年龄相关基因IGFBP7调控人骨髓间充质干细胞成骨分化的研究

发布时间：2018-02-05 01:36

本文关键词： 年龄骨髓间充质干细胞 IGFBP7 成骨分化 Wnt/β-catenin信号通路干细胞膜片　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：人类正迈入一个老龄化的社会。老龄化带来的骨质疏松及骨质疏松骨折的增加是一个亟需重视的问题。大量的证据证明老年性骨质疏松症是一种多基因疾病,基因间的互相调控在骨代谢中发挥了重要的作用。而且老年性的骨质疏松是骨低转换的疾病,以骨合成减少为主。骨的合成主要是由干细胞成骨分化而来,所以年龄相关基因对干细胞的调控与骨合成代谢密切相关。明确年龄相关基因对骨合成代谢的影响及对干细胞成骨分化的调控机制,对了解骨质疏松的发病机制,治疗和预防骨质疏松骨折起到重要作用。既往的研究表明,胰岛素样生长因子结合蛋白7(isulin-like growth factor bondingprotein7,IGFBP7)与衰老和骨代谢密切相关。但至今,IGFBP7在不同年龄的骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)中的表达情况,以及在BMSCs成骨分化中的作用及机制仍不清楚。所以,本研究旨在探讨IGFBP7基因对BMSCs成骨分化的影响,希望为骨合成代谢提供新的靶点。本研究通过筛选不同年龄组的BMSCs中IGFBP7的表达情况明确其与年龄的关系。同时,体外实验,通过在BMSCs中过表达和敲低IGFBP7的表达,来明确IGFBP7在BMSCs成骨分化中的作用,并探索其信号通路调控机制。此外,体内实验,利用IGFBP7修饰的BMSCs修复骨缺损模型,来验证其对骨缺损的修复影响。(?)本研究共分为以下3个部分:(1)IGFBP7在不同年龄组中BMSCs中的表达情况;(2)IGFBP7对BMSCs成骨分化的作用及其机制研究;(3)IGFBP7修饰的BMSCs膜片修复大鼠胫骨骨缺损的研究。第一章IGFBP7在不同年龄组中BMSCs中的表达情况目的:探索IGFBP7在不同年龄组的BMSCs中表达情况。方法:通过梯度离心的方法分离培养人BMSCs,通过流式分析鉴定提取细胞表面抗原,并通过对提取细胞进行成脂、成骨、成软骨诱导鉴定其多向分化能力。将不同年龄人分为年轻组(16-22岁)和高龄组(≥70岁);年轻组15人,高龄组13人。通过ELISA检测不同年龄组的血清中IGFBP7的表达情况。通过RT-qPCR、Western blot分析等比较两组的BMSCs的成骨特异性的基因、IGFBP7和β-catenin的表达情况。同时比较BMSCs不同代数之间成骨特异性的基因、IGFBP7和β-catenin的表达情况。结果:通过流式鉴定显示提取的细胞CD73阳性率为99.8%,CD109阳性率为99.9%,CD105 阳性率为 98.5%,CD34 阳性率为 0.042%,CD45 阳性率为 0.06%,CD31阳性率为0.051%。所提取的细胞能成功进行成骨分化、成脂分化、成软骨分化。ELISA检测显示高龄老人血清中的IGFBP7蛋白的表达明显高于年轻人(P0.05)。老年人的BMSCs中成骨特异性的基因(RUNX2、OCN、SP7)和β-catenin表达是降低的(P0.05),但是IGFBP7的表达是增加的(P0.05)。随着传代的增加,成骨标志物和β-catenin表达降低(P0.05),IGFBP7的表达明显增高(P0.05)。结论:在BMSCs中,随着年龄的增长IGFBP7的表达增加,而成骨特异性的基因和Wnt/β-catenin信号通路的表达降低。第二章IGFBP7对BMSCs成骨分化的作用及其机制研究目的:探索IGFBP7对BMSCs成骨分化的作用及其机制。方法:在BMSCs中,通过慢病毒载体过表达和敲低IGFBP7的表达。将BMSCs分为过表达IGFBP7组、过表达对照组、敲低IGFBP7和敲低对照组。通过台盼蓝染色鉴定IGFBP7的表达对BMSCs增殖能力的影响。通过RT-qPCR、Western blot分析和免疫荧光,检测不同组间的成骨特异性基因表达差异。通过ALP染色和ALP活性检测,不同分组间的ALP的表达情况和活性情况。通过茜素红染色和Von Kossa染色检测不同分组间矿化水平的差异。同时添加外源性重组IGFBP7蛋白检测其对BMSCs的成骨分化的影响。通过Western blot分析和免疫荧光分析检测常见成骨分化信号通路的变化。并通过抑制剂和siRNA干扰信号通路,验证其对IGFBP7成骨影响的变化。结果:与对照组相比,敲低IGFBP7组的BMSCs的细胞活力明显减低(P0.05),而过表达IGFBP7组的BMSCs的细胞活力无明显变化(P0.05)。在成骨分化诱导的第3天和第7天,RT-qPCR显示较对照组,过表达IGFBP7组BMSCs成骨特异性基因(ALP、RUNX2、SP7、COL1A1、SP、OCN)的表达显著升高(P0.05);敲低IGFBP7组BMSCs成骨特异性基因的表达显著降低(P0.05)。免疫荧光和Western blot检测分析显示:在诱导的第1天,较对照组,过表达IGFBP7组成骨特异性蛋白(RUNX2、SP7、COL1A1)的表达显著升高;敲低IGFBP7组成骨特异性蛋白(RUNX2、SP7、COL]A1)的表达显著降低。ALP染色和ALP活性检测结果显示:成骨诱导3天和7天,过表达IGFBP7组ALP活性明显升高,敲低IGFBP7组ALP的活性明显减低(P0.05)。茜素红和Von Kossa染色显示:成骨分化诱导第10天,过表达IGFBP7组钙结节积累明显增多(P0.05),敲低IGFBP7组钙结节的积累明显减少(P0.05)。外源性的重组IGFBP7蛋白对BMSCs的成骨分化作用呈现出浓度依赖性,当浓度≥500ng/mL时,其可以加强BMSCs的成骨分化(P0.05)。信号通路检测结果显示:成骨分化第1天,MAPK(ERK/p-38/JNK)信号通路和PI3K/Akt信号通路无明显变化,而过表达IGFBP7可以明显增加总β-catenin蛋白的量,而且激活状态的β-catenin蛋白也增加。应用特异性的Wnt/β-catenin信号通路的抑制剂DKK1(1000ng/mL),可以明显减弱因为IGFBP7过表达增加的成骨分化效应。应用特异性的β-catenin信号siRNA,也可以明显减弱因为IGFBP7过表达增加的成骨分化效应。结论:在BMSCs中,IGFBP7通过Wnt/β-catenin信号通路调控BMSCs的成骨分化。第三章IGFBP7基因修饰的BMSCs膜片对大鼠胫骨骨缺损愈合的影响目的:探索过表达IGFBP7的BMSCs膜片对大鼠胫骨骨缺损愈合的影响。方法:通过体外培养构建BMSCs细胞膜片。将30只SD大鼠随机分配为3组,分别为空白组(blank组)、对照组细胞膜片组(Ctrl组)、过表达IGFBP7干细胞膜片组(exIg7组),每组10只。应用膜片修复大鼠胫骨骨缺损。术后8周,安乐死动物后,取标本。以X线和Mirco-CT评估骨缺损愈合情况。同时,用HE染色、番红速绿、Masson三色染色和免疫组化评估骨缺损愈合情况。GFP免疫组化追踪移植细胞存留情况。结果:X线和Mirco-CT结果显示:空白组(Blank)骨折间隙仍然清晰可见,未见明显连续的骨痂形成;对照组(Ctrl)骨折线模糊,可见明显的骨痂生长,矿化组织已开始连续,骨折线已经模糊,骨缺损区域未完全愈合;过表达IGFBP7组(exIg7)骨折区域矿化组织已完全填充,骨缺损已接近愈合。HE染色、番红速绿和Masson三色染色显示:较空白组相比,BMSCs膜片明显加骨速缺损愈合,过表达IGFBP7组愈合的最快。COL1A1和OPN的免疫组化也显示相似的结果。GFP的免疫组化显示少量的骨组织残留GFP蛋白。结论:过表达IGFBP7的BMSCs膜片可以加速骨缺损的修复。
[Abstract]:Mankind is entering an aging society. To increase the aging of osteoporosis and osteoporotic fracture is an urgent need to pay attention to the problem. A lot of evidence of senile osteoporosis is a polygenic disease, mutual regulation between genes play an important role in bone metabolism and the elderly. Osteoporosis is a bone disease with low conversion, synthetic bone decreased. Bone synthesis is mainly come from stem cells, osteogenic differentiation, so the age related genes on the regulation of stem cell and bone metabolism are closely related to specific age related genes on bone anabolic effects and regulation mechanism of differentiation the stem cells, to understand the pathogenesis of osteoporosis, treatment and prevention of osteoporotic fractures play an important role. Previous studies indicated that insulin-like growth factor binding protein 7 (isulin-like growth factor bonding Protein7, IGFBP7) is closely associated with aging and bone metabolism. But so far, IGFBP7 in different age of bone marrow mesenchymal stem cells (bone marrow derived mesenchymal stem cells, BMSCs) the expression, as well as in the BMSCs into effect and mechanism of bone differentiation remains unclear. The purpose of this study was to investigate IGFBP7 gene effects of osteogenic differentiation of BMSCs, hoping to provide a new target for bone anabolism. In this study the expression of IGFBP7 through the screening of different age groups in BMSCs to clarify its relationship with age. At the same time, through in vitro experiments, overexpression and knockdown of IGFBP7 in BMSCs, to clear IGFBP7 in BMSCs the role of osteogenic differentiation, and to explore the signal transduction mechanism. In addition, the in vivo experiment, using BMSCs IGFBP7 to repair bone defect model modification, to verify the effect of repair of the bone defect. (?) this study is divided into the following 3 parts: ( 1) the expression of IGFBP7 BMSCs in different age groups in; (2) study the effect and mechanism of differentiation of IGFBP7 to BMSCs; (3) study on repairing bone defect of tibia in rats with BMSCs IGFBP7 modified diaphragm. The purpose of the first chapter of IGFBP7 BMSCs expression in different age groups: To investigate the expression of IGFBP7 in different age groups in BMSCs. Methods: Cultured BMSCs by gradient centrifugation separation by flow cytometry analysis of isolated cell surface antigens, and through the extraction cell adipogenic, osteogenic, chondrogenic differentiation ability. The identification of the different age were divided into young group (16-22 years old) and old group (aged 70); young group of 15 people, 13 people in old age group. The expression of ELISA by detection of serum IGFBP7 in different age groups. Through RT-qPCR, bone specific genes Western blot analysis to compare two groups of BMSCs, IGFBP7 and beta -caten The expression of in. At the same time comparison between different BMSCs algebra of osteoblast specific gene expression of IGFBP7 and beta -catenin. Results: by flow cytometry showed that the positive rate of CD73 cell extraction was 99.8%, the positive rate of CD109 was 99.9%, the positive rate of CD105 was 98.5%, the positive rate of CD34 was 0.042%, the positive rate of CD45 0.06%, the positive rate of CD31 was extracted 0.051%. cells can successfully osteogenic, adipogenic differentiation and chondrogenic differentiation of.ELISA showed that the expression of IGFBP7 protein in serum of elderly patients was significantly higher than that of young people (P0.05). The elderly BMSCs in osteoblast specific genes (RUNX2, OCN, and SP7) beta -catenin expression is lower (P0.05), but the expression of IGFBP7 was increased (P0.05). With the passage of the increased osteogenic markers and decreased expression of -catenin beta (P0.05), IGFBP7 expression was significantly higher (P0.05). Conclusion: in BMSCs, with the increasing of age Increased expression of IGFBP7, and the expression of specific genes and Wnt/ beta -catenin signaling pathway decreased. The second chapter IGFBP7 of BMSCs objective to study the effect and mechanism of differentiation of BMSCs into IGFBP7: To explore the effect and mechanism of bone differentiation. Methods: in BMSCs, the lentiviral vector expression and knockdown of IGFBP7. Overexpression of BMSCs were divided into IGFBP7 group, expression control group, knockdown of IGFBP7 knockdown and control group. Affected by trypan blue staining and identification of IGFBP7 expression on the proliferation ability of BMSCs. Through RT-qPCR, Western blot analysis and immunofluorescence detection, the expression of different groups. Specific gene differences. Detected by ALP staining and ALP activity, expression and activity of ALP in different groups. The difference between the different groups of mineralization level detection stained by alizarin red staining and Von Kossa. The addition of exogenous IG and heavy group Detection of FBP7 protein on BMSCs osteogenic differentiation. Through Western blot analysis and immunofluorescence detection and analysis of common signal pathway of osteoblast differentiation. The inhibitor and siRNA interference pathway, verify its osteogenic effect on IGFBP7 changes. Results: compared with the control group, the knockdown of IGFBP7 group BMSCs the cell viability was significantly decreased (P0.05), and the expression of IGFBP7 group of BMSCs cell activity (P0.05). No significant changes in osteogenic differentiation induced by third days and seventh days, RT-qPCR showed that compared with the control group, over expression of IGFBP7 group BMSCs osteoblast specific genes (ALP, RUNX2, SP7, COL1A1. SP, OCN) was significantly higher (P0.05); group BMSCs knockdown of IGFBP7 expression of specific genes was significantly decreased (P0.05). Immunofluorescence and Western blot analysis showed that: in first days of induction, compared with the control group, over expression of bone specific protein composition of IGFBP7 (RUNX2, SP7, C OL1A1) the knockdown of IGFBP7 expression was significantly increased; the composition of bone specific protein (RUNX2, SP7, COL]A1) was significantly reduced by.ALP staining and ALP activity assay showed that the osteogenic induction for 3 days and 7 days, over expression of IGFBP7 in ALP group was significantly increased, knockdown of IGFBP7 group ALP activity was significantly decreased (P0.05). Alizarin red and Von Kossa staining showed that the osteogenic differentiation induced by tenth days, over expression of IGFBP7 group significantly increased the accumulation of calcium nodules (P0.05), knockdown of IGFBP7 group significantly reduced the accumulation of calcium nodules (P0.05). Exogenous recombinant IGFBP7 protein on BMSCs osteogenic differentiation effect showed a concentration dependence. When the concentration is 500ng/mL, which can enhance the osteogenic differentiation of BMSCs (P0.05) signal pathway. Test results show that: the osteogenic differentiation of the first day, MAPK (ERK/p-38/JNK) pathway and the PI3K/Akt pathway had no obvious change, while overexpression of IGFBP7 could significantly increase the total beta -catenin protein The amount and the activation status of -catenin protein also increased. Wnt/ beta -catenin signaling pathway by specific inhibitors of DKK1 (1000ng/mL), can be significantly reduced because of the overexpression of IGFBP7 increased osteogenic differentiation effect. Beta -catenin signal siRNA application specific, can also be significantly weakened because of the overexpression of IGFBP7 increased the osteogenic differentiation effect. Conclusion: in BMSCs, IGFBP7 through the osteogenic differentiation of Wnt/ beta -catenin signaling pathway of BMSCs. The effects of BMSCs gene modified IGFBP7 membrane third chapter on the healing of bone defect of tibia in rats Objective: To explore the effect of overexpression of BMSCs IGFBP7 membrane on the healing of bone defect of tibia in rats. Methods: in vitro culture construction of BMSCs cell membrane. 30 SD rats were randomly divided into 3 groups, namely control group (blank group) and control group (group Ctrl) cell membrane group, overexpression of IGFBP7 stem cell membrane group (exIg7 group, n = 10) Only. The repair of the tibial bone defect in rats using patch. After 8 weeks, animal euthanasia after specimens. With X-ray and Mirco-CT evaluation of bone defect healing. At the same time, using HE staining, safranin fast green, Masson trichrome staining and immunohistochemical evaluation of bone defect healing.GFP immunohistochemistry to track transplanted cells retained. Results: the X-ray and Mirco-CT showed that the blank group (Blank) the fracture gap is still clearly visible, no obvious continuous bone callus formation; the control group (Ctrl) fracture lines blurred, visible callus growth obviously, mineralized tissue has begun continuously, the fracture line was blurred, the bone defect area had not healed completely; the expression of IGFBP7 group (exIg7) fracture zone of mineralized tissue has been completely filled, has been close to the bone defect healing.HE staining, safranin fast green and Masson trichrome staining showed that: compared with the blank group, BMSCs and bone defect rate of diaphragm was healing, healing of the over expression of IGFBP7 group The immunohistochemistry of.COL1A1 and OPN also showed similar results. Immunohistochemical staining of.GFP showed a small amount of residual GFP protein in bone tissue. Conclusion: over expression of IGFBP7 BMSCs patch can accelerate bone defect repair.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68

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