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国家自然科学基金(30901637)

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相关作者:王晔臧新杰更多>>
相关机构:山东省医学科学院更多>>
发文基金:国家自然科学基金山东省优秀中青年科学家科研奖励基金更多>>
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p16^INK4a基因及其抑制基因Bmil在不同年龄人角膜内皮细胞中的表达及其意义
2013年
背景p16^INK4a基因在细胞老化和早衰过程中发挥着重要作用,是目前已知的细胞衰老的主导基因。角膜内皮细胞(CECs)周期停滞于G。期且体内缺乏增生能力,是否与细胞衰老有关尚未得知。目的探讨不同年龄的正常人CECs中p16^INK4a基因及其抑制基因Bmil的表达。方法收集临床上DX液保存的行角膜移植手术后剩余的供体周边环状角膜组织,记录供体年龄。经锥虫蓝一茜素红双染法观察CECs的活性;行苏木精一伊红染色以证实角膜材料的组织结构正常。将收集到的角膜环材料按照年龄分为〈30岁组、30~50岁组和〉50岁组,每组30个角膜环,其中15个用于总RNA提取,另外15个角膜环等分为3份,分别用于免疫组织化学检测、免疫荧光检测和CECs活性的观察。提取每个角膜环的总RNA,行实时荧光定量PCR检测,观察p16^INK4amRNA、BmilmRNA和Ki67mRNA在CECs中的表达。行冰冻切片免疫组织化学染色,检测p16^INK4a、Bmil和Ki67蛋白在CECs中的表达。免疫荧光法检测p16”“。在CECs的表达。结果苏木精一伊红染色结果显示,供体角膜上皮、基质和内皮结构完整,排列规则。实时荧光定量PCR检测显示随年龄增长,p16^INK4a。mRNA的表达增强,而BmilmRNA和Ki67mRNA的表达减弱,差异均有统计学意义(F=5.703,P=0.014;F=3.950,P=0.042;F=548.500,P=0.000),其中与〈30岁组比较,〉50岁组p16^INK4amRNA在CECs中的表达量明显升高,而BmilmRNA和Ki67mRNA的表达均明显降低,差异均有统计学意义(P=0.006、0.013、0.000)。免疫组织化学结果可见,p16^INK4a。蛋白在各组CECs中均有表达,主要位于细胞核内,而Bmil与Ki67在年老供体的表达强度均弱于年轻供体,与实时荧光定量PCR的结果基本一致。免疫荧光染色显示,年老供体CECs p16^INK4a的表达强度强于年轻供体。结论细胞衰老主导基因p16^INK4a的表达随�
臧新杰王晔
关键词:角膜内皮细胞衰老P16^INK4A基因
Age-related maculopathy susceptibility 2 participates in the phagocytosis functions of the retinal pigment epithelium被引量:1
2012年
AIM: Age-related macular degeneration (AMD) is a multifactorial disease and a prevalent cause of visual impairment in developed countries. Many studies suggest that age-related maculopathy susceptibility 2 (ARMS2) is a second major susceptibility gene for AMD. At present, there is no functional information on this gene. Therefore, the purpose of the present study was to detect the expression of ARMS2 in retinal pigment epithelium (RPE) cells and to investigate the effect of ARMS2 on the phagocytosis function of RPE cells. METHODS: Immunofluorescence and reverse transcriptase PCR were used to demonstrate the presence and location of ARMS2 in ARPE-19 (human retinal pigment epithelial cell line, ATCC, catalog No.CRL-2302) cells. siRNA was used to knock down ARMS2 mRNA, and the effects of the knockdown on the phagocytosis function of the ARPE-19 cells were evaluated via Fluorescence Activated Cell Sorting (FACS). RESULTS: ARMS2 was present in ARPE-19 cells, localized in the cytosol of the perinuclear region. The expression of ARMS2 mRNA (messenger RNA) in ARPE-19 cells transfected with ARMS2-siRNA (small interfering RNA, 0.73+/- 0.08) was decreased compared with normal cells (1.00+/- 0.00) or with cells transfected with scrambled siRNA (0.95+/- 0.13) (P<0.05). After incubation of RPE cells with a latex beads medium for 12, 18, or 24 hours, the fluorescence intensities were 38.04 +/- 1.02, 68.92 +/- 0.92, and 78.00 +/- 0.12 in the ARMS2-siRNA-transfected groups, respectively, and 77.98 +/- 5.43, 94.87 +/- 0.60, and 98.30 +/- 0.11 in the scrambled siRNA-transfected groups, respectively. The fluorescent intensities of the same time points in the two groups were compared using Student's t-test, and the p values were all less than 0.001 at the three different time points. CONCLUSION: There is endogenous expression of ARMS2 in ARPE-19 cells. ARMS2 plays a role in the phagocytosis function of RPE cells, and this role may be one of the mechanisms that participates in the development of AMD.
Yi-Ting Xu,Hai-Feng Xu
关键词:PHAGOCYTOSIS
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