MiR-17-5p调控非创伤性股骨头坏死骨髓基质干细胞成骨分化与增殖的相关研究

发布时间：2018-06-07 00:08

本文选题：非创伤性股骨头坏死 + microRNA　；参考：《华中科技大学》2015年博士论文

【摘要】：目的：观察非创伤性股骨头坏死患者与骨性关节炎患者骨髓基质干细胞中主要miRNA的表达差异,寻找在非创伤性股骨头坏死发病过程中起重要调节作用的miRNA。方法：大量查阅文献后找到参与调节骨髓基质干细胞成骨分化,成脂分化,增殖相关过程的主要备选miRNA10个,分别为：miR-20a, miR-17-5p, miR-27a-3p, miR-96-5p, miR-124-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p。临床上收集了20例非创伤性股骨头坏死患者原代骨髓基质干细胞以及10例对照组患者原代骨髓基质干细胞,使用实时荧光定量RT-PCR方法检测上述10个miRNA的在实验组及对照组中的表达。结果：miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p在实验组表达较对照组显著降低,有统计学意义(p0.05),miR-96-5p, miR-124-3p在对照组和实验组间无明显差异(p0.05)。结论：miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p可能通过靶向于不同的mRNA调节骨髓基质干细胞的分化与增殖,在非创伤性股骨头坏死病理过程中起作用。目的：miR-17-5p在非创伤性股骨头坏死患者MSC中表达水平显著低于对照组,本部分实验目的在于通过生物信息学预测与荧光素酶报告基因检测找到并确定miR-17-5p的靶基因,并检测其在非创伤性股骨头坏死患者MSC标本中的表达水平以及在HMSC-bm成骨分化过程中的表达水平变化规律。方法：使用TargetScan6.2与Pictar网站进行miR-17-5p靶基因预测。实时定量PCR检测第一部分中20个非创伤性股骨头坏死患者MSC标本中SMAD7表达水平,以及其与miR-17-5p表达水平是否具有相关性。实时定量PCR检测BMP-2诱导的HMSC-bm成骨分化过程中第0,1,2,4,6天miR-17-5p及SMAD7表达水平变化趋势(与不加入BMP-2的对照组进行比较)。在HMSC-bm细胞中使用miR-17-5p类似物与miR-17-5p抑制剂调整miR-17-5p表达水平,实时定量PCR检测SMAD7表达水平,Western蛋白印迹试验检测Smad7浓度。采用荧光素酶报告基因检测方法,检测miR-17-5p与SMDA7是否具有调控关系。结果：SMAD7通过生物信息学预测可能是miR-17-5p的靶基因,结合SMAD7的生理作用我们认为miR-17-5p可能通过靶向抑制SMAD7在非创伤性股骨头坏死病理过程中起调控作用。Real-time PCR结果显示在非创伤性股骨头坏死患者MSC标本中miR-17-5p与SMAD7表达水平呈负相关(R2=0.5440,p=0.0002)。在BMP-2诱导的HMSC-bm成骨分化过程中miR-17-5p的表达水平自诱导后第一天起显著高于对照组(p0.05),SMAD7的表达水平自诱导后第二天起显著低于对照组(p0.05)。miR-17-5p类似物组SMAD7表达水平与Smad7蛋白浓度显著高于阴性对照组,miR-17-5p抑制剂组SMAD7表达水平与Smad7蛋白浓度显著低于阴性对照组,差异均有统计学意义(p0.05)。荧光素酶报告基因检测示miR-17-5p通过靶向抑制SMAD7下调其表达。结论：miR-17-5p通过不完全互补结合SMAD7的3'UTR端靶向抑制其表达,在非创伤性股骨头坏死发病过程及MSC成骨分化过程中发挥调控作用。目的：研究]miR-17-5p提高HMSC-bm细胞系增殖与成骨分化的机制。方法：在HMSC-bm细胞中转染miR-17-5p类似物与miR-17-5p抑制剂调整miR-17-5p表达水平并设立阴性对照,转染4小时后加入100ng/ml BMP-2进行成骨诱导。6天后实时定量PCR观察RUNX2以及Ⅰ型胶原表达水平改变,通过ALP染色观察HMSC-bm细胞内碱性磷酸酶表达强度,通过CCK-8试剂盒检测细胞增殖的改变。为了证明HMSC-bm细胞增殖及成骨分化增强是通过SMAD7表达水平下降而导致的,我们通过转染pcDNA3.1-SMAD7人为提高SMAD7表达水平,观察以上效应是否减弱及逆转。结果：在BMP-2诱导的HMSC-bm成骨分化过程中,转染miR-17-5p类似物组RUNX2, COL1A1的表达水平显著高于阴性对照组(p0.05),碱性磷酸酶表达显著高于对照组,细胞增殖显著高于对照组(p0.05)。转染miR-17-5p抑制剂组RUNX2,Ⅰ型胶原的表达水平显著低于阴性对照组(p0.05),碱性磷酸酶表达显著低于对照组,细胞增殖显著低于对照组(p0.05)。通过转染pcDNA3.1-SMAD7人为提高SMAD7表达水平后可见以上效应被部分削弱。结论：miR-17-5p可提高HMSC-bm细胞的成骨分化及增殖,这种调控通过靶向抑制SMAD7表达起作用。
[Abstract]:Objective: To observe the difference in the expression of main miRNA in the bone marrow stromal cells of patients with non traumatic osteonecrosis of femoral head and osteoarthritis, and to find the important regulatory role of miRNA. in the pathogenesis of non traumatic femoral head necrosis.
Methods: after consulting a large number of literature, we found the main alternative miRNA10 to regulate the osteogenic differentiation, lipid differentiation and proliferation related processes of bone marrow stromal cells, which were miR-20a, miR-17-5p, miR-27a-3p, miR-96-5p, miR-124-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, and miR-199a-5p., 20 cases of non traumatic cases were collected. The original bone marrow stromal cells of the patients with femoral head necrosis and 10 cases of the primary bone marrow stromal cells in the control group were used to detect the expression of the 10 miRNA in the experimental group and the control group by real time fluorescence quantitative RT-PCR.
Results: the expression of miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p in the experimental group was significantly lower than that in the control group. There was a significant difference (P0.05), miR-96-5p, miR-124-3p between the control group and the experimental group (P0.05).
Conclusion: miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p may regulate the differentiation and proliferation of bone marrow stromal stem cells by targeting different mRNA, and play a role in the pathological process of non traumatic femoral head necrosis.
Objective: the expression level of miR-17-5p in the patients with non traumatic avascular necrosis of the femoral head was significantly lower than that of the control group. The purpose of this study was to find and determine the target gene of miR-17-5p through bioinformatics prediction and luciferase reporter gene detection, and to detect the expression level of the MSC in the non traumatic patients with femoral head necrosis and the expression of the target gene in the MSC specimens. The expression level of HMSC-bm during osteogenic differentiation.
Methods: TargetScan6.2 and Pictar sites were used to predict the target gene of miR-17-5p. Real-time quantitative PCR was used to detect the level of SMAD7 expression in the MSC specimens of 20 non traumatic patients with avascular necrosis of the femoral head, and whether it was related to the expression level of miR-17-5p. Real-time quantitative PCR was used to detect the number of BMP-2 induced HMSC-bm in the process of osteogenesis. 0,1,2,4,6 days miR-17-5p and SMAD7 expression level changes (compared with those that do not join BMP-2). MiR-17-5p analogues and miR-17-5p inhibitors are used in HMSC-bm cells to adjust miR-17-5p expression level, real-time quantitative PCR detection SMAD7 expression level, Western protein blot test for Smad7 concentration. Using luciferase reporter base The detection method is used to detect whether miR-17-5p has a regulatory relationship with SMDA7.
Results: SMAD7 may be the target gene of miR-17-5p through bioinformatics. Combining the physiological role of SMAD7, we believe that miR-17-5p may play a regulatory role in the pathological process of non traumatic femoral head necrosis by targeting SMAD7, and.Real-time PCR results show miR-17-5p and SMAD7 in MSC specimens of non traumatic patients with necrosis of the femoral head. The expression level was negatively correlated (R2=0.5440, p=0.0002). The expression level of miR-17-5p in BMP-2 induced HMSC-bm osteogenesis was significantly higher than that of the control group (P0.05) from the first day after induction. The expression level of SMAD7 was significantly lower than the control group (P0.05).MiR-17-5p analogue group SMAD7 expression level and Smad7 protein concentration from the control group (P0.05) in the second day after induction. Significantly higher than the negative control group, the level of SMAD7 expression and Smad7 protein concentration in the miR-17-5p inhibitor group were significantly lower than that of the negative control group, and the difference was statistically significant (P0.05). The luciferase reporter gene detection showed that the expression of miR-17-5p was down regulated by the target inhibition SMAD7.
Conclusion: miR-17-5p plays a regulatory role in the process of non traumatic avascular necrosis of the femoral head and the process of MSC osteogenesis in the process of non traumatic necrosis of the femoral head through incomplete complementarity and the inhibition of the expression of the 3'UTR end target of the SMAD7.
Objective: To study the mechanism of]miR-17-5p enhancing the proliferation and osteogenic differentiation of HMSC-bm cell line.
Methods: transfection of miR-17-5p analogue and miR-17-5p inhibitor in HMSC-bm cells to adjust miR-17-5p expression level and set up negative control. After 4 hours transfection, 100ng/ml BMP-2 was added to.6 days after.6 days to observe RUNX2 and the expression level of type I collagen, and ALP staining was used to observe the alkaline phosphatase in HMSC-bm cells. In order to prove that the proliferation of HMSC-bm cells and the enhancement of osteogenic differentiation are caused by the decrease of the expression level of SMAD7, the expression level of SMAD7 is enhanced by transfection of pcDNA3.1-SMAD7, and the effects of the above effects are observed to see if the above effects are weakened and reversed by the CCK-8 kit.
Results: in the process of BMP-2 induced HMSC-bm osteogenesis, the expression of COL1A1 was significantly higher than that of the negative control group (P0.05), and the expression of alkaline phosphatase was significantly higher than that of the control group (P0.05). The cell proliferation was significantly higher than that of the control group (P0.05). The expression of miR-17-5p inhibitor group was significantly lower than that of the control group (P0.05). The expression level of type I collagen was significantly lower. In the negative control group (P0.05), the expression of alkaline phosphatase was significantly lower than that of the control group, and the cell proliferation was significantly lower than that of the control group (P0.05). The above effects were partially weakened after transfection of pcDNA3.1-SMAD7 to improve the expression of SMAD7.
Conclusion: miR-17-5p can enhance the osteogenic differentiation and proliferation of HMSC-bm cells. This regulation can play a role in inhibiting SMAD7 expression by targeting.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R681.8

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