| Objective: Observe the change of apoptosis rate of RLE-6TN cells,over expression Derlin-1, that treatment with cigarette smoke extract(CSE) at different time.Exploring the relationship between Derlin-1 and apoptosis induced by endoplasmic reticulum stress.Methods: overexpress of Derlin-1 gene in RLE-6TN cells by transfecting with lentiviral that encoding Derlin-1 sequence; transfect blank lentivirus as control group; treat the cells of control and expression group at 0h, 3h, 6h, 12 h, 24 h with 10% CSE;flow cytometry detect the cell apoptosis rate;western blot was used to detect the cell,s protein expression of derlin-1, IRE1, p-IRE1, JNK, p-JNK, chop and caspase-12; realtime PCR detect the cell Derlin-1m RNA expression.1.The detection of the cell that over expression derlin-1 by lentivirus:the expression of Derlin-1 protein that detected by Western blot in the over expression group was higher than that in the control group, and the difference has statistically significance(p<0.05).The expression of Derlin-1m RNA was detected by Realtime-PCR were in agreement with the Western blot results.2.Cell apoptosis was detected by flow cytometry:treat cells of control and over expression groups with 10%CSE 0h, 3h,6h, 12 h, 24 h and then collecte. Cell,s apoptosis rate was detected after cell treat with V-APC/7-AAD Annexin apoptosis detection kit. The apoptosis rate of control and over expression groups increased with the extend of treatment time. Compared with the control group, the apoptosis of the over expression group were significantly lower,the difference was statistically significant between the two groups at the same time(p<0.05).3.Derlin-1 expression in each group of cells: Western blot results show the expression of Derlin-1 in control group show a increasing trend with CSE treatment time increased,and the differences between group and group have statistically significance(P < 0.05).In the over expression group, the expression of Derlin-1 also showed an increasing trend with time increased, and there were significant differences between the two groups(p<0.05).But the expression level of each group was significantly higher than that of the control group, the difference has statistically significance(p<0.05). The expression of Derlin-1m RNA in the two groups was detected by Real time-PCR, which was consistent with the results of Western blot.4.Effect of Derlin-1 on ERS pathway:Western Blot results showed that the total IRE1 expression in the control and over expression groups are same at all time points, and the difference is not statistically significant(p>0.05).The p-IRE1 of the two groups showed an increasing trend with the prolongation of treatment time, and the comparison of each time point is statistically significant(p<0.05).But the expression of p-IRE1 in the expression group was lower than that in the control group, and the difference is statistically significant(p<0.05).5.Effect of Derlin-1 on ERSIA pathway: Western Blot results showed that the expression of total JNK in the control group and over expression group had no significant change at all time points, the difference is not statistically significant(p>0.05). The expression of p-JNK and CHOP in the two groups are gradually increasing trend, time dependence, the difference between the time points with statistical significance(p<0.05).The expression of p-JNK and CHOP in the two groups are gradually increasing trend, and showed a time dependence, differences between each group are statistical significant(p<0.05). Compared with the control group, the expression of the two proteins in the overexpression group is decreased, and the difference is statistically significant(p<0.05).The expression of caspase12 in the control group increased from 3h, 12 h reached the peak and 24 h decreased, but still higher than the 0h group, the differences are statistically significant(p<0.05) at different time points(p<0.05).In the over expression group,the expression of caspase12 increased with time, but the expression in each time point is decreased compared with the control group, and the differences are statistically significant(p<0.05).Conclusion:CSE induced RLE-6TN cells to ERS and promote the occurrence of ERSIA. Derlin-1 upregulation can ease the ERS and then protect cells from apoptosis. |
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