)出顯著性差異:與模型組(31.51±4.34)相比,MI+CSCs+HIF1α組(43.99±4.95)和MI+CSCs組(39.41±3.72)的LVEF顯著升高,其中以MI+CSCs+HIF1α組變化更為顯著(P0.05);5移植4周后,MI+CSCs+HIF1α組心臟干細胞存活率(12.391±1.881)顯著高于MI+CSCs組(6.756±1.181)(P0.05);移植前后左心室射血分數(shù)差值與梗死邊緣區(qū)CSCs存活率呈正相關(guān)(r=0.867,P0.01);6移植4周后,3組均可見凋亡細胞,MI+CSCs+HIF1α組細胞凋亡率(47.40±3.06)%和MI+CSCs組(53±3.65)%比較顯著降低(P0.05),而MI+CSCs組細胞凋亡率又明顯低于MI組(61.20±3.91)%(P0.05);7術(shù)后4周CSC+HIF1α+MI組(4.747±0.969)比MI組(1.17±0.088)VEGF蛋白表達水平顯著增加,具有統(tǒng)計學意義(P0.01);MI+CSCs+HIF1α組毛細血管密度(12.82±0.86)顯著高于MI+CSCs組(8.86±1.08),具有統(tǒng)計學意義(P0.05)。結(jié)論:1 HIF1α基因修飾的CSCs移植和單純CSCs移植均可減少心肌梗死大鼠心肌細胞凋亡、促進梗死周邊區(qū)域血管再生,改善心功能,其中HIF1α基因修飾的CSCs效果更加顯著。2移植前后LVEF差值與CSCs存活率呈正相關(guān)。3 HIF-1α腺病毒載體感染心臟干細胞后可以增加心臟干細胞中HIF1αmRNA的表達和蛋白含量。
[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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R542.22;R541.6

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