人臍帶Wharton膠支架負載脂肪干細胞構(gòu)建新型組織工程軟骨的實驗研究
發(fā)布時間：2018-10-26 19:13
【摘要】：目的:探索人臍帶Wharton膠支架負載脂肪干細胞構(gòu)建新型組織工程軟骨的可行性,并評價該組織工程軟骨修復(fù)兔膝關(guān)節(jié)全層軟骨缺損的效果。方法:⑴從兔頸背部皮下脂肪分離得到脂肪干細胞,培養(yǎng)至P3代后通過流式細胞學(xué)鑒定其表面標(biāo)記物,明確其干細胞來源。⑵普通培養(yǎng)至P3代后更換為誘導(dǎo)液(DMEM中加入10%FBS,10ng/ml TGF-β1,25ng/ml b FGF,10-7M地塞米松)成軟骨方向誘導(dǎo)兩周,收集細胞并調(diào)整終濃度為1×107/ml,將細胞接種至Wharton膠支架上繼續(xù)三維誘導(dǎo)一周構(gòu)建組織工程軟骨。FDA-PI染色及Hoechst33258染色評估細胞在支架上的活力及增殖情況,掃描電鏡觀察細胞在支架上分布及黏附情況,甲苯胺藍、番紅O、Ⅱ型膠原免疫組化染色觀察細胞外基質(zhì)分泌情況。⑶于兔膝關(guān)節(jié)軟骨滑車處形成1個直徑4mm,深2mm的環(huán)形全層軟骨缺損,設(shè)置空白組、單純支架材料組及細胞支架復(fù)合體組。術(shù)后24周取材,缺損修復(fù)區(qū)行大體觀察、組織學(xué)染色、糖胺多糖、總膠原含量檢測及生物力學(xué)測定分析評價缺損修復(fù)情況。結(jié)果:⑴流式細胞學(xué)鑒定顯示CD73、CD90、CD105陽性,CD34、CD45、HLA-DR陰性,提示所取細胞為脂肪來源干細胞;⑵FDA-PI染色及Hoechst33258染色可見支架上粘附的細胞分布均勻,活細胞較多,死細胞較少,支架深層也有部分細胞粘附。掃描電鏡觀察可見誘導(dǎo)后大量細胞黏附于支架孔隙內(nèi),呈圓形或橢圓形。組織學(xué)染色陽性提示誘導(dǎo)后細胞分泌大量細胞外基質(zhì);⑶術(shù)后24周細胞支架復(fù)合體組缺損區(qū)大部分被修復(fù),缺損部位被軟骨組織填充,修復(fù)區(qū)表面光滑。組織學(xué)染色顯示有典型的透明軟骨樣結(jié)構(gòu),GAG、總膠原定量測定提示可達正常值的90%以上,生物力學(xué)測定提示與正常軟骨相似。單純支架材料組雖然缺損區(qū)大部分被修復(fù),但修復(fù)區(qū)多為纖維軟骨,僅有少量透明軟骨樣結(jié)構(gòu)�？瞻捉M未能修復(fù)關(guān)節(jié)軟骨缺損,纖維組織部分填充缺損,表面覆蓋薄層纖維組織。結(jié)論:脂肪干細胞作為軟骨組織工程種子細胞具有可行性,人臍帶Wharton膠支架可作為良好軟骨組織工程支架材料,實驗構(gòu)建的組織工程軟骨在遠期能夠有效的修復(fù)關(guān)節(jié)軟骨缺損。
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【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08;R68
本文編號：2296691
[Abstract]:% E7%9B%AE%E7%9A%84%3A%E6%8E%A2%E7%B4%A2%E4%BA%E8%84%90%E5%B8%A6Wharton%E8%83%B6%E6%94%AF%E6%9E%B6%E8%B4%9F%E8 % BD%E8%84%82%E8%82%AA%E5%B9%B2%E7%BB%86%E8%83%9E%E6%9E%84%E5%BB%BA%E6%96%B0%E5%9E%8B%E7%BB%84%E7%BB%87 % E5%B7%A5%E7%A8%8B%E8%BD%AF%E9%AA%A8%E7%9A%84%E5%8F%AF%E8%A1%8C%E6%80%A7%2C%E5%B9%B6%E8%AF%84%E4%BB%B7%E8 % AF%A5%E7%BB%84%E7%BB%87%E5%B7%A5%E7%A8%8B%E8%BD%AF%E9%AA%A8%E4%BF%AE%E5%A4%8D%E5%85%94%E8%86%9D%E5%85%B3 % E88A8A82B1B _ 1, E88A8A82B _ 1, E88A8A _ 8B _ 1, E88A _ 8A _ 8B _ 8A _ 8A _ 8B _ 7A _ 7F _ 9F _ 7AAE _ 7e _ 7e _ 7e _ 7F _ _ _ 2 _ _ _ 2 _ _ _ B395A E2, B395A, E882, E888, A838, E838, E832, E882, and E882, including E8882, A882, E588, and E8888, including E588, including E585, A882, E882, E888, and E8882, including E8882, A882, E888, E882, E888, 86E7A7A682B0, including E8887B3P3%, including E884882, E582, E886, E882, and E8882, including E884B982, B582, E582, and E8886, E832, E882, and E582, including E8887, B575, BBE887, B3, P3,%, E4BBA908, E4BBA9809A, E4BBA8FE589B5ADA6FB586E83E5ADA88FE5809A, including E6FE787, E6FB589B5ADA883E5ADA8, including E6BF878E589, E5809A, E9E5809A, and E9E9A9A9A589, including E585%, right, right. 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E6%9D%A5%E6%BA%90.%E2%91%B5%E6%99%AE%E9%80%9A%E5%9F%B9%E5%85%BB%E8%87%B3P3%E4%BB%A3%E5%90%8E%E6%9B%B4%E6%8D % A2E4E4B8B8batter, including BCE6B6B1E51E550, including BCE6B6E51E550, including BCE6B6B1E51, and the baton of BCE6B6B1E551, including BCE6B6B1E51E52DMEM8A8A0A0A0A0FB4FS2C10Ng2ADE5NG2F0TGF-885A510FB8B8B8B8B1B1B2F20A8FG2FGF2C10TGF-B2B2B2F20Ng2FGF2C10- 7Mm, right and right, E5D9C9C9E7 and E7E7, including E8AFB1, E5AFB1A5AFB1, E5AFB1, E5AFBC%, E5AFB1, E5AFBC%, E4B8A8A82C82CE62C2C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C82C8888A888E888E889A888A888E8888E888A8888A8882C82C82C82C82C82C82C82C82C82C8 BA%A6%E4%B8%BA1%C3%97107%2Fml%2C%E5%B0%86%E7%BB%86%E8%83%9E%E6%8E%A5%E7%A7%8D%E8%87%B3Wharton%E8%83%B6%E6%94%AF%E6%9E% B6E4E8B8A8A, B6E4B88A8A88A8889, A4E8889, A4E888E889, AFB4B880E880E81AFB88888B888A88B88B888A888B888A888B8888A88B88888E888A888B888A888B888A88B888A88B888A88E8888A888 BB%84%E7%BB%87%E5%B7%A5%E7%A8%8B%E8%BD%AF%E9%AA%A8.FDA-PI%E6%9F%93%E8%89%B2%E5%8F%8AHoechst33258%E6%9F%93%E8%89%B2%E8% AF%84%E4%BC%B0%E7%BB%86%E8%83%9E%E5%9C%A8%E6%94%AF%E6%9E%B6%E4%B8%8A%E7%9A%84%E6%B4%BB%E5%8A%9B%E5%8F%8A% E5A2A29E8389, including E832, including E885%, E88F8F8FE789, E689, E789, E689, A689, E686, 892, E689, and E686, including E586, E689, and E689, including the side of the bank, including the head of the bank, the other side. 839E9E9E9E9E88A88A88A88A88A88F88A88A88A88F88A88A88A88A88A88A88E88A8888A88E888A88A88888E888E88B8888A8888A888A88A888A88A888A88888888888888888888888888888888 2CE77B2C7E7B83, including E833, E883, E585%, including E833F9FE585%, including BE83B83C2C2C2E285A1, E78BAA2O2E795, E795, E88B, and B83B8B8B883, including the head of BAE83B883, E883, E583, E585%, E585%. 8DE79ABA8C8C8C8C8C8C8C8C8C8C8C8C8C8C8C8C8C8888889 E7889A782E886E889A8888888888888888888888888889A88888888888888888888888A8888888888888888888888888A8888888A88 B4%A8%E5%88%86%E6%B3%8C%E6%83%85%E5%86%B5.%E2%91%B6%E4%BA%8E%E5%85%94%E8%86%9D%E5%85%B3%E8%8A%82%E8%BD%AF % E9%AA%A8%E6%BB%91%E8%BD%A6%E5%A4%84%E5%BD%A2%E6%88%901%E4%B8%AA%E7%9B%B4%E5%BE%844mm%2C%E6%B7%B12mm%E7%9A%84%E7 % 8E%AF%E5%BD%A2%E5%85%A8%E5%B1%82%E8%BD%AF%E9%AA%A8%E7%BC%BA%E6%8D%9F%2C%E8%AE%BE%E7%BD%AE%E7%A9%BA%E7%99 % BD%E7%BB%84%2C%E5%8D%95%E7%BA%AF%E6%94%AF%E6%9E%B6%E6%9D%90%E6%96%99%E7%BB%84%E5%8F%8A%E7%BB%86%E8%83%9E % E694 E6908A445, including the side of E694, including E6908E5908CAFE5908E7BBB, 84E69088E68B6908N8D5908F8E5908F88D5908F8E5908F8E5908F88D5908F8E5908F8A5908F8A5908F8A5908F8E5908F8D5902C5902C% and including the side of the side. 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【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08;R68
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