Mir-205调节骨髓间充质干细胞成骨分化中的机制研究

发布时间：2018-04-25 17:31

本文选题：mir-205 + 骨髓间充质干细胞　；参考：《中国人民解放军医学院》2015年博士论文

【摘要】：背景骨髓间充质干细胞是由骨髓中分离而来的,由于其具有多种分化潜能,因此受到多个领域的广泛关注。骨髓间充质干细胞能在一定条件下诱导分化成多种成熟细胞,其中包括成骨细胞、脂肪细胞和软骨细胞,诱导骨髓间充质干细胞朝向成骨细胞分化的研究一直是本领域研究的热点。在组织损伤修复以及种植骨结合方面BMSCs也发挥重要功能：迁移到缺损组织部位,分化为成骨干细胞。SATB2全称为特异性AT富集序列结合蛋白2,是特异性AT富集序列结合蛋白家族中的一员,其主要与核基质蛋白结合。SATB2能够与唾液蛋白(BSP)和骨钙蛋白(OCN)的基因相互结合,并发挥调控其转录的作用。同时SATB2还能够增加Runt相关的转录因子2(Runx2)和活化转录因子4(ATF4)的转录活性,由此,SATB2在调节成骨分化中的重要作用渐渐被人们所认识。最近,SATB2被认为是一个成骨分化的新的标志物。SATB2不仅能够激活成骨相关转录蛋白的活性,并且能够增强其他转录因子的功能,因此,我们的研究目标是,深入探究SATB2在成骨分化中的作用。microRNA是小RNA家族的一种,能够结合信使RNA的非编码区域,通过降解或者抑制mRNA从而调节mRNA的活性,起到调节蛋白表达的作用。microRNA参与调节细胞多项生理功能,包括细胞的增殖、分化、调亡以及自噬等各种生理过程。最近的研究显示,多种microRNA能够参与骨髓间充质干细胞的分化过程,其中包括mir-31,34c,204,338-3p等等。mir-205之前被作为一个肿瘤抑制因子被广泛研究,前期的研究显示mir-205能够抑制肿瘤细胞的细胞增殖以及细胞迁移,然而最近的研究显示,mir-205在调节骨髓间充质干细胞的分化中也发挥着十分重要的作用。然而,相应的具体机制仍不完全清楚,并且mir-205在骨髓间充质干细胞中的作用也未得到证实,因此我们拟通过体外细胞实验,检测mir-205在骨髓间充质干细胞中的重要作用。目的通过体外原代培养骨髓间充质干细胞,检测mir-205在细胞增殖和细胞分化过程中改变。通过mir-205的抑制剂和模拟物抑制mir-205在骨髓间充质干细胞中的表达,观察改变mir-205之后,骨髓间充质干细胞分化过程中的改变。同时观察Runx2和SATB2的表达改变,揭示mir-205与SATB2和Runx2的关系,为阐明microRNA调节骨髓间充质干细胞功能提供有力的证据和新的思路。方法1.mir-205在大鼠骨髓间充质干细胞成骨分化中的作用。采用骨髓间充质干细胞原代培养的方法观察mir-205在其成骨分化中的作用。运用RT-PCR实验检测mir-205在成骨分化中的表达。通过加入mir-205抑制剂和模拟物来改变骨髓间充质干细胞中mir-205的表达,通过检测成骨关键蛋白BSP和OPN的表达来检测骨髓间充质干细胞的成骨分化功能。2.mir-205对SATB2和Runx2的调节作用。我们首先通过生物信息学分析,发现mir-205与SATB2的3’UTR区存在碱基互补配对,提示两者可能存在密切的关系。通过Western blot实验检测发现,高表达mir-205能降低SATB2的蛋白表达。通过RT-PCR实验检测发现Runx2也是mir-205的靶mRNA。3. SATB2调节mir-205介导骨髓间充质干细胞的成骨分化。我们首先构建PEGFP-N1质粒,使SATB2在骨髓间充质干细胞中表达升高。通过Western blot实验检测Runx2蛋白的改变。另外我们通过ALP和OCN检测升高SATB2是否能有效增加骨髓间充质干细胞的成骨分化能力。4. MAPK信号通路在mir-205调节骨髓间充质干细胞中的作用。通过Western blot检测MAPK信号通路磷酸化程度的改变,干预mir-205后观察MAPK信号通路的改变。结果1.mir-205在骨髓间充质干细胞成骨分化中逐渐下降,而在细胞增殖过程中,无明显变化。RT-PCR实验结果显示,mir-205在骨髓间充质干细胞分化过程中呈时间依赖性下降,而在细胞增殖过程中却不发生变化。为了更加明确mir-205在骨髓间充质干细胞分化中的改变情况,我们采用Real time-PCR验证之前的结果,结果显示加入分化培养基后能有效降低mir-205的表达,统计学分析显示,差异有统计学意义(P0.05)。而加入增殖分化培养基mir-205却不会发生降低,并且随着培养时间的延长,mir-205的表达下降。2.抑制mir-205的表达能增加骨髓间充质干细胞的成骨分化。我们通过加入mir-205抑制剂和模拟物来改变骨髓间充质干细胞中mir-205的表达,通过BSP和OPN的蛋白表达改变来反映骨髓间充质干细胞的分化情况。结果显示,当加入分化培养基48h后,我们发现加入mir-205模拟物能有效抑制BSP和OCN的蛋白表达水平。而加入mir-205的抑制剂能显著增加BSP和OCN的蛋白表达。同时,ALP活性实验和OCN分泌量实验结果显示,降低mir-205的表达能增加ALP的活性,同时增加OCN向周围的分泌量,而高表达mir-205能降低ALP活性和减少OCN分泌量。3.mir-205能够调节骨髓间充质干细胞中SATB2和Runx2的表达我们首先通过生物信息学的预测,发现mir-205与SATB2的3’UTR区存在碱基互补配对,提示两者可能存在密切的关系。为了进一步验证mir-205和SATB2之间的结合,我们通过点突变制造了突变型SATB2的3’UTR区。荧光素酶结果显示给予骨髓间充质干细胞mir-205模拟物能显著降低野生型SATB2的表达,而不能降低突变型SATB2。另外,通过Western blot实验检测发现,高表达mir-205能降低SATB2的蛋白表达。另外我们发现Runx2也是mir-205的靶mRNA,升高mir-205能够降低Runx2的表达,而降低mir-205能够升高Runx2的表达。我们的实验结果提示,mir-205可能是通过调节SATB2和Runx2影响骨髓间充质干细胞的成骨分化。4. SATB2能够调节mir-205介导骨髓间充质干细胞的成骨分化我们采用PEGFP-N1质粒升高SATB2的表达进一步证明SATB2在骨髓间充质干细胞分化的作用。通过Western blot实验我们发现,转染PEGFP-N1-SATB2质粒后,我们发现Runx2的蛋白表达明显升高。ALP和OCN检测结果发现,升高SATB2能有效增加骨髓间充质干细胞的成骨分化能力。以上实验结果提示,SATB2能调节mir-205介导的骨髓间充质干细胞的成骨分化。5.抑制mir-205能够增加ERK和MAPK的磷酸化水平ERK和MAPK通路在调节骨髓间充质干细胞的成骨分化中起到非常重要的作用,之前的研究显示MAPK在调节microRNA中也起到重要作用。因此我们接下来的实验检测ERK和MAPK信号通路在mir-205介导的成骨分化中的作用。结果显示在分化过程中,ERK和MAPK在分化过程中磷酸化程度逐渐加强,而加入mir-205能显著抑制ERK和MAPK的磷酸化。我们的结果显示mir-205可能是通过抑制ERK和MAPK的磷酸化来抑制骨髓间充质干细胞的成骨分化。结论在我们的实验中,我们通过体外原代培养骨髓间充质干细胞检测mir-205在细胞增殖和细胞分化过程中改变,之后通过mir-205的抑制剂和模拟物改变mir-205的表达,观察改变mi-205之后,细胞分化过程中的改变。通过检测Runx2和SATB2的表达改变,观察mir-205与SATB2和Runx2的关系。另外。我们通过检测分化过程中ERK和P38MAPK通路的相关变化,发现mir-205可能通过调节MAPK通路影响骨髓间充质干细胞的分化。我们的研究为理解microRNA调节骨髓间充质干细胞功能提供了有力的证据和新的思路。
[Abstract]:Background bone marrow mesenchymal stem cells are isolated from bone marrow. Because of their multiple differentiation potential, bone marrow mesenchymal stem cells are widely concerned in many fields. Bone marrow mesenchymal stem cells can be induced to differentiate into many mature cells under certain conditions, including osteoblasts, adipocytes and chondrocytes, and induce bone marrow mesenchymal stem cells. The study of osteoblast differentiation has been a hot spot in this field. BMSCs also plays an important role in tissue damage repair and implant bone binding. Migration to the defect tissue site, differentiated into diaphysis.SATB2 as a specific AT enrichment sequence binding protein 2, is a specific AT enriched sequence binding protein family. One member, which combines.SATB2 with the nuclear matrix protein, can combine with the genes of saliva protein (BSP) and Osteocalcin (OCN), and plays a role in regulating its transcription. Meanwhile, SATB2 can also increase the transcriptional activity of Runt related transcription factor 2 (Runx2) and activated transcription factor 4 (ATF4). Thus, SATB2 plays an important role in regulating osteogenesis. SATB2 has recently been recognized as a new marker of osteogenesis,.SATB2 not only activates the activity of bone related transcriptional proteins, but also enhances the function of other transcription factors. Therefore, our goal is to explore the role of SATB2 in the osteogenic differentiation of.MicroRNA as one of the small RNA family. The non coding region of the messenger RNA, which regulates the activity of mRNA by degrading or inhibiting mRNA, plays a role in regulating the expression of protein, and.MicroRNA participates in the regulation of cell multiple physiological functions, including cell proliferation, differentiation, autophagy, and autophagy. Recent studies have shown that a variety of microRNA can participate in bone. The differentiation process of medullary mesenchymal stem cells, including mir-31,34c, 204338-3p and so on, was widely studied as a tumor suppressor before.Mir-205. Previous studies showed that mir-205 could inhibit cell proliferation and cell migration in tumor cells. However, recent studies have shown that mir-205 regulates bone marrow mesenchymal stem cells. However, the specific mechanisms are still not completely clear, and the role of mir-205 in bone marrow mesenchymal stem cells has not been confirmed. Therefore, we intend to detect the important role of mir-205 in bone marrow mesenchymal stem cells through in vitro cell experiments. The changes in the proliferation and differentiation of mir-205 were detected in the cell proliferation and cell differentiation. The expression of mir-205 in bone marrow mesenchymal stem cells was inhibited by mir-205 inhibitors and simulants. The changes in the differentiation of bone marrow mesenchymal stem cells after the change of mir-205 were observed. The changes in the expression of Runx2 and SATB2 were observed, and mir-205 and SATB2 were revealed. The relationship with Runx2 provides powerful evidence and new ideas to clarify the function of microRNA in regulating the function of bone marrow mesenchymal stem cells. Method the role of 1.mir-205 in the osteogenesis of bone marrow mesenchymal stem cells in rats. The role of mir-205 in the osteogenic differentiation of bone marrow mesenchymal stem cells was observed by the method of RT-PCR test. The expression of mir-205 in osteogenic differentiation. The expression of mir-205 in bone marrow mesenchymal stem cells was changed by adding mir-205 inhibitors and simulants. The expression of the key bone protein BSP and OPN was detected to detect the regulation of the osteogenic differentiation of bone marrow mesenchymal stem cells (.2.mir-205) on SATB2 and Runx2. We first passed the biology. Informatics analysis found that mir-205 and the 3 'UTR region of SATB2 are complementary to the base pairs, suggesting that there may be a close relationship between the two. Through the Western blot test, the high expression of mir-205 can reduce the protein expression of SATB2. It is found that Runx2 is also the target mRNA.3. SATB2 of mir-205 through RT-PCR experiment. Osteogenic differentiation of stem cells. We first constructed the PEGFP-N1 plasmid to increase the expression of SATB2 in bone marrow mesenchymal stem cells. The Western blot test was used to detect the changes in the Runx2 protein. In addition, we detected whether the increase of SATB2 can effectively increase the osteogenic differentiation of bone marrow mesenchymal stem cells by ALP and OCN and the.4. MAPK signaling pathway in the mir-20 is in mir-20. 5 modulate the role of bone marrow mesenchymal stem cells. The changes in the degree of phosphorylation of MAPK signaling pathway were detected by Western blot, and the changes of MAPK signaling pathway were observed after mir-205 intervention. Results 1.mir-205 decreased gradually in bone marrow mesenchymal stem cells, and there was no obvious change of.RT-PCR experimental results in the process of cell proliferation. Mir -205 has a time-dependent decline in the differentiation of bone marrow mesenchymal stem cells, but does not change during cell proliferation. In order to make more clear of the changes in the differentiation of mir-205 in bone marrow mesenchymal stem cells, we used Real time-PCR to verify the results. The results show that mir-205 can effectively reduce mir-205 after the differentiation medium. The statistical analysis showed that the difference was statistically significant (P0.05), but the proliferation and differentiation medium mir-205 did not decrease, and with the prolongation of the culture time, the expression of mir-205 decreased by the expression of.2. and the expression of mir-205 could increase the osteogenic differentiation of bone marrow mesenchymal stem cells. We added mir-205 inhibitors and simulants. To change the expression of mir-205 in bone marrow mesenchymal stem cells and to reflect the differentiation of bone marrow mesenchymal stem cells by BSP and OPN protein expression changes. The results show that after adding the differentiation medium 48h, we found that adding mir-205 mimics can effectively inhibit the protein expression level of BSP and OCN. The inhibitors with mir-205 can be significant. The protein expression of BSP and OCN was increased. At the same time, the results of ALP activity and OCN secretion showed that the decrease of mir-205 expression could increase the activity of ALP and increase the secretion of OCN to the surrounding area, while the high expression of mir-205 could reduce ALP activity and reduce OCN secreted quantity.3.mir-205 can regulate the expression of SATB2 in bone marrow mesenchymal stem cells. Through bioinformatics prediction, we found that mir-205 and the 3 'UTR region of SATB2 are complementary to each other, suggesting that there may be a close relationship. In order to further verify the combination of mir-205 and SATB2, we made the 3' UTR region of the mutant SATB2 by point mutation. The results of luciferase show that the bone marrow stroma is given. The stem cell mir-205 mimics can significantly reduce the expression of wild type SATB2, but can not reduce the mutant SATB2.. The high expression of mir-205 can reduce the protein expression of SATB2 by the Western blot test. Furthermore, we found that Runx2 is the target mRNA of mir-205, and the increase mir-205 can reduce the expression of Runx2. Runx2 expression. Our experimental results suggest that mir-205 may regulate the osteogenic differentiation of bone marrow mesenchymal stem cells by regulating SATB2 and Runx2,.4. SATB2 can regulate the osteogenesis of bone marrow mesenchymal stem cells mediated by mir-205. We use PEGFP-N1 plasmids to increase the expression of SATB2 to prove SATB2 in bone marrow mesenchymal stem cells. Through the Western blot experiment, we found that after transfection of PEGFP-N1-SATB2 plasmid, we found that the expression of Runx2 protein was significantly increased by.ALP and OCN detection results, and that elevated SATB2 could effectively increase the osteogenic differentiation of bone marrow mesenchymal stem cells. These results suggest that SATB2 can regulate the mir-205 mediated bone marrow mesenchymal stem cells. Osteogenic differentiation of cells.5. inhibits mir-205, which increases the phosphorylation level of ERK and MAPK, ERK and MAPK pathways play a very important role in regulating bone marrow mesenchymal stem cells' osteogenesis. Previous studies showed that MAPK plays an important role in regulating microRNA. Therefore, our next experiment detected ERK and MAPK signaling pathways. The effect of mir-205 mediated osteogenic differentiation showed that during differentiation, the degree of phosphorylation of ERK and MAPK increased gradually during the differentiation process, while mir-205 could significantly inhibit the phosphorylation of ERK and MAPK. Our results suggest that mir-205 may inhibit the osteogenesis of mesenchymal stem cells by inhibiting the phosphorylation of ERK and MAPK. Conclusion in our experiment, in our experiment, we detected the changes of mir-205 in cell proliferation and cell differentiation through primary cultured bone marrow mesenchymal stem cells in vitro, then changed the expression of mir-205 through mir-205 inhibitors and simulants, observed changes in the process of cell differentiation after the change of mi-205. By detecting Runx2 and SATB2 The relationship between mir-205 and SATB2 and Runx2 was observed. In addition, we found that mir-205 may affect the differentiation of bone marrow mesenchymal stem cells by regulating the MAPK pathway by detecting the related changes in the ERK and P38MAPK pathways during the differentiation. Our study provides a powerful evidence for understanding the function of microRNA to regulate bone marrow mesenchymal stem cells. According to the new idea.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R78

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