miR-21对SD大鼠骨髓间充质干细胞治疗心肌梗死的影响实验研究

发布时间：2018-06-18 22:04

本文选题：骨髓间充质干细胞 + miRNA-21　；参考：《苏州大学》2015年硕士论文

【摘要】：目的:通过过表达或抑制micro RNA-21(mi R-21)在SD大鼠骨髓间充质干细胞SD-BMSCs(MSCs)内的表达,评估其在治疗心肌梗死的效果上的变化,并研究其作用机制。方法:采用全骨髓差异贴壁法分离培养SD大鼠MSCs至第4代,使用脂质体转染法转染mi R21-agomir(过表达)及mi R-21 antagomir(抑制)调控mi R-21在MSCs内的表达,转染成功后收集细胞并收集条件培养基,用于后续研究。体内实验中,建立SD大鼠心肌梗死模型后,采用心肌内注射方法,分别将MSCsmi R-21 agomir组、MSCsmi R-21antagomir组、MSCs未转染组及PBS移植至心梗及周边区域,通过检测大鼠心梗后心功能变化及心梗面积评估治疗效果。体外实验中,购买原代脐静脉内皮细胞(HUVEC),心肌细胞系H9C2细胞,子宫颈癌细胞系Hela细胞。以Hela细胞为载体,通过双荧光素酶报告验证mi R-21的靶点是否为Jagged1。同时利用收集的MSCs转染组产生的培养基和MSCs未转染组产生的培养基及含有10%FBS+双抗的DMEM/HIGH培养基(空白对照),用作以下两个实验,一是处理HUVEC细胞72小时后在Matrigel基质胶上培养,于12小时观察成管样结构;二是处理H9C2细胞72h后加入CCK-8溶液孵育1个小时,检测细胞增殖情况。结果:体内实验中:MSCsmi R-21 antagomir组平均LVEF(%)变化值:17.14±2.63,MSCs mi R-21 agomir组LVEF(%)变化值:13.09±1.74,MSCs未转染组LVEF(%)变化值:13.34±2.25,三组与对照组LVEF(%)变化值:7.09±4.23相比,心功能恢复效果更佳,差异有统计学意义(P0.05),三组之中,MSCsmi R-21antagomir组与心功能恢复效果最佳,与其他两组相比差异有统计学意义(P0.05)。通过Masson染色分析心梗面积发现心梗区域纤维化组织与正常心肌面积所占横截面外周径比值(%):MSCsmi R-21 antagomir组最小,为10.50±3.15,与MSCsmi R-21 agomir15.91+4.12、MSCs未转染组20.34±3.25、PBS组37.09±6.23均有统计学差异(P0.05)。心梗区域纤维化组织与正常心肌面积所占横截面面积比值(%)MSCsmi R-21 antagomir组同样最小,为8.31±2.35,与MSCsmi R-21 agomir组19.91+4.93、MSCs普通组25.14±2.19、PBS组31.04±5.93均有统计学差异(P0.05)。通过双荧光素酶报告基因验证,我们发现mi R-21可与Jagged1的m RNA 3’UTR段结合,说明mi R-21对Jagged1具有调控作用。q RT-PCR显示,转染mi R-21 antagomir的MSCs内的Jagged1、NOTCH、VEGF等基因全部上调。体外管样生成实验中,MSCsmi R-21 agomir培养基组体外管样结构连接长度(11976.14±866.62)与MSCsmi R-21 antagomir培养基组(12196.52±858.00)、MSCs未转染培养基组(12275.09±561.74)之间均无统计学差异(P0.05),但三组与空白组之间均有统计学差异(P0.01);CCK8实验中,抑制了mi R-21在MSCs内的表达后,其条件培养基促心肌增殖的能力得到了明显的增强。结论:抑制了mi R-21在MSCs内的表达后,其治疗心肌梗死的能力得到了增强。mi R-21可与Jagged1 m RNA结合,对其产生调控。抑制了mi R-21在SD大鼠MSCs的表达后,Jagged1,Notch,VEGF等基因上调。MSCs产生的条件培养基具有促进心肌细胞增殖的能力,抑制了mi R-21在其内的表达后,这种能力得到了提升。MSCs产生的条件培养基也具有促进管样结构生成的能力,但这种能力与mi R-21的表达无关。
[Abstract]:Objective: to express or inhibit the expression of micro RNA-21 (MI R-21) in SD rat bone marrow mesenchymal stem cells SD-BMSCs (MSCs), evaluate its effect in the treatment of myocardial infarction, and study its mechanism. Methods: the whole bone marrow differential adherence method was used to separate the MSCs to fourth generations of cultured SD rats, and the liposome transfection method was used to transfect mi R21. -agomir (overexpressed) and MI R-21 antagomir (inhibition) regulated the expression of MI R-21 in MSCs. After the transfection, the cells were collected and the conditioned medium was collected and used for follow-up study. In vivo, the myocardial infarction model of SD rats was established, and the MSCsmi R-21 agomir group and MSCsmi group were not transfected. The group and PBS were transplanted to the myocardial infarction and the surrounding area to evaluate the effect of cardiac function changes and myocardial infarction area after myocardial infarction. In vitro, the primary umbilical vein endothelial cells (HUVEC), myocardial cell line H9C2 cells, and cervical cancer cell line Hela cells were used to verify mi R-21 by the double Luciferase Report with the Hela cell as the carrier. Whether the target is Jagged1. and the culture medium produced by the MSCs transfection group and the culture medium produced by the MSCs untransfected group and the DMEM/HIGH culture medium containing 10%FBS+ double resistance (blank control), it is used as the following two experiments. One is to treat HUVEC cells after 72 hours to culture on the Matrigel matrix, and to observe the tube like structure in 12 hours; two After treating H9C2 cell 72h after adding CCK-8 solution to incubate for 1 hours, the cell proliferation was detected. Results: in the body experiment, the average LVEF (%) change value of MSCsmi R-21 antagomir group was: 17.14 + 2.63, MSCs mi R-21 agomir group LVEF (%) change value: 13.09 + 1.74, 13.34 + 2.25, three group and control group (%) change value: 7.09 Compared with 4.23, the effect of cardiac function recovery was better, the difference was statistically significant (P0.05). Among the three groups, the effect of MSCsmi R-21antagomir group and cardiac function recovery was the best, compared with the other two groups, the difference was statistically significant (P0.05). Through Masson staining analysis of myocardial infarction area, the section of myocardial infarction area and normal myocardial area accounted for the cross section. The ratio of peripheral diameter (%) was the smallest in MSCsmi R-21 antagomir group, 10.50 + 3.15, MSCsmi R-21 agomir15.91+4.12, 20.34 + 3.25 in MSCs untransfected group and 37.09 + 6.23 in PBS group (P0.05). The ratio of fibrotic tissue to normal myocardial area (%) MSCsmi R-21 antagomir group was the same as 8.31 + 2.35. MSCsmi R-21 agomir group 19.91+4.93, MSCs common group 25.14 + 2.19, PBS group 31.04 + 5.93 (P0.05). Through double luciferase reporter gene verification, we found mi R-21 can be combined with Jagged1 m RNA 3. Agged1, NOTCH, VEGF and other genes were all up-regulated. In vitro tube generation experiments, the tube like structure connection length of MSCsmi R-21 agomir medium group (11976.14 + 866.62) and MSCsmi R-21 antagomir medium group (12196.52 + 858), MSCs untransfected medium group (12275.09 + 561.74) had no statistical difference (P0.05), but the three group and the blank group There were statistically significant differences (P0.01); in the CCK8 experiment, the ability of MI R-21 to promote myocardial proliferation was significantly enhanced after inhibiting the expression of MI R-21 in MSCs. Conclusion: after the inhibition of the expression of MI R-21 in MSCs, the ability to treat myocardial infarction is enhanced by the combination of.Mi R-21 with Jagged1 m, and regulation of it. After inhibiting the expression of MI R-21 in the MSCs of SD rats, the conditioned medium of Jagged1, Notch, VEGF and other genes up-regulated.MSCs has the ability to promote the proliferation of cardiac myocytes. After the inhibition of the expression of MI R-21 in it, the ability to enhance the conditioned medium of ascension.MSCs also has the ability to promote tubular structure formation, but this ability It has nothing to do with the expression of MI R-21.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.22

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