本文選題：肝纖維化　切入點(diǎn)：肝星狀細(xì)胞　出處：《華北理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的探討腺病毒介導(dǎo)的sh RNA下調(diào)第10號(hào)染色體缺失的磷酸酶張力蛋白同源物基因(phosphatase and tensin homology deleted on chromosome Ten,PTEN)表達(dá)對(duì)體外培養(yǎng)的活化肝星狀細(xì)胞(hepatic stellate cells,HSC)骨架蛋白肌動(dòng)蛋白(actin)及紐蛋白(vinculin)的影響。方法通過(guò)反復(fù)感染293A細(xì)胞的方法擴(kuò)增攜帶靶向PTEN的RNA干擾序列[短發(fā)夾RNA(short hairpin RNA,sh RNA)]并表達(dá)綠色熒光蛋白(green fluorescent protein,GFP)的重組腺病毒Ad-sh RNA/PTEN及僅表達(dá)GFP的對(duì)照空病毒Ad-GFP,并測(cè)定其滴度;體外培養(yǎng)活化大鼠肝星狀細(xì)胞系HSC-T6,以腺病毒為載體將靶向PTEN的sh RNA瞬時(shí)轉(zhuǎn)染活化HSC;采用Western blot及實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)HSC的PTEN蛋白及m RNA表達(dá);借助激光掃描共聚焦顯微鏡(laser scanning confocal microscope,LSCM),應(yīng)用四甲基異硫氰酸羅丹明(Tetramethylrhodamine isothiocyanate,TRITC)標(biāo)記的鬼筆環(huán)肽檢測(cè)HSC的形態(tài)、actin的分布、纖維狀肌動(dòng)蛋白(filamentous actin,F-actin)的熒光強(qiáng)度、偽足以及應(yīng)力纖維的變化,采用免疫熒光法檢測(cè)HSC的vinculin變化,并采用鈣熒光探針Rhod-2/AM負(fù)載檢測(cè)HSC內(nèi)鈣離子(Ca~(2+))濃度變化。采用SPSS17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)處理,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(?)表示,多組間均數(shù)比較采用單因素方差分析(one-way ANOVA),組間比較采用LSD檢驗(yàn),P0.05即認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。實(shí)驗(yàn)分組:(1)Control組:在腺病毒轉(zhuǎn)染步驟以DMEM代替腺病毒液;(2)Ad-GFP組:轉(zhuǎn)染僅表達(dá)GFP的對(duì)照空病毒Ad-GFP;(3)Ad-sh RNA/PTEN組:轉(zhuǎn)染重組腺病毒Ad-sh RNA/PTEN。結(jié)果1通過(guò)反復(fù)感染293A細(xì)胞的方法,獲得實(shí)驗(yàn)所需腺病毒Ad-GFP、Ad-sh RNA/PTEN,滴度分別為1.6′109pfu/m L、1.3′109pfu/m L。2將腺病毒以感染倍數(shù)(multiplicity of infection,MOI)100感染體外活化HSC,感染48 h計(jì)數(shù)GFP陽(yáng)性表達(dá)細(xì)胞數(shù),測(cè)得Ad-GFP、Ad-sh RNA/PTEN轉(zhuǎn)染效率均大于80%。3腺病毒感染HSC 48 h,應(yīng)用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)各組HSC的PTEN m RNA表達(dá),以Control組PTEN的m RNA表達(dá)量為1,則Ad-GFP組及Ad-sh RNA/PTEN組的PTEN m RNA相對(duì)Control組的表達(dá)倍數(shù)分別為0.92倍、0.64倍,Ad-sh RNA/PTEN組PTEN m RNA表達(dá)量明顯低于Control組及Ad-GFP組(P0.05),而Control組與Ad-GFP組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。應(yīng)用Western blot法檢測(cè)HSC的PTEN蛋白表達(dá),Ad-sh RNA/PTEN組(1.088±0.036)明顯低于Control組(1.438±0.038)及Ad-GFP組(1.413±0.058),P0.05,而Control組與Ad-GFP組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4 TRITC標(biāo)記的鬼筆環(huán)肽檢測(cè)顯示:Control組與Ad-GFP組HSC內(nèi)可見(jiàn)應(yīng)力纖維,纖維絲短而稀疏,排列不規(guī)則,很少偽足形成;轉(zhuǎn)染攜帶靶向PTEN的sh RNA的重組腺病毒48 h,可見(jiàn)HSC呈星形向四周伸展,actin排列緊密規(guī)則,數(shù)量增多,偽足充分向外伸展,應(yīng)力纖維增長(zhǎng)增粗。Ad-sh RNA/PTEN組F-actin的熒光強(qiáng)度(1146.10±28.57)較Control組(393.49±21.72)及Ad-GFP組(380.11±19.29)顯著增強(qiáng)(P0.05),而Control組與Ad-GFP組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。5鈣熒光探針Rhod-2/AM檢測(cè)HSC內(nèi)Ca~(2+)濃度顯示:Ad-sh RNA/PTEN組(1363.63±26.06)較Control組(334.62±16.97)及Ad-GFP組(348.05±19.99)明顯增強(qiáng)(P0.05),而Control組與Ad-GFP組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。6免疫熒光法檢測(cè)HSC的vinculin表達(dá)顯示:Ad-sh RNA/PTEN組(770.77±20.12)較Control組(381.13±9.12)及Ad-GFP組(383.87±12.15)顯著增強(qiáng)(P0.05),而Control組與Ad-GFP組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1 PTEN表達(dá)下調(diào)可明顯促進(jìn)體外活化肝星狀細(xì)胞骨架蛋白actin的形成及細(xì)胞骨架的重構(gòu)。2 PTEN表達(dá)下調(diào)可顯著增加活化肝星狀細(xì)胞內(nèi)鈣離子濃度。3 PTEN表達(dá)下調(diào)可顯著上調(diào)活化HSC的vinculin表達(dá)。
[Abstract]:Objective to investigate the SH RNA adenovirus mediated downregulation of chromosome tenth deletion phosphatase and tensin homologue (phosphatase and tensin homology deleted on chromosome Ten, PTEN) on the expression of activation of hepatic stellate cells in vitro (hepatic stellate cells, HSC) (actin) and actin cytoskeleton protein vinculin (vinculin) effect through the method of repeated infection. Methods 293A cells were carrying RNA interference targeting sequence [PTEN RNA (short clip short hairpin RNA, sh RNA) and the expression of green fluorescent protein (green fluorescent, protein, GFP) of the recombinant adenovirus Ad-sh RNA/PTEN and Ad-GFP GFP expression only control empty vector, and the titer of virus was determined; in vitro activation of rat hepatic stellate cell line HSC-T6 by adenovirus vector targeting sh RNA transient transfection of PTEN activated HSC; using Western blot and real-time fluorescence quantitative PCR detection of HSC The expression of PTEN protein and m RNA; by confocal laser scanning microscope (laser scanning confocal microscope, LSCM), using four methyl isothiocyanate Luo Danming (Tetramethylrhodamine isothiocyanate, TRITC) HSC labeled phalloidin to detect the morphology, distribution of actin, F-actin (filamentous actin, F-actin) of the fluorescence intensity. Filopodia, change of stress fibers, vinculin changes were detected by immunofluorescence and HSC, using calcium fluorescent probe Rhod-2/AM HSC load detection calcium ion (Ca~ (2+)) concentration. Statistical analysis was carried out using SPSS17.0 software, the measurement data to mean + standard deviation (?) said Multi-X group compared with single factor analysis of variance (one-way ANOVA), were compared by LSD test, P0.05 is considered statistically significant. Experimental groups: (1): Control group with DMEM instead of in adenovirus transfection steps Adenovirus; (2) Ad-GFP group: transfection only expressed control empty virus Ad-GFP GFP; (3) Ad-sh group: RNA/PTEN transfected with recombinant adenovirus Ad-sh RNA/PTEN. results of the 1 methods by repeated infection of 293A cells, obtained the required adenovirus Ad-GFP, Ad-sh RNA/PTEN, 109pfu/m L titers were 1.6 ', 1.3' 109pfu/m L.2 adenovirus infection ratio (multiplicity of infection, MOI 100) infection in vitro activated HSC, infection of 48 h counts the number of GFP positive cells, measured by Ad-GFP, the transfection efficiency of Ad-sh RNA/PTEN was greater than 48 h HSC 80%.3 adenovirus infection, the expression of PTEN m of RNA HSC was detected using real-time fluorescent quantitative PCR. The m RNA Control group the expression of PTEN was 1, the expression of multiple Ad-GFP group and Ad-sh group RNA/PTEN PTEN m RNA relative to the Control group were 0.92 times, 0.64 times, the expression of Ad-sh RNA/PTEN PTEN m RNA group was significantly lower than that of Control group and Ad-GFP group. (P0.05), while there was no significant difference between Control group and Ad-GFP group (P0.05). The application of Western blot method to detect the expression of HSC PTEN protein, Ad-sh RNA/PTEN group (1.088 + 0.036) was significantly lower than that of Control group (1.438 + 0.038) and Ad-GFP group (1.413 + 0.058), P0.05, and there was no significant difference between Control group and Ad-GFP group (P0.05) showed phalloidin to detect.4 TRITC markers: Control group and Ad-GFP group HSC in stress fibers, filaments short and sparse, irregular, rarely pseudopod formation; GFP targeting recombinant adenovirus sh RNA PTEN 48 h, HSC were seen to star the surrounding stretch, actin closely arranged rules, to increase the number of pseudopodia fully extend outward, should the fluorescence intensity of stress fibers thickening growth.Ad-sh RNA/PTEN group F-actin (1146.10 + 28.57) than in group Control (393.49 + 21.72) and Ad-GFP group (380.11 + 19.29) was significantly increased (P0.05), and Control group No statistically significant differences between groups Ad-GFP (P0.05).5 calcium fluorescent probe Rhod-2/AM detection of HSC Ca~ (2+) concentration showed: Ad-sh group RNA/PTEN (1363.63 + 26.06) than in group Control (334.62 + 16.97) and Ad-GFP group (348.05 + 19.99) increased significantly (P0.05), while there was no significant difference between Control group and group Ad-GFP (P0.05) detection of HSC.6 immunofluorescence showed that Ad-sh vinculin expression in RNA/PTEN group (770.77 + 20.12) than in group Control (381.13 + 9.12) and Ad-GFP group (383.87 + 12.15) was significantly increased (P0.05), while there was no significant difference between Control group and Ad-GFP group (P0.05). Conclusion the expression of 1 the down-regulation of PTEN could significantly promote the expression significantly increased activation expression significantly increased the activation of HSC vinculin expression of calcium ion concentration of.3 PTEN in the hepatic stellate cell formation and cytoskeleton in vitro activation of hepatic stellate cell skeleton protein actin reconstruction.2 PTEN.

【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575

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