本文關(guān)鍵詞： 年齡 骨髓間充質(zhì)干細(xì)胞 IGFBP7 成骨分化 Wnt/β-catenin信號(hào)通路 干細(xì)胞膜片　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：人類正邁入一個(gè)老齡化的社會(huì)。老齡化帶來(lái)的骨質(zhì)疏松及骨質(zhì)疏松骨折的增加是一個(gè)亟需重視的問(wèn)題。大量的證據(jù)證明老年性骨質(zhì)疏松癥是一種多基因疾病,基因間的互相調(diào)控在骨代謝中發(fā)揮了重要的作用。而且老年性的骨質(zhì)疏松是骨低轉(zhuǎn)換的疾病,以骨合成減少為主。骨的合成主要是由干細(xì)胞成骨分化而來(lái),所以年齡相關(guān)基因?qū)Ω杉?xì)胞的調(diào)控與骨合成代謝密切相關(guān)。明確年齡相關(guān)基因?qū)呛铣纱x的影響及對(duì)干細(xì)胞成骨分化的調(diào)控機(jī)制,對(duì)了解骨質(zhì)疏松的發(fā)病機(jī)制,治療和預(yù)防骨質(zhì)疏松骨折起到重要作用。既往的研究表明,胰島素樣生長(zhǎng)因子結(jié)合蛋白7(isulin-like growth factor bondingprotein7,IGFBP7)與衰老和骨代謝密切相關(guān)。但至今,IGFBP7在不同年齡的骨髓間充質(zhì)干細(xì)胞(bone marrow derived mesenchymal stem cells,BMSCs)中的表達(dá)情況,以及在BMSCs成骨分化中的作用及機(jī)制仍不清楚。所以,本研究旨在探討IGFBP7基因?qū)MSCs成骨分化的影響,希望為骨合成代謝提供新的靶點(diǎn)。本研究通過(guò)篩選不同年齡組的BMSCs中IGFBP7的表達(dá)情況明確其與年齡的關(guān)系。同時(shí),體外實(shí)驗(yàn),通過(guò)在BMSCs中過(guò)表達(dá)和敲低IGFBP7的表達(dá),來(lái)明確IGFBP7在BMSCs成骨分化中的作用,并探索其信號(hào)通路調(diào)控機(jī)制。此外,體內(nèi)實(shí)驗(yàn),利用IGFBP7修飾的BMSCs修復(fù)骨缺損模型,來(lái)驗(yàn)證其對(duì)骨缺損的修復(fù)影響。(?)本研究共分為以下3個(gè)部分:(1)IGFBP7在不同年齡組中BMSCs中的表達(dá)情況;(2)IGFBP7對(duì)BMSCs成骨分化的作用及其機(jī)制研究;(3)IGFBP7修飾的BMSCs膜片修復(fù)大鼠脛骨骨缺損的研究。第一章IGFBP7在不同年齡組中BMSCs中的表達(dá)情況目的:探索IGFBP7在不同年齡組的BMSCs中表達(dá)情況。方法:通過(guò)梯度離心的方法分離培養(yǎng)人BMSCs,通過(guò)流式分析鑒定提取細(xì)胞表面抗原,并通過(guò)對(duì)提取細(xì)胞進(jìn)行成脂、成骨、成軟骨誘導(dǎo)鑒定其多向分化能力。將不同年齡人分為年輕組(16-22歲)和高齡組(≥70歲);年輕組15人,高齡組13人。通過(guò)ELISA檢測(cè)不同年齡組的血清中IGFBP7的表達(dá)情況。通過(guò)RT-qPCR、Western blot分析等比較兩組的BMSCs的成骨特異性的基因、IGFBP7和β-catenin的表達(dá)情況。同時(shí)比較BMSCs不同代數(shù)之間成骨特異性的基因、IGFBP7和β-catenin的表達(dá)情況。結(jié)果:通過(guò)流式鑒定顯示提取的細(xì)胞CD73陽(yáng)性率為99.8%,CD109陽(yáng)性率為99.9%,CD105 陽(yáng)性率為 98.5%,CD34 陽(yáng)性率為 0.042%,CD45 陽(yáng)性率為 0.06%,CD31陽(yáng)性率為0.051%。所提取的細(xì)胞能成功進(jìn)行成骨分化、成脂分化、成軟骨分化。ELISA檢測(cè)顯示高齡老人血清中的IGFBP7蛋白的表達(dá)明顯高于年輕人(P0.05)。老年人的BMSCs中成骨特異性的基因(RUNX2、OCN、SP7)和β-catenin表達(dá)是降低的(P0.05),但是IGFBP7的表達(dá)是增加的(P0.05)。隨著傳代的增加,成骨標(biāo)志物和β-catenin表達(dá)降低(P0.05),IGFBP7的表達(dá)明顯增高(P0.05)。結(jié)論:在BMSCs中,隨著年齡的增長(zhǎng)IGFBP7的表達(dá)增加,而成骨特異性的基因和Wnt/β-catenin信號(hào)通路的表達(dá)降低。第二章IGFBP7對(duì)BMSCs成骨分化的作用及其機(jī)制研究目的:探索IGFBP7對(duì)BMSCs成骨分化的作用及其機(jī)制。方法:在BMSCs中,通過(guò)慢病毒載體過(guò)表達(dá)和敲低IGFBP7的表達(dá)。將BMSCs分為過(guò)表達(dá)IGFBP7組、過(guò)表達(dá)對(duì)照組、敲低IGFBP7和敲低對(duì)照組。通過(guò)臺(tái)盼藍(lán)染色鑒定IGFBP7的表達(dá)對(duì)BMSCs增殖能力的影響。通過(guò)RT-qPCR、Western blot分析和免疫熒光,檢測(cè)不同組間的成骨特異性基因表達(dá)差異。通過(guò)ALP染色和ALP活性檢測(cè),不同分組間的ALP的表達(dá)情況和活性情況。通過(guò)茜素紅染色和Von Kossa染色檢測(cè)不同分組間礦化水平的差異。同時(shí)添加外源性重組IGFBP7蛋白檢測(cè)其對(duì)BMSCs的成骨分化的影響。通過(guò)Western blot分析和免疫熒光分析檢測(cè)常見(jiàn)成骨分化信號(hào)通路的變化。并通過(guò)抑制劑和siRNA干擾信號(hào)通路,驗(yàn)證其對(duì)IGFBP7成骨影響的變化。結(jié)果:與對(duì)照組相比,敲低IGFBP7組的BMSCs的細(xì)胞活力明顯減低(P0.05),而過(guò)表達(dá)IGFBP7組的BMSCs的細(xì)胞活力無(wú)明顯變化(P0.05)。在成骨分化誘導(dǎo)的第3天和第7天,RT-qPCR顯示較對(duì)照組,過(guò)表達(dá)IGFBP7組BMSCs成骨特異性基因(ALP、RUNX2、SP7、COL1A1、SP、OCN)的表達(dá)顯著升高(P0.05);敲低IGFBP7組BMSCs成骨特異性基因的表達(dá)顯著降低(P0.05)。免疫熒光和Western blot檢測(cè)分析顯示:在誘導(dǎo)的第1天,較對(duì)照組,過(guò)表達(dá)IGFBP7組成骨特異性蛋白(RUNX2、SP7、COL1A1)的表達(dá)顯著升高;敲低IGFBP7組成骨特異性蛋白(RUNX2、SP7、COL]A1)的表達(dá)顯著降低。ALP染色和ALP活性檢測(cè)結(jié)果顯示:成骨誘導(dǎo)3天和7天,過(guò)表達(dá)IGFBP7組ALP活性明顯升高,敲低IGFBP7組ALP的活性明顯減低(P0.05)。茜素紅和Von Kossa染色顯示:成骨分化誘導(dǎo)第10天,過(guò)表達(dá)IGFBP7組鈣結(jié)節(jié)積累明顯增多(P0.05),敲低IGFBP7組鈣結(jié)節(jié)的積累明顯減少(P0.05)。外源性的重組IGFBP7蛋白對(duì)BMSCs的成骨分化作用呈現(xiàn)出濃度依賴性,當(dāng)濃度≥500ng/mL時(shí),其可以加強(qiáng)BMSCs的成骨分化(P0.05)。信號(hào)通路檢測(cè)結(jié)果顯示:成骨分化第1天,MAPK(ERK/p-38/JNK)信號(hào)通路和PI3K/Akt信號(hào)通路無(wú)明顯變化,而過(guò)表達(dá)IGFBP7可以明顯增加總β-catenin蛋白的量,而且激活狀態(tài)的β-catenin蛋白也增加。應(yīng)用特異性的Wnt/β-catenin信號(hào)通路的抑制劑DKK1(1000ng/mL),可以明顯減弱因?yàn)镮GFBP7過(guò)表達(dá)增加的成骨分化效應(yīng)。應(yīng)用特異性的β-catenin信號(hào)siRNA,也可以明顯減弱因?yàn)镮GFBP7過(guò)表達(dá)增加的成骨分化效應(yīng)。結(jié)論:在BMSCs中,IGFBP7通過(guò)Wnt/β-catenin信號(hào)通路調(diào)控BMSCs的成骨分化。第三章IGFBP7基因修飾的BMSCs膜片對(duì)大鼠脛骨骨缺損愈合的影響目的:探索過(guò)表達(dá)IGFBP7的BMSCs膜片對(duì)大鼠脛骨骨缺損愈合的影響。方法:通過(guò)體外培養(yǎng)構(gòu)建BMSCs細(xì)胞膜片。將30只SD大鼠隨機(jī)分配為3組,分別為空白組(blank組)、對(duì)照組細(xì)胞膜片組(Ctrl組)、過(guò)表達(dá)IGFBP7干細(xì)胞膜片組(exIg7組),每組10只。應(yīng)用膜片修復(fù)大鼠脛骨骨缺損。術(shù)后8周,安樂(lè)死動(dòng)物后,取標(biāo)本。以X線和Mirco-CT評(píng)估骨缺損愈合情況。同時(shí),用HE染色、番紅速綠、Masson三色染色和免疫組化評(píng)估骨缺損愈合情況。GFP免疫組化追蹤移植細(xì)胞存留情況。結(jié)果:X線和Mirco-CT結(jié)果顯示:空白組(Blank)骨折間隙仍然清晰可見(jiàn),未見(jiàn)明顯連續(xù)的骨痂形成;對(duì)照組(Ctrl)骨折線模糊,可見(jiàn)明顯的骨痂生長(zhǎng),礦化組織已開(kāi)始連續(xù),骨折線已經(jīng)模糊,骨缺損區(qū)域未完全愈合;過(guò)表達(dá)IGFBP7組(exIg7)骨折區(qū)域礦化組織已完全填充,骨缺損已接近愈合。HE染色、番紅速綠和Masson三色染色顯示:較空白組相比,BMSCs膜片明顯加骨速缺損愈合,過(guò)表達(dá)IGFBP7組愈合的最快。COL1A1和OPN的免疫組化也顯示相似的結(jié)果。GFP的免疫組化顯示少量的骨組織殘留GFP蛋白。結(jié)論:過(guò)表達(dá)IGFBP7的BMSCs膜片可以加速骨缺損的修復(fù)。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R68

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本文編號(hào)：1491739