本文選題：生長(zhǎng)分化因子　切入點(diǎn)：慢病毒感染　出處：《中國(guó)臨床藥理學(xué)雜志》2017年10期 　論文類型：期刊論文

【摘要】：目的通過(guò)構(gòu)建生長(zhǎng)分化因子15(GDF15)穩(wěn)定過(guò)表達(dá)的心肌細(xì)胞株,探討其對(duì)H9c2心肌細(xì)胞缺血/再灌注(I/R)損傷凋亡和自噬的影響。方法建立H9c2 I/R損傷模型,用靶向GDF15過(guò)表達(dá)慢病毒和陰性空載體病毒轉(zhuǎn)染H9c2細(xì)胞,篩選出GDF15穩(wěn)定過(guò)表達(dá)細(xì)胞株。分為3組:對(duì)照組、空載體組和GDF15過(guò)表達(dá)組。用MTT法檢測(cè)I/R后細(xì)胞存活率,以LDH試劑盒檢測(cè)細(xì)胞培養(yǎng)上清液中乳酸脫氫酶活力,以免疫印跡法檢測(cè)凋亡相關(guān)蛋白Caspase-3、Bcl-2、Bax和自噬相關(guān)蛋白Beclin-1、LC3-B的表達(dá)。結(jié)果 I/R 2 h,對(duì)照組、空載體組和過(guò)表達(dá)組的細(xì)胞存活率分別為(60.47±5.03)%,(63.21±17.13)%,(74.93±10.03)%,過(guò)表達(dá)組細(xì)胞活性較對(duì)照組與空載體組均增高,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。對(duì)照組和過(guò)表達(dá)組LDH活力值分別為108.72±7.20,50.29±10.05,與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)。在I/R干預(yù)下,凋亡相關(guān)蛋白Caspase-3在對(duì)照組和過(guò)表達(dá)組的灰度值分別為99.73±0.61,67.68±0.47,過(guò)表達(dá)組低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。Bcl-2/Bax在對(duì)照組、空載體組和過(guò)表達(dá)組的灰度值分別為72.52±2.05,39.55±0.46,82.52±1.39,過(guò)表達(dá)組明顯高于對(duì)照組和空載體組,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。在I/R后,自噬相關(guān)蛋白Beclin-1在對(duì)照組、空載體組和GDF15過(guò)表達(dá)組的灰度值分別為58.93±0.64,36.71±0.35,88.39±0.72,過(guò)表達(dá)組明顯高于對(duì)照組和空載體組,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。LC3-Ⅱ/LC3-Ⅰ在空載體組和GDF15過(guò)表達(dá)組的灰度值分別為2.11±0.02,3.32±0.04,過(guò)表達(dá)組明顯高于空載體組,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論上調(diào)H9c2細(xì)胞的GDF15水平可保護(hù)H9c2大鼠心肌細(xì)胞,其作用機(jī)制可能通過(guò)促進(jìn)細(xì)胞的自噬作用得以實(shí)現(xiàn)。
[Abstract]:Objective to study the effects of growth differentiation factor 15 (GDF15) on apoptosis and autophagy in H9c2 myocardial ischemia / reperfusion injury by constructing a stable overexpression cardiomyocyte line of GDF15. Methods A model of H9c2I / R injury was established. H9c2 cells were transfected with target GDF15 overexpression lentivirus and negative empty vector virus, and stable overexpression cell lines of GDF15 were selected. The cells were divided into three groups: control group, empty vector group and GDF15 overexpression group. The survival rate of H9c2 cells after I / R was detected by MTT method. The activity of lactate dehydrogenase in the supernatant of cell culture was detected by LDH kit, the expression of apoptosis-related protein Caspase-3Bcl-2nBax and the autophagy associated protein Beclin-1 (LC3-B) were detected by Western blotting. The cell survival rate of empty vector group and overexpression group were 60.47 鹵5.03 and 63.21 鹵17.13 respectively. The cell activity of overexpressed group was higher than that of control group and empty vector group. The activity of LDH in the control group and overexpression group was 108.72 鹵7.20 鹵50.29 鹵10.05, which was significantly higher than that in the control group. The grayscale value of apoptosis-related protein Caspase-3 in control group and overexpression group was 99.73 鹵0.61U 67.68 鹵0.47, respectively, which was significantly lower than that in control group (P 0.01). Bcl-2 / Bax was significantly higher in control group. The gray values of empty vector group and overexpression group were 72.52 鹵2.05t 39.55 鹵0.46U 82.52 鹵1.39, respectively, which were significantly higher than those of control group and empty vector group (P 0.01). After I / R, autophagy related protein Beclin-1 was found in control group. The gray values of the empty vector group and the GDF15 overexpression group were 58.93 鹵0.64U 36.71 鹵0.35 鹵88.39 鹵0.72, respectively, which were significantly higher than those of the control group and the empty vector group. The grayscale values of P0.01 + LC3- 鈪，

本文編號(hào)：1601916