腺病毒介导的shRNA下调PTEN表达对活化肝星状细胞骨架蛋白actin及vinculin的影响

发布时间：2018-03-21 12:40

本文选题：肝纤维化　切入点：肝星状细胞　出处：《华北理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨腺病毒介导的sh RNA下调第10号染色体缺失的磷酸酶张力蛋白同源物基因(phosphatase and tensin homology deleted on chromosome Ten,PTEN)表达对体外培养的活化肝星状细胞(hepatic stellate cells,HSC)骨架蛋白肌动蛋白(actin)及纽蛋白(vinculin)的影响。方法通过反复感染293A细胞的方法扩增携带靶向PTEN的RNA干扰序列[短发夹RNA(short hairpin RNA,sh RNA)]并表达绿色荧光蛋白(green fluorescent protein,GFP)的重组腺病毒Ad-sh RNA/PTEN及仅表达GFP的对照空病毒Ad-GFP,并测定其滴度;体外培养活化大鼠肝星状细胞系HSC-T6,以腺病毒为载体将靶向PTEN的sh RNA瞬时转染活化HSC;采用Western blot及实时荧光定量PCR技术检测HSC的PTEN蛋白及m RNA表达;借助激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM),应用四甲基异硫氰酸罗丹明(Tetramethylrhodamine isothiocyanate,TRITC)标记的鬼笔环肽检测HSC的形态、actin的分布、纤维状肌动蛋白(filamentous actin,F-actin)的荧光强度、伪足以及应力纤维的变化,采用免疫荧光法检测HSC的vinculin变化,并采用钙荧光探针Rhod-2/AM负载检测HSC内钙离子(Ca~(2+))浓度变化。采用SPSS17.0软件进行统计学处理,计量资料以均数±标准差(?)表示,多组间均数比较采用单因素方差分析(one-way ANOVA),组间比较采用LSD检验,P0.05即认为差异有统计学意义。实验分组:(1)Control组:在腺病毒转染步骤以DMEM代替腺病毒液;(2)Ad-GFP组:转染仅表达GFP的对照空病毒Ad-GFP;(3)Ad-sh RNA/PTEN组:转染重组腺病毒Ad-sh RNA/PTEN。结果1通过反复感染293A细胞的方法,获得实验所需腺病毒Ad-GFP、Ad-sh RNA/PTEN,滴度分别为1.6′109pfu/m L、1.3′109pfu/m L。2将腺病毒以感染倍数(multiplicity of infection,MOI)100感染体外活化HSC,感染48 h计数GFP阳性表达细胞数,测得Ad-GFP、Ad-sh RNA/PTEN转染效率均大于80%。3腺病毒感染HSC 48 h,应用实时荧光定量PCR技术检测各组HSC的PTEN m RNA表达,以Control组PTEN的m RNA表达量为1,则Ad-GFP组及Ad-sh RNA/PTEN组的PTEN m RNA相对Control组的表达倍数分别为0.92倍、0.64倍,Ad-sh RNA/PTEN组PTEN m RNA表达量明显低于Control组及Ad-GFP组(P0.05),而Control组与Ad-GFP组之间差异无统计学意义(P0.05)。应用Western blot法检测HSC的PTEN蛋白表达,Ad-sh RNA/PTEN组(1.088±0.036)明显低于Control组(1.438±0.038)及Ad-GFP组(1.413±0.058),P0.05,而Control组与Ad-GFP组之间差异无统计学意义(P0.05)。4 TRITC标记的鬼笔环肽检测显示:Control组与Ad-GFP组HSC内可见应力纤维,纤维丝短而稀疏,排列不规则,很少伪足形成;转染携带靶向PTEN的sh RNA的重组腺病毒48 h,可见HSC呈星形向四周伸展,actin排列紧密规则,数量增多,伪足充分向外伸展,应力纤维增长增粗。Ad-sh RNA/PTEN组F-actin的荧光强度(1146.10±28.57)较Control组(393.49±21.72)及Ad-GFP组(380.11±19.29)显著增强(P0.05),而Control组与Ad-GFP组之间差异无统计学意义(P0.05)。5钙荧光探针Rhod-2/AM检测HSC内Ca~(2+)浓度显示:Ad-sh RNA/PTEN组(1363.63±26.06)较Control组(334.62±16.97)及Ad-GFP组(348.05±19.99)明显增强(P0.05),而Control组与Ad-GFP组之间差异无统计学意义(P0.05)。6免疫荧光法检测HSC的vinculin表达显示:Ad-sh RNA/PTEN组(770.77±20.12)较Control组(381.13±9.12)及Ad-GFP组(383.87±12.15)显著增强(P0.05),而Control组与Ad-GFP组之间差异无统计学意义(P0.05)。结论1 PTEN表达下调可明显促进体外活化肝星状细胞骨架蛋白actin的形成及细胞骨架的重构。2 PTEN表达下调可显著增加活化肝星状细胞内钙离子浓度。3 PTEN表达下调可显著上调活化HSC的vinculin表达。
[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.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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