本文選題：骨髓間充質(zhì)干細(xì)胞 + 心肌細(xì)胞�。� 參考：《廣西醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的探討體外大鼠骨髓間充質(zhì)干細(xì)胞(marrow mesenchyma stem cells, MSCs)和心肌細(xì)胞(cardiomyocytes, CM)的分離、培養(yǎng)及5-氮胞苷(5-Aza)和共培養(yǎng)兩種方法對(duì)MSCs誘導(dǎo)分化為CM的作用；不同方法誘導(dǎo)MSCs分化后,MSCs在不同時(shí)間段分化為CM的能力及表達(dá)心肌細(xì)胞特異性標(biāo)記物縫隙連接蛋白43(Cx43)、肌鈣蛋白T(cTnT)的差別,為MSCs移植治療急性心肌梗死(acute myocardial infarction,AMI)提供一定的理論基礎(chǔ)。 方法在無(wú)菌條件下分離Wistar大鼠股骨骨髓,采用密度梯度離心與貼壁培養(yǎng)法相結(jié)合進(jìn)行MSCs培養(yǎng)、純化和擴(kuò)增,并行透射電鏡鑒定。在無(wú)菌條件下分離同種乳鼠心臟,采用差速貼壁法進(jìn)行CM培養(yǎng)、純化并行Cx43、cTnT的免疫細(xì)胞化學(xué)檢測(cè)。在獲得穩(wěn)定的細(xì)胞系后,選取生長(zhǎng)良好的第三代MSCs用4',6-二脒基-2-苯基吲哚(DAPI)標(biāo)記,分三組：①正常對(duì)照組：DAPI-MSCs在普通培養(yǎng)基中生長(zhǎng)；②5-Aza誘導(dǎo)組：DAPI-MSCs加入5-Aza誘導(dǎo),用不同濃度5-Aza分別作用不同時(shí)長(zhǎng),以觀察最佳濃度和作用時(shí)間并行免疫組化鑒定cTnT、Cx43;③共培養(yǎng)組：與培養(yǎng)第3天的CM共培養(yǎng)。培養(yǎng)后1w、2w、3w、4w在倒置相差顯微鏡下觀察各組細(xì)胞形態(tài)結(jié)構(gòu)變化及免疫細(xì)胞化學(xué)染色鑒定cTnT、Cx43,并計(jì)算其誘導(dǎo)陽(yáng)性率。 結(jié)果Wistar大鼠MSCs經(jīng)過分離、貼壁、純化能在體外進(jìn)行增殖并保持良好的未分化潛能。同種乳鼠CM分離、貼壁后1天即能出現(xiàn)自發(fā)性搏動(dòng),培養(yǎng)2-3d可長(zhǎng)成單層并形成細(xì)胞簇,并出現(xiàn)成簇細(xì)胞的同步搏動(dòng)。經(jīng)10μmol/L 5-Aza持續(xù)作用15天的MSCs,4w時(shí)呈現(xiàn)出心肌細(xì)胞的典型改變,并有部分細(xì)胞出現(xiàn)自發(fā)搏動(dòng),頻率為15～20次／分。MSCs傳至第3代時(shí)用DAPI標(biāo)記,標(biāo)記效率為100%,DAPI-MSCs在正常培養(yǎng)基中未見有細(xì)胞收縮,cTnT、Cx43表達(dá)陰性；在誘導(dǎo)組和共培養(yǎng)組,培養(yǎng)至4w時(shí)細(xì)胞排列方向(?)致,成肌性排列,部分細(xì)胞呈現(xiàn)搏動(dòng),誘導(dǎo)1w時(shí)cTnT、Cx43表達(dá)陰性,誘導(dǎo)2w時(shí)cTnT陽(yáng)性率為(9.98±1.67)%,Cx43陽(yáng)性率為(13.38±2.15)%,培養(yǎng)至4w時(shí)陽(yáng)性率升高,而且與3w,2w相比(P0.01)；共培養(yǎng)組第5天cTnT、Cx43就開始表達(dá)了,隨著培養(yǎng)時(shí)間延長(zhǎng),陽(yáng)性率逐漸升高,到第4w時(shí)cTnT、Cx43陽(yáng)性率分別為(88.3±1.33)%,(90.38±1.87)%,與3w,2w,1w相比(P0.01)；相同周數(shù)內(nèi)比較,共培養(yǎng)組的陽(yáng)性率均高于誘導(dǎo)組(P0.01)。 結(jié)論MSCs在體外有良好的增殖能力并保持其未分化潛能特性,CM經(jīng)差速貼壁法分離純化,細(xì)胞純度可達(dá)95%以上。MSCs經(jīng)適宜濃度5-Aza誘導(dǎo)和與CM共培養(yǎng),可轉(zhuǎn)化為心肌細(xì)胞并表達(dá)心肌細(xì)胞特異性標(biāo)記物cTnT、Cx43,且共培養(yǎng)MSCs分化為心肌細(xì)胞的能力比5-Aza誘導(dǎo)強(qiáng)。
[Abstract]:Objective To investigate the effects of two methods on the induction and differentiation of bone marrow mesenchymal stem cells ( MSCs ) and cardiomyocytes ( MSCs ) and myocardial cells ( CM ) in vitro .
After inducing MSCs to differentiate , MSCs differentiated into CM at different time intervals and expressed myocardial cell specific marker gap junction protein 43 ( Cx43 ) and troponin T , which provided a theoretical basis for MSCs transplantation for acute myocardial infarction ( AMI ) .


Methods The bone marrow of Wistar rats was isolated under aseptic conditions . MSCs were cultured , purified and amplified by density gradient centrifugation and adherent culture method .
( 2 ) 5 - Aza - induced group : DAPI - MSCs were induced by 5 - Aza , and different concentrations of 5 - Aza were used to observe the optimal concentration and the time of action .


Results MSCs were isolated , adherent and purified in Wistar rats to proliferate and maintain good undifferentiated potential in vitro .
The positive rate was ( 9.98 鹵 1.67 ) % , the positive rate of Cx43 was ( 13.38 鹵 2.15 ) % , and the positive rate of Cx43 was ( 13.38 鹵 2.15 ) % , and the positive rate of Cx43 was higher than that of 3w and 2w ( P0.01 ) .
At the 5th day of the coculture , Cx43 began to express , and the positive rate gradually increased with the increase of culture time . The positive rate was ( 88.3 鹵 1.33 ) % and ( 90.38 鹵 1 . 87 ) % , respectively , compared with 3w , 2w and 1w ( P0.01 ) .
The positive rate of co - culture group was higher than that in the induction group ( P0.01 ) .


Conclusion MSCs can proliferate in vitro and maintain their undifferentiation potential . The purity of MSCs can reach more than 95 % . MSCs can be transformed into cardiomyocytes and express cardiac muscle cell - specific markers for cardiac muscle cells . MSCs can be transformed into cardiomyocytes and express cardiac muscle cell - specific markers . The ability of MSCs to differentiate into cardiomyocytes is stronger than that of 5 - Aza .

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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