本文選題：骨髓基質(zhì)干細(xì)胞 + 肌腱細(xì)胞　； 參考：《大連醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的肌腱病的治療是臨床骨科的難點(diǎn),骨髓基質(zhì)干細(xì)胞的應(yīng)用為肌腱病的治療提供了新思路。骨髓基質(zhì)干細(xì)胞具有多向分化潛能并可以被誘導(dǎo)分化為多種細(xì)胞,但由于骨髓基質(zhì)干細(xì)胞的分化機(jī)制及在治療過程中的作用方式尚未明確,限制了其在肌腱病治療中的應(yīng)用。本課題通過體外構(gòu)建骨髓基質(zhì)干細(xì)胞與自體肌腱細(xì)胞的間接共培養(yǎng)體系,在單純間接共培養(yǎng)環(huán)境下與間接共培養(yǎng)同時(shí)添加自體富血小板血漿進(jìn)行干預(yù)的情況下研究間接共培養(yǎng)環(huán)境對(duì)骨髓基質(zhì)干細(xì)胞的分化誘導(dǎo)作用,以探索骨髓基質(zhì)干細(xì)胞的分化機(jī)制并為骨髓基質(zhì)干細(xì)胞在肌腱病治療中的應(yīng)用提供參考。 方法應(yīng)用組織塊培養(yǎng)法分離培養(yǎng)大鼠肌腱細(xì)胞。體外采集大鼠骨髓基質(zhì)干細(xì)胞并擴(kuò)增。采集大鼠血液制備富血小板血漿并激活以獲取富血小板血漿萃取液。利用孔徑0.4μm的細(xì)胞共培養(yǎng)嵌盒與6孔板構(gòu)建培養(yǎng)液相通但細(xì)胞不直接接觸的間接共培養(yǎng)體系。將生長狀態(tài)良好的第三代(P3)大鼠肌腱細(xì)胞及第四代大鼠骨髓基質(zhì)干細(xì)胞(P4)用于實(shí)驗(yàn),實(shí)驗(yàn)組骨髓基質(zhì)干細(xì)胞被接種于間接共培養(yǎng)體系外室,將同體肌腱細(xì)胞種植于內(nèi)室,另外單獨(dú)設(shè)置一組間接共培養(yǎng)組并加入富血小板血漿萃取液。于間接共培養(yǎng)后第3天、第7天及第10天,分別提取各組間接共培養(yǎng)體系內(nèi)外室的細(xì)胞進(jìn)行形態(tài)學(xué)觀察,并利用RT-PCR及免疫組化染色技術(shù)分別對(duì)各組肌腱細(xì)胞與骨髓基質(zhì)干細(xì)胞的Ⅰ型膠原與腱調(diào)蛋白(TNMD)進(jìn)行基因?qū)用媾c蛋白層面的檢測,以分析間接共培養(yǎng)環(huán)境對(duì)骨髓基質(zhì)干細(xì)胞分化與細(xì)胞外基質(zhì)表達(dá)的影響。 結(jié)果應(yīng)用組織塊培養(yǎng)法成功分離出細(xì)胞,并經(jīng)鑒定證實(shí)為肌腱細(xì)胞。應(yīng)用肌腱細(xì)胞與骨髓基質(zhì)干細(xì)胞間接共培養(yǎng)3天后,間接共培養(yǎng)組的骨髓基質(zhì)干細(xì)胞在RT-PCR檢測與免疫組化染色檢測中均未表達(dá)Ⅰ型膠原與腱調(diào)蛋白(TNMD)。間接共培養(yǎng)7天后,間接共培養(yǎng)組干細(xì)胞在Ⅰ型膠原的PCR電泳圖像中出現(xiàn)模糊條帶,同時(shí)Ⅰ型膠原免疫組化顯示部分細(xì)胞呈弱陽性染色,單純間接共培養(yǎng)組與富血小板血漿干預(yù)組之間結(jié)果無顯著差異。間接共培養(yǎng)組干細(xì)胞在各種檢測中未出現(xiàn)對(duì)腱調(diào)蛋白(TNMD)的表達(dá)。間接共培養(yǎng)10天后,間接共培養(yǎng)組干細(xì)胞在Ⅰ型膠原的PCR電泳圖像中出現(xiàn)清晰條帶,但亮度低于肌腱細(xì)胞對(duì)照組。同時(shí)Ⅰ型膠原免疫組化顯示間接共培養(yǎng)組干細(xì)胞呈廣泛弱陽性染色,通過測定免疫組化結(jié)果的平均光密度進(jìn)行統(tǒng)計(jì)學(xué)分析,結(jié)果顯示間接共培養(yǎng)組骨髓基質(zhì)干細(xì)胞Ⅰ型膠原表達(dá)量顯著高于干細(xì)胞陰性對(duì)照組,但低于肌腱細(xì)胞對(duì)照組。間接共培養(yǎng)組干細(xì)胞在腱調(diào)蛋白(TNMD)的PCR電泳圖像中表現(xiàn)為模糊條帶,但腱調(diào)蛋白(TNMD)免疫組化染色結(jié)果為陰性。 結(jié)論通過組織塊培養(yǎng)法可以在體外成功分離并培養(yǎng)大鼠肌腱細(xì)胞。與肌腱細(xì)胞間接共培養(yǎng)的培養(yǎng)環(huán)境可以誘導(dǎo)骨髓基質(zhì)干細(xì)胞表達(dá)Ⅰ型膠原。間接共培養(yǎng)環(huán)境下骨髓基質(zhì)干細(xì)胞未出現(xiàn)明顯的腱調(diào)蛋白表達(dá)。根據(jù)實(shí)驗(yàn)結(jié)果,不能認(rèn)為富血小板血漿的干預(yù)對(duì)間接共培養(yǎng)環(huán)境誘導(dǎo)后骨髓基質(zhì)干細(xì)胞細(xì)胞外基質(zhì)的分泌產(chǎn)生影響。尚不能認(rèn)為間接共培養(yǎng)環(huán)境可以誘導(dǎo)骨髓基質(zhì)干細(xì)胞向肌腱細(xì)胞分化。
[Abstract]:Objective To study the differentiation mechanism of bone marrow stromal cells ( MSCs ) and autologous tendon cells , and to investigate the differentiation of bone marrow stromal cells ( MSCs ) in the treatment of tendon diseases .



Methods The rat bone marrow stromal cells were isolated from rat bone marrow stromal cells and amplified by tissue block culture method . The rat bone marrow stromal cells were collected and activated to obtain platelet - rich plasma extract . The third generation ( P3 ) rat tendon cells and the fourth generation rat bone marrow stromal cells ( TNMD ) were cultured in the indirect co - culture system .



Results The expression of bone marrow stromal cells in indirect co - culture group was significantly higher than that of control group . The results showed that the expression of collagen type 鈪，

本文編號(hào)：1904017