【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)粘附與遷移的影響及其可能的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制。方法利用AD293細(xì)胞反復(fù)的被腺病毒感染的方法擴(kuò)增實(shí)驗(yàn)所需的腺病毒(AdGFP、Ad-sh RNA/PTEN),并且測(cè)定兩組病毒滴度;體外培養(yǎng)活化大鼠HSC系(HSC-T6),以腺病毒為載體將靶向PTEN的sh RNA干擾重組體瞬時(shí)轉(zhuǎn)染至活化HSC;采用Western blot方法檢測(cè)HSC的PTEN、細(xì)胞外信號(hào)調(diào)節(jié)激酶1(extracellular signal-regulated kinase1,ERK1)、磷酸化ERK1(phosphorylated ERK1,p-ERK1)蛋白表達(dá);采用四甲基偶氮唑鹽(MTT)比色法與甲苯胺藍(lán)染色法測(cè)定HSC的粘附能力;采用劃痕修復(fù)實(shí)驗(yàn)測(cè)定HSC的平面遷移能力、Transwell小室測(cè)定HSC的跨膜遷移能力。所有資料應(yīng)用Excel 2010建立數(shù)據(jù)庫(kù),應(yīng)用SPSS19.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,多組間比較應(yīng)用單因素方差分析(one-way ANOVA),組間比較應(yīng)用LSD檢驗(yàn),P0.05為差異有統(tǒng)計(jì)學(xué)意義。實(shí)驗(yàn)分組如下:(1)Control組:在轉(zhuǎn)染病毒步驟加入無(wú)血清無(wú)抗生素的DMEM代替病毒液;(2)Ad-GFP組:在轉(zhuǎn)染步驟加入只表達(dá)綠色熒光蛋白(green fluorescent protein,GFP)的空病毒;(3)Ad-sh RNA/PTEN組:轉(zhuǎn)染靶向PTEN的sh RNA干擾序列并同時(shí)表達(dá)GFP的重組腺病毒。結(jié)果1通過(guò)反復(fù)感染AD293細(xì)胞的方法獲得實(shí)驗(yàn)所需腺病毒液Ad-GFP、Adsh RNA/PTEN,其滴度分別為:1.3×109pfu/m L、1.5×109pfu/m L。2感染倍數(shù)(Multiplicity of infection,M.O.I.)20時(shí),腺病毒感染HSC后48h,計(jì)數(shù)GFP熒光陽(yáng)性的表達(dá)細(xì)胞,測(cè)的Ad-GFP、Ad-sh RNA/PTEN組腺病毒轉(zhuǎn)染率均80%。3腺病毒轉(zhuǎn)染后48h,實(shí)時(shí)熒光定量PCR檢測(cè)各組HSC的PTEN m RNA表達(dá),并采用2-△△Ct相對(duì)定量法比較,其中以Control組PTEN基因表達(dá)量為1,Ad-GFP組、Ad-sh RNA/PTEN組較Control組的PTEN m RNA的相對(duì)表達(dá)量分別為0.92倍和0.64倍,Ad-sh RNA/PTEN組PTEN m RNA表達(dá)明顯低于Control組及Ad-GFP組(P0.05);Control組與Ad-GFP組PTEN m RNA表達(dá)無(wú)明顯差異(P0.05)。應(yīng)用Western blot技術(shù)檢測(cè)各組HSC的PTEN蛋白表達(dá)顯示:Adsh RNA/PTEN組(1.088±0.036)顯著低于Control組(1.438±0.038)及Ad-GFP(1.413±0.058),P0.05;Control組與Ad-GFP組之間無(wú)明顯性差異(P0.05)。4甲苯胺藍(lán)染色法檢測(cè)HSC粘附能力:腺病毒感染HSC 48 h,與Control組粘附的細(xì)胞數(shù)(495.00±13.23)、Ad-GFP組粘附細(xì)胞數(shù)(500.00±18.03)相比,Ad-sh RNA/PTEN組粘附細(xì)胞數(shù)(592.00±12.53)明顯增多(P0.05);而Ad-GFP組與Control組之間粘附細(xì)胞數(shù)無(wú)明顯差異(P0.05)。5 MTT比色法測(cè)定HSC粘附能力:腺病毒感染HSC 48 h,與Control組的吸光度(Absorbance,A)值(0.279±0.015)相比,Ad-GFP組A值(0.285±0.011),兩組間A值無(wú)明顯差異(P0.05),Ad-GFP組HSC粘附率為102.67±8.29%;Ad-sh RNA/PTEN組A值(0.335±0.013),較Control組及Ad-GFP組明顯增高(P0.05),Adsh RNA/PTEN組HSC粘附率為120.34±7.26%。6劃痕修復(fù)實(shí)驗(yàn)顯示:劃痕后12h各組HSC均向中間有所遷移,但遷移距離經(jīng)計(jì)算無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);劃痕后24 h HSC向中間遷移的距離,Control組(207.78±3.24μm)、Adsh RNA/PTEN組(225.00±4.48μm)、Ad-GFP組(207.95±3.54μm),Adsh RNA/PTEN組與Ad-GFP組及Control組相比有顯著差異(P0.05),Ad-GFP組與Control組相比無(wú)顯著差異(P0.05);劃痕后48 h HSC向中間遷移的距離,Control組(284.33±2.52μm)、Ad-sh RNA/PTEN組(312.87±2.25μm)、Ad-GFP組(281.98±4.34μm),Ad-sh RNA/PTEN組與Control組及Ad-GFP組相比有顯著性差異(P0.05),Control組及Ad-GFP組無(wú)差異(P0.05)。7Transwell小室檢測(cè)HSC跨膜遷移結(jié)果示:Ad-sh RNA/PTEN組跨膜細(xì)胞數(shù)(101.67±3.06)較Ad-GFP組跨膜細(xì)胞數(shù)(67.67±1.53)及Control組跨膜細(xì)胞數(shù)(69.33±2.52)顯著增多(P0.05),Control組與Ad-GFP組之間無(wú)明顯差異(P0.05)。8應(yīng)用Western blot和實(shí)時(shí)熒光定量PCR方法檢測(cè)腺病毒感染HSC48 h后各組HSC ERK1蛋白及其m RNA表達(dá),其結(jié)果表明各組ERK1蛋白及其m RNA表達(dá)均無(wú)明顯變化(P0.05);應(yīng)用Western blot檢測(cè)各組p-ERK1蛋白表達(dá),Ad-sh RNA/PTEN組(1.067±0.047)、Control組(0.710±0.023)及AdGFP組(0.692±0.037),Ad-sh RNA/PTEN組較Control組及Ad-GFP組表達(dá)明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而Control組及Ad-GFP組之間無(wú)顯著差異(P0.05)。結(jié)論1 PTEN低表達(dá)可促進(jìn)體外活化HSC的粘附;2 PTEN低表達(dá)可促進(jìn)體外活化HSC的平面遷移及跨膜遷移;3 ERK信號(hào)通路參與了PTEN對(duì)活化HSC粘附、遷移的調(diào)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.
【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】：2016
【分類號(hào)】：R575.2

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