本文選題：骨髓間充質(zhì)干細(xì)胞 + miR-21�。� 參考：《第四軍醫(yī)大學(xué)》2012年博士論文

【摘要】：在炎癥性骨疾病中，骨形成能力受損是造成骨丟失的主要原因之一。提高骨形成能力是治療這類疾病的關(guān)鍵問題。骨髓間充質(zhì)干細(xì)胞（BMMSCs）是成骨細(xì)胞和骨細(xì)胞的前體細(xì)胞。在炎癥性骨疾病如骨質(zhì)疏松，類風(fēng)濕性關(guān)節(jié)炎中，BMMSCs分化為成骨細(xì)胞的能力減弱可能導(dǎo)致骨形成障礙。因此，研究調(diào)控BMMSCs的成骨分化機(jī)制，提高骨髓間充質(zhì)成骨分化能力對于治療炎癥性骨疾病具有重要意義。 MicroRNAs (miRNA)是一類短小的非編碼RNA，它們通過翻譯后抑制或者降解靶基因的mRNA在細(xì)胞的多種生理病理過程中發(fā)揮重要的調(diào)控作用，例如細(xì)胞的增殖、分化、凋亡和癌癥的發(fā)生進(jìn)展。研究表明，miRNAs能夠調(diào)控間充質(zhì)干細(xì)胞和成骨細(xì)胞的成骨分化。本課題組前期結(jié)果和文獻(xiàn)報道均表明，，miR-21與人骨髓間充質(zhì)干細(xì)胞(hBMMSCs)成骨分化以及絕經(jīng)后骨質(zhì)疏松相關(guān)，但miR-21是否調(diào)控了hBMMSCs的成骨分化，以及miR-21如何調(diào)控hBMMSCs的成骨分化，它們的表達(dá)和功能是否在絕經(jīng)后骨質(zhì)疏松環(huán)境中發(fā)生變化，目前還未知曉。研究miR-21對hBMMSCs成骨分化的調(diào)控作用以及在骨質(zhì)疏松環(huán)境下表達(dá)和功能的異常，對于理解BMMSCs成骨分化的機(jī)制和開發(fā)治療骨質(zhì)疏松新的治療策略具有重要意義。 研究目的 1.探討miR-21在hBMMSCs成骨分化過程中的表達(dá)。 2.探討miR-21在hBMMSCs體外與體內(nèi)成骨分化過程中的調(diào)控作用。 3.探討miR-21調(diào)控hBMMSCs成骨分化的分子機(jī)制。 4.探討miR-21在絕經(jīng)后骨質(zhì)疏松骨髓間充質(zhì)干細(xì)胞（PMOP-hBMMSC）中表達(dá)和功能的異常。 研究方法 1.利用密度梯度離心和全骨髓貼壁相結(jié)合的方法分離培養(yǎng)hBMMSCs，流式細(xì)胞技術(shù)檢測表面分子表達(dá)；克隆集落形成、MTT、細(xì)胞周期分析檢測細(xì)胞增殖能力。成骨、成脂、成神經(jīng)誘導(dǎo)檢測hBMMSCs多向分化能力。Real time RT-PCR分析成骨成脂誘導(dǎo)后標(biāo)志性基因的表達(dá)。 2.利用Real time RT-PCR檢測miR-21在hBMMSCs成骨分化中的表達(dá)變化。通過轉(zhuǎn)染技術(shù)過表達(dá)或者抑制miR-21的表達(dá)，體外檢測hBMMSCs成骨分化能力。ALP、茜素紅染色觀察堿性磷酸酶和鈣化結(jié)節(jié)的表達(dá)。Real time RT-PCR和Western Blot檢測成骨標(biāo)志性基因Runx2和Osterix的表達(dá)，并且將miR-21不同表達(dá)水平的hBMMSCs植入裸鼠皮下，8周后取材，固定后脫鈣兩周石蠟包埋切片，HE染色和Masson三色染色觀測體內(nèi)類骨質(zhì)形成。 3.生物信息學(xué)預(yù)測結(jié)合熒光素酶報告檢測和Western Blot蛋白分析，確定miR-21在hBMMSCs成骨分化中作用的主要靶基因Sprouty1(Spry1)。Western Blot檢測Spry1在成骨過程的表達(dá)。構(gòu)建Spry1慢病毒表達(dá)載體，感染hBMMSCs。ALP、茜素紅染色、 RT-PCR和Western Blot分析Spry1高表達(dá)后，hBMMSCs成骨能力的改變。 4.分離培養(yǎng)正常人（H-hBMMSCs）和絕經(jīng)后骨質(zhì)疏松患者(PMOP-hBMMSCs)的骨髓間充質(zhì)干細(xì)胞，并對兩組細(xì)胞的成骨能力進(jìn)行比較。Real time RT-PCR比較miR-21在兩組細(xì)胞間的表達(dá)差異，同時檢測兩組hBMMSCs中Spry1基因和蛋白水平表達(dá)的差異。利用轉(zhuǎn)染的方法上調(diào)PMOP-hBMMSCs中miR-21的水平，檢測其成骨能力的改變及Spry1的表達(dá)變化。 實驗結(jié)果 1.分離培養(yǎng)的hBMMSCs表達(dá)間充質(zhì)表面標(biāo)志物，而不表達(dá)造血系標(biāo)志物�？寺〖浜蚆TT結(jié)果顯示hBMMSCs具有自我更新和復(fù)制的能力。成骨、成脂、成軟骨誘導(dǎo)分化，證明培養(yǎng)的hBMMSCs具有多向分化能力。 2. miR-21在hBMMSCs成骨過程中表達(dá)量升高。通過對miR-21的功能分析發(fā)現(xiàn)，高表達(dá)miR-21能夠促進(jìn)hBMMSCs體外成骨分化，并且也能夠促進(jìn)hBMMSCs體內(nèi)異位成骨能力，而沉默miR-21能夠抑制hBMMSCs體外成骨分化，降低hBMMSCs體內(nèi)異位成骨能力。 3.通過生物信息學(xué)預(yù)測的方法篩選出了miR-21的靶基因Spry1。熒光素酶報告檢測和蛋白分析證明Spry1是miR-21的靶基因。通過對Spry1在成骨過程中表達(dá)譜分析發(fā)現(xiàn)Spry1在成骨過程中表達(dá)量下降而與miR-21在成骨分化中的表達(dá)相反。Spry1功能分析發(fā)現(xiàn)，上調(diào)Spry1能夠抑制hBMMSCs的成骨分化。 4.與H-hBMMSCs相比PMOP-hBMMSCs成骨能力明顯減弱，而且miR-21表達(dá)下降，Spry1表達(dá)上升。通過轉(zhuǎn)染miR-21，能夠部分恢復(fù)PMOP-hBMMSCs的成骨分化能力，下調(diào)Spry1。 結(jié)論： 1. miR-21能夠促進(jìn)hBMMSCs的體外成骨分化能力。 2. miR-21能夠增強(qiáng)hBMMSCs體內(nèi)異位成骨的能力。 3. miR-21通過抑制其靶基因Spry1的表達(dá)來促進(jìn)hBMMSCs的成骨分化。 4. PMOP-hBMMSCs成骨能力減弱，miR-21-Spry1功能軸受到抑制。上調(diào)miR-21-Spry1功能軸，能夠部分恢復(fù)PMOP-hBMMSCs的成骨分化能力。
[Abstract]:Bone formation ability is one of the main causes of bone loss in inflammatory bone diseases . Bone formation ability is a key issue in the treatment of these diseases . Bone marrow mesenchymal stem cells ( BMMSCs ) are precursors of osteoblasts and osteocytes . In inflammatory bone diseases such as osteoporosis and rheumatoid arthritis , the ability of BMMSCs to differentiate into osteoblasts may lead to bone formation disorders .

MicroRNAs ( miRNA ) are a kind of short non - coding RNA , which plays an important role in regulating the differentiation of human bone marrow mesenchymal stem cells ( hBMMSCs ) and the progression of postmenopausal osteoporosis . It is shown that miR - 21 regulates the osteogenic differentiation of human bone marrow mesenchymal stem cells ( hBMMSCs ) and the postmenopausal osteoporosis .

Purpose of study

1 . To investigate the expression of miR - 21 in bone differentiation of hBMMSCs .

2 . To investigate the role of miR - 21 in vitro and in vivo osteogenic differentiation of hBMMSCs .

3 . To investigate the molecular mechanism of miR - 21 regulating the osteogenic differentiation of hBMMSCs .

4 . To investigate the expression and function of miR - 21 in postmenopausal osteoporosis bone marrow mesenchymal stem cells ( PMOP - hBMMSC ) .

Research Methods

1 . separating and culturing hBMMSCs by a method of combining density gradient centrifugation and full - bone marrow adherent cells , and performing flow cytometry to detect surface molecule expression ;
The proliferation of hBMMSCs was detected by colony forming , MTT and cell cycle analysis . The expression of marker genes after bone formation was analyzed by Real time RT - PCR .

2 . The expression of miR - 21 in bone differentiation of hBMMSCs was detected by Real time RT - PCR . The expression of miR - 21 was detected in vitro by transfection . ALP and alizarin red staining were used to observe the expression of alkaline phosphatase and calcification nodules . Real time RT - PCR and Western Blot were used to detect the expression of bone marker Runx2 and Osterix .

3 . The expression of miR - 21 in osteogenic differentiation of hBMMSCs was determined by bioinformatics prediction combined with luciferase reporter assay and Western Blot analysis .

4 . The bone marrow mesenchymal stem cells of normal human ( H - hBMMSCs ) and postmenopausal osteoporosis ( PMOP - hBMMSCs ) were isolated and compared .

experimental results

1 . The isolated cultured hBMMSCs express mesenchymal surface markers without expressing hematopoietic markers . The clones and MTT results show that hBMMSCs possess the ability to self - renew and replicate . These results show that the cultured hBMMSCs have multi - directional differentiation ability .

2 . The expression of miR - 21 in bone formation of hBMMSCs is increased . Through the functional analysis of miR - 21 , high expression of miR - 21 can promote bone differentiation in vitro of hBMMSCs , and also can promote ectopic osteogenesis in hBMMSCs , while silencing miR - 21 can inhibit the differentiation of hBMMSCs in vitro and reduce the ectopic osteogenesis in hBMMSCs .

3 . The target gene of miR - 21 was screened by bioinformatics prediction . Spryl was the target gene of miR - 21 . The expression of Spryl was decreased in the process of osteogenic differentiation compared with the expression of miR - 21 in osteogenic differentiation . Spryl function analysis showed that the up - regulation of Spryl inhibited the osteogenic differentiation of hBMMSCs .

4 . Compared with H - hBMMSCs , the osteogenic ability of PMOP - hBMMSCs decreased significantly , and the expression of miR - 21 decreased , and the expression of Spryl increased . Through transfection of miR - 21 , the osteogenic differentiation ability of PMOP - hBMMSCs can be partially recovered , and Spryl can be regulated down .

Conclusion :

1 . miR - 21 can promote the in vitro osteogenic differentiation capability of hBMMSCs .

2 . miR - 21 has the capability of enhancing the ectopic osteogenesis in hBMMSCs .

3 . miR - 21 promotes the osteogenic differentiation of hBMMSCs by inhibiting the expression of its target gene Spryl .

4 . The osteogenic ability of PMOP - hBMMSCs was weakened , and the function axis of miR - 21 - Spryl was inhibited . The osteogenic differentiation ability of PMOP - hBMMSCs could be partially restored by up - regulation of the miR - 21 - Spryl functional axis .
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

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