腺病毒介导突变型HIF-1α基因对体外诱导环境人骨髓间充质干细胞分化成心肌样细胞的影响

发布时间：2018-01-01 02:24

  本文关键词：腺病毒介导突变型HIF-1α基因对体外诱导环境人骨髓间充质干细胞分化成心肌样细胞的影响　出处：《南方医科大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 重组腺病毒 低氧诱导因子-1α 人骨髓间充质干细胞 诱导分化 脂肪细胞 成骨细胞 5-氮胞苷 心肌样细胞

【摘要】： 研究背景目的： 缺血性心脏病目前治疗主要是常规的药物治疗、冠脉介入(PCI)及冠脉旁路移植术(CABG)，然而相当一部分患者药物治疗难以见效，也不适于常规血运再通术处理，所以必须寻求其它新的治疗途径。近年来对干细胞理论及技术研究的逐渐深入，发展速度超然，，已经取得了初步的实验成果。细胞移植成为治疗心肌梗死的新策略。多种细胞可以作为细胞移植治疗的种子细胞。人骨髓间充质干细胞(Human Mesenchymal Stem Cells，hMSCs)是一种具有高度自我更新和多向分化潜能的细胞群，hMSCs易于获取及体外培养，适合自体移植，使之成为细胞治疗的合适种子细胞。1999年Makino等将hMSCs连续传代4个月以上，得到永生细胞(immortalized cells)，其在5-氮胞苷(5-azacytidine，5-aza)的作用下诱导分化，筛选出部分细胞类似胎儿的心肌细胞，此后国内外研究证实多种胚胎及成体干细胞在适当的培养环境中加入某种有效成份时可在体外诱导分化为心肌样细胞。这为hMSCs的心脏细胞移植提供了理论基础。 低氧诱导因子-1(hypoxia inducible factor 1，HIF-1)是细胞对低氧／缺氧作出适应性反应的关键性转录因子，在缺氧条件下可稳定表达，它可通过对VEGF等60多种靶基因的转录调控而促进这些基因表达，参与了心肌细胞生成、红细胞生成、糖代谢等病理生理过程。临床前期研究表明，应用具有组成型表达活性的HIF-1α基因治疗可诱导生理功能完整的血管新生。近年来在缺血性心脏病等涉及治疗性血管新生的疾病研究中，HIF-1α对心肌、血管新生的作用受到广泛关注，因此HIF-1α被认为是最具有前途的治疗基因之一。腺病毒载体较其他病毒载体具有制备容易、感染谱广、病毒滴度高、不引起插入突变等优点而成为治疗性血管新生最具临床应用价值的载体。 关于HIF-1α是否对5-aza诱导hMSCs成为心肌样细胞产生影响，目前国内外尚未见报道。基于这样一个研究背景，本实验构建了携突变型HIF-1α基因的重组腺病毒载体，通过转染体外诱导培养hMSCs，研究HIF-1α表达或增强其表达后对5-aza诱导hMSCs成为心肌样细胞过程的影响，以进一步明确HIF-1α的心脏保护活性，为进一步研究HIF-1α基因及其突变型对缺血性心脏病的血管新生作用及以后的临床应用奠定基础。本课题分四个部分进行研究。 方法 第一部分：本课题组所构建的重组腺病毒载体Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)连同对照病毒Ad-LacZ，在HEK293细胞内大量扩增后终点稀释实验法测定病毒滴度，再经氯化铯梯度离心法纯化；采用PCR方法对纯化后病毒所携突变型HIF-1α基因进行完整性鉴定；采用扫描电镜对纯化后病毒进行形态学鉴定；以Ad-LacZ感染HEK293细胞，经X-ga1染色确定转染效率；重组腺病毒Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)体外转染Hela细胞评价其生物安全性。 第二部分：取本院成人检查骨髓正常结果的骨髓标本。按Pittenger的方法，分离骨髓标本，取单个核细胞层培养、传代。取第1、5、10代的贴壁细胞达到80-90％融合后，流式细胞仪Becton Dickinson FACScan检测FITC-Anti-HLA-DR／CD13-PE；FITC-CD45／CD34-PE；FITC-CD44／CD29-PE的表达。1μmol／L地塞米松、0.5mmol／L IBMX，10μg／ml胰岛素的条件定向诱导hMSCs向脂肪细胞分化，第14d实验组与对照组均行油红O染色。0.1μmol／L地塞米松，10mmol／Lβ-甘油磷酸，50μmol／L Vit.C条件定向诱导hMSCs向成骨细胞分化。第28 d Von Kossa's染色及茜素红染色显示钙盐沉着。 第三部分：用终浓度为10μmol／L 5-aza对第5代达到对数生长期的hMSCs进行诱导培养，作用24h，诱导后培养使用含5％胎牛血清DMEM-LG培养基，每3-4d酌情换液。诱导培养第28 d收获细胞，用免疫细胞化学方法检测肌系细胞的标记抗原及心肌特异性标记抗原：α-Actin、Connexin 43和心肌肌钙蛋白T(cardiac troponin T，cTnT)。 第四部分：Ad-LacZ感染hMSCs，经X-gal染色确定转染效率；重组腺病毒Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)以最佳转染复数转染hMSCs后用5-aza对其进行诱导培养，分别于诱导培养后的第14、28、32d，提取细胞浆蛋白行cTnI检测。诱导的第28 d提取细胞浆蛋白，用Western blot方法检测HIF-1α和VEGF蛋白表达水平。 结果 第一部分：在HEK293细胞内扩增后的重组腺病毒Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)的滴度分别为1.99×10~(12) pfu／ml、6.31×10~(12) pfu／ml、3.98×10~(12) pfu／ml、10.0×10~(12) pfu／ml和1.26×10~(12)pfu／ml；经氯化铯梯度离心法纯化后滴度分别为(7.755±0.477)×10~(11)OPU／ml、(5.077±0.188)×10~(11)OPU／ml、(6.848±0.129)×10~(11)OPU／ml、(6.292±0.260)×10~(11)OPU／ml和(6.193±0.221)×10~(11)OPU／ml；纯化后病毒PCR检测腺病毒骨架及所携HIF-1α基因均得到预期的287bp、380bp、460bp和214bp扩增产物，且DNA测序结果符合实验设计要求；电镜结果显示腺病毒形态学特点，无支原体、衣原体污染；转染HEK293后X-gal染色显示，最佳转染复数为50 pfu／cell。 第二部分：(一)、hMSCs的分离、培养：Ficoll分离液(1.077g／ml)分离得到的单个核细胞24 h后贴壁，细胞呈圆形，48-72 h后呈纺锤形、梭形、多角形，增殖迅速，原代培养约14-20 d可达到80-90％融合。传代细胞24 h内完全贴壁，5-7 d内达到80-90％融合。并传代至第7-10代。(二)、流式细胞仪检测表面标记物：生长良好的贴壁细胞在第1、5、10代时行表面标记物检测。第5代细胞均一性达到94％左右。CD34、CD45、HLA-DR阴性但CD13，CD29和CD44阳性。(三)、定向诱导为脂肪细胞：第3 d起光镜下可见脂滴沉着的细胞，外形变为圆形，椭圆形，第14 d油红O染色示胞核呈蓝色，脂滴呈橙红色。随着时间延长，脂肪细胞比例逐渐增高。对照组无脂滴出现。(四)、定向诱导为成骨细胞：细胞呈现立方形外观。Von Kossa's染色及茜素红染色均能显示矿化物(钙盐)的沉积，而且随着诱导时间的延长不断增高。对照组无矿化物(钙盐)的沉积。 第三部分：10μmol／L 5-aza诱导后，细胞形态变大，排列方向更趋一致，细胞增殖减慢，部分细胞脱壁死亡，2-3 W可见少数细胞胞体粗大，呈长方形。5-aza诱导培养28 d的爬片细胞经免疫细胞化学染色检测肌系细胞标志，显示部分诱导细胞表达α-Actin，Connexin 43和cTnT，阳性细胞率分别为(32.27±13.17)％、(29.51±5.22)％和(19.96±4.01)％。在未经5-aza诱导的同培养天数的hMSCs中未见表达。 第四部分：随着MOI值的升高，Ad-LacZ的转染效率和基因表达增强，在MOI值为75 OPU／cell时，Ad-LacZ转染心肌细胞的效率高于90％。诱导的第14、28、32 d，Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)四组均促进hMSCs向心肌样细胞转化，与对照Ad-LacZ组相比差异有显著性(P=0.000)，Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)和Ad-HIF-1α_(564／402)三组cTnI值，与Ad-HIF-1α_(564／803)相比，亦有显著性差异，P值分别为0.001、0.001以及0.021。但是Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)和Ad-HIF-1α_(564／402)组间差异无显著性，P值分别为0.974、0.229和0.217。Western blot结果显示Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)、Ad-HIF-1α_(564／803)四组HIF-1α蛋白表达水平较Ad-LacZ组高。VEGF蛋白表达也上升。 结论 第一部分：本课题组构建的重组腺病毒载体Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)滴度高、转染效率高且安全，为下一步实验提供了保证。 第二部分：骨髓分离、培养hMSCs获得成功，依据如下：(一)、从骨髓分离、培养单个核细胞呈贴壁生长，成纤维细胞样外观。(二)、高表达CD13、CD29和CD44，CD34、CD45和HLA-DR表达极低。(三)、可以被定向诱导分化为脂肪细胞和成骨细胞。 第三部分：5-aza可以诱导纯化培养的hMSCs向心肌样细胞转化，表达肌系蛋白及心肌特异性蛋白。含5％胎牛血清DMEM-LG培养基适合于hMSCs向心肌样细胞诱导分化的体外实验研究。 第四部分：野生型HIF-1α基因及其突变型能促进5-aza诱导的hMSCs向心肌细胞转化，为HIF-1α进一步应用于心肌再生奠定了实验基础。
[Abstract]:Research background purpose:
At present the treatment of ischemic heart disease is the main routine drug treatment, percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG), however, a considerable number of patients with drug treatment is difficult to bear fruit, is not suitable for routine blood recanalization treatment, so we must seek other new therapeutic approaches. In recent years for stem cell research and theory technology gradually, the development speed of detachment, has achieved preliminary results of the experiment. Cell transplantation has become a new strategy for the treatment of myocardial infarction. Many kinds of cells can be used as seed cells for cell transplantation. Bone marrow mesenchymal stem cells (Human Mesenchymal Stem Cells, hMSCs) is a highly self renewing and differentiation the cell group, hMSCs are easy to be got and cultured in vitro for autologous transplantation, make it become the appropriate source of cells for cell therapy in.1999 Makino hMSCs will be continuously passaged for more than 4 months to get The living cells (immortalized cells), the 5- azacytidine (5-azacytidine, 5-aza) induced differentiation, select the cells similar to fetal cardiomyocytes, then the domestic and foreign research confirmed a variety of embryonic and adult stem cells into a kind of effective component in the training environment when appropriate can be induced to differentiate into cardiomyocyte like cells in vitro. This provides a theoretical basis for cardiac cell transplantation of hMSCs.
Hypoxia inducible factor -1 (hypoxia inducible factor 1, HIF-1) is a key transcription factor in cell adaptive response to hypoxia / anoxia, under hypoxic conditions can be expressed stably, it can be through the transcriptional regulation of the VEGF and other 60 kinds of target genes and promote the expression of these genes, involved in myocardial cell generation, the generation of red blood cells. The pathophysiological process of glucose metabolism. The clinical preliminary study shows that the application has the constitutive expression of HIF-1 alpha gene therapy activity can induce physiological angiogenesis in ischemic heart disease. In recent years, involved in disease treatment study of newborn blood vessels, HIF-1 alpha on myocardial angiogenesis, thus received extensive attention. Alpha HIF-1 is considered to be one of the most promising gene therapy. Adenovirus vector than other viral vectors with easy preparation, broad spectrum of infection, high viral titers, does not cause insertion mutation It has become the most valuable carrier of therapeutic angiogenesis.
About HIF-1 to 5-aza alpha induced hMSCs become affect cardiomyocytes, has not yet been reported. Based on such a background, we constructed the recombinant adenovirus vector carrying the mutant HIF-1 gene, induced by transfection of hMSCs in vitro, the expression of HIF-1 alpha or enhance the expression of 5-aza induced by hMSCs become cardiomyocytes process, to further clarify the cardioprotective activity of HIF-1 alpha, which lays a foundation for further study of HIF-1 gene and its clinical application of angiogenesis mutant of ischemic heart disease and later. The topic is divided into four parts to conduct the research.
Method
The first part: the recombinant adenovirus vector Ad-HIF-1 alpha _ constructed by our group (nature), Ad-HIF-1 (564), _ alpha Ad-HIF-1 alpha _ (564 / 402) and Ad-HIF-1 alpha _ (564 / 803) and the control virus Ad-LacZ amplified in HEK293 cells after the end point detection of the virus titer dilution test then, by cesium chloride gradient centrifugation method; PCR method is used to complete the identification of the gene mutated HIF-1 alpha carrying the purified virus; morphological identification of the purified virus by using scanning electron microscope; Ad-LacZ HEK293 cells infected by X-ga1 staining to determine the transfection efficiency of recombinant adenovirus; Ad-LacZ, Ad-HIF-1 alpha _ (nature). Ad-HIF-1 _ (564), alpha Ad-HIF-1 alpha _ (564 / 402) and Ad-HIF-1 alpha _ (564 / 803) were transfected into Hela cells in vitro evaluation of the biological safety.
The second part: bone marrow specimens were collected in our normal adult bone marrow examination results. According to the method of Pittenger, isolated from bone marrow specimens from mononuclear cells cultured and passaged. The 1,5,10 generation of adherent cells reached 80-90% after fusion, flow cytometry Becton Dickinson FACScan detection of FITC-Anti-HLA-DR / CD13-PE / CD34-PE / FITC-CD44; FITC-CD45; the expression of CD29-PE.1 mol / L 0.5mmol / L IBMX, dexamethasone, condition directed 10 u g / ml insulin induced hMSCs cell differentiation into adipocytes, 14d of the experimental group and the control group was stained by oil red O.0.1 mol / L 10mmol / L dexamethasone, beta glycerophosphate, mol / 50 L Vit.C induced osteogenic differentiation of hMSCs. Twenty-eighth D Von Kossa's staining and alizarin red staining showed calcinosis.
The third part: the final concentration of 10 mol / L 5-aza for fifth generations to reach the logarithmic growth phase hMSCs were induced and cultured, 24h after induction culture containing 5% fetal bovine serum DMEM-LG medium, 3-4d medium change. Each appropriate induced twenty-eighth D cells were harvested, labeled antigen and cardiac specific marker antigen immunocytochemical method for detection of muscle cells: alpha -Actin, Connexin 43 and T (cardiac troponin cardiac troponin T, cTnT).
The fourth part: the infection of Ad-LacZ hMSCs, by X-gal staining to determine the transfection efficiency of recombinant adenovirus; Ad-LacZ, Ad-HIF-1 alpha _ (nature), Ad-HIF-1 (564), _ alpha Ad-HIF-1 alpha _ (564 / 402) and Ad-HIF-1 alpha _ (564 / 803) with the best transfection complex after transfection of hMSCs with 5-aza on the induced culture the 14,28,32d, respectively after induction, extraction of cTnI for detection of protein induced by the cytoplasm. Twenty-eighth D cytoplasmic protein extraction, using Western blot method to detect HIF-1 alpha and VEGF protein expression.
Result
绗�涓�閮ㄥ垎锛氬湪HEK293缁嗚優鍐呮墿澧炲悗鐨勯噸缁勮吅鐥呮瘨Ad-LacZ,Ad-HIF-1伪_(nature),Ad-HIF-1伪_(564),Ad-HIF-1伪_(564锛

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