MG132抑制人晶状体上皮细胞增殖、移行和分化的实验研究

发布时间：2018-05-28 19:01

本文选题：晶状体上皮细胞 + 后发性白内障　；参考：《山西医科大学》2010年硕士论文

【摘要】： 目的：通过体外细胞培养研究蛋白酶体抑制剂MG132对HLEC增殖、移行和分化的抑制作用,初步探索MG132防治后发性白内障的机制。方法： 1、采用组织块培养法,对人晶状体前囊膜进行培养,并利用形态学观察和体外培养HLEC特异性的“晶状体小体”进行细胞鉴定。 2、MTT比色法：传代的HLEC分别加入FGF-2(10ng/ml); MG132(10nM); MG132 +FGF-2(FGF-2, 10ng/ml; MG132, 10μM)并作对照,培养24h后以MTT法测定吸光度(OD)值,计算其增生率。 3、移行能力测定：传代的HLEC分别加入FGF-2(10ng/ml); MG132(10μM); MG132 +FGF-2(FGF-2, 10ng/ml; MG132, 10μM)并作对照,用无菌棉棒在细胞层中划出细胞裸露区,培养24h后对行至裸露区的细胞计数,计算移行能力。 4、细胞免疫组化法：收集传代的HLEC分别加入TGF-β2(10ng/ml); MG132(10μM); MG132+TGF-β2(TGF-β2, 10ng/ml; MG132,10μM)并作对照,培养24h后免疫组化检测胞浆中FN的表达。结果： 1、HLEC原代培养中,细胞接种后24h内,即有部分上皮细胞自囊膜片生长移出,贴壁生长。长期培养的HLEC中,可观察到细胞呈同心圆环绕的小球体状结构,即体外培养LEC特征性的“晶状体小体”。 2、10ng/ml FGF-2能诱导HLEC增殖24.2%；10μM的MG132能够有效抑制HLEC增殖,增殖率为-25.4%且能够有效抑制FGF-2诱导的HLEC增殖,增殖率为-20.1%。MG132+FGF-2组和FGF-2组对HLEC的作用有显著性差异(P0.05)。 3、10ng/ml FGF-2能诱导HLEC移行,移行能力为33.6%,10μM的MG132能够强有效地抑制FGF-2诱导HLEC的移行作用。MG132+FGF-2组和FGF-2组对HLEC的作用有显著性差异(P0.05)。 4、TGF-β2组可见HLEC胞浆中多量黄褐色沉积物；而MG132+TGF-β2组FN的表达明显下降,与对照组相比无明显差异；MG132组接近对照组。结论：1.本实验成功地进行了HLEC的体外培养,获得了高纯度的HLEC细胞,可以作为后发障的细胞学研究的实验对象。2.无论FGF-2、TGF-β2存在与否,MG132能强有效抑制HLEC增殖、移行和分化。
[Abstract]:Aim: to investigate the inhibitory effect of proteasome inhibitor MG132 on the proliferation, migration and differentiation of HLEC in vitro, and to explore the mechanism of MG132 in preventing and treating post-cataract. Methods: 1. The anterior capsule of human lens was cultured by tissue mass culture, and the HLEC specific "lens bodies" were identified by morphological observation and in vitro culture. 2MTT colorimetric method: the HLEC was added to FGF-2G / ml; MG132N / 10nMN; MG132 FGF-2FGF-2FGF-2FGF-2FGF-2, 10 渭 M) and compared with the control. After 24 hours of culture, the absorbance of HLEC was determined by MTT method, and the proliferation rate was calculated. 3. Translocation ability: HLEC was added into FGF-2G / ml; MG132(10 渭 Mel; MG132 FGF-2FGF-2FGF-2,10ng / ml; MG132,10 渭 Mrespectively. The bare areas of cells were marked out in the cell layer with sterile cotton rods. After 24 hours of culture, the number of cells moving to the exposed area was counted and the transference ability was calculated. (4) Immunohistochemical method: the cultured HLEC was added to TGF- 尾 _ 2G / ml; MG132(10 渭 M; MG132 TGF- 尾 _ 2 to TGF- 尾 _ 2, 10 ng / ml; MG132C _ (10 渭 M), respectively. The expression of FN in the cytoplasm was detected by immunohistochemistry for 24 hours. Results: 1 in primary culture of HLEC, within 24 hours after inoculation, some epithelial cells grew out of the capsule and grew on the wall. In HLEC cultured for a long time, it was observed that the cells were concentric round small globular structure, that is, the characteristic "lens bodies" of LEC cultured in vitro. 210 ng / ml FGF-2 could induce HLEC proliferation 24.2um 10 渭 M MG132 could effectively inhibit the proliferation of HLEC, the proliferation rate was -25.4% and could effectively inhibit the HLEC proliferation induced by FGF-2. The proliferation rate was -20.1. MG132 FGF-2 group and FGF-2 group had significant effect on HLEC (P 0.05). 310 ng / ml FGF-2 could induce HLEC migration. MG132 with a migration capacity of 33.6 渭 M and 10 渭 M could strongly and effectively inhibit HLEC migration induced by FGF-2. There was significant difference between MG132 FGF-2 group and FGF-2 group in HLEC. 4TGF- 尾 2 group showed a large amount of yellow brown sediment in the cytoplasm of HLEC, while the expression of FN in MG132 TGF- 尾 2 group was significantly decreased, and there was no significant difference between MG132 group and control group. Conclusion 1. In this experiment, HLEC cells were successfully cultured in vitro, and high purity HLEC cells were obtained. Whether FGF-2TGF- 尾 2 exists or not, MG132 can effectively inhibit the proliferation, migration and differentiation of HLEC.
【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】：R776.1

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