本文選題：大鼠骨髓間充質干細胞 + 5-氮胞苷�。� 參考：《第三軍醫(yī)大學》2005年碩士論文

【摘要】：背景與目的各種病因造成心肌組織不可逆損傷均可導致心肌細胞數量的減少、心肌丟失。成年心肌再生能力差,壞死心肌無法通過自身的增殖、分化進行修復,丟失的心肌被瘢痕組織替代,造成心臟重構、心功能下降,最終導致慢性心力衰竭的發(fā)生。成體心肌細胞的再生是醫(yī)學界急待解決的難題之一。骨髓間充質干細胞(mensenchymal stem cell,MSCs)所具有的多能分化特性為心肌細胞再生的研究提供了新的方向和思路。骨髓中MSCs 的含量非常少,必需在體外分離純化、培養(yǎng)擴增才能滿足要求,而如何獲得大量純化的MSCs 則成為臨床利用這些細胞治療疾病的前提。1999 年Makino 等首次體外給予3μmol/ L 的5-氮胞苷(5-Azacytidine, 5-aza)誘導MSCs 定向分化心肌樣細胞獲得成功,開創(chuàng)了MSCs 體外誘導分化心肌樣細胞研究的先河。但仍然存在著誘導出功能性心肌樣細胞的重復性差、誘導比率低、誘導條件不成熟等問題。已有研究證實,脈沖電磁場(pulsed electromagnetic fields, PEMFs)可以促進MSCs 向成骨方向分化。但其對MSCs 向心肌樣細胞分化有無影響尚不清楚。因此本研究擬在用5-aza 體外誘導MSCs 分化為心肌樣細胞的基礎上,探討電磁場對MSCs的增殖及向心肌樣細胞分化的影響。 方法 1. 通過對比不同鼠齡、不同純化方法對大鼠MSCs 集落形成和生長特性的影響,篩選大鼠MSCs 分離、培養(yǎng)的條件,選取CD44、CD105 細胞免疫組化方法對大鼠MSCs進行初步鑒定。 2. 取第三代大鼠MSCs 隨機分A、B、C 為三組,分別加入5μmol/L、10μmol/L 和20μmol/L 的5-aza;每組分別孵育12h、24h 和48h,繼續(xù)培養(yǎng)28d。相差顯微鏡觀察細胞形態(tài)變化以及電境觀察誘導前后超微結構變化,細胞免疫化學染色檢測α-肌動蛋白(α-actin)和心肌肌鈣蛋白T(cardiac troponinT ,cTnT) 表達,并通過Western 印跡法對細胞cTnT 進行半定量分析。 3. 50Hz 的PEMFs 干預5-aza 誘導7d 后的大鼠MSCs,0.5mT、1 mT 和5 mT 三種感應強度分別為A、B、C 三組,各組根據暴露時間10min/d、20min/d、30min/d 及60min/d 又分1、2、3、4 亞組,作用4 d 后采用MTT 法測定細胞增殖變化,作用14d
[Abstract]:Background & objective the irreversible injury of myocardial tissue caused by various causes can lead to the decrease of myocardial cell number and myocardial loss. The regeneration ability of adult myocardium is poor, the necrotic myocardium can not be repaired through its own proliferation, differentiation and repair, the lost myocardium is replaced by scar tissue, resulting in cardiac remodeling, cardiac function decline, and eventually lead to the occurrence of chronic heart failure. The regeneration of adult cardiomyocytes is one of the urgent problems in medical field. The pluripotent differentiation of bone marrow mesenchymal stem cells provides a new direction and thought for the study of cardiomyocyte regeneration. The content of MSCs in bone marrow is very small. It is necessary to isolate and purify in vitro, culture and amplify to meet the requirements. However, how to obtain a large number of purified MSCs has become the precondition for the clinical treatment of these cells. In 1999, Makino et al were given 3 渭 mol/ L 5-Azacytidine (5-aza) for the first time in vitro to induce MSCs directionally differentiated cardiomyocytes. The study of MSCs induced differentiation of cardiomyoid cells in vitro was initiated. However, there are still some problems such as poor reproducibility, low induction rate and immature induction conditions. It has been proved that pulsed electromagnetic fields (PEMFs) can promote the differentiation of MSCs into osteogenesis. However, it is not clear whether it has any effect on the differentiation of MSCs into cardiomyocytes. Therefore, the purpose of this study was to investigate the effects of electromagnetic fields on the proliferation and differentiation of MSCs into cardiomyocyte-like cells on the basis of the induction of 5-aza into cardiomyocyte-like cells in vitro. Method 1. By comparing the effects of different ages and purification methods on the colony formation and growth characteristics of rat MSCs, the conditions for isolation and culture of rat MSCs were screened, and CD44-CD105 cells were selected to identify rat MSCs by immunohistochemical method. 2. The third generation MSCs rats were randomly divided into three groups, 5 渭 mol / L (10 渭 mol/L) and 20 渭 mol/L (5-aza), each group was incubated for 12 h for 24 h and 48 h for 28 days. Phase contrast microscopy was used to observe the changes of cell morphology and ultrastructure before and after induction. The expression of 偽 -actin (偽 -actin) and cardiac troponin (T(cardiac troponinT) was detected by immunocytochemical staining. The cTnT of cells was analyzed by Western blotting. 3. The three induction intensities of 5-aza induced by PEMFs of 50Hz for 7 days were as follows: 1 Mt and 5 Mt, respectively. According to the exposure time of 10 min / d, 20 min / d and 30 min / d respectively, each group was divided into two groups: 1 / 2 / 30 min / d and 1 / 2 / 3 / 4 subgroup respectively. After 4 days of treatment, the cell proliferation was measured by MTT method, and the effect was 14 days.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R35

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