本文關(guān)鍵詞： PERK SREBP1c PERK信號(hào)通路 內(nèi)質(zhì)網(wǎng)應(yīng)激 骨肉瘤細(xì)胞 自噬　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的探討固醇調(diào)節(jié)元件結(jié)合蛋白1c(sterol regulating element binding protein 1c,SREBP1c)是否與人蛋白激酶樣內(nèi)質(zhì)網(wǎng)激酶(protein kinase-like endoplasmic reticulum kinase,PERK)基因啟動(dòng)子結(jié)合,并通過調(diào)控PERK基因轉(zhuǎn)錄與表達(dá),影響骨肉瘤細(xì)胞saos2和RCS發(fā)生增殖、凋亡和自噬的可能機(jī)制。方法構(gòu)建人PERK基因啟動(dòng)子全長及一系列截短體報(bào)告基因載體,與內(nèi)參質(zhì)粒p RL-SV40和pc DNA3.1-SREBP1c、pc DNA3.1-SREBP1cm共轉(zhuǎn)染骨肉瘤細(xì)胞后檢測熒光素酶活性;運(yùn)用Ch IP和EMSA方法檢測SREBP1c與PERK啟動(dòng)子的結(jié)合作用;應(yīng)用p WPT-GFP慢病毒載體系統(tǒng),構(gòu)建并包裝慢病毒SREBP1c、SREBP1cm和PERK。感染細(xì)胞后,通過RT-PCR和蛋白質(zhì)印跡法檢測骨肉瘤細(xì)胞中PERK m RNA和蛋白的表達(dá)。應(yīng)用衣霉素(Tunicamycin,TM)建立內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress,ERS)模型,檢測和探討ERS狀態(tài)下,SREBP1c、SREBP1cm與PERK對(duì)骨肉瘤細(xì)胞的增殖、凋亡和自噬的調(diào)控作用及可能的分子機(jī)制。結(jié)果pc DNA3.1-SREBP1c、pc DNA3.1-SREBP1cm上調(diào)PERK啟動(dòng)子的活性;Ch IP和EMSA證明轉(zhuǎn)錄因子SREBP1c直接與PERK基因啟動(dòng)子區(qū)域結(jié)合并調(diào)節(jié)PERK的轉(zhuǎn)錄與表達(dá);成功構(gòu)建并獲得慢病毒SREBP1c、SREBP1cm顆粒;成功建立內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress,ERS)模型;在ERS狀態(tài)下,SREBP1c、SREBP1cm分組感染后,與對(duì)照組GFP相比較,SREBP1c、SREBP1cm可以不同程度的促進(jìn)骨肉瘤細(xì)胞PERK及p-PERK的表達(dá)(P0.05);過表達(dá)SREBP1c、SREBP1cm及PERK在未經(jīng)TM誘導(dǎo)的情況下同樣可以誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激的發(fā)生;在ERS狀態(tài)下,過表達(dá)SREBP1c、SREBP1cm和PERK都可以抑制骨肉瘤細(xì)胞的增殖、促進(jìn)其凋亡和自噬;其中SREBP1c/1cm可以通過上調(diào)PERK的表達(dá)及磷酸化增強(qiáng)PERK對(duì)細(xì)胞增殖的抑制作用、促進(jìn)PERK介導(dǎo)的細(xì)胞凋亡和細(xì)胞自噬。而在加入PERK信號(hào)通路抑制劑GSK2606414后,可以有效逆轉(zhuǎn)SREBP1cm對(duì)PERK各項(xiàng)生物學(xué)功能的促進(jìn)作用,即:GSK2606414有效逆轉(zhuǎn)SREBP1cm促進(jìn)PERK對(duì)細(xì)胞增殖的抑制作用、促進(jìn)PERK介導(dǎo)的細(xì)胞凋亡和細(xì)胞自噬,證明SREBP1c調(diào)節(jié)細(xì)胞增殖、凋亡和自噬均是通過上調(diào)PERK表達(dá)及磷酸化激活相應(yīng)的信號(hào)通路來實(shí)現(xiàn)的。結(jié)論SREBP1c轉(zhuǎn)錄因子通過直接與PERK基因啟動(dòng)子結(jié)合調(diào)節(jié)PERK基因的轉(zhuǎn)錄與表達(dá);SREBP1c/1cm增強(qiáng)PERK對(duì)細(xì)胞增殖的抑制作用,促進(jìn)PERK介導(dǎo)的凋亡和自噬;SREBP1c/1cm的這一作用依賴于PERK信號(hào)通路。
[Abstract]:Objective to investigate sterol regulating element binding protein 1c. Whether SREBP1c is associated with human protein kinase-like endoplasmic reticulum kinase (SREBP1c). Protein kinase-like endoplasmic reticulum kinase. Perk) gene promoter binds and regulates the transcription and expression of PERK gene to affect the proliferation of saos2 and RCS in osteosarcoma cells. Methods the full-length promoter of human PERK gene and a series of truncated reporter gene vectors were constructed. The luciferase activity of osteosarcoma cells was detected after co-transfection with p#en0# and pcDNA3.1-SREBP1cpc DNA3.1-SREBP1cm. The interaction between SREBP1c and PERK promoter was detected by Ch IP and EMSA. Using p WPT-GFP lentivirus vector system, we constructed and packaged the cells infected with lentivirus SREBP1ctr SREBP1cm and PERK. The expression of PERK m RNA and protein in osteosarcoma cells was detected by RT-PCR and Western blotting. The endoplasmic reticulum stress reticulum stress (ERS) model was established to detect and study SREBP1c under ERS condition. Effects of SREBP1cm and PERK on proliferation, apoptosis and autophagy of osteosarcoma cells and their possible molecular mechanisms. Results PC DNA3.1-SREBP1c. PC DNA3.1-SREBP1cm upregulated the activity of PERK promoter. Ch IP and EMSA demonstrated that the transcription factor SREBP1c binds directly to the promoter region of PERK gene and regulates the transcription and expression of PERK. The lentivirus SREBP1ctr SREBP1cm particles were successfully constructed and obtained. The endoplasmic reticulum stress reticulum stress model was successfully established. Under the condition of ERS, SREBP1c was infected with SREBP1cm, and compared with control group (GFP). SREBP1cm could promote the expression of PERK and p-PERK in osteosarcoma cells in varying degrees. Overexpression of SREBP1cncnsSREBP1cm and PERK could also induce endoplasmic reticulum stress without TM induction. Under the condition of ERS, the overexpression of SREBP1cnsSREBP1cm and PERK could inhibit the proliferation of osteosarcoma cells and promote the apoptosis and autophagy of osteosarcoma cells. SREBP1c/1cm can up-regulate the expression of PERK and increase the inhibitory effect of PERK on cell proliferation by phosphorylation. Promote PERK mediated apoptosis and autophagy, but after adding PERK signaling pathway inhibitor GSK2606414. It can effectively reverse the role of SREBP1cm in promoting the biological functions of PERK. That is, GSK2606414 can effectively reverse the inhibitory effect of SREBP1cm on cell proliferation, promote apoptosis and autophagy mediated by PERK. It is proved that SREBP1c regulates cell proliferation. Apoptosis and autophagy are achieved by up-regulation of PERK expression and activation of corresponding signal pathways by phosphorylation. Conclusion SREBP1c transcription factors regulate PERK by directly binding to PERK gene promoter. Transcription and expression of genes; SREBP1c/1cm enhanced the inhibitory effect of PERK on cell proliferation and promoted PERK mediated apoptosis and autophagy. This role of SREBP1c/1cm depends on the PERK signaling pathway.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R738

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本文編號(hào)：1465297