miRNA-146a在人正常软骨细胞压力损伤模型中的功能及机制研究

发布时间：2018-07-17 02:00

【摘要】：miRNA-146a是最早发现的与骨性关节炎相关的差异性miRNA。我们通过miRNA芯片技术研究发现其在急性压力损伤后的软骨细胞中表达也会上调。然而miRNA-146a在软骨细胞压力损伤中的作用尚不清楚。本研究的目的就是希望找到miRNA-146a的靶基因，并阐明其在软骨细胞压力损伤中的作用机制。 miRNA芯片技术研究显示，相对于正常软骨细胞，压力损伤后的软骨细胞miRNA-146a表达上调，采用关联分析的方法，发现生物信息学分析预测的miRNA-146a的靶基因SMAD4，在联合应用的全基因组基因芯片结果中低表达。因此，我们推测miRNA-146a通过SMAD4介导的方式来调控软骨细胞的压力损伤。研究目的：在这一假设的基础上，本实验拟通过研究阐明miRNA-146a在软骨细胞压力损伤中的调控作用及其分子机制；证明SMAD4是miRNA-146a的直接靶基因，并参与介导miRNA-146a对软骨细胞压力损伤的调控。我们希望此研究可以为临床上急性关节损伤的治疗带来新的研究思路和切入点。研究方法： 1.原代分离及培养人软骨细胞和骨性关节炎细胞，使用光镜下观察其生长情况，MTT法绘制生长曲线，采用甲苯胺蓝染色、II型胶原免疫组化染色等方法对比两种细胞的差别。 2.研制一套具有自主知识产权的实用多功能细胞压力加载系统，为进行细胞力学相关实验奠定基础。使用该系统构建软骨细胞压力损伤模型，力求尽量模拟高能损伤的力学特点。 3.联合应用miRNA芯片和全基因组基因芯片筛选正常及压力损伤后软骨细胞的差异miRNA和基因。采用Realtime RT-PCR分析的方法，，验证压力损伤组特异性高表达的miRNA-146a表达情况是否与芯片结果一致，采用Realtime RT-PCR的方法及Western Blot的方法，验证芯片结果中SMAD4和VEGF的差异表达。 4.通过向细胞内转染miRNA-146a的模拟物、抑制物、无关序列和空白对照组的方式，调控miRNA-146a的表达，进一步研究其功能。 5.观察调控miRNA-146a的表达后，对软骨细胞生物学特性的影响，并使用实时定量RT-PCR和Western-blot检测各组中SMAD4和VEGF的表达情况。 6.观察miRNA-146a的上调和下调后对软骨细胞压力损伤作用的影响 7.采用双荧光素酶报告基因系统证明SMAD4的mRNA3’UTR和miRNA-146a之间可以直接结合，验证了SMAD4是miRNA-146a的直接靶基因。研究结果： 1.通过细胞表型和生物学活性的检测，保证原代培养所得细胞为状态良好的软骨细胞，可以进行下一步实验。 2.根据对比MTT等试验结果，选择对细胞生物学特性影响加大，且大部分存活的参数，10Mpa60min作为成模参数。 3. miRNA-146a是芯片结果中差异最显著的一个，联合基因芯片结果并根据生物信息学方法预测其靶基因，找出了在芯片结果中与预测结果相一致的潜在靶基因SMAD4，并成功验证了芯片结果。 4.转染外源性miRNA-146a mimics后，人正常软骨细胞中miRNA-146a的表达水平显著上升。转染外源性miRNA-146a inhibitor后，人正常软骨细胞中miRNA-146a的表达水平显著下调。 5.在正常人软骨细胞中上调miRNA-146a的表达，可以使SMAD4的表达下调并使VEGF的表达上调，miRNA-146a的表达下调时可引起相反的作用。 6.实验结果显示miRNA-146a具有抑制细胞增殖，促进压力损伤软骨细胞的凋亡的能力。 7. SMAD4是miRNA-146a的直接靶基因。研究结论： 1.软骨细胞压力损伤后，miRNA-146a的表达升高，SMAD4表达下调而VEGF的表达升高。 2. SMAD4是miRNA-146a的直接靶基因。 3. miRNA-146a可以通过SMAD4介导的方式调控VEGF的表达，具有促进软骨细胞凋亡的作用。 4. miRNA-146a可能通过下调SMAD4的方式阻断TGF-β通路，上调VEGF的表达水平，促进骨性关节炎的发生。
[Abstract]:MiRNA-146a is the first identified differential miRNA. associated with osteoarthritis. We found that the expression in the cartilage cells after acute stress injury is also up regulated by miRNA chip technology. However, the role of miRNA-146a in cartilage pressure damage is not clear. The purpose of this study is to find the target base of miRNA-146a. To elucidate its mechanism in the stress injury of chondrocytes.
MiRNA chip technology studies show that the expression of miRNA-146a in cartilage cells after stress damage is up regulation relative to normal cartilage cells. Using correlation analysis, the target gene SMAD4 of miRNA-146a, predicted by bioinformatics analysis, is low expressed in the results of the combined whole genome gene chip. Therefore, we speculate that miRNA-146a passes through the results. SMAD4 mediated the regulation of stress damage in chondrocytes.
On the basis of this hypothesis, this study intends to elucidate the regulatory role of miRNA-146a in cartilage cell stress damage and its molecular mechanism. It is proved that SMAD4 is the direct target gene of miRNA-146a and is involved in the regulation of miRNA-146a's regulation of cartilage cell stress damage. We hope this study can be a clinical acute joint injury. Treatment brings new research ideas and breakthrough points.
Research methods:
1. the human chondrocytes and osteoarthritis cells were isolated and cultured in the original generation. The growth of the cells was observed under the light microscope. The growth curve was drawn by MTT method. The difference of the two cells was compared with the methods of toluidine blue staining and the immunohistochemical staining of type II collagen.
2. a practical multifunction cell pressure loading system with independent intellectual property rights is developed to lay the foundation for the experiment of cell mechanics. The system is used to construct the model of cartilage cell stress damage, and try to simulate the mechanical characteristics of high energy damage.
3. miRNA chip and whole genome microarray were used to screen the difference miRNA and gene of cartilage cells after normal and stress damage. The method of Realtime RT-PCR analysis was used to verify whether the specific high expression of miRNA-146a expression in the stress group was in accordance with the results of the chip. The method of using Realtime RT-PCR and the prescription of Western Blot were used. Methods to verify the differential expression of SMAD4 and VEGF in the chip results.
4. to regulate the expression of miRNA-146a by transfecting miRNA-146a mimic, inhibitor, irrelevant sequence and blank control group to further study its function.
5. the effects of the expression of miRNA-146a on the biological characteristics of chondrocytes were observed, and the expression of SMAD4 and VEGF in each group was detected by real-time quantitative RT-PCR and Western-blot.
6. to observe the effect of miRNA-146a up regulation and down regulation on the stress injury of chondrocytes.
7. dual luciferase reporter gene system is used to prove that mRNA3 'UTR and miRNA-146a can directly bind to SMAD4, which proves that SMAD4 is the direct target gene of miRNA-146a.
The results of the study:
1. through the detection of cell phenotype and biological activity, we can ensure that primary cultured cells are well preserved chondrocytes, and we can carry out the next experiment.
2. based on the comparison of MTT and other experimental results, 10Mpa60min was chosen as the model parameter to increase the influence of cell biological characteristics and most of the survival parameters.
3. miRNA-146a is the most significant difference in the result of the chip. Combining the results of the gene chip and predicting the target gene according to the bioinformatics method, the potential target gene SMAD4 which is consistent with the prediction results in the result of the chip is found, and the result of the chip is verified successfully.
After transfection of exogenous miRNA-146a mimics, the expression level of miRNA-146a in normal human cartilage cells increased significantly. After transfection of exogenous miRNA-146a inhibitor, the expression level of miRNA-146a in normal human cartilage cells decreased significantly.
5. up regulation of the expression of miRNA-146a in normal human chondrocytes can reduce the expression of SMAD4 and increase the expression of VEGF. The down regulation of miRNA-146a can cause the opposite effect.
6. the experimental results showed that miRNA-146a could inhibit cell proliferation and promote the ability of stress to damage chondrocyte apoptosis.
7. SMAD4 is the direct target gene of miRNA-146a.
The conclusions are as follows:
1. the expression of miRNA-146a increased and the expression of SMAD4 was downregulated and the expression of VEGF increased after the stress injury of chondrocytes.
2. SMAD4 is the direct target gene of miRNA-146a.
3. miRNA-146a can regulate the expression of VEGF through SMAD4 mediated manner, and has the effect of promoting chondrocyte apoptosis.
4. miRNA-146a may block the TGF- beta pathway by up regulating SMAD4, up regulate the expression level of VEGF, and promote the occurrence of osteoarthritis.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R684.3

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