5-氮胞苷联合流体切应力体外诱导大鼠骨髓间充质干细胞分化为心肌样细胞的初步研究

发布时间：2018-05-11 05:17

本文选题：大鼠骨髓间充质干细胞 + 5-氮胞苷　；参考：《山西医科大学》2011年硕士论文

【摘要】：第一部分大鼠骨髓间充质干细胞的分离、鉴定、培养目的:体外分离、鉴定、培养BMSCs。方法:贴壁法从大鼠骨髓中分离骨髓间充质干细胞,进行纯化传代培养,利用倒置相差显微镜对细胞形态进行观察,同时流式细胞仪测定CD29 CD34 CD44 CD105及细胞周期。结果:⒈倒置相差显微镜下可见培养的大鼠骨髓间充质干细胞,原代培养细胞呈多角形,成集落生长趋势。传代细胞逐代伸展呈长梭形,排列具有方向性。 ⒉流式细胞仪检测结果显示,第4代BMSCs,CD29表达率为99.8%,CD44表达率为99.9%,CD105表达率为99.4%,而CD34表达率为1.1%,表明实验用的第4代BMSCs是纯化的细胞。 ⒊细胞周期检测结果显示,第4代BMSCs,G1期细胞为80.049%,S期为14.832%。说明大多数是处于静止期细胞,是一群未分化的非定向细胞。第二部分5-氮胞苷(5-Aza)体外诱导BMSCs向心肌样细胞分化目的:利用5-Aza体外诱导BMSCs向心肌样细胞的分化。方法:取第4代纯化BMSCs以含3、5、10、15、20μmol/l 5-氮胞苷的培养液分别诱导12、24、48h。诱导后第21天免疫荧光细胞化学技术鉴定α-横纹肌肌动蛋白(α-actin)、结蛋白(desmin)、心肌特异性肌钙蛋白I(cTnI)。考虑5-Aza的细胞毒性,结合细胞存活情况,统计分析α-横纹肌肌动蛋白表达率,确立10 umol/L作用24小时为最佳诱导条件。采用最佳浓度10μmol/l,最佳作用时间24h进行实验。在第7、14、21、28天用免疫荧光细胞化学技术鉴定心肌细胞α-横纹肌肌动蛋白(α-actin)、结蛋白(desmin)、心肌特异性肌钙蛋白I(cTnI)表达。结果:⒈根据细胞存活情况和免疫荧光细胞化学技术鉴定α-横纹肌肌动蛋白阳性表达率,确定最佳浓度为10μmol/l,最佳作用时间为24h。 ⒉5-Aza诱导后细胞体积及细胞核增大,伸出不同大小、不同方向的突起,类似于心肌样细胞生长。 ⒊在用10 umol/L作用24小时后,第7天进行免疫荧光细胞化学技术鉴定,未见有α-横纹肌肌动蛋白(α-actin)、结蛋白(desmin)、心肌特异性肌钙蛋白I(cTnI)表达。14天少量细胞α-横纹肌肌动蛋白(α-actin)、结蛋白(desmin)阳性表达,心肌特异性肌钙蛋白I(cTnI)阴性表达。21天表达α-横纹肌肌动蛋白(α-actin)、结蛋白(desmin)的细胞数量增多,并可见少量细胞阳性表达心肌特异性肌钙蛋白I(cTnI)。28天α-横纹肌肌动蛋白(α-actin)、结蛋白(desmin)、心肌特异性肌钙蛋白I(cTnI)阳性表达细胞数量进一步增多。第三部分流体切应力对5-Aza诱导BMSCs成心肌样细胞影响的初步研究目的:观察体外对5-Aza诱导BMSCs成心肌样细胞的影响。方法:取第4代生长活跃的BMSCs接种于载玻片上(硫酸过夜,表面涂多聚赖氨酸),用终浓度为10μmol/l 5-Aza诱导干预24h后,超净工作台内将载玻片置于平行平板流动腔中固定,利用加载装置,通过改变循环液的流量来改变剪切力的大小。分组:实验组流体切应力为5 dyne/cm2、15 dyne/cm2、25 dyne/cm2的力学刺激24h,对照组不接受力学刺激。力学加载干预后,倒置显微镜观察细胞形态的变化和排列方向的改变。21天免疫荧光细胞化学技术鉴定心肌特异性肌钙蛋白I(cTnI)的阳性表达率。干预7天后RT-PCR测定心肌发育相关基因Nkx2.5的表达。干预21天后RT-PCR测定cTnI的基因表达。结果:⒈力学加载干预后,细胞间隙增宽,形态变长,细胞沿流动腔长轴方向伸长,细胞排列接近平行于力的方向。 ⒉免疫荧光细胞化学技术鉴定结果显示,力学刺激干预后,心肌特异性肌钙蛋白I(cTnI)的阳性表达率均增加,15 dyne/cm2的效果最明显。 ⒊RT-PCR结果显示,Nkx2.5、cTnI均阳性表达,但经过力学刺激后阳性条带更为明显,15 dyne/cm2的力学刺激效果最为明显,但是25 dyne/cm2的效果并没有随着力的增大而增强。结论:⒈大鼠骨髓中可以分离出骨髓间充质干细胞并且可以在体外传代培养。⒉5-Aza可以在体外诱导大鼠骨髓间充质干细胞分化为心肌样细胞。 ⒊5-氮胞苷(5-Aza)可联合适当的流体切应力诱导BMSCs向心肌样细胞分化,且诱导效果优于单独使用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 伪 - 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 伪 - actin ( 伪 - actin ) , the positive expression of 伪 - actin ( 伪 - actin ) , and the positive expression of 伪 - actin ( 伪 - actin ) in 14 days , the positive expression of 伪 - actin ( 伪 - actin ) , and the positive expression of 伪 - actin ( 伪 - actin ) , and the number of positive expression cells of 伪 - actin ( 伪 - 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 ) .

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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