【摘要】：目的：分別探討成人脂肪間充質干細胞（ADMSCs）和成人外周血內皮祖細胞（EPCs）體外分離、培養(yǎng)的方法；證實ADMSCs和EPCs經(jīng)5-氮胞苷（5-aza）的誘導可分化為心肌樣細胞；初步觀察心肌特異性轉錄因子GATA-4和Nkx2.5在誘導過程中表達的變化，并對兩種干細胞的心肌樣分化特性進行比較，為干細胞移植治療冠心病時，選擇合適的種子細胞提供初步的基礎實驗支持。 方法：（1）①ADMSCs的分離與培養(yǎng)：外科手術中取成人皮下脂肪組織，利用膠原酶Ⅰ消化法及細胞貼壁法體外分離培養(yǎng)。取生長狀態(tài)較好的第3代細胞，進行CD29，CD44，Ⅷ因子和人類白細胞抗原（HLA-DR）等表面抗原的間接免疫熒光鑒定，同時設立磷酸鹽緩沖溶液（PBS）的空白對照。②ADMSCs的體外誘導：用5-aza對生長狀態(tài)較好的第3代細胞進行誘導，并對誘導后的細胞進行心肌特異表面標志物結蛋白（Desmin）和肌鈣蛋白T（cTnT）的間接免疫熒光鑒定。（2）①EPCs的分離與培養(yǎng)：無菌條件下采集成人外周靜脈血，利用密度梯度離心法及細胞貼壁法體外分離培養(yǎng)。原代細胞培養(yǎng)至7d時，進行荊豆凝集素（FITC-UEA-Ⅰ）和乙�；兔芏戎鞍祝―il-ac-LDL）免疫雙熒光鑒定；同時以PBS為空白對照，進行CD133，CD34，KDR等表面抗原的免疫細胞化學染色鑒定。②EPCs的體外誘導：原代細胞培養(yǎng)至7d時，，用5-aza進行誘導，并對誘導后的細胞進行心肌特異表面標志物Desmin和cTnT的間接免疫熒光鑒定。（3）分別在ADMSCs和EPCs誘導后的第7、14、21d，采用PCR方法檢測誘導過程中心肌特異性轉錄因子GATA-4和Nkx2.5的表達情況，并進行比較。 結果：（1）成人脂肪組織中含有長梭形的間充質干細胞，易于在體外分離培養(yǎng)。免疫熒光鑒定CD29，CD44呈陽性表達，Ⅷ因子和HLA-DR呈陰性表達。5-aza誘導ADMSCs后，細胞形態(tài)逐漸發(fā)生改變，同時增殖速度顯著減慢。誘導7d時，細胞呈較為均一的長梭形；誘導至14d時，細胞排列方向逐漸一致，并有聚集成團的傾向；誘導21d時，細胞體積較前增大，呈球形或不規(guī)則形，有的有突起伸出，胞漿不均。免疫熒光鑒定Desmin和cTnT呈陽性表達。（2）成人外周血中含有EPCs，可以在體外分離培養(yǎng)。免疫雙熒光鑒定呈陽性表達，免疫細胞化學染色鑒定CD133，CD34，KDR呈陽性表達。5-aza誘導EPCs后，細胞形態(tài)發(fā)生改變，細胞長度增加，逐漸呈均勻一致的長梭形，同時細胞排列方向也漸趨一致；誘導后期有的細胞呈球形或不規(guī)則形，有聚集成團傾向。誘導14d時免疫熒光鑒定Desmin和cTnT呈陽性表達。（3）ADMSCs和EPCs誘導后的PCR產(chǎn)物條帶可見GATA-4和Nkx2.5基因表達，并且隨著誘導時間的逐漸延長，相對表達量增多；同一誘導時間段比較，ADMSCs誘導后的心肌特異基因相對表達顯著高于EPCs組(p0.05)。 結論：（1）5-氮胞苷能將體外培養(yǎng)的脂肪間充質干細胞誘導為心肌樣細胞；（2）5-氮胞苷能將外周血內皮祖細胞誘導為心肌樣細胞，但成功率較低。（3）與內皮祖細胞比較，脂肪間充質干細胞誘導相對容易，誘導后心肌樣細胞分化較多，是干細胞移植治療冠心病時更為理想的種子細胞。
[Abstract]:Objective: To investigate the isolation and culture of adult adipose mesenchymal stem cells (ADMSCs) and adult peripheral blood endothelial progenitor cells (EPCs) in vitro, and to confirm that ADMSCs and EPCs can be differentiated into myocardial like cells by the induction of 5- azytidine (5-aza), and the changes of the expression of GATA-4 and Nkx2.5 in the induction of cardiac specific transcription factors are observed. The comparison of the myocardial differentiation characteristics of two kinds of stem cells is made to provide preliminary basic experimental support for selecting suitable seed cells for stem cell transplantation in the treatment of coronary heart disease.
Methods: (1) the isolation and culture of ADMSCs: taking adult subcutaneous adipose tissue during surgery, using collagenase I digestion method and cell adherence method to isolate and culture in vitro. Third generation cells with better growth status were selected for indirect immunofluorescence identification of surface antigens such as CD29, CD44, factor VIII and human leukocyte antigen (HLA-DR), and were established at the same time. The blank control of phosphate buffer solution (PBS). (2) the induction of ADMSCs in vitro: the induction of third generation cells with better growth state by 5-aza and indirect immunofluorescence identification of the induced cell specific surface marker protein (Desmin) and troponin T (cTnT). (2) the isolation and culture of EPCs: aseptic conditions The adult peripheral venous blood was collected and cultured in vitro by density gradient centrifugation and cell adherence method. When the primary cells were cultured to 7d, the double fluorescence identification of FITC-UEA- I and acetylated low density lipoprotein (Dil-ac-LDL) was carried out, and the immune cells of CD133, CD34, KDR and other surface antigens were carried out with PBS as blank control. Chemical staining identification. (2) induction of EPCs in vitro: induction of primary cell culture to 7d, induced by 5-aza, and indirect immunofluorescence identification of specific surface markers Desmin and cTnT for the induced cells. (3) ADMSCs and EPCs induced 7,14,21d, respectively, to detect the specific transcriptional cause of the central muscle in the induction process by PCR method. The expressions of GATA-4 and Nkx2.5 are compared and compared.
Results: (1) the adult adipose tissue contains long spindle shaped mesenchymal stem cells, which are easy to be isolated and cultured in vitro. The immunofluorescent identification of CD29, CD44 is positive. The cell morphology changes gradually and the proliferation rate slows markedly at the same time when the negative expression of.5-aza is induced by factor VIII and the negative expression of HLA-DR. At the same time, the cells are more homogeneous in the induction of 7D. Spindle shape, when induced to 14d, the direction of cell arrangement is consistent and there is a tendency to gather together. When inducement of 21d, the cell volume is larger than before, it is spherical or irregular, some protruding and uneven cytoplasm. Immunofluorescence identification of Desmin and cTnT is positive. (2) the adult peripheral blood contains EPCs, and can be isolated and cultured in vitro. Double fluorescence identification showed positive expression. Immunocytochemical staining identified CD133, CD34, KDR positive expression of.5-aza induced EPCs, cell morphology changed, cell length increased, and gradually showed uniform long spindle shape, and the direction of cell arrangement gradually became consistent; some cells in the post induction period were spherical or irregular, and there were aggregation groups tilting. The positive expression of Desmin and cTnT was expressed by immunofluorescence in the induction of 14d. (3) the expression of GATA-4 and Nkx2.5 gene in the PCR product bands induced by ADMSCs and EPCs was visible, and the relative expression increased with the gradual prolongation of the induction time. Compared with the same induction time, the relative expression of the specific gene of myocardium after the induction of ADMSCs was significantly higher than that of the EPCs group. (P0.05).
Conclusion: (1) 5- nitrocytidine can induce adipose mesenchymal stem cells cultured in vitro into cardiomyocyte like cells. (2) 5- azacytidine can induce endothelial progenitor cells of peripheral blood into cardiomyocyte like cells, but the success rate is low. (3) compared with endothelial progenitor cells, the inducement of adipose mesenchymal stem cells is relatively easy, and the differentiation of cardiomyocyte like cells is more than that of endothelial progenitor cells. Cell transplantation is a more ideal seed cell for the treatment of coronary heart disease.
【學位授予單位】：寧波大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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