【摘要】：目的 用不同劑量紫外線(Ultraviolet, UV)照射體外培養(yǎng)人晶狀體上皮細胞(Human lens epithelial cell, HLEC)通過對HLEC凋亡凋亡調(diào)控基因(Bax、Bcl-2)、細胞周期及ALDH,蛋白表達變化的觀察,探討紫外線誘導HLEC凋亡的細胞及分子生物學機制。方法 本試驗以實驗室培養(yǎng)的HLEC細胞株為研究模型,采用同一紫外線光源(UVA照度為0.29mw/cm2,UVB照度為0.09 mw/cm2),對HLEC進行照射。按UV照射時間將HLEC分為Omin、5min、10min、15min及30min組,UV照射后HLEC放回二氧化碳培養(yǎng)箱繼續(xù)培養(yǎng)12小時后再進行各項檢測。 1.UV誘導HLEC凋亡的檢測：采用Hoechst 33342染色、瓊脂糖凝膠電泳對HLEC凋亡進行定性檢測；AO-EB染色及Annexin-V +PI雙染流式細胞計數(shù)對HLEC凋亡進行定量檢測,并分析UV照射劑量與凋亡率間的關系。 2.UV照射誘導HLEC凋亡基因的表達及細胞周期的變化：用原位雜交的方法檢測各組Bax、Bcl-2 mRNA表達,流式細胞術(shù)檢測細胞周期的變化。 3.UV照射對HLEC內(nèi)ALDH1蛋白表達的影響：用免疫組化的方法觀察ALDH1在對照組及各實驗組的表達,用Western Blot對其蛋白含量進行測定。結(jié)果 1.UV照射與HLEC凋亡：Hoechst 33342染色及AO-EB染色UV處理后各實驗組HLEC均有凋亡特征性形態(tài)改變,如細胞質(zhì)濃縮,細胞核致密濃染或呈碎塊狀形成凋亡小體等。瓊脂糖凝膠電泳法檢測除5min組外其余各組都出現(xiàn)DNA ladder,上述三種方法定性證實UV可誘導HLEC凋亡。AO-EB染色法檢測HLEC凋亡率在Omin、5min、l0min、15min、30min組分別為1.82±0.53%、13.15±2.32%、17.58±1.62%、31.16±3.03%、29.25±2.53%(F=146.10,P0.05)。Annexin-V+PI雙染流式細胞計數(shù)檢測以上各組凋亡率分別為1.98±0.84%、11.90±3.21%、16.15±3.05%、33.93±3.74%、22.72±6.05%(F=34.16,P0.05)。兩種方法定性分析均表明隨UV照射時間的延長凋亡率增加。 2.UV照射與Bax上調(diào)和Bcl-2下調(diào)：隨UV照射時間延長,Bcl-2陽性細胞率逐漸降低,而Bax陽性細胞率逐漸增加。經(jīng)配對資料T檢驗,組與組間差異有顯著性(P0.05)。HLEC凋亡率與Bcl-2/Bax比率呈負相關(r=-0.874,P0.05)。 UV照射與HLEC細胞周期阻滯：各實驗組G2/M期細胞與Omin組比較差異有顯著性(P0.05),但各實驗組間差別無顯著性(P0.05)；經(jīng)單因素方差分析,S期細胞隨UV照射時間延長逐漸減少(F=40.34,P0.05)。 3.UV照射與ALDH1:ALDH1免疫組化染色陽性細胞率在Omin、5min、10min、15min及30min組分別為39.23±5.34%、30.57±4.45%、17.91±4.28%、10.25±3.01%、3.83±0.83%,各組陽性細胞率的兩兩比較除Omin組與5min組間差異無顯著性外(P0.05),其余各組間兩兩比較差異均有顯著意義(P0.05)；ALDH1免疫組化染色陽性細胞率隨著UV照射時間延長而降低(F=68.827,P0.05)；Western blot灰度比值隨UV照射時間延長逐漸下降(F=17.256,P0.05)。結(jié)論 1.UV照射可誘導HLEC凋亡,凋亡率在一定時間內(nèi)與UV照射劑量和時間呈正相關。 2.UV照射可誘導HLEC凋亡調(diào)控基因Bax上調(diào)和Bcl-2下調(diào)。 3.UV照射可誘導HLEC細胞周期阻滯,與劑量呈正相關。 4.UV可導致HLEC內(nèi)ALDH1含量下降。
[Abstract]:Purpose Human lens epithelial cell (HLEC) was irradiated with different doses of ultraviolet (UV), and the expression of the expression of Bax, Bcl-2, cell cycle and ALDH and protein of HLEC was changed. Observation on the Cell and Molecular Biology of the Apoptosis of the HLEC Induced by UV mechanism Methods The HLE cell line cultured in the laboratory was used as the study model, and the same ultraviolet light source was used (the UVA illumination intensity was 0.29mw/ cm2, and the UVB illumination was 0.09 mw/ cm2). C. The HLEC is divided into Omin, 5min, 10min, 15min and 30min under the irradiation time of UV. After UV irradiation, the HLEC is returned to the carbon dioxide incubator to continue to be cultured for 12 hours, and then the HLEC is fed into the carbon dioxide incubator for 12 hours. Line item detection.1. UV-induced HLEC apoptosis: HLEC apoptosis was qualitatively detected by Hoechst 33342 staining and agarose gel electrophoresis; AO-EB staining and Annexin-V + PI double staining flow cytometry were used for quantitative detection of HLEC apoptosis, and the UV irradiation dose was analyzed. The relationship between the expression of the apoptosis gene and the cell cycle of the HLEC induced by UV irradiation: the expression of Bax and Bcl-2 mRNA in each group was detected by in situ hybridization. 3. The effect of UV irradiation on the expression of ALDH1 in the HLEC: The expression of ALDH1 in the control group and each experimental group was observed by the method of immunohistochemistry. Bl The results were as follows:1. UV irradiation and HLEC apoptosis: Hoechst 33342 staining and AO-EB staining, the HLEC in each experimental group had a characteristic morphological change, such as cytoplasmic concentration, cells, The DNA ladder was detected in the other groups except the 5-min group by agarose gel electrophoresis. Methods The apoptosis rate of HLEC could be induced by UV. The apoptosis rate of HLEC was 1.82% 0.53%, 13.15% 2.32%, 17.58% 1.62%, 31.16% 3.03%, 29.25% 2.53%, respectively. The apoptosis rate was 1.98% 0.84%, 11.90% 3.21%, 16.15% 3.05%, 33.93% 3.74%, 22.72% 6.0, respectively. 5% (F = 34.16, P0.05). The increase of the apoptosis rate with the UV irradiation time was shown.2. UV irradiation and Bax upregulation and the down-regulation of Bcl-2: The expression of Bcl-2 was fine with the time of UV irradiation. The rate of apoptosis and the ratio of Bcl-2/ Bax were significant (P0.05). There was a negative correlation (r =-0.874, P0.05). The UV irradiation and the cell cycle arrest of the HLEC were significant (P <0.05), but there was no significant difference between the experimental groups (P <0.05), and the S-phase cells were irradiated with UV in the single-factor analysis of variance. The positive rate of the positive cells was 39.23% 5.34%, 30.57% 4.45%, 17.91% 4.28%, 10.25% 3.01%, 3.83% and 0.83%, respectively. There was no significant difference between the Omin group and the 5-min group (P0.05). The positive cell rate of the positive staining of ALDH1 was decreased with the time of UV irradiation (F = 68.827, P0.05). irradiation Conclusion 1. UV irradiation can induce H. The apoptosis and apoptosis rate of LEC were positively correlated with the dose and time of UV irradiation. 2. The expression of Bax and Bcl-2 can be induced by UV irradiation. -2 down-regulation.3. UV irradiation can induce HLEC fine
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

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