本文關鍵詞：腺病毒介導突變型HIF-1α基因?qū)w外誘導環(huán)境人骨髓間充質(zhì)干細胞分化成心肌樣細胞的影響　出處：《南方醫(yī)科大學》2007年碩士論文　論文類型：學位論文

  更多相關文章： 重組腺病毒 低氧誘導因子-1α 人骨髓間充質(zhì)干細胞 誘導分化 脂肪細胞 成骨細胞 5-氮胞苷 心肌樣細胞

【摘要】： 研究背景目的： 缺血性心臟病目前治療主要是常規(guī)的藥物治療、冠脈介入(PCI)及冠脈旁路移植術(CABG)，然而相當一部分患者藥物治療難以見效，也不適于常規(guī)血運再通術處理，所以必須尋求其它新的治療途徑。近年來對干細胞理論及技術研究的逐漸深入，發(fā)展速度超然，，已經(jīng)取得了初步的實驗成果。細胞移植成為治療心肌梗死的新策略。多種細胞可以作為細胞移植治療的種子細胞。人骨髓間充質(zhì)干細胞(Human Mesenchymal Stem Cells，hMSCs)是一種具有高度自我更新和多向分化潛能的細胞群，hMSCs易于獲取及體外培養(yǎng)，適合自體移植，使之成為細胞治療的合適種子細胞。1999年Makino等將hMSCs連續(xù)傳代4個月以上，得到永生細胞(immortalized cells)，其在5-氮胞苷(5-azacytidine，5-aza)的作用下誘導分化，篩選出部分細胞類似胎兒的心肌細胞，此后國內(nèi)外研究證實多種胚胎及成體干細胞在適當?shù)呐囵B(yǎng)環(huán)境中加入某種有效成份時可在體外誘導分化為心肌樣細胞。這為hMSCs的心臟細胞移植提供了理論基礎。 低氧誘導因子-1(hypoxia inducible factor 1，HIF-1)是細胞對低氧／缺氧作出適應性反應的關鍵性轉錄因子，在缺氧條件下可穩(wěn)定表達，它可通過對VEGF等60多種靶基因的轉錄調(diào)控而促進這些基因表達，參與了心肌細胞生成、紅細胞生成、糖代謝等病理生理過程。臨床前期研究表明，應用具有組成型表達活性的HIF-1α基因治療可誘導生理功能完整的血管新生。近年來在缺血性心臟病等涉及治療性血管新生的疾病研究中，HIF-1α對心肌、血管新生的作用受到廣泛關注，因此HIF-1α被認為是最具有前途的治療基因之一。腺病毒載體較其他病毒載體具有制備容易、感染譜廣、病毒滴度高、不引起插入突變等優(yōu)點而成為治療性血管新生最具臨床應用價值的載體。 關于HIF-1α是否對5-aza誘導hMSCs成為心肌樣細胞產(chǎn)生影響，目前國內(nèi)外尚未見報道�；谶@樣一個研究背景，本實驗構建了攜突變型HIF-1α基因的重組腺病毒載體，通過轉染體外誘導培養(yǎng)hMSCs，研究HIF-1α表達或增強其表達后對5-aza誘導hMSCs成為心肌樣細胞過程的影響，以進一步明確HIF-1α的心臟保護活性，為進一步研究HIF-1α基因及其突變型對缺血性心臟病的血管新生作用及以后的臨床應用奠定基礎。本課題分四個部分進行研究。 方法 第一部分：本課題組所構建的重組腺病毒載體Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)連同對照病毒Ad-LacZ，在HEK293細胞內(nèi)大量擴增后終點稀釋實驗法測定病毒滴度，再經(jīng)氯化銫梯度離心法純化；采用PCR方法對純化后病毒所攜突變型HIF-1α基因進行完整性鑒定；采用掃描電鏡對純化后病毒進行形態(tài)學鑒定；以Ad-LacZ感染HEK293細胞，經(jīng)X-ga1染色確定轉染效率；重組腺病毒Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)體外轉染Hela細胞評價其生物安全性。 第二部分：取本院成人檢查骨髓正常結果的骨髓標本。按Pittenger的方法，分離骨髓標本，取單個核細胞層培養(yǎng)、傳代。取第1、5、10代的貼壁細胞達到80-90％融合后，流式細胞儀Becton Dickinson FACScan檢測FITC-Anti-HLA-DR／CD13-PE；FITC-CD45／CD34-PE；FITC-CD44／CD29-PE的表達。1μmol／L地塞米松、0.5mmol／L IBMX，10μg／ml胰島素的條件定向誘導hMSCs向脂肪細胞分化，第14d實驗組與對照組均行油紅O染色。0.1μmol／L地塞米松，10mmol／Lβ-甘油磷酸，50μmol／L Vit.C條件定向誘導hMSCs向成骨細胞分化。第28 d Von Kossa's染色及茜素紅染色顯示鈣鹽沉著。 第三部分：用終濃度為10μmol／L 5-aza對第5代達到對數(shù)生長期的hMSCs進行誘導培養(yǎng)，作用24h，誘導后培養(yǎng)使用含5％胎牛血清DMEM-LG培養(yǎng)基，每3-4d酌情換液。誘導培養(yǎng)第28 d收獲細胞，用免疫細胞化學方法檢測肌系細胞的標記抗原及心肌特異性標記抗原：α-Actin、Connexin 43和心肌肌鈣蛋白T(cardiac troponin T，cTnT)。 第四部分：Ad-LacZ感染hMSCs，經(jīng)X-gal染色確定轉染效率；重組腺病毒Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)以最佳轉染復數(shù)轉染hMSCs后用5-aza對其進行誘導培養(yǎng)，分別于誘導培養(yǎng)后的第14、28、32d，提取細胞漿蛋白行cTnI檢測。誘導的第28 d提取細胞漿蛋白，用Western blot方法檢測HIF-1α和VEGF蛋白表達水平。 結果 第一部分：在HEK293細胞內(nèi)擴增后的重組腺病毒Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)的滴度分別為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；經(jīng)氯化銫梯度離心法純化后滴度分別為(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；純化后病毒PCR檢測腺病毒骨架及所攜HIF-1α基因均得到預期的287bp、380bp、460bp和214bp擴增產(chǎn)物，且DNA測序結果符合實驗設計要求；電鏡結果顯示腺病毒形態(tài)學特點，無支原體、衣原體污染；轉染HEK293后X-gal染色顯示，最佳轉染復數(shù)為50 pfu／cell。 第二部分：(一)、hMSCs的分離、培養(yǎng)：Ficoll分離液(1.077g／ml)分離得到的單個核細胞24 h后貼壁，細胞呈圓形，48-72 h后呈紡錘形、梭形、多角形，增殖迅速，原代培養(yǎng)約14-20 d可達到80-90％融合。傳代細胞24 h內(nèi)完全貼壁，5-7 d內(nèi)達到80-90％融合。并傳代至第7-10代。(二)、流式細胞儀檢測表面標記物：生長良好的貼壁細胞在第1、5、10代時行表面標記物檢測。第5代細胞均一性達到94％左右。CD34、CD45、HLA-DR陰性但CD13，CD29和CD44陽性。(三)、定向誘導為脂肪細胞：第3 d起光鏡下可見脂滴沉著的細胞，外形變?yōu)閳A形，橢圓形，第14 d油紅O染色示胞核呈藍色，脂滴呈橙紅色。隨著時間延長，脂肪細胞比例逐漸增高。對照組無脂滴出現(xiàn)。(四)、定向誘導為成骨細胞：細胞呈現(xiàn)立方形外觀。Von Kossa's染色及茜素紅染色均能顯示礦化物(鈣鹽)的沉積，而且隨著誘導時間的延長不斷增高。對照組無礦化物(鈣鹽)的沉積。 第三部分：10μmol／L 5-aza誘導后，細胞形態(tài)變大，排列方向更趨一致，細胞增殖減慢，部分細胞脫壁死亡，2-3 W可見少數(shù)細胞胞體粗大，呈長方形。5-aza誘導培養(yǎng)28 d的爬片細胞經(jīng)免疫細胞化學染色檢測肌系細胞標志，顯示部分誘導細胞表達α-Actin，Connexin 43和cTnT，陽性細胞率分別為(32.27±13.17)％、(29.51±5.22)％和(19.96±4.01)％。在未經(jīng)5-aza誘導的同培養(yǎng)天數(shù)的hMSCs中未見表達。 第四部分：隨著MOI值的升高，Ad-LacZ的轉染效率和基因表達增強，在MOI值為75 OPU／cell時，Ad-LacZ轉染心肌細胞的效率高于90％。誘導的第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)四組均促進hMSCs向心肌樣細胞轉化，與對照Ad-LacZ組相比差異有顯著性(P=0.000)，Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)和Ad-HIF-1α_(564／402)三組cTnI值，與Ad-HIF-1α_(564／803)相比，亦有顯著性差異，P值分別為0.001、0.001以及0.021。但是Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)和Ad-HIF-1α_(564／402)組間差異無顯著性，P值分別為0.974、0.229和0.217。Western blot結果顯示Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)、Ad-HIF-1α_(564／803)四組HIF-1α蛋白表達水平較Ad-LacZ組高。VEGF蛋白表達也上升。 結論 第一部分：本課題組構建的重組腺病毒載體Ad-LacZ、Ad-HIF-1α_(nature)、Ad-HIF-1α_(564)、Ad-HIF-1α_(564／402)和Ad-HIF-1α_(564／803)滴度高、轉染效率高且安全，為下一步實驗提供了保證。 第二部分：骨髓分離、培養(yǎng)hMSCs獲得成功，依據(jù)如下：(一)、從骨髓分離、培養(yǎng)單個核細胞呈貼壁生長，成纖維細胞樣外觀。(二)、高表達CD13、CD29和CD44，CD34、CD45和HLA-DR表達極低。(三)、可以被定向誘導分化為脂肪細胞和成骨細胞。 第三部分：5-aza可以誘導純化培養(yǎng)的hMSCs向心肌樣細胞轉化，表達肌系蛋白及心肌特異性蛋白。含5％胎牛血清DMEM-LG培養(yǎng)基適合于hMSCs向心肌樣細胞誘導分化的體外實驗研究。 第四部分：野生型HIF-1α基因及其突變型能促進5-aza誘導的hMSCs向心肌細胞轉化，為HIF-1α進一步應用于心肌再生奠定了實驗基礎。
[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
絎竴閮ㄥ垎錛氬湪HEK293緇嗚優(yōu)鍐呮墿澧炲悗鐨勯噸緇勮吅鐥呮瘨Ad-LacZ,Ad-HIF-1偽_(nature),Ad-HIF-1偽_(564),Ad-HIF-1偽_(564錛

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