發(fā)布時間：2018-05-10 07:09

  本文選題：PC12細胞 + H_2O_2�。� 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]用H202氧化應(yīng)激損傷PC12細胞,復(fù)制神經(jīng)細胞損傷模型,初步探索低溫、硫化氫(hydrogen sulfide,H2S)聯(lián)合干預(yù)后,對氧化應(yīng)激損傷的PC12細胞的保護作用,以及低溫聯(lián)合H2S在對抗神經(jīng)細胞的氧化應(yīng)激損傷方面的可能機制。[方法]按照常規(guī)方法培養(yǎng)PC12細胞,選取貼壁80%-90%、生長狀態(tài)良好的細胞,調(diào)整H202濃度為10Oμmol/L,過氧化損傷PC12細胞12h,得到神經(jīng)細胞氧化應(yīng)激模型,采用RT-QPCR方法分別檢測氧化應(yīng)激損傷前后PC12細胞IRAK1和TNF-α兩個指標(biāo)mRNA的表達,獲得本次實驗的目標(biāo)細胞;分組(n=3),空白對照(37℃,5%的C02,培養(yǎng)24h);低溫干預(yù)組:將目標(biāo)細胞分別置于37℃、32℃、27℃,置于5%的C02條件下培養(yǎng)24h;低溫聯(lián)合H2S干預(yù)組:以NaHS為H2S供體,向培養(yǎng)基中加入NaHS,調(diào)整終濃度至100μmol/L,分別置于37℃、32℃、27℃,5%的C02培養(yǎng)箱條件下培養(yǎng)24h;NaHS對照組:調(diào)整NaHS的濃度到100μmol/L,37℃,5%的C02培養(yǎng)條件下培養(yǎng)24h。CCK-8檢細胞增殖;Hoechst33258染色觀察核形態(tài)變化;FITC、PI雙染,FCM檢測細胞凋亡;通過電泳、蛋白質(zhì)印跡(Western Blot)技術(shù)和RT-QPCR技術(shù)檢測Bcl-2和GSK-3β在不同干預(yù)組的蛋白和mRNA含量變化情況,用SPSS17.0對結(jié)果進行統(tǒng)計學(xué)分析。[結(jié)果]1.細胞在27℃-37℃內(nèi)生長良好;2.PC12細胞H202氧化應(yīng)激模型,有效可行;3.單純低溫干預(yù),能減弱H202對Bcl-2的蛋白、mRNA表達量的下調(diào)作用,H2S聯(lián)合低溫可進一步加強這種作用;4.亞低溫能減弱H202對GSK-3β的蛋白表達的上調(diào)作用,在深低溫組和亞低溫組差異不明顯,H2S聯(lián)合低溫可進一步減輕H202對GSK-3β蛋白表達的上調(diào)作用;低溫能減輕H202對GSK-3β mRNA的上調(diào)作用,H2S聯(lián)合低溫能進一步減輕這種作用。[結(jié)論](1)體外培養(yǎng)大鼠嗜鉻細胞瘤(PC12)細胞模型操作簡便,重復(fù)性好,能模擬氧化應(yīng)激損傷后低溫治療過程,是一種較好的體外神經(jīng)元低溫模型。(2)PC12細胞在氧化應(yīng)激損傷過后,Bcl-2的蛋白和mRNA的表達量下調(diào),GSK-3β蛋白和mRNA表達上調(diào)。(3)在27℃-37℃范圍內(nèi),隨著溫度的降低Bcl-2蛋白和mRNA表達量逐漸升高,表明低溫能促進Bcl-2蛋白轉(zhuǎn)錄、表達,減輕細胞凋亡,H2S可加強這種作用。(4)在27℃-37℃范圍內(nèi),隨著溫度的降低GSK-3β蛋白和mRNA表達量逐漸減少,H2S可加強這種作用。(5).低溫聯(lián)合H2S對GSK-3 β和Bcl-2蛋白質(zhì)和mRNA表達量的影響可能是低溫腦保護的作用機制之一。
[Abstract]:[objective] to study the protective effects of hypothermia and hydrogen sulfide (H2S) on PC12 cells injured by oxidative stress (H202), and to establish a model of nerve cell injury, and to explore the protective effect of H202 on PC12 cells injured by oxidative stress. And the possible mechanism of hypothermia combined with H _ 2S in antagonizing oxidative stress injury of nerve cells. [methods] PC12 cells were cultured according to the conventional method. The cells with good growth state were selected from 80 to 90 cells attached to the wall. The oxidative stress model of nerve cells was obtained by adjusting the concentration of H202 to 10 O 渭 mol / L for 12 h after peroxidation injury of PC12 cells. The expression of IRAK1 and TNF- 偽 in PC12 cells before and after oxidative stress injury was detected by RT-QPCR method, and the target cells were obtained. They were divided into three groups, the control group was incubated with C02 at 37 鈩，

本文編號：1868318