腺病毒介导的shRNA下调PTEN表达对体外活化肝星状细胞粘附与迁移的影响
发布时间：2018-08-08 11:35
【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)粘附与迁移的影响及其可能的信号转导机制。方法利用AD293细胞反复的被腺病毒感染的方法扩增实验所需的腺病毒(AdGFP、Ad-sh RNA/PTEN),并且测定两组病毒滴度;体外培养活化大鼠HSC系(HSC-T6),以腺病毒为载体将靶向PTEN的sh RNA干扰重组体瞬时转染至活化HSC;采用Western blot方法检测HSC的PTEN、细胞外信号调节激酶1(extracellular signal-regulated kinase1,ERK1)、磷酸化ERK1(phosphorylated ERK1,p-ERK1)蛋白表达;采用四甲基偶氮唑盐(MTT)比色法与甲苯胺蓝染色法测定HSC的粘附能力;采用划痕修复实验测定HSC的平面迁移能力、Transwell小室测定HSC的跨膜迁移能力。所有资料应用Excel 2010建立数据库,应用SPSS19.0软件进行统计学分析,计量资料以均数±标准差(x±s)表示,多组间比较应用单因素方差分析(one-way ANOVA),组间比较应用LSD检验,P0.05为差异有统计学意义。实验分组如下:(1)Control组:在转染病毒步骤加入无血清无抗生素的DMEM代替病毒液;(2)Ad-GFP组:在转染步骤加入只表达绿色荧光蛋白(green fluorescent protein,GFP)的空病毒;(3)Ad-sh RNA/PTEN组:转染靶向PTEN的sh RNA干扰序列并同时表达GFP的重组腺病毒。结果1通过反复感染AD293细胞的方法获得实验所需腺病毒液Ad-GFP、Adsh RNA/PTEN,其滴度分别为:1.3×109pfu/m L、1.5×109pfu/m L。2感染倍数(Multiplicity of infection,M.O.I.)20时,腺病毒感染HSC后48h,计数GFP荧光阳性的表达细胞,测的Ad-GFP、Ad-sh RNA/PTEN组腺病毒转染率均80%。3腺病毒转染后48h,实时荧光定量PCR检测各组HSC的PTEN m RNA表达,并采用2-△△Ct相对定量法比较,其中以Control组PTEN基因表达量为1,Ad-GFP组、Ad-sh RNA/PTEN组较Control组的PTEN m RNA的相对表达量分别为0.92倍和0.64倍,Ad-sh RNA/PTEN组PTEN m RNA表达明显低于Control组及Ad-GFP组(P0.05);Control组与Ad-GFP组PTEN m RNA表达无明显差异(P0.05)。应用Western blot技术检测各组HSC的PTEN蛋白表达显示:Adsh RNA/PTEN组(1.088±0.036)显著低于Control组(1.438±0.038)及Ad-GFP(1.413±0.058),P0.05;Control组与Ad-GFP组之间无明显性差异(P0.05)。4甲苯胺蓝染色法检测HSC粘附能力:腺病毒感染HSC 48 h,与Control组粘附的细胞数(495.00±13.23)、Ad-GFP组粘附细胞数(500.00±18.03)相比,Ad-sh RNA/PTEN组粘附细胞数(592.00±12.53)明显增多(P0.05);而Ad-GFP组与Control组之间粘附细胞数无明显差异(P0.05)。5 MTT比色法测定HSC粘附能力:腺病毒感染HSC 48 h,与Control组的吸光度(Absorbance,A)值(0.279±0.015)相比,Ad-GFP组A值(0.285±0.011),两组间A值无明显差异(P0.05),Ad-GFP组HSC粘附率为102.67±8.29%;Ad-sh RNA/PTEN组A值(0.335±0.013),较Control组及Ad-GFP组明显增高(P0.05),Adsh RNA/PTEN组HSC粘附率为120.34±7.26%。6划痕修复实验显示:划痕后12h各组HSC均向中间有所迁移,但迁移距离经计算无统计学意义(P0.05);划痕后24 h HSC向中间迁移的距离,Control组(207.78±3.24μm)、Adsh RNA/PTEN组(225.00±4.48μm)、Ad-GFP组(207.95±3.54μm),Adsh RNA/PTEN组与Ad-GFP组及Control组相比有显著差异(P0.05),Ad-GFP组与Control组相比无显著差异(P0.05);划痕后48 h HSC向中间迁移的距离,Control组(284.33±2.52μm)、Ad-sh RNA/PTEN组(312.87±2.25μm)、Ad-GFP组(281.98±4.34μm),Ad-sh RNA/PTEN组与Control组及Ad-GFP组相比有显著性差异(P0.05),Control组及Ad-GFP组无差异(P0.05)。7Transwell小室检测HSC跨膜迁移结果示:Ad-sh RNA/PTEN组跨膜细胞数(101.67±3.06)较Ad-GFP组跨膜细胞数(67.67±1.53)及Control组跨膜细胞数(69.33±2.52)显著增多(P0.05),Control组与Ad-GFP组之间无明显差异(P0.05)。8应用Western blot和实时荧光定量PCR方法检测腺病毒感染HSC48 h后各组HSC ERK1蛋白及其m RNA表达,其结果表明各组ERK1蛋白及其m RNA表达均无明显变化(P0.05);应用Western blot检测各组p-ERK1蛋白表达,Ad-sh RNA/PTEN组(1.067±0.047)、Control组(0.710±0.023)及AdGFP组(0.692±0.037),Ad-sh RNA/PTEN组较Control组及Ad-GFP组表达明显升高,差异有统计学意义(P0.05),而Control组及Ad-GFP组之间无显著差异(P0.05)。结论1 PTEN低表达可促进体外活化HSC的粘附;2 PTEN低表达可促进体外活化HSC的平面迁移及跨膜迁移;3 ERK信号通路参与了PTEN对活化HSC粘附、迁移的调控。
[Abstract]:Objective to investigate the effect of adenovirus mediated sh RNA on the expression of phosphatase and tensin homology deleted on chromosome Ten, PTEN) on the adhesion and migration of activated hepatic stellate cells (tensin homology deleted on chromosome Ten, PTEN) and its possible signal transduction mechanism. Methods the adenovirus (AdGFP, Ad-sh RNA/PTEN) needed in the experiment was amplified by repeated adenovirus infection of AD293 cells, and the two groups of virus titers were measured. The HSC system (HSC-T6) was activated in vitro, and the adenovirus was used as the carrier to instantaneously transfect the SH RNA interfering recombinant of the PTEN target to activated HSC, and the Western blot method was used to detect the HSC. PTEN, extracellular signal regulated kinase 1 (extracellular signal-regulated kinase1, ERK1), phosphorylated ERK1 (phosphorylated ERK1, p-ERK1) protein expression; the adhesion ability of HSC was measured with four methazolazoles (MTT) colorimetry and toluidine blue staining. The transmembrane migration ability of HSC was determined. All data were used to establish a database with Excel 2010, and SPSS19.0 software was used for statistical analysis. The measurement data were expressed with mean standard deviation (x + s). Single factor variance analysis (one-way ANOVA) was used in multiple groups. LSD test was used among groups. P0.05 was statistically significant. The experimental group was as follows: (1) ) Control group: transfection of the virus step into the serum-free and non antibiotic DMEM instead of the virus; (2) group Ad-GFP: an empty virus that only expresses green fluorescent protein (green fluorescent protein, GFP) in the transfection step; (3) Ad-sh RNA/PTEN group: transfection of the SH RNA interference sequence to target PTEN and simultaneously expressing the recombinant adenovirus. Results 1 pass through the inverse The method of reinfection of AD293 cells obtained the experimental adenovirus Ad-GFP, Adsh RNA/PTEN, and its titers were 1.3 x 109pfu/m L, 1.5 x 109pfu/m L.2 infection multiple (Multiplicity of infection, M.O.I.) 20, and the adenovirus positive cells were counted. The transfection rate of adenovirus was 80 The expression of PTEN m RNA in HSC was detected by real-time fluorescence quantitative PCR after transfection of%.3 adenovirus, and the expression of PTEN m RNA in each group was compared with 2- Delta Delta Ct relative quantitative method. The expression of PTEN gene in Control group was 1, and the relative expression of Ad-GFP group was 0.92 times and 0.64 times respectively. The expression of PTEN m RNA in group Control and Ad-GFP group was significantly lower than that of group Control and group Ad-GFP (P0.05). The expression of protein expression of HSC in each group was detected by Western blot technique. P0.05.4 toluidine blue staining was used to detect HSC adhesion: adenovirus infected HSC 48 h, the number of cells adhered to Control group (495 + 13.23), and the number of adhesion cells in Ad-GFP group (500 + 18.03), the number of adhesion cells in Ad-sh RNA/PTEN group (592 + 12.53) increased significantly (P0.05), while the number of adhesion cells between Ad-GFP group and Control group was not clear. P0.05.5 MTT colorimetric assay was used to determine the adhesion ability of HSC: adenovirus infection HSC 48 h, compared with the absorbance (Absorbance, A) value of Control group (0.279 + 0.015), A value of Ad-GFP group (0.285 + 0.011). The A values between the two groups were not significantly different (102.67 + 8.29%, 0.335 + 0.013). The P group was significantly higher (P0.05), and the HSC adhesion rate of the Adsh RNA/PTEN group was 120.34 + 7.26%.6 scratch repair experiment, and the HSC of the 12h groups was migrated to the middle after the scratch, but the migration distance was not statistically significant (P0.05), the distance of the 24 h HSC to the middle after scratch, the Control group (207.78 + 3.24 mu), and 225 + 4.48 micron. Group FP (207.95 + 3.54 mu m), Adsh RNA/PTEN group and Ad-GFP group and Control group have significant difference (P0.05), Ad-GFP group has no significant difference compared with Control group (P0.05); the distance of 48 h HSC after scratch (284.33 + 2.52 mu), 312.87 + 2.25 micron (281.98 + 4.34 Mu). There was significant difference between group trol and group Ad-GFP (P0.05), and there was no difference between group Control and Ad-GFP group (P0.05), the transmembrane migration of HSC in.7Transwell compartment showed that the number of transmembrane cells in Ad-sh RNA/PTEN group (101.67 + 3.06) was more than that of Ad-GFP group (67.67 + 1.53) and the number of transmembrane cells in Control group increased significantly (69.33 + 2.52). There was no significant difference between the -GFP groups (P0.05).8 application Western blot and real-time fluorescent quantitative PCR method to detect the HSC ERK1 protein and m RNA expression after adenovirus infection. The results showed that there were no significant changes in the expression of HSC48 h in each group. 7 + 0.047), group Control (0.710 + 0.023) and group AdGFP (0.692 + 0.037), the expression of Ad-sh RNA/PTEN group was significantly higher than that of Control and Ad-GFP group, and there was no significant difference (P0.05), but there was no significant difference between group Control and Ad-GFP group (P0.05). Conclusion 1 PTEN low expression can promote the adhesion of activated HSC in vitro; 2 low expression can promote activation in vitro. SC migration and transmembrane migration; the 3 ERK signaling pathway is involved in the regulation of PTEN on the adhesion and migration of HSC.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.2
[Abstract]:Objective to investigate the effect of adenovirus mediated sh RNA on the expression of phosphatase and tensin homology deleted on chromosome Ten, PTEN) on the adhesion and migration of activated hepatic stellate cells (tensin homology deleted on chromosome Ten, PTEN) and its possible signal transduction mechanism. Methods the adenovirus (AdGFP, Ad-sh RNA/PTEN) needed in the experiment was amplified by repeated adenovirus infection of AD293 cells, and the two groups of virus titers were measured. The HSC system (HSC-T6) was activated in vitro, and the adenovirus was used as the carrier to instantaneously transfect the SH RNA interfering recombinant of the PTEN target to activated HSC, and the Western blot method was used to detect the HSC. PTEN, extracellular signal regulated kinase 1 (extracellular signal-regulated kinase1, ERK1), phosphorylated ERK1 (phosphorylated ERK1, p-ERK1) protein expression; the adhesion ability of HSC was measured with four methazolazoles (MTT) colorimetry and toluidine blue staining. The transmembrane migration ability of HSC was determined. All data were used to establish a database with Excel 2010, and SPSS19.0 software was used for statistical analysis. The measurement data were expressed with mean standard deviation (x + s). Single factor variance analysis (one-way ANOVA) was used in multiple groups. LSD test was used among groups. P0.05 was statistically significant. The experimental group was as follows: (1) ) Control group: transfection of the virus step into the serum-free and non antibiotic DMEM instead of the virus; (2) group Ad-GFP: an empty virus that only expresses green fluorescent protein (green fluorescent protein, GFP) in the transfection step; (3) Ad-sh RNA/PTEN group: transfection of the SH RNA interference sequence to target PTEN and simultaneously expressing the recombinant adenovirus. Results 1 pass through the inverse The method of reinfection of AD293 cells obtained the experimental adenovirus Ad-GFP, Adsh RNA/PTEN, and its titers were 1.3 x 109pfu/m L, 1.5 x 109pfu/m L.2 infection multiple (Multiplicity of infection, M.O.I.) 20, and the adenovirus positive cells were counted. The transfection rate of adenovirus was 80 The expression of PTEN m RNA in HSC was detected by real-time fluorescence quantitative PCR after transfection of%.3 adenovirus, and the expression of PTEN m RNA in each group was compared with 2- Delta Delta Ct relative quantitative method. The expression of PTEN gene in Control group was 1, and the relative expression of Ad-GFP group was 0.92 times and 0.64 times respectively. The expression of PTEN m RNA in group Control and Ad-GFP group was significantly lower than that of group Control and group Ad-GFP (P0.05). The expression of protein expression of HSC in each group was detected by Western blot technique. P0.05.4 toluidine blue staining was used to detect HSC adhesion: adenovirus infected HSC 48 h, the number of cells adhered to Control group (495 + 13.23), and the number of adhesion cells in Ad-GFP group (500 + 18.03), the number of adhesion cells in Ad-sh RNA/PTEN group (592 + 12.53) increased significantly (P0.05), while the number of adhesion cells between Ad-GFP group and Control group was not clear. P0.05.5 MTT colorimetric assay was used to determine the adhesion ability of HSC: adenovirus infection HSC 48 h, compared with the absorbance (Absorbance, A) value of Control group (0.279 + 0.015), A value of Ad-GFP group (0.285 + 0.011). The A values between the two groups were not significantly different (102.67 + 8.29%, 0.335 + 0.013). The P group was significantly higher (P0.05), and the HSC adhesion rate of the Adsh RNA/PTEN group was 120.34 + 7.26%.6 scratch repair experiment, and the HSC of the 12h groups was migrated to the middle after the scratch, but the migration distance was not statistically significant (P0.05), the distance of the 24 h HSC to the middle after scratch, the Control group (207.78 + 3.24 mu), and 225 + 4.48 micron. Group FP (207.95 + 3.54 mu m), Adsh RNA/PTEN group and Ad-GFP group and Control group have significant difference (P0.05), Ad-GFP group has no significant difference compared with Control group (P0.05); the distance of 48 h HSC after scratch (284.33 + 2.52 mu), 312.87 + 2.25 micron (281.98 + 4.34 Mu). There was significant difference between group trol and group Ad-GFP (P0.05), and there was no difference between group Control and Ad-GFP group (P0.05), the transmembrane migration of HSC in.7Transwell compartment showed that the number of transmembrane cells in Ad-sh RNA/PTEN group (101.67 + 3.06) was more than that of Ad-GFP group (67.67 + 1.53) and the number of transmembrane cells in Control group increased significantly (69.33 + 2.52). There was no significant difference between the -GFP groups (P0.05).8 application Western blot and real-time fluorescent quantitative PCR method to detect the HSC ERK1 protein and m RNA expression after adenovirus infection. The results showed that there were no significant changes in the expression of HSC48 h in each group. 7 + 0.047), group Control (0.710 + 0.023) and group AdGFP (0.692 + 0.037), the expression of Ad-sh RNA/PTEN group was significantly higher than that of Control and Ad-GFP group, and there was no significant difference (P0.05), but there was no significant difference between group Control and Ad-GFP group (P0.05). Conclusion 1 PTEN low expression can promote the adhesion of activated HSC in vitro; 2 low expression can promote activation in vitro. SC migration and transmembrane migration; the 3 ERK signaling pathway is involved in the regulation of PTEN on the adhesion and migration of HSC.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.2
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