UV诱导HLEC凋亡的细胞及分子机制的实验研究

发布时间：2019-05-20 09:10

【摘要】：目的用不同剂量紫外线(Ultraviolet, UV)照射体外培养人晶状体上皮细胞(Human lens epithelial cell, HLEC)通过对HLEC凋亡凋亡调控基因(Bax、Bcl-2)、细胞周期及ALDH,蛋白表达变化的观察,探讨紫外线诱导HLEC凋亡的细胞及分子生物学机制。方法本试验以实验室培养的HLEC细胞株为研究模型,采用同一紫外线光源(UVA照度为0.29mw/cm2,UVB照度为0.09 mw/cm2),对HLEC进行照射。按UV照射时间将HLEC分为Omin、5min、10min、15min及30min组,UV照射后HLEC放回二氧化碳培养箱继续培养12小时后再进行各项检测。 1.UV诱导HLEC凋亡的检测：采用Hoechst 33342染色、琼脂糖凝胶电泳对HLEC凋亡进行定性检测；AO-EB染色及Annexin-V +PI双染流式细胞计数对HLEC凋亡进行定量检测,并分析UV照射剂量与凋亡率间的关系。 2.UV照射诱导HLEC凋亡基因的表达及细胞周期的变化：用原位杂交的方法检测各组Bax、Bcl-2 mRNA表达,流式细胞术检测细胞周期的变化。 3.UV照射对HLEC内ALDH1蛋白表达的影响：用免疫组化的方法观察ALDH1在对照组及各实验组的表达,用Western Blot对其蛋白含量进行测定。结果 1.UV照射与HLEC凋亡：Hoechst 33342染色及AO-EB染色UV处理后各实验组HLEC均有凋亡特征性形态改变,如细胞质浓缩,细胞核致密浓染或呈碎块状形成凋亡小体等。琼脂糖凝胶电泳法检测除5min组外其余各组都出现DNA ladder,上述三种方法定性证实UV可诱导HLEC凋亡。AO-EB染色法检测HLEC凋亡率在Omin、5min、l0min、15min、30min组分别为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双染流式细胞计数检测以上各组凋亡率分别为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)。两种方法定性分析均表明随UV照射时间的延长凋亡率增加。 2.UV照射与Bax上调和Bcl-2下调：随UV照射时间延长,Bcl-2阳性细胞率逐渐降低,而Bax阳性细胞率逐渐增加。经配对资料T检验,组与组间差异有显著性(P0.05)。HLEC凋亡率与Bcl-2/Bax比率呈负相关(r=-0.874,P0.05)。 UV照射与HLEC细胞周期阻滞：各实验组G2/M期细胞与Omin组比较差异有显著性(P0.05),但各实验组间差别无显著性(P0.05)；经单因素方差分析,S期细胞随UV照射时间延长逐渐减少(F=40.34,P0.05)。 3.UV照射与ALDH1:ALDH1免疫组化染色阳性细胞率在Omin、5min、10min、15min及30min组分别为39.23±5.34%、30.57±4.45%、17.91±4.28%、10.25±3.01%、3.83±0.83%,各组阳性细胞率的两两比较除Omin组与5min组间差异无显著性外(P0.05),其余各组间两两比较差异均有显著意义(P0.05)；ALDH1免疫组化染色阳性细胞率随着UV照射时间延长而降低(F=68.827,P0.05)；Western blot灰度比值随UV照射时间延长逐渐下降(F=17.256,P0.05)。结论 1.UV照射可诱导HLEC凋亡,凋亡率在一定时间内与UV照射剂量和时间呈正相关。 2.UV照射可诱导HLEC凋亡调控基因Bax上调和Bcl-2下调。 3.UV照射可诱导HLEC细胞周期阻滞,与剂量呈正相关。 4.UV可导致HLEC内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
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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