【摘要】：目的： 探討激活素A(activinA, ACTA)對(duì)人晶狀體上皮細(xì)胞株SRA01/04(human lens epithelial cell,HLEC SRA01/04)增殖的抑制作用及作用機(jī)制的研究,及其與時(shí)間—效應(yīng)的關(guān)系。 方法： (1)細(xì)胞培養(yǎng):HLECs用10%FBS的LDMEM培養(yǎng)液培養(yǎng),并收集狀態(tài)良好的對(duì)數(shù)生長(zhǎng)期細(xì)胞進(jìn)行下列實(shí)驗(yàn); (2)四甲基偶氮唑鹽(methyl thiazolyl tetrazolium, MTT)比色法：檢測(cè)10ng/mlACTA作用于HLECs 24h、48h、72h后細(xì)胞生長(zhǎng)抑制率; (3)流式細(xì)胞術(shù)(flow cytometrc analysis, FCM):檢測(cè)10ng/ml的ACTA作用于HLECs 24h、48h、72h后早期細(xì)胞凋亡率; (4) DAPI免疫熒光染色(4,6—diamidino—2—phenylindole dihydro-chloride, DAPI):觀察lOng/ml的ACTA作用于HLECs 48h后的形態(tài)學(xué)改變; (5)免疫熒光：觀察lOng/ml的ACTA作用于HLECs 24h、48h、72h后,檢測(cè)caspase-3蛋白的表達(dá)量。實(shí)驗(yàn)結(jié)果均行統(tǒng)計(jì)學(xué)分析。 結(jié)果： (1)MTT:10ng/mlACTA溶液作用于HLECs 24h、48h、72h后其抑制率分別為：10.02%、10.91%、18.11%,說(shuō)明ACTA對(duì)HLECs有明顯的抑制作用,并隨作用時(shí)間增加而增加,差異有統(tǒng)計(jì)學(xué)意義(P均0.05)。 (2)FCM:ACTA作用于HLECs 24h、48h、72h后,早期細(xì)胞凋亡率分別為2.3%、6.2%、9.4%,而空白對(duì)照組的早期細(xì)胞凋亡率為1.4%,說(shuō)明ACTA對(duì)HLECs有誘導(dǎo)凋亡的作用,各組間差異有統(tǒng)計(jì)學(xué)意義(p0.05),并呈時(shí)間一效應(yīng)關(guān)系。 (3)DAPI:ACTA作用于HLECs 48h后細(xì)胞變小變圓、細(xì)胞膜發(fā)生皺縮、染色質(zhì)濃縮、染色質(zhì)斷裂、聚集、核碎裂等凋亡的形態(tài)學(xué)改變。對(duì)照組細(xì)胞形態(tài)規(guī)則,細(xì)胞核飽滿,熒光染色均一。 (4)免疫熒光:ACTA作用于HLECs 24h、48h、72h后,caspase-3蛋白表達(dá)隨時(shí)間增加而增加。差異有統(tǒng)計(jì)學(xué)意義(p0.05)。 結(jié)論： 激活素A對(duì)人晶狀體上皮細(xì)胞的增殖有抑制作用及誘導(dǎo)凋亡的作用,并呈時(shí)間—效應(yīng)關(guān)系,因此可望成為防治后發(fā)性白內(nèi)障的有效藥物。
[Abstract]:Aim: to investigate the inhibitory effect of activin A (activinA, ACTA) on the proliferation of human lens epithelial cell line SRA01/04 (human lens epithelial cell line HLEC SRA01/04 and its relationship with time-effect. Methods: (1) the cells were cultured in 10 S LDMEM medium. The following experiments were carried out: (2) tetramethylazolium (methyl thiazolyl tetrazolium, MTT) colorimetry: to detect the inhibition rate of cell growth by 10ng/mlACTA on HLECs 24 h, 48 h and 72 h; (3) flow cytometry. (4) DAPI immunofluorescence staining (46-diamidino-2-phenylindole dihydro-chloride (DAPI): the morphological changes of lOng/ml ACTA on HLECs 48 h were observed. (5) Immunofluorescence: the effects of ACTA of lOng/ml on HLECs for 24 h and 48 h after 72 h were observed. The expression of caspase-3 protein was detected. The experimental results were analyzed statistically. Results: (1) after treated with MTT:10ng/mlACTA solution for 24 h or 48 h or 72 h, the inhibition rates of ACTA were 10.02 and 10.91, respectively, indicating that ACTA had obvious inhibitory effect on HLECs and increased with the increase of time. The difference was statistically significant (P0. 05). (2). After treated with FCM:ACTA for 24 h or 48 h or 72 h, the rate of early apoptosis was 2. 3% and 6. 2%, respectively, while that of the blank control group was 1. 4%, indicating that ACTA could induce apoptosis of HLECs. There were significant differences among the groups (p0. 05). (3) after DAPI:ACTA was treated with HLECs for 48 h, the cells became smaller and rounded, the cell membrane shrank, chromatin condensed, chromatin breaks and aggregates. Morphological changes of apoptosis such as nuclear fragmentation. In the control group, the cell morphology was regular, the nucleus was full and the fluorescence staining was uniform. (4) the expression of caspase-3 protein increased with the increase of time after the treatment of HLECs 24 h, 48 h and 72 h with immunofluorescence: ACTA. The difference was statistically significant (p0.05). Conclusion: Activin A can inhibit the proliferation of human lens epithelial cells and induce apoptosis in a time-dependent manner, so it is expected to be an effective drug for the prevention and treatment of post-cataract.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R776.1

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 趙瑞苓;外源表達(dá)人激活要素受體ⅡA對(duì)人晶狀體上皮細(xì)胞增殖的影響[D];吉林大學(xué);2012年

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本文編號(hào)：2153615