發(fā)布時間：2018-01-26 13:19

  本文關(guān)鍵詞： 骨髓間充質(zhì)干細胞 胚胎干細胞 心肌細胞 5-氮胞苷 肝細胞生長因子 細胞分化　出處：《青島大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 第一部分 研究目的： 觀察大鼠骨髓間充質(zhì)干細胞(Mesenchymal stem cells MSCs)的分離和培養(yǎng)，，通過不同濃度的5-氮胞苷(5-aza-dC)誘導(dǎo)大鼠MSCs分化為心肌細胞的比較，確定合適的的誘導(dǎo)濃度。 研究方法： 抽取大鼠骨髓，采用貼壁培養(yǎng)法分離MSCs，進行培養(yǎng)、傳代，在第4代MSCs中添加誘導(dǎo)劑5-氮胞苷(5-aza)對其進行誘導(dǎo)分化，其濃度分別為：0μmol／L、10μmol／L、30μmol／L，觀察細胞形態(tài)學(xué)的變化，熒光免疫組織化學(xué)方法進行鑒定。 研究結(jié)果： 5-aza-dC作用24h后，隨著時間的延長，加入終濃度為10μmol／L 5-aza-dC的大鼠MSCs，10d后細胞之間出現(xiàn)連接，排列方向漸趨一致，有肌管形成，部分細胞則死亡。誘導(dǎo)28d后，細胞變稀，周圍伸出多個長的突起，細胞形態(tài)大小不一。而加入終濃度為30μmol／L 5-aza-dC的MSCs，在誘導(dǎo)后第5d，細胞則全部死亡。 結(jié)論： 大鼠MSCs體外在5-氮胞苷作用下可定向分化為心肌樣細胞。 第二部分 研究目的： 探討5-氮胞苷(5-aza-dC)、肝細胞生長因子(Hepatocyte growth factor HGF)作為誘導(dǎo)劑，體外誘導(dǎo)人類胚胎干細胞(Human embryonic stem cell hESC)分化為心肌細胞的最佳誘導(dǎo)分化濃度。 研究方法： 1．將傳代的hESC(PKU-1-1)接種到低粘附六孔板中進行懸浮培養(yǎng)形成擬胚體(Embryonic Bodies EBs)。7d后，分別將EBs接種到四孔板中貼壁培養(yǎng)，分別以5-aza-dC、HGF為分化誘導(dǎo)劑加入分化培養(yǎng)基中，組合成幾種分化培養(yǎng)基，分別為無誘導(dǎo)物組；5-aza-dC組，其濃度分別為：2.5μmol／L、5μmol／L、10μmol／L、20μmol／L；HGF組，其濃度分別為：1ng/ml、5ng/ml、10ng/ml、30ng/ml。通過觀察出現(xiàn)的自發(fā)搏動的EBs的數(shù)量，計算各組分化成功的EBs數(shù)。 2．光鏡動態(tài)觀察細胞形態(tài)學(xué)的變化，熒光免疫組織化學(xué)，RT-PCR以及電鏡方法對分化細胞進行鑒定。組間比較用方差分析及t檢驗，以p＜0.05，為差異有顯著性，比較各組之間的分化比率。 研究結(jié)果： 1．5-aza-dC終濃度為0-10μmol／L時均能在體外誘導(dǎo)hESC向心肌細胞分化，2.5-5μmol／L為最佳誘導(dǎo)濃度。Troponin T及Connexin43的表達呈陽性，并可見清晰肌小節(jié)。RT-PCR檢測MLC-2A、MLC-2V、hANP、β-Actin mRNA表達陽性。電鏡下可見細胞內(nèi)有肌節(jié)樣結(jié)構(gòu)。細胞分化率比較：2.5μmol／L、5μmol／L組之間無明顯差異(p＞0.05)，均明顯高于0μmol／L、10μmol／L(p＜0.05)，此外0μmol／L、10μmol／L組之間也沒有明顯差異(p＞0.05)。而20μmol／L組的細胞大部分出現(xiàn)死亡。 2．HGF終濃度為1ng/ml、5ng/ml、10ng／ml、30ng／ml時均能在體外誘導(dǎo)hESC向心肌細胞分化，Troponin T及Connexin43的表達呈陽性，并可見清晰肌小節(jié)。RT-PCR檢測MLC-2A、MLC-2V、hANP、NKx2.5、GATA-4、a-MHC、β-Actin mRNA表達陽性。細胞分化率比較：5ng／ml、10ng／ml組之間無明顯差異(p＞0.05)，均明顯高于1ng／ml、30ng／ml組(p＜0.05)。 結(jié)論： 5-aza-dC、HGF均能誘導(dǎo)人類胚胎干細胞向心肌細胞分化，2.5μmol／L-5μmol／L的5-aza-dC效果最理想；5ng／ml-10ng／ml的HGF為最佳濃度區(qū)間。
[Abstract]:Part one
The purpose of the study is:
Objective To observe the isolation and culture of rat bone marrow mesenchymal stem cells (Mesenchymal stem cells MSCs), and compare the induction of MSCs differentiation into cardiomyocytes by 5- azacytidine (5-aza-dC) at different concentrations, and determine the appropriate induced concentration.
Research methods:
From rat bone marrow by adherent culture method isolated MSCs were cultured and passaged, adding inducer of 5- azacytidine in the fourth generation of MSCs (5-aza) to induce differentiation, the concentrations were 0 mol / L 10 mol / L 30 mol / L, to observe the change of cell the morphology, immunohistochemistry methods were identified.
The results of the study:
The role of 5-aza-dC 24h, with the extension of time, with a final concentration of 10 mol / L 5-aza-dC rat MSCs, appeared a connection between 10d cells, orientation is consistent, a part of myotube formation, cell death induced by 28d cells. After thinning, a plurality of protrusions around the protruding length, cell morphology is not the same size and adding a final concentration of 30 mol / L 5-aza-dC MSCs in 5D after induction, cells all died.
Conclusion:
Rat MSCs can differentiate into cardiomyocytes in vitro under the action of 5- azacytidine.
The second part
The purpose of the study is:
Objective to investigate the optimal differentiation concentration of 5- 5-aza-dC, Hepatocyte growth factor HGF as an inducer to induce the differentiation of human embryonic stem cells (Human embryonic stem cell hESC) into cardiomyocytes in vitro.
Research methods:
1.. HESC (PKU-1-1) was inoculated into the culture of embryoid bodies were suspended in low adhesion plate (Embryonic Bodies EBs).7d, respectively, EBs were inoculated into four well plate adherent culture, respectively by 5-aza-dC, HGF induced differentiation agent into the differentiation medium, combined into several differentiation medium. Respectively no inducer group; group 5-aza-dC, the concentrations were 2.5 mol / L 5 mol / L 10 mol / L 20 mol / L; group HGF, the concentrations were 1ng/ml, 5ng/ml, 10ng/ml, the number of spontaneous beating 30ng/ml. by observing the EBs and calculate the number of EBs groups successfully differentiated.
2., the morphological changes of cells were observed dynamically under light microscope. The differentiated cells were identified by fluorescence immunohistochemistry, RT-PCR and electron microscopy. The difference between groups was statistically significant with P < 0.05, and the ratio of differentiation between groups was compared by T analysis.
The results of the study:
HESC can be induced to differentiation into cardiomyocytes in vitro 1.5-aza-dC final concentration of 0-10 mol / L, mol / 2.5-5 L for the expression of.Troponin T and optimal concentration of Connexin43 was positive, and clear sarcomere.RT-PCR detection of MLC-2A, MLC-2V, hANP, -Actin expression of mRNA positive cells under electron microscope. Sarcomere structure. The rate of cell differentiation: 2.5 mol / L, no significant difference between the 5 mol / L group (P > 0.05), were significantly higher than that of 0 mol / L 10 mol / L (P < 0.05), in addition to 0 mol / L, there is no obvious difference between the 10 mol / L group (P > 0.05). And 20 mol / L group most of the cells died.
The final concentration of 2.HGF is 1ng/ml, 5ng/ml, 10NG / ml, were able to induce hESC differentiation into cardiomyocytes in vitro by 30ng / ml, the expression of Troponin T and Connexin43 were positive and clear sarcomere.RT-PCR detection of MLC-2A, MLC-2V, hANP, NKx2.5, GATA-4, a-MHC, -Actin. MRNA positive expression of beta cell rate differentiation: 5ng / ml, there is no significant difference between 10NG / ml group (P > 0.05), were significantly higher than 1ng / ml, 30ng / ml group (P < 0.05).
Conclusion:
5-aza-dC and HGF can induce human embryonic stem cells to differentiate into cardiomyocytes. The 5-aza-dC effect of 2.5 mol / L-5 mol / L is the best, and 5ng / ml-10ng / ml HGF is the best concentration range.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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