【摘要】：目的:觀察過(guò)表達(dá)Derlin-1的RLE-6TN細(xì)胞經(jīng)香煙煙霧提取物(cigarette smoke extrac,CSE)不同時(shí)間處理后的凋亡率的變化。探索其與內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)性凋亡(Endoplasmic reticulum stress induced apoptosis,ERSIA)的關(guān)系。方法:攜帶Derlin-1編碼序列的慢病毒轉(zhuǎn)染RLE-6TN細(xì)胞過(guò)表達(dá)Derlin-1基因;轉(zhuǎn)染空白慢病毒作為對(duì)照組;制備CSE;用10%CSE處理對(duì)照組、過(guò)表達(dá)組細(xì)胞0h、3h、6h、12h、24h;流式細(xì)胞儀檢測(cè)各組細(xì)胞的凋亡率;Western blot技術(shù)檢測(cè)各組細(xì)胞Derlin-1、IRE1、p-IRE1、JNK、P-JNK,CHOP、caspase12蛋白的表達(dá);Realtime-PCR技術(shù)檢測(cè)各組細(xì)胞Derlin-1m RNA的表達(dá)。結(jié)果:1.慢病毒過(guò)表達(dá)Derlin-1穩(wěn)定株的檢測(cè):Western blot檢測(cè)過(guò)表達(dá)組Derlin-1蛋白的表達(dá)較對(duì)照組增加,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。Realtime-PCR檢測(cè)Derlin-1m RNA的表達(dá)結(jié)果與Western blot結(jié)果一致。2.流式細(xì)胞技術(shù)檢測(cè)細(xì)胞凋亡:用10%CSE處理對(duì)照組、過(guò)表達(dá)組細(xì)胞0h,3h,6h,12h,24h后收集細(xì)胞,用Annexin V-APC/7-AAD細(xì)胞凋亡檢測(cè)試劑盒處理后在流式細(xì)胞儀上檢測(cè)細(xì)胞凋亡率。對(duì)照組及過(guò)表達(dá)組凋亡率隨著處理時(shí)間延長(zhǎng)而增加,每組各時(shí)間點(diǎn)之間兩兩比較差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。過(guò)表達(dá)組凋亡率較對(duì)照組均明顯降低,同一時(shí)間點(diǎn)的兩組比較差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。3.Derlin-1在各組細(xì)胞中的表達(dá):Western blot結(jié)果顯示對(duì)照組Derlin-1的表達(dá)隨CSE處理時(shí)間的延長(zhǎng)呈逐漸增加的趨勢(shì),且組與組之間差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。在過(guò)表達(dá)組,Derlin-1的表達(dá)同樣呈現(xiàn)出隨時(shí)間延長(zhǎng)的增長(zhǎng)趨勢(shì),各組間的兩兩比較均有統(tǒng)計(jì)學(xué)差異(p0.05)。但每組較對(duì)照組表達(dá)量明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。Realtime-PCR檢測(cè)兩組Derlin-1m RNA的表達(dá)結(jié)果與Western blot結(jié)果一致。4.Derlin-1對(duì)ERS通路的影響:Western blot結(jié)果顯示在對(duì)照組及過(guò)表達(dá)組中總的IRE1的表達(dá)在各時(shí)間點(diǎn)的變化不大,差異沒(méi)有統(tǒng)計(jì)學(xué)意義(p0.05)。兩組的p-IRE1均有隨處理時(shí)間的延長(zhǎng)呈現(xiàn)增加趨勢(shì),每組各時(shí)間點(diǎn)的比較具有統(tǒng)計(jì)學(xué)意義(p0.05)。但過(guò)表達(dá)組的p-IRE1的表達(dá)量較對(duì)照組有所下降,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)5.Derlin-1對(duì)ERSIA通路的影響:Western blot結(jié)果顯示在對(duì)照組和過(guò)表達(dá)組中總JNK的表達(dá)量在各時(shí)間點(diǎn)的表達(dá)無(wú)明顯變化,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。p-JNK與CHOP的表達(dá)量在兩組中均有逐漸增加的趨勢(shì),呈時(shí)間依賴性,每組各時(shí)間點(diǎn)兩兩比較差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。兩種蛋白過(guò)表達(dá)組較對(duì)照組表達(dá)量均下降,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。caspase12的表達(dá)量在對(duì)照組從3h升高,12h達(dá)高峰,24h有所下降,但仍高于0h組,各時(shí)間點(diǎn)的兩兩比較差異有統(tǒng)計(jì)學(xué)意義(p0.05)。在過(guò)表達(dá)組中,caspase12的表達(dá)隨時(shí)間的延長(zhǎng)而增加,但各時(shí)間點(diǎn)表達(dá)量較對(duì)照組均下降,且差異有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:CSE誘導(dǎo)RLE-6TN細(xì)胞發(fā)生ERS,促進(jìn)ERSIA的發(fā)生。Derlin-1的上調(diào)可以緩解ERS,保護(hù)細(xì)胞免于凋亡。
[Abstract]:Aim: to observe the changes of apoptosis rate of RLE-6TN cells which expressed Derlin-1 after treated with cigarette smoke extract (cigarette smoke extrac,CSE) at different time. To explore the relationship between endoplasmic reticulum stress-induced apoptosis (Endoplasmic reticulum stress induced apoptosis,ERSIA). Methods: lentivirus carrying Derlin-1 coding sequence was transfected into RLE-6TN cells to overexpression Derlin-1 gene, blank lentivirus was transfected as control group, and CSE; was treated with 10%CSE for 24 h. Flow cytometry was used to detect the apoptosis rate of cells in each group. The expression of Derlin-1,IRE1,p-IRE1,JNK,P-JNK,CHOP,caspase12 protein was detected by; Western blot and the expression of Derlin-1m RNA was detected by Realtime-PCR technique. Results: 1. Detection of lentivirus overexpression Derlin-1 stable strain the expression of Derlin-1 protein in: Western blot overexpression group was significantly higher than that in control group (p0.05). The expression of Derlin-1m RNA by Realtime-PCR was consistent with that of Western blot. 2. Flow cytometry was used to detect apoptosis: the control group was treated with 10%CSE, and the cells in the overexpression group were collected after 24 hours after 0 h, 3 h, 6 h and 12 h. Apoptosis rate was detected by flow cytometry after treated with Annexin V-APC/7-AAD cell apoptosis detection kit. The apoptotic rate of the control group and the overexpression group increased with the prolongation of the treatment time, and there were significant differences between the two groups at each time point (p0.05). The rate of apoptosis in overexpression group was significantly lower than that in control group. There were significant differences between the two groups at the same time point (p0.05). The expression of: Western blot in the cells of each group showed that the expression of Derlin-1 in the control group increased gradually with the prolongation of the time of CSE treatment. The difference between the two groups was statistically significant (p 0.05). In the overexpression group, the expression of Derlin-1 also showed an increasing trend with time, and there was statistical difference between the two groups (p0. 05). However, the expression of protein in each group was significantly higher than that in the control group. The difference was statistically significant (p0.05). The expression of Derlin-1m RNA in the two groups by Realtime-PCR was consistent with that of Western blot. The effect of 4.Derlin-1 on the ERS pathway was found in the control group and the overexpression group. The expression of IRE1 changed little at different time points. The difference was not statistically significant (p0.05). The p-IRE1 of both groups showed an increasing trend with the prolongation of treatment time, and the comparison of each time point in each group was statistically significant (p0. 05). However, the expression of p-IRE1 in the overexpression group was lower than that in the control group. The difference was statistically significant (p0.05) the effect of 5.Derlin-1 on the ERSIA pathway. The results of: Western blot showed that the expression of total JNK in control group and overexpression group had no significant change at each time point. There was no significant difference (p0.05). The expression of p-JNK and CHOP increased gradually in both groups in a time-dependent manner, and the differences were statistically significant at each time point (p0.05). The expression of caspase12 increased from 3 h to 12 h, peaked at 12 h, decreased at 24 h, but still higher than that in 0 h group. There was significant difference between the two groups at each time point (p 0.05). In the overexpression group, the expression of caspase12 increased with the prolongation of time, but the expression of caspase12 at each time point was lower than that of the control group, and the difference was statistically significant (p0.05). Conclusion: CSE induces the occurrence of ERS, in RLE-6TN cells, and the up-regulation of Derlin-1 can alleviate the apoptosis of ERS,.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R56

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9 蘇志剛;組蛋白乙酰化修飾在香煙煙霧致BEAS-2B細(xì)胞惡性轉(zhuǎn)化過(guò)程中的作用[D];蘇州大學(xué);2016年

10 陳文瓊;過(guò)表達(dá)Derlin-1對(duì)香煙煙霧提取物誘導(dǎo)Ⅱ型肺泡上皮細(xì)胞凋亡的作用研究[D];南華大學(xué);2016年

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