本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：肌腱干細(xì)胞　出處：《中國人民解放軍醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:肌腱組織由于其獨(dú)特的解剖和組織結(jié)構(gòu),因此缺乏自身修復(fù)能力,愈合后損傷部位以常以瘢痕組織代替。為解決此問題,組織工程化肌腱成為研究熱點(diǎn),但目前使用的人工生物材料載體存在老化、排異等缺點(diǎn)。骨髓間充質(zhì)干細(xì)胞(BMSCs)作為老牌種子細(xì)胞在組織再生領(lǐng)域應(yīng)用廣泛,但有報(bào)道稱其應(yīng)用后出現(xiàn)了異位鈣化、干細(xì)胞形成腫瘤等情況。肌腱干細(xì)胞(TDSCs)由于其來源,用于肌腱修復(fù)研究理論上更具優(yōu)勢。結(jié)締組織生長因子(CTGF)可誘導(dǎo)兩種細(xì)胞向肌腱細(xì)胞方向分化。因此,課題組使用CTGF和這兩種細(xì)胞進(jìn)行體外實(shí)驗(yàn),構(gòu)建出一種不含其它材料的組織工程化肌腱用于肌腱損傷的修復(fù),并通過對比兩種細(xì)胞選取更適合肌腱損傷修復(fù)的種子細(xì)胞。目的:1、驗(yàn)證結(jié)締組織生長因子(CTGF)誘導(dǎo)骨髓間充質(zhì)干細(xì)胞(BMSCs)和肌腱干細(xì)胞(TDSCs)成肌腱分化能力,并對比兩種干細(xì)胞誘導(dǎo)前后的成肌腱潛能。2、利用結(jié)締組織生長因子(CTGF)誘導(dǎo)成肌腱分化能力,利用這兩種干細(xì)胞體外構(gòu)建組織工程化肌腱。3、初步探索組織工程化肌腱在肌腱損傷修復(fù)中的作用,并對比兩種組織工程化肌腱的效果。方法:1、分離培養(yǎng)綠熒光大鼠的骨髓間充質(zhì)干細(xì)胞(BMSCs)和肌腱干細(xì)胞(TDSCs),并進(jìn)行鑒定。2、取第3代的骨髓間充質(zhì)干細(xì)胞(BMSCs)和肌腱干細(xì)胞(TDSCs), 25ng/ml的CTGF進(jìn)行誘導(dǎo)分化培養(yǎng)2周,選取scleraxis和tenomodulin這兩個成肌腱相關(guān)基因,利用RT-qPCR進(jìn)行mRNA表達(dá)檢測。驗(yàn)證CTGF誘導(dǎo)BMSCs和TDSCs成肌腱分化能力,并對比兩種干細(xì)胞誘導(dǎo)前后的成肌腱潛能。3、取第3代的BMSCs和TDSCs,體外誘導(dǎo)成肌腱分化2周,并構(gòu)建出干細(xì)胞組織工程化肌腱。4、將BMSCs和TDSCs構(gòu)建的組織工程化肌腱分別植入裸鼠皮下,8周、12周后取材,進(jìn)行組織形態(tài)學(xué)檢測、免疫組化染色。結(jié)果:1、RT-qPCR結(jié)果顯示,CTGF能顯著增加BMSCs和TDSCs中tenomodulin和scleraxis的mRNA表達(dá)(p0.05),對比發(fā)現(xiàn),誘導(dǎo)分化前后TDSCs中tenomodulin 和 scleraxis 的 mRNA 表達(dá)均明顯高于 BMSCs (p0.05)。2、利用CTGF誘導(dǎo)BMSCs和TDSCs體外構(gòu)建兩種組織工程化肌腱,組織形態(tài)學(xué)分析結(jié)果顯示兩種組織工程化肌腱均具有不成熟肌腱樣結(jié)構(gòu),TDSCs組結(jié)構(gòu)更加理想。3、組織形態(tài)學(xué)和免疫組織化學(xué)分析結(jié)果顯示,兩種組織工程化肌腱均可在裸鼠體內(nèi)形成肌腱樣組織,其中TDSCs組種植后結(jié)構(gòu)成熟更快,細(xì)胞外基質(zhì)成分更加接近正常肌腱。結(jié)論:1、CTGF配合抗壞血酸可有效誘導(dǎo)BMSCs和TDSCs成肌腱分化,TDSCs比BMSCs有著更大的成肌腱分化潛能。2、僅利用二維條件進(jìn)行細(xì)胞培養(yǎng),利用CTGF誘導(dǎo)BMSCs和TDSCs的成肌腱分化作用,兩種細(xì)胞均可在體外構(gòu)建出一種不含任何合成人工材料的組織工程化肌腱。3、體外構(gòu)建出兩種組織工程化肌腱在裸鼠體內(nèi)種植均可形成類肌腱樣組織。TDSCs構(gòu)建的組織工程化肌腱優(yōu)于BMSCs。 TDSCs可能比BMSCs更適合在組織工程學(xué)領(lǐng)域用于肌腱損傷修復(fù)。
[Abstract]:Background: because of its unique anatomy and tissue structure, tendon tissue lacks the ability to repair itself. Scar tissue is often used to replace the injured site after healing. In order to solve this problem, tissue engineered tendon has become a hot research topic. However, the artificial biomaterial carriers used at present have some disadvantages, such as aging and rejection. Bone marrow mesenchymal stem cells (BMSCs) are widely used in the field of tissue regeneration as old seed cells, but there are reports of ectopic calcification after their application. Because of its origin, TDSCs have a theoretical advantage in tendon repair. Connective tissue growth factor (CTGFs) can induce two kinds of cells to differentiate into tendon cells. CTGF and these two kinds of cells were used to construct a kind of tissue engineered tendon without other materials to repair tendon injury. By comparing the two kinds of cells, seed cells were selected to repair tendon injury. Objective: 1 to test the ability of bone marrow mesenchymal stem cells (BMSCs) and tendon stem cells (TDSCs) induced by connective tissue growth factor (CTGF) to differentiate tendon. The ability of tendon differentiation induced by connective tissue growth factor (CTGF) was compared between the two kinds of stem cells before and after induction. The two kinds of stem cells were used to construct tissue engineered tendon in vitro to explore the role of tissue engineered tendon in the repair of tendon injury. Methods the bone marrow mesenchymal stem cells (BMSCs) and the tendon stem cells (TDSCs1) of green fluorescent rats were isolated and cultured. TDS cells were cultured for 2 weeks with 25 ng / ml CTGF. Scleraxis and tenomodulin were selected to detect the expression of mRNA by RT-qPCR. The ability of CTGF to induce the differentiation of BMSCs and TDSCs tendons was verified. The tendon potential of the two stem cells before and after induction was compared. The third generation of BMSCs and TDSCs were taken and induced into tendon differentiation for 2 weeks in vitro. The tissue engineered tendon of stem cells was constructed. The tissue engineered tendons constructed by BMSCs and TDSCs were implanted into nude mice for 8 weeks and 12 weeks, respectively. Results the expression of mRNA in tenomodulin and scleraxis in BMSCs and TDSCs was significantly increased by RT qPCR. The results showed that CTGF could significantly increase the mRNA expression of tenomodulin and scleraxis in BMSCs and TDSCs. The mRNA expression of tenomodulin and scleraxis in TDSCs was significantly higher than that in BMSCs p0.05. 2before and after differentiation. CTGF was used to induce BMSCs and TDSCs to construct two kinds of tissue engineered tendons in vitro. The results of histomorphology analysis showed that both of them had immature tendonoid structures. The structure of TDSCs was more ideal. The results of histomorphology and immunohistochemical analysis showed that the two kinds of tissue engineered tendons had immature tendon-like structures. Both kinds of tissue engineered tendons could form tendon-like tissues in nude mice. In TDSCs group, the structure matured more quickly after implantation. Conclusion BMSCs and TDSCs can induce tendon differentiation of BMSCs and TDSCs more effectively than that of BMSCs. TSCs can only be cultured under two-dimensional conditions, and the differentiation potential of TSCs is higher than that of BMSCs, and the extracellular matrix components are closer to normal tendons. Conclusion: TSCs can induce tendon differentiation of BMSCs and TDSCs effectively, and the differentiation potential of TSCs is higher than that of BMSCs, and only two dimensional conditions can be used for cell culture. The tendon differentiation of BMSCs and TDSCs was induced by CTGF. Both kinds of cells could construct a kind of tissue engineered tendon without any synthetic material in vitro. In vitro, two kinds of tissue engineered tendons could be implanted in nude mice to form tendonoid tissue. TDSCs could be constructed. Engineering tendons are superior to BMSCs. TDSCs may be more suitable for tendon injury repair than BMSCs.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R686

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