發(fā)布時間：2018-07-06 21:01

  本文選題：骨髓間充質(zhì)干細胞 + 心肌細胞　； 參考：《山西醫(yī)科大學》2008年碩士論文

【摘要】： 目的 體外模擬心肌環(huán)境,誘導骨髓間充質(zhì)干細胞(mesenchymal stem cells,MSCs)向心肌細胞分化,觀察其誘導作用,同時與5-氮雜胞苷(5-azacytidine,5-aza)的誘導作用進行比較。 方法 自成年Wister大鼠的骨髓分離MSCs,新生Wister乳鼠分離心肌細胞并制備心肌細胞凍融液作為培養(yǎng)基體外模擬心肌環(huán)境,分四組培養(yǎng)MSCs。A組為對照組即用普通培養(yǎng)基培養(yǎng)細胞;B組為5-aza組;C組為心肌細胞凍融液與5-aza共同培養(yǎng);D組為心肌細胞凍融液培養(yǎng)。觀察細胞形態(tài)變化,利用免疫細胞化學技術鑒定心肌特異性蛋白T(Troponin T,cTnT),連接蛋白43(Connexin43),a一橫紋肌動蛋白(a—Sarcomeric Actin),CD31的表達情況。在光鏡下隨機選擇8個非重疊視野,圖像分析分別計算陽性細胞所占比例。數(shù)據(jù)采用SPSS13.0統(tǒng)計軟件,測量數(shù)據(jù)以均數(shù)±標準差( x±s)表示,作成組比較t檢驗。P0.05有統(tǒng)計學意義。 結果 ①體外模擬心肌微環(huán)境情況:培養(yǎng)7d時心肌細胞形成細胞簇或細胞單層,呈放射狀排列的同心圓狀或峰谷樣生長,細胞簇搏動呈明顯同步性。傳代后的心肌細胞仍具有自主收縮的特性。 ②骨髓間充質(zhì)干細胞的體外誘導結果:誘導處理1周后,5-氮雜胞苷組多數(shù)細胞呈桿狀,緊密平行排列生長,細胞體積變大,可見肌絲樣結構形成;心肌細胞凍融液組細胞有聚集生長趨勢,形成大量的子細胞,可見大量的肌絲樣的結構;5-氮雜胞苷+心肌細胞凍融液組脫落或降解的細胞數(shù)明顯少于5-氮雜胞苷組,也形成肌絲樣結構,但部分細胞內(nèi)有脂肪空泡形成。 ③免疫細胞化學鑒定結果:誘導培養(yǎng)1周后,血清對照組僅表達α-橫紋肌肌動蛋白;5-氮雜胞苷組表達心肌特異性蛋白T、α-橫紋肌肌動蛋白、連接蛋白43,其陽性率分別為20%,28%,25%,抗CD31染色呈陰性;心肌細胞凍融液組上述蛋白表達陽性率分別為23%,32%,28%,明顯高于5-氮雜胞苷組與5-氮雜胞苷+心肌細胞凍融液組(P0.05),同時抗CD31染色呈陽性;5-氮雜胞苷+心肌細胞凍融液組上述蛋白表達陽性率分別為21%,28%,24%,與5-氮雜胞苷組相比無明顯差異(P0.05),同時抗CD31染色呈陽性 結論 ①以心肌細胞凍融液體外模擬心肌微環(huán)境,可高效誘導骨髓間充質(zhì)干細胞分化為心肌細胞。 ②部分骨髓間充質(zhì)干細胞表達CD31,即可向血管內(nèi)皮細胞分化,提示與5-氮雜胞苷單一的肌細胞誘導作用相比,心肌細胞凍融液更能提供一個心肌再生所需的自然條件。
[Abstract]:Objective to induce the differentiation of bone marrow mesenchymal stem cells (mesenchymal stem cells) into cardiomyocytes by simulating myocardial environment in vitro, and to observe the induction effect of 5-azacytidineine 5-aza (5-azacytidineine 5-aza). Methods Myocardial cells were isolated from bone marrow of adult Wister rats and cardiomyocytes were isolated from newborn Wister rats. Cardiomyocyte freeze-thawed solution was used as culture medium to simulate myocardial environment in vitro. MSCs.A group was divided into four groups as control group: normal culture medium group B was 5-aza group C group was cardiomyocyte freezing-thawing solution and 5-aza co-culture group D group was cardiomyocyte freeze-thawing solution culture. The expression of cardiac specific protein T (Troponin TnT) and Connexin 43 (Connexin 43) and a-Sarcomeric Actin (a-sarcomeric Actin) CD31 were detected by immunocytochemistry. Eight unoverlapped visual fields were randomly selected under light microscope and the proportion of positive cells was calculated by image analysis. The data were measured by SPSS 13.0 software and the data were expressed as mean 鹵standard deviation (x 鹵s). Results 1 simulated myocardial microenvironment in vitro: after 7 days of culture, cardiomyocytes formed cell clusters or monolayers, which grew radially in concentric circles or peaks and valleys, and cell cluster pulsation showed obvious synchronism. In vitro induction of bone marrow mesenchymal stem cells: after one week of induction, most of the cells in the 5-azacytidine group were in the shape of rods and grew in close parallel order. The size of the cells became larger and the myofilm-like structure was formed, and the cells in the cardiomyocyte freezing and thawing fluid group had the tendency of aggregation and growth, forming a large number of daughter cells. It can be seen that a large number of myofilm-like structure, 5-azacytidine cardiomyocyte freeze-thaw solution group, the number of cells dropped or degraded significantly less than 5-azacytidine group, but also formed a myofilament structure. But adipose vacuoles formed in some cells. 3 Immunocytochemical identification results: 1 week after induction, In the serum control group, only 偽 -rhabdomyosin 5 azacytidine expressed myocardial specific protein T, 偽 -rhabdomydomycin 43, the positive rates were 20% and 28% 25%, respectively, and the anti-CD31 staining was negative. The positive rate of the above protein expression in the cardiomyocyte freeze-thaw solution group was 2332 / 28, which was significantly higher than that in the 5-azacytidine group and 5-azacytidine cardiomyocyte freeze-thaw group (P0.05), and the anti-CD31 staining was positive in the 5-azacytidine cardiomyocyte freezing and thawing group (P0.05). The positive rates of these proteins were 21% and 28%, respectively, and there was no significant difference compared with the 5-azacytidine group (P0.05). At the same time, the anti-CD31 staining showed positive conclusion: 1 the myocardial microenvironment was simulated by cardiomyocyte freeze-thawing fluid, and no significant difference was found between 5-azacytidine group and 5-azacytidine group. Bone marrow mesenchymal stem cells can be induced to differentiate into cardiomyocytes. 2 some of bone marrow mesenchymal stem cells express CD31, which can differentiate into vascular endothelial cells, suggesting that compared with 5-azacytidine single myocyte induction, some bone marrow mesenchymal stem cells can differentiate into vascular endothelial cells. Cardiomyocyte freeze-thawing fluid can provide a natural condition for myocardial regeneration.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

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