【摘要】： 目的探討以豬角膜前板層基質(zhì)為載體誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞(Mesenchymal Stem Cells, MSCs)治療兔角膜損傷的可能性。 方法用全骨髓貼壁法分離純化人MSCs,將細(xì)胞接種于含10%胎牛血清(FBS)的DMEM培養(yǎng)基中培養(yǎng)擴(kuò)增,傳代,流式細(xì)胞儀檢測(cè)免疫表型及誘導(dǎo)成脂、成骨分化,采用油紅O及硝酸銀染色鑒定。自動(dòng)板層角膜刀鉆取直徑約7.5 mm,厚160μm的去上皮豬角膜基質(zhì)片。28只新西蘭白兔隨機(jī)分為2組,制作角膜廣泛損傷模型。實(shí)驗(yàn)組取第3代MSCs接種于去上皮和全周角膜板層緣的豬角膜基質(zhì)上,培養(yǎng)4天后移植到廣泛損傷的兔角膜上,對(duì)照組單純移植去上皮豬角膜基質(zhì)。術(shù)后2、4、8周觀察實(shí)驗(yàn)眼角膜透明度和新生血管并評(píng)價(jià)分析。術(shù)后2、4、8周,取各實(shí)驗(yàn)眼行組織學(xué)檢查,觀察移植的MSCs及豬角膜基質(zhì)的存活、轉(zhuǎn)歸及移植局部的反應(yīng)。免疫組織化學(xué)、免疫熒光染色檢測(cè)移植后角膜上皮細(xì)胞標(biāo)志物細(xì)胞角蛋白12(Cytokeratin 12, CK12)的表達(dá)。 結(jié)果獲得的細(xì)胞流式細(xì)胞儀檢測(cè)：CD29陽(yáng)性率為95.97%,CD44為96.49%,CD90為92.79%,CD105為94.66%,CD34為0.59%,CD45為0.36%。符合MCSs的免疫表型,并可以誘導(dǎo)成脂及成骨分化。接種到去上皮豬角膜基質(zhì)后貼附、生長(zhǎng)迅速。術(shù)后植片在植床上存活良好,未見明顯排斥反應(yīng),實(shí)驗(yàn)組角膜較對(duì)照組明顯透明,新生血管少。實(shí)驗(yàn)組角膜免疫組織化學(xué)及免疫熒光均檢測(cè)出CK12陽(yáng)性細(xì)胞。 結(jié)論人骨髓MSC可以在體外成功進(jìn)行原代和傳代培養(yǎng),體外培養(yǎng)的人骨髓MSC有很強(qiáng)的增殖能力。種植MSCs的豬角膜基質(zhì)移植到損傷兔角膜后可以存活并減少兔角膜新生血管、增加透明度,本實(shí)驗(yàn)條件下MSCs可以分化為角膜上皮樣細(xì)胞,可能具有構(gòu)建組織工程角膜的潛能。
[Abstract]:Objective to investigate the possibility of rabbit corneal injury induced by human bone marrow mesenchymal stem cell (Mesenchymal Stem Cells, MSCs) with porcine anterior lamellar matrix. Methods Human MSCs, was isolated and purified by whole bone marrow adherent method. The cells were cultured and amplified in DMEM medium containing 10% fetal bovine serum (FBS). The immunophenotype, adipose induction and osteogenic differentiation were detected by flow cytometry. Oil red O and silver nitrate staining were used. The epitheliized porcine corneal stroma with the diameter of 7.5 mm, and 160 渭 m was obtained by automatic lamellar keratectomy. 28 New Zealand white rabbits were randomly divided into two groups to make extensive corneal injury model. The third generation of MSCs was inoculated on the porcine corneal stroma which was epithelially removed and the entire limbus cornea was cultured for 4 days, then transplanted to the widely damaged rabbit cornea, while the control group was only transplanted to the epithelial porcine corneal stroma. Corneal transparency and neovascularization were observed and evaluated 8 weeks after operation. Four weeks after operation, the experimental eyes were examined by histological examination to observe the survival, prognosis and local reaction of transplanted MSCs and porcine corneal stroma. Immunohistochemistry and immunofluorescence staining were used to detect the expression of Cytokeratin 12 (CK12) in corneal epithelial cells after transplantation. Results the positive rate of CD29 was 95.97 and the CD44 was 96.499.The CD90 was 92.79 and the CD105 was 94.666.The CD34 was 0.59and the CD45 was 0.360.The results showed that the positive rate of CD29 was 95.97% and the CD44 was 96.49% and the CD90 was 92.79%. It conforms to the immunophenotype of MCSs and can induce adipogenic and osteogenic differentiation. After inoculation to epithelial porcine corneal stroma, it grows rapidly. After operation, the grafts survived well in the bed, and there was no obvious rejection. The cornea in the experimental group was more transparent than that in the control group, and the neovascularization was less than that in the control group. CK12 positive cells were detected by immunohistochemistry and immunofluorescence in the experimental group. Conclusion Human bone marrow MSC can be successfully cultured in vitro. Human bone marrow MSC has strong proliferative ability. The porcine corneal stroma implanted with MSCs can survive and reduce the corneal neovascularization and increase the transparency. Under this condition MSCs can differentiate into corneal epithelioid cells which may have the potential to construct tissue engineered cornea.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R772.2

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