本文選題：mir-205 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2015年博士論文

【摘要】：背景骨髓間充質(zhì)干細(xì)胞是由骨髓中分離而來(lái)的,由于其具有多種分化潛能,因此受到多個(gè)領(lǐng)域的廣泛關(guān)注。骨髓間充質(zhì)干細(xì)胞能在一定條件下誘導(dǎo)分化成多種成熟細(xì)胞,其中包括成骨細(xì)胞、脂肪細(xì)胞和軟骨細(xì)胞,誘導(dǎo)骨髓間充質(zhì)干細(xì)胞朝向成骨細(xì)胞分化的研究一直是本領(lǐng)域研究的熱點(diǎn)。在組織損傷修復(fù)以及種植骨結(jié)合方面BMSCs也發(fā)揮重要功能：遷移到缺損組織部位,分化為成骨干細(xì)胞。SATB2全稱為特異性AT富集序列結(jié)合蛋白2,是特異性AT富集序列結(jié)合蛋白家族中的一員,其主要與核基質(zhì)蛋白結(jié)合。SATB2能夠與唾液蛋白(BSP)和骨鈣蛋白(OCN)的基因相互結(jié)合,并發(fā)揮調(diào)控其轉(zhuǎn)錄的作用。同時(shí)SATB2還能夠增加Runt相關(guān)的轉(zhuǎn)錄因子2(Runx2)和活化轉(zhuǎn)錄因子4(ATF4)的轉(zhuǎn)錄活性,由此,SATB2在調(diào)節(jié)成骨分化中的重要作用漸漸被人們所認(rèn)識(shí)。最近,SATB2被認(rèn)為是一個(gè)成骨分化的新的標(biāo)志物。SATB2不僅能夠激活成骨相關(guān)轉(zhuǎn)錄蛋白的活性,并且能夠增強(qiáng)其他轉(zhuǎn)錄因子的功能,因此,我們的研究目標(biāo)是,深入探究SATB2在成骨分化中的作用。microRNA是小RNA家族的一種,能夠結(jié)合信使RNA的非編碼區(qū)域,通過(guò)降解或者抑制mRNA從而調(diào)節(jié)mRNA的活性,起到調(diào)節(jié)蛋白表達(dá)的作用。microRNA參與調(diào)節(jié)細(xì)胞多項(xiàng)生理功能,包括細(xì)胞的增殖、分化、調(diào)亡以及自噬等各種生理過(guò)程。最近的研究顯示,多種microRNA能夠參與骨髓間充質(zhì)干細(xì)胞的分化過(guò)程,其中包括mir-31,34c,204,338-3p等等。mir-205之前被作為一個(gè)腫瘤抑制因子被廣泛研究,前期的研究顯示mir-205能夠抑制腫瘤細(xì)胞的細(xì)胞增殖以及細(xì)胞遷移,然而最近的研究顯示,mir-205在調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞的分化中也發(fā)揮著十分重要的作用。然而,相應(yīng)的具體機(jī)制仍不完全清楚,并且mir-205在骨髓間充質(zhì)干細(xì)胞中的作用也未得到證實(shí),因此我們擬通過(guò)體外細(xì)胞實(shí)驗(yàn),檢測(cè)mir-205在骨髓間充質(zhì)干細(xì)胞中的重要作用。目的通過(guò)體外原代培養(yǎng)骨髓間充質(zhì)干細(xì)胞,檢測(cè)mir-205在細(xì)胞增殖和細(xì)胞分化過(guò)程中改變。通過(guò)mir-205的抑制劑和模擬物抑制mir-205在骨髓間充質(zhì)干細(xì)胞中的表達(dá),觀察改變mir-205之后,骨髓間充質(zhì)干細(xì)胞分化過(guò)程中的改變。同時(shí)觀察Runx2和SATB2的表達(dá)改變,揭示mir-205與SATB2和Runx2的關(guān)系,為闡明microRNA調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞功能提供有力的證據(jù)和新的思路。方法1.mir-205在大鼠骨髓間充質(zhì)干細(xì)胞成骨分化中的作用。采用骨髓間充質(zhì)干細(xì)胞原代培養(yǎng)的方法觀察mir-205在其成骨分化中的作用。運(yùn)用RT-PCR實(shí)驗(yàn)檢測(cè)mir-205在成骨分化中的表達(dá)。通過(guò)加入mir-205抑制劑和模擬物來(lái)改變骨髓間充質(zhì)干細(xì)胞中mir-205的表達(dá),通過(guò)檢測(cè)成骨關(guān)鍵蛋白BSP和OPN的表達(dá)來(lái)檢測(cè)骨髓間充質(zhì)干細(xì)胞的成骨分化功能。2.mir-205對(duì)SATB2和Runx2的調(diào)節(jié)作用。我們首先通過(guò)生物信息學(xué)分析,發(fā)現(xiàn)mir-205與SATB2的3’UTR區(qū)存在堿基互補(bǔ)配對(duì),提示兩者可能存在密切的關(guān)系。通過(guò)Western blot實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn),高表達(dá)mir-205能降低SATB2的蛋白表達(dá)。通過(guò)RT-PCR實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)Runx2也是mir-205的靶mRNA。3. SATB2調(diào)節(jié)mir-205介導(dǎo)骨髓間充質(zhì)干細(xì)胞的成骨分化。我們首先構(gòu)建PEGFP-N1質(zhì)粒,使SATB2在骨髓間充質(zhì)干細(xì)胞中表達(dá)升高。通過(guò)Western blot實(shí)驗(yàn)檢測(cè)Runx2蛋白的改變。另外我們通過(guò)ALP和OCN檢測(cè)升高SATB2是否能有效增加骨髓間充質(zhì)干細(xì)胞的成骨分化能力。4. MAPK信號(hào)通路在mir-205調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞中的作用。通過(guò)Western blot檢測(cè)MAPK信號(hào)通路磷酸化程度的改變,干預(yù)mir-205后觀察MAPK信號(hào)通路的改變。結(jié)果1.mir-205在骨髓間充質(zhì)干細(xì)胞成骨分化中逐漸下降,而在細(xì)胞增殖過(guò)程中,無(wú)明顯變化。RT-PCR實(shí)驗(yàn)結(jié)果顯示,mir-205在骨髓間充質(zhì)干細(xì)胞分化過(guò)程中呈時(shí)間依賴性下降,而在細(xì)胞增殖過(guò)程中卻不發(fā)生變化。為了更加明確mir-205在骨髓間充質(zhì)干細(xì)胞分化中的改變情況,我們采用Real time-PCR驗(yàn)證之前的結(jié)果,結(jié)果顯示加入分化培養(yǎng)基后能有效降低mir-205的表達(dá),統(tǒng)計(jì)學(xué)分析顯示,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。而加入增殖分化培養(yǎng)基mir-205卻不會(huì)發(fā)生降低,并且隨著培養(yǎng)時(shí)間的延長(zhǎng),mir-205的表達(dá)下降。2.抑制mir-205的表達(dá)能增加骨髓間充質(zhì)干細(xì)胞的成骨分化。我們通過(guò)加入mir-205抑制劑和模擬物來(lái)改變骨髓間充質(zhì)干細(xì)胞中mir-205的表達(dá),通過(guò)BSP和OPN的蛋白表達(dá)改變來(lái)反映骨髓間充質(zhì)干細(xì)胞的分化情況。結(jié)果顯示,當(dāng)加入分化培養(yǎng)基48h后,我們發(fā)現(xiàn)加入mir-205模擬物能有效抑制BSP和OCN的蛋白表達(dá)水平。而加入mir-205的抑制劑能顯著增加BSP和OCN的蛋白表達(dá)。同時(shí),ALP活性實(shí)驗(yàn)和OCN分泌量實(shí)驗(yàn)結(jié)果顯示,降低mir-205的表達(dá)能增加ALP的活性,同時(shí)增加OCN向周圍的分泌量,而高表達(dá)mir-205能降低ALP活性和減少OCN分泌量。3.mir-205能夠調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞中SATB2和Runx2的表達(dá)我們首先通過(guò)生物信息學(xué)的預(yù)測(cè),發(fā)現(xiàn)mir-205與SATB2的3’UTR區(qū)存在堿基互補(bǔ)配對(duì),提示兩者可能存在密切的關(guān)系。為了進(jìn)一步驗(yàn)證mir-205和SATB2之間的結(jié)合,我們通過(guò)點(diǎn)突變制造了突變型SATB2的3’UTR區(qū)。熒光素酶結(jié)果顯示給予骨髓間充質(zhì)干細(xì)胞mir-205模擬物能顯著降低野生型SATB2的表達(dá),而不能降低突變型SATB2。另外,通過(guò)Western blot實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn),高表達(dá)mir-205能降低SATB2的蛋白表達(dá)。另外我們發(fā)現(xiàn)Runx2也是mir-205的靶mRNA,升高mir-205能夠降低Runx2的表達(dá),而降低mir-205能夠升高Runx2的表達(dá)。我們的實(shí)驗(yàn)結(jié)果提示,mir-205可能是通過(guò)調(diào)節(jié)SATB2和Runx2影響骨髓間充質(zhì)干細(xì)胞的成骨分化。4. SATB2能夠調(diào)節(jié)mir-205介導(dǎo)骨髓間充質(zhì)干細(xì)胞的成骨分化我們采用PEGFP-N1質(zhì)粒升高SATB2的表達(dá)進(jìn)一步證明SATB2在骨髓間充質(zhì)干細(xì)胞分化的作用。通過(guò)Western blot實(shí)驗(yàn)我們發(fā)現(xiàn),轉(zhuǎn)染PEGFP-N1-SATB2質(zhì)粒后,我們發(fā)現(xiàn)Runx2的蛋白表達(dá)明顯升高。ALP和OCN檢測(cè)結(jié)果發(fā)現(xiàn),升高SATB2能有效增加骨髓間充質(zhì)干細(xì)胞的成骨分化能力。以上實(shí)驗(yàn)結(jié)果提示,SATB2能調(diào)節(jié)mir-205介導(dǎo)的骨髓間充質(zhì)干細(xì)胞的成骨分化。5.抑制mir-205能夠增加ERK和MAPK的磷酸化水平ERK和MAPK通路在調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞的成骨分化中起到非常重要的作用,之前的研究顯示MAPK在調(diào)節(jié)microRNA中也起到重要作用。因此我們接下來(lái)的實(shí)驗(yàn)檢測(cè)ERK和MAPK信號(hào)通路在mir-205介導(dǎo)的成骨分化中的作用。結(jié)果顯示在分化過(guò)程中,ERK和MAPK在分化過(guò)程中磷酸化程度逐漸加強(qiáng),而加入mir-205能顯著抑制ERK和MAPK的磷酸化。我們的結(jié)果顯示mir-205可能是通過(guò)抑制ERK和MAPK的磷酸化來(lái)抑制骨髓間充質(zhì)干細(xì)胞的成骨分化。結(jié)論在我們的實(shí)驗(yàn)中,我們通過(guò)體外原代培養(yǎng)骨髓間充質(zhì)干細(xì)胞檢測(cè)mir-205在細(xì)胞增殖和細(xì)胞分化過(guò)程中改變,之后通過(guò)mir-205的抑制劑和模擬物改變mir-205的表達(dá),觀察改變mi-205之后,細(xì)胞分化過(guò)程中的改變。通過(guò)檢測(cè)Runx2和SATB2的表達(dá)改變,觀察mir-205與SATB2和Runx2的關(guān)系。另外。我們通過(guò)檢測(cè)分化過(guò)程中ERK和P38MAPK通路的相關(guān)變化,發(fā)現(xiàn)mir-205可能通過(guò)調(diào)節(jié)MAPK通路影響骨髓間充質(zhì)干細(xì)胞的分化。我們的研究為理解microRNA調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞功能提供了有力的證據(jù)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.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R78

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