角膜基质载体培养的角膜缘干细胞的增龄性变化及相关性研究

发布时间：2018-03-01 01:33

本文关键词： 角膜缘干细胞增龄角膜基质 △Np63 ABCG2　出处：《山西医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：通过将不同年龄兔角膜缘干细胞种植于不同年龄兔角膜基质载体上,观察同种异体角膜基质载体培养的兔角膜缘干细胞生物学特性随增龄的变化,探讨了增龄对在角膜基质载体上培养的LSCs增殖能力的影响,同时检测了角膜基质载体培养的角膜缘干细胞的相关性标记物ΔNp63、ABCG2的表达量随增龄的变化,探讨其与载体培养的干细胞老化有无关系,为组织工程化角膜种子细胞选择的成因开辟了新途径。方法：对照实验研究。用酶消化法将不同年龄新西兰兔角膜缘组织制成细胞悬液培养,以相同的细胞密度种植于不同年龄的新西兰兔的角膜基质上,观察角膜基质载体培养的角膜缘干细胞的生长情况,原位杂交检测ABCG2、ΔNp63的表达,鉴定LSCs, IPP5.1软件分析图像,流式细胞术测细胞周期,逆转录聚合酶链反应(RT-PCR)技术检测角膜基质载体培养的细胞中ΔNp63、ABCG2蛋白的表达量,运用SPSS13.0软件对析因设计资料进行方差分析。结果：种植于不同年龄角膜基质载体上的青年角膜缘干细胞较其他组增殖快,潜伏期短,形态较小,以圆形、椭圆形和多边形为主,形成的集落较大。传代培养于角膜基质载体上的LSCs经ABCG2、p63原位杂交检测,DAB染色大部分细胞呈阳性反应,细胞胞浆着色呈棕黄色,胞核为蓝色。角膜缘干细胞供体年龄各水平差异有统计学意义,相同时间内角膜基质载体培养的青年兔角膜缘干细胞增殖期细胞所占比例大,ΔNp63、ABCG2表达量高；而角膜基质供体年龄各水平差异与角膜缘干细胞和角膜基质的交互作用均无统计学意义。结论：角膜基质载体培养的角膜缘干细胞的增殖能力随着角膜缘干细胞供体年龄的增加而降低。ΔNp63、ABCG2蛋白的表达具有明显的年龄相关性,随着增龄ΔNp63、ABCG2蛋白表达量下调可能是载体培养的角膜缘干细胞老化的分子机制之一
[Abstract]:Objective: to observe the biological characteristics of corneal limbal stem cells cultured with different age of rabbit corneal stromal cells, and to observe the changes of biological characteristics of corneal limbal stem cells cultured with allogeneic corneal stromal cells on the corneal stromal cells of rabbits of different ages. The effect of aging on the proliferation of LSCs cultured on corneal stromal carrier was studied. The expression of 螖 Np63 ABCG2, a relative marker of limbal stem cells cultured on corneal stroma carrier, was detected with age. To explore whether it is related to the aging of stem cells in carrier culture, and to open a new way for the selection of corneal seed cells in tissue engineering. Methods: the control experiment was carried out. The corneal limbus tissue of New Zealand rabbits of different ages was cultured with cell suspension by enzyme digestion and implanted on the corneal stroma of New Zealand rabbits of different ages with the same cell density. The growth of corneal limbal stem cells cultured with corneal stromal media was observed. The expressions of ABCG2 and 螖 Np63 were detected by in situ hybridization, and the images were identified by IPP5.1 software. The cell cycle was measured by flow cytometry. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of 螖 Np63 ABCG2 protein in the cells cultured with corneal stromal vector. The variance analysis of factorial design data was performed with SPSS13.0 software. Results: the proliferation of corneal limbal stem cells planted on corneal stromal carrier of different ages was faster, the incubation period was shorter, the shape was smaller, and the cells were mainly round, elliptical and polygonal. The LSCs cultured on the corneal stromal carrier was detected by in situ hybridization with ABCG2P63 to detect the positive reaction of most of the cells, and the cytoplasm of the cells was brownish yellow. The nucleus was blue. The age of corneal limbal stem cells was significantly different. In the same time, the percentage of proliferative cells of corneal limbal stem cells was large and the expression of 螖 Np63 ABCG2 was high in young rabbit corneal limbal stem cells cultured with corneal stromal cells at the same time. However, there was no significant difference between corneal stromal donor age and corneal limbal stem cells and corneal stroma. Conclusion: the proliferation ability of corneal limbal stem cells cultured with corneal stromal cells decreased with the age of corneal limbal stem cells, and the expression of 螖 Np63 ABCG2 protein was significantly age-related. The down-regulation of 螖 Np63 ABCG2 protein expression with aging may be one of the molecular mechanisms of corneal limbal stem cell aging in vector culture.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R779.65

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