發(fā)布時(shí)間：2018-05-17 19:17

  本文選題：草酸鈣 + 人腎小管上皮細(xì)胞　； 參考：《沈陽醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的內(nèi)質(zhì)網(wǎng)是蛋自翻譯后修飾的場(chǎng)所,內(nèi)質(zhì)網(wǎng)應(yīng)激被視為誘導(dǎo)細(xì)胞發(fā)生凋亡的第二條信號(hào)通路,是誘發(fā)多種慢性、代謝性疾病發(fā)病的重要途徑。本項(xiàng)目研究腎結(jié)石形成與內(nèi)質(zhì)網(wǎng)應(yīng)激的關(guān)系。方法本研究采用100μg/mL的草酸鈣結(jié)晶刺激HK-2人腎小管上皮細(xì)胞3h、6h、12h和24h,Oh為對(duì)照組,用Real-time PCR法檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志基因BIP/GRP78,同時(shí)檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)信號(hào)通路中CHOP、PERK、JNK、P38和CasPase-3等基因mRNA的表達(dá)情況,用Western blot法檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白BIP/GRP78、CHOP、P-PERK、P-JNK、P-P38、XBP1、Cleaved-CasPase-3 和CasPase-12的表達(dá)情況,用TUNEL法檢測(cè)草酸鈣結(jié)晶刺激24h時(shí)細(xì)胞凋亡的情況。結(jié)果用100μg/mL的草酸鈣結(jié)晶刺激HK-2細(xì)胞后,內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志基因BIP/GRP78和CHOP基因的mRNA表達(dá)升高,內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路中的PERK、JNK、P38基因的mRNA表達(dá)均升高。當(dāng)用草酸鈣結(jié)晶刺激HK-2細(xì)胞6h,BIP/GRP78蛋白表達(dá)升高,暴露12h,CHOP蛋白表達(dá)升高與對(duì)照組Oh相比具有統(tǒng)計(jì)學(xué)差異,P-PERK、P-JNK、P-P38和XBP1蛋白分別在草酸鈣結(jié)晶刺激3h、3h、6h和3h時(shí)蛋白表達(dá)與對(duì)照組相比升高,Western blot檢測(cè)結(jié)果與Real-time RCR結(jié)果一致。TUNEL檢測(cè)發(fā)現(xiàn)24小時(shí)結(jié)晶暴露后細(xì)胞發(fā)生凋亡,凋亡率約達(dá)25%。Western blot顯示內(nèi)質(zhì)網(wǎng)應(yīng)激特異性蛋白CasPase-12和Cleaved-CasPase-3的表達(dá)升高。結(jié)論我們研究發(fā)現(xiàn)草酸鈣結(jié)晶誘導(dǎo)腎小管上皮細(xì)胞發(fā)生了內(nèi)質(zhì)網(wǎng)應(yīng)激,結(jié)晶誘導(dǎo)的腎小管細(xì)胞凋亡是通過線粒體凋亡通路和內(nèi)質(zhì)網(wǎng)凋亡通路共同引起的。這些發(fā)現(xiàn)提示內(nèi)質(zhì)網(wǎng)應(yīng)激可能是腎結(jié)石形成過程的重要發(fā)病機(jī)制。
[Abstract]:Objective the endoplasmic reticulum (ER) is a place where eggs are modified after translation. Endoplasmic reticulum stress is regarded as the second signal pathway to induce apoptosis, and it is an important way to induce many chronic and metabolic diseases. The purpose of this study was to study the relationship between renal stone formation and endoplasmic reticulum stress. Methods in this study, 100 渭 g/mL calcium oxalate crystals were used to stimulate HK-2 human renal tubular epithelial cells for 12 h and 24 h respectively as control group. The expression of the endoplasmic reticulum stress marker gene BIP-GRP78, the expression of CHOPPERKP38 and CasPase-3 genes in the endoplasmic reticulum stress-related signaling pathway and the expression of BIPP / GRP78 CHOPP-PERKP-JNKP-P38 and XBP1Cleaved-CasPase-3 and CasPase-12 were detected by Real-time PCR method, and the expression of BIP-PERKP-JNKP-38-XBP1Cleaved-Caspase-3 and CasPase-12 were detected by Western blot method. The apoptosis of calcium oxalate after 24 h was detected by TUNEL method. Results after 100 渭 g/mL calcium oxalate was used to stimulate HK-2 cells, the mRNA expression of endoplasmic reticulum stress marker gene BIP/GRP78 and CHOP gene was increased, and the mRNA expression of PERKN JNKN P38 gene in endoplasmic reticulum stress signaling pathway was increased. The expression of BIPP / GRP78 protein was increased in HK-2 cells stimulated by calcium oxalate crystallization for 6 h. There were significant differences in the expression of chop protein between the control group and the control group at 12 h after exposure. The protein expression of P-PERKU P-JNKP-P38 and XBP1 was increased at 3h and 3h after the crystallization of calcium oxalate, respectively. The results of Western blot and Real-time RCR were consistent with those of the control. It was found that cell apoptosis occurred after 24 hours of crystallization exposure. Apoptosis rate up to 25%.Western blot showed increased expression of ER stress-specific proteins CasPase-12 and Cleaved-CasPase-3. Conclusion We found that calcium oxalate crystallization induced endoplasmic reticulum stress in renal tubular epithelial cells. The apoptosis of renal tubular cells induced by calcium oxalate was induced by mitochondrial apoptosis pathway and endoplasmic reticulum apoptosis pathway. These findings suggest that endoplasmic reticulum stress may play an important role in the formation of renal calculi.
【學(xué)位授予單位】：沈陽醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692.4

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本文編號(hào)：1902536