miR-21及其靶基因Sprouty1调控人骨髓间充质干细胞成骨分化的机制研究

发布时间：2018-07-06 08:40

  本文选题：骨髓间充质干细胞 + miR-21　； 参考：《第四军医大学》2012年博士论文

【摘要】：在炎症性骨疾病中，骨形成能力受损是造成骨丢失的主要原因之一。提高骨形成能力是治疗这类疾病的关键问题。骨髓间充质干细胞（BMMSCs）是成骨细胞和骨细胞的前体细胞。在炎症性骨疾病如骨质疏松，类风湿性关节炎中，BMMSCs分化为成骨细胞的能力减弱可能导致骨形成障碍。因此，研究调控BMMSCs的成骨分化机制，提高骨髓间充质成骨分化能力对于治疗炎症性骨疾病具有重要意义。 MicroRNAs (miRNA)是一类短小的非编码RNA，它们通过翻译后抑制或者降解靶基因的mRNA在细胞的多种生理病理过程中发挥重要的调控作用，例如细胞的增殖、分化、凋亡和癌症的发生进展。研究表明，miRNAs能够调控间充质干细胞和成骨细胞的成骨分化。本课题组前期结果和文献报道均表明，，miR-21与人骨髓间充质干细胞(hBMMSCs)成骨分化以及绝经后骨质疏松相关，但miR-21是否调控了hBMMSCs的成骨分化，以及miR-21如何调控hBMMSCs的成骨分化，它们的表达和功能是否在绝经后骨质疏松环境中发生变化，目前还未知晓。研究miR-21对hBMMSCs成骨分化的调控作用以及在骨质疏松环境下表达和功能的异常，对于理解BMMSCs成骨分化的机制和开发治疗骨质疏松新的治疗策略具有重要意义。 研究目的 1.探讨miR-21在hBMMSCs成骨分化过程中的表达。 2.探讨miR-21在hBMMSCs体外与体内成骨分化过程中的调控作用。 3.探讨miR-21调控hBMMSCs成骨分化的分子机制。 4.探讨miR-21在绝经后骨质疏松骨髓间充质干细胞（PMOP-hBMMSC）中表达和功能的异常。 研究方法 1.利用密度梯度离心和全骨髓贴壁相结合的方法分离培养hBMMSCs，流式细胞技术检测表面分子表达；克隆集落形成、MTT、细胞周期分析检测细胞增殖能力。成骨、成脂、成神经诱导检测hBMMSCs多向分化能力。Real time RT-PCR分析成骨成脂诱导后标志性基因的表达。 2.利用Real time RT-PCR检测miR-21在hBMMSCs成骨分化中的表达变化。通过转染技术过表达或者抑制miR-21的表达，体外检测hBMMSCs成骨分化能力。ALP、茜素红染色观察碱性磷酸酶和钙化结节的表达。Real time RT-PCR和Western Blot检测成骨标志性基因Runx2和Osterix的表达，并且将miR-21不同表达水平的hBMMSCs植入裸鼠皮下，8周后取材，固定后脱钙两周石蜡包埋切片，HE染色和Masson三色染色观测体内类骨质形成。 3.生物信息学预测结合荧光素酶报告检测和Western Blot蛋白分析，确定miR-21在hBMMSCs成骨分化中作用的主要靶基因Sprouty1(Spry1)。Western Blot检测Spry1在成骨过程的表达。构建Spry1慢病毒表达载体，感染hBMMSCs。ALP、茜素红染色、 RT-PCR和Western Blot分析Spry1高表达后，hBMMSCs成骨能力的改变。 4.分离培养正常人（H-hBMMSCs）和绝经后骨质疏松患者(PMOP-hBMMSCs)的骨髓间充质干细胞，并对两组细胞的成骨能力进行比较。Real time RT-PCR比较miR-21在两组细胞间的表达差异，同时检测两组hBMMSCs中Spry1基因和蛋白水平表达的差异。利用转染的方法上调PMOP-hBMMSCs中miR-21的水平，检测其成骨能力的改变及Spry1的表达变化。 实验结果 1.分离培养的hBMMSCs表达间充质表面标志物，而不表达造血系标志物。克隆集落和MTT结果显示hBMMSCs具有自我更新和复制的能力。成骨、成脂、成软骨诱导分化，证明培养的hBMMSCs具有多向分化能力。 2. miR-21在hBMMSCs成骨过程中表达量升高。通过对miR-21的功能分析发现，高表达miR-21能够促进hBMMSCs体外成骨分化，并且也能够促进hBMMSCs体内异位成骨能力，而沉默miR-21能够抑制hBMMSCs体外成骨分化，降低hBMMSCs体内异位成骨能力。 3.通过生物信息学预测的方法筛选出了miR-21的靶基因Spry1。荧光素酶报告检测和蛋白分析证明Spry1是miR-21的靶基因。通过对Spry1在成骨过程中表达谱分析发现Spry1在成骨过程中表达量下降而与miR-21在成骨分化中的表达相反。Spry1功能分析发现，上调Spry1能够抑制hBMMSCs的成骨分化。 4.与H-hBMMSCs相比PMOP-hBMMSCs成骨能力明显减弱，而且miR-21表达下降，Spry1表达上升。通过转染miR-21，能够部分恢复PMOP-hBMMSCs的成骨分化能力，下调Spry1。 结论： 1. miR-21能够促进hBMMSCs的体外成骨分化能力。 2. miR-21能够增强hBMMSCs体内异位成骨的能力。 3. miR-21通过抑制其靶基因Spry1的表达来促进hBMMSCs的成骨分化。 4. PMOP-hBMMSCs成骨能力减弱，miR-21-Spry1功能轴受到抑制。上调miR-21-Spry1功能轴，能够部分恢复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 .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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