本文選題：大鼠骨髓間充質(zhì)干細(xì)胞 + 5-氮胞苷　； 參考：《山西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：第一部分大鼠骨髓間充質(zhì)干細(xì)胞的分離、鑒定、培養(yǎng) 目的:體外分離、鑒定、培養(yǎng)BMSCs。 方法:貼壁法從大鼠骨髓中分離骨髓間充質(zhì)干細(xì)胞,進(jìn)行純化傳代培養(yǎng),利用倒置相差顯微鏡對(duì)細(xì)胞形態(tài)進(jìn)行觀察,同時(shí)流式細(xì)胞儀測(cè)定CD29 CD34 CD44 CD105及細(xì)胞周期。 結(jié)果:⒈倒置相差顯微鏡下可見(jiàn)培養(yǎng)的大鼠骨髓間充質(zhì)干細(xì)胞,原代培養(yǎng)細(xì)胞呈多角形,成集落生長(zhǎng)趨勢(shì)。傳代細(xì)胞逐代伸展呈長(zhǎng)梭形,排列具有方向性。 ⒉流式細(xì)胞儀檢測(cè)結(jié)果顯示,第4代BMSCs,CD29表達(dá)率為99.8%,CD44表達(dá)率為99.9%,CD105表達(dá)率為99.4%,而CD34表達(dá)率為1.1%,表明實(shí)驗(yàn)用的第4代BMSCs是純化的細(xì)胞。 ⒊細(xì)胞周期檢測(cè)結(jié)果顯示,第4代BMSCs,G1期細(xì)胞為80.049%,S期為14.832%。說(shuō)明大多數(shù)是處于靜止期細(xì)胞,是一群未分化的非定向細(xì)胞。 第二部分5-氮胞苷(5-Aza)體外誘導(dǎo)BMSCs向心肌樣細(xì)胞分化 目的:利用5-Aza體外誘導(dǎo)BMSCs向心肌樣細(xì)胞的分化。 方法:取第4代純化BMSCs以含3、5、10、15、20μmol/l 5-氮胞苷的培養(yǎng)液分別誘導(dǎo)12、24、48h。誘導(dǎo)后第21天免疫熒光細(xì)胞化學(xué)技術(shù)鑒定α-橫紋肌肌動(dòng)蛋白(α-actin)、結(jié)蛋白(desmin)、心肌特異性肌鈣蛋白I(cTnI)。考慮5-Aza的細(xì)胞毒性,結(jié)合細(xì)胞存活情況,統(tǒng)計(jì)分析α-橫紋肌肌動(dòng)蛋白表達(dá)率,確立10 umol/L作用24小時(shí)為最佳誘導(dǎo)條件。采用最佳濃度10μmol/l,最佳作用時(shí)間24h進(jìn)行實(shí)驗(yàn)。在第7、14、21、28天用免疫熒光細(xì)胞化學(xué)技術(shù)鑒定心肌細(xì)胞α-橫紋肌肌動(dòng)蛋白(α-actin)、結(jié)蛋白(desmin)、心肌特異性肌鈣蛋白I(cTnI)表達(dá)。 結(jié)果:⒈根據(jù)細(xì)胞存活情況和免疫熒光細(xì)胞化學(xué)技術(shù)鑒定α-橫紋肌肌動(dòng)蛋白陽(yáng)性表達(dá)率,確定最佳濃度為10μmol/l,最佳作用時(shí)間為24h。 ⒉5-Aza誘導(dǎo)后細(xì)胞體積及細(xì)胞核增大,伸出不同大小、不同方向的突起,類(lèi)似于心肌樣細(xì)胞生長(zhǎng)。 ⒊在用10 umol/L作用24小時(shí)后,第7天進(jìn)行免疫熒光細(xì)胞化學(xué)技術(shù)鑒定,未見(jiàn)有α-橫紋肌肌動(dòng)蛋白(α-actin)、結(jié)蛋白(desmin)、心肌特異性肌鈣蛋白I(cTnI)表達(dá)。14天少量細(xì)胞α-橫紋肌肌動(dòng)蛋白(α-actin)、結(jié)蛋白(desmin)陽(yáng)性表達(dá),心肌特異性肌鈣蛋白I(cTnI)陰性表達(dá)。21天表達(dá)α-橫紋肌肌動(dòng)蛋白(α-actin)、結(jié)蛋白(desmin)的細(xì)胞數(shù)量增多,并可見(jiàn)少量細(xì)胞陽(yáng)性表達(dá)心肌特異性肌鈣蛋白I(cTnI)。28天α-橫紋肌肌動(dòng)蛋白(α-actin)、結(jié)蛋白(desmin)、心肌特異性肌鈣蛋白I(cTnI)陽(yáng)性表達(dá)細(xì)胞數(shù)量進(jìn)一步增多。 第三部分流體切應(yīng)力對(duì)5-Aza誘導(dǎo)BMSCs成心肌樣細(xì)胞影響的初步研究 目的:觀察體外對(duì)5-Aza誘導(dǎo)BMSCs成心肌樣細(xì)胞的影響。 方法:取第4代生長(zhǎng)活躍的BMSCs接種于載玻片上(硫酸過(guò)夜,表面涂多聚賴(lài)氨酸),用終濃度為10μmol/l 5-Aza誘導(dǎo)干預(yù)24h后,超凈工作臺(tái)內(nèi)將載玻片置于平行平板流動(dòng)腔中固定,利用加載裝置,通過(guò)改變循環(huán)液的流量來(lái)改變剪切力的大小。分組:實(shí)驗(yàn)組流體切應(yīng)力為5 dyne/cm2、15 dyne/cm2、25 dyne/cm2的力學(xué)刺激24h,對(duì)照組不接受力學(xué)刺激。力學(xué)加載干預(yù)后,倒置顯微鏡觀察細(xì)胞形態(tài)的變化和排列方向的改變。21天免疫熒光細(xì)胞化學(xué)技術(shù)鑒定心肌特異性肌鈣蛋白I(cTnI)的陽(yáng)性表達(dá)率。干預(yù)7天后RT-PCR測(cè)定心肌發(fā)育相關(guān)基因Nkx2.5的表達(dá)。干預(yù)21天后RT-PCR測(cè)定cTnI的基因表達(dá)。 結(jié)果:⒈力學(xué)加載干預(yù)后,細(xì)胞間隙增寬,形態(tài)變長(zhǎng),細(xì)胞沿流動(dòng)腔長(zhǎng)軸方向伸長(zhǎng),細(xì)胞排列接近平行于力的方向。 ⒉免疫熒光細(xì)胞化學(xué)技術(shù)鑒定結(jié)果顯示,力學(xué)刺激干預(yù)后,心肌特異性肌鈣蛋白I(cTnI)的陽(yáng)性表達(dá)率均增加,15 dyne/cm2的效果最明顯。 ⒊RT-PCR結(jié)果顯示,Nkx2.5、cTnI均陽(yáng)性表達(dá),但經(jīng)過(guò)力學(xué)刺激后陽(yáng)性條帶更為明顯,15 dyne/cm2的力學(xué)刺激效果最為明顯,但是25 dyne/cm2的效果并沒(méi)有隨著力的增大而增強(qiáng)。 結(jié)論:⒈大鼠骨髓中可以分離出骨髓間充質(zhì)干細(xì)胞并且可以在體外傳代培養(yǎng)。⒉5-Aza可以在體外誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞分化為心肌樣細(xì)胞。 ⒊5-氮胞苷(5-Aza)可聯(lián)合適當(dāng)?shù)牧黧w切應(yīng)力誘導(dǎo)BMSCs向心肌樣細(xì)胞分化,且誘導(dǎo)效果優(yōu)于單獨(dú)使用5-氮胞苷(5-Aza)。
[Abstract]:Isolation , identification and culture of bone marrow mesenchymal stem cells from the first part of rats

Objective : To isolate , identify and culture the bone marrow cells in vitro .

Methods : The bone marrow mesenchymal stem cells were isolated from rat bone marrow by the adherent method . The cell morphology was observed by reversed phase contrast microscope , and the CD29 CD34 CD44 CD105 and cell cycle were measured by flow cytometry .

Results : The cultured rat bone marrow mesenchymal stem cells were observed under the microscope of inverted phase contrast microscope . The primary cultured cells were polygonal and had a tendency of colony growth .

The expression rate of CD29 was 99.8 % , the expression rate of CD44 was 99.9 % , the expression rate of CD105 was 99 . 4 % , while that of CD34 was 1.1 % .

The results of cell cycle test showed that in 4th generation , the cells in G1 phase were 80.049 % and 14.832 % in S phase . Most of them were in quiescent period , and were a group of undifferentiated non - directional cells .

In vitro induction of second part 5 - azacytidine ( 5 - Aza ) into myocardial - like cells

Objective : To investigate the differentiation of bone marrow cells induced by 5 - Aza in vitro .

Methods : The culture medium containing 3 , 5 , 10 , 15 , 20 渭mol / l of 5 - azacytidine was induced by the 4th generation of purified bone marrow cells for 12 hours , 24 hours and 48 hours .

Results : According to cell survival and immunofluorescence cytochemistry technique , the positive rate of 偽 - striated actin was determined . The optimal concentration was 10 渭mol / l and the optimal time was 24 h .

The cell volume and cell nucleus increased after 5 - Aza induction , extending in different sizes and different directions , similar to the growth of myocardial - like cells .

The positive expression of 偽 - actin ( 偽 - actin ) , the positive expression of 偽 - actin ( 偽 - actin ) , and the positive expression of 偽 - actin ( 偽 - actin ) in 14 days , the positive expression of 偽 - actin ( 偽 - actin ) , and the positive expression of 偽 - actin ( 偽 - actin ) , and the number of positive expression cells of 偽 - actin ( 偽 - actin ) .

Effect of Fluid Shear Stress on Myocardial - like Cells Induced by 5 - Aza

Objective : To observe the effect of 5 - Aza on myocardial - like cells in vitro .

Methods : The 4th generation growth - active bone marrow cells were seeded onto slides ( overnight in sulfuric acid , surface coated with poly - lysine ) . After 24 hours of intervention with a final concentration of 10 渭mol / l 5 - Aza , the size of shear force was changed by changing the flow rate of circulating fluid .

Results : After the intervention of biomechanical loading , the cell gap was widened , the morphology became longer , the cells were elongated in the direction of the long axis of the flow lumen , and the cells were arranged close to the direction of force .

The results showed that after the intervention of mechanical stimulation , the positive expression rate of cardiac troponin I was increased and the effect of 15 dyne / cm2 was the most obvious .

The results of RT - PCR showed that the positive expression of the positive bands was found in the positive bands after mechanical stimulation , but the effect of 15 dyne / cm2 was the most obvious , but the effect of 25 dyne / cm2 did not increase with the increase of force .

Conclusion : Bone marrow mesenchymal stem cells can be isolated from rat bone marrow and can be cultured in vitro .

5 - Aza - 5 - azacytidine ( 5 - Aza ) can be combined with appropriate fluid shear stress to differentiate into myocardial - like cells , and the induction effect is better than that of 5 - azacytidine alone ( 5 - Aza ) .

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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本文編號(hào)：1872554