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目的:构建携带抑癌基因PTEN的腺病毒载体,探讨PTEN基因对前列腺癌细胞株PC-3增殖及cyclin D1、p21表达的影响。方法:采用RT-PCR方法从大鼠海马神经元扩增目的基因PTEN,连接入pENTR2A载体,在293A细胞中进行重组腺病毒的包装及扩增。以携带PTEN基因的腺病毒载体感染体外培养的前列腺癌细胞株PC-3。采用间接免疫荧光法检测细胞中PTEN的过表达情况。Western印迹检测PTEN、cyclin D1和p21表达的变化,通过细胞生长实验、平板克隆实验检测PTEN对PC-3细胞增殖能力的影响。结果:成功构建重组腺病毒载体Ad-PTEN;PC-3细胞经Ad-PTEN感染后PTEN蛋白表达水平明显增加。Western印迹实验所得条带进行灰度值分析后与β-actin求比值,PTEN蛋白在Ad-PTEN感染组细胞中表达(0.215±0.065)明显高于对照组[(0.052±0.009),t=4.30,P<0.05]和Ad-LacZ组[(0.056±0.008),t=4.21,P<0.05]。cyclin D1蛋白在Ad-PTEN感染组细胞中表达(0.256±0.072)明显低于对照组[(0.502±0.087,t=3.77,P<0.05)]和Ad-LacZ组[(0.498±0.081,t=3.87,P<0.05)]。p21蛋白在Ad-PTEN感染组细胞中表达(0.589±0.076)明显高于对照组[(0.146±0.026,t=9.55,P<0.01)]和Ad-LacZ组[(0.163±0.024,t=9.26,P<0.01)]。MTT实验显示Ad-PTEN对PC-3细胞的抑制作用自48h后(21.98%)有显著性(t=6.80,P<0.01)。平板克隆实验显示Ad-PTEN组克隆形成率[(37.4±4.18)%]明显低于对照组[(54.9±4.81)%,t=4.76,P<0.01]及Ad-LacZ组[(56.5±5.42)%,t=4.83,P<0.01]。结论:通过腺病毒载体Ad-PTEN使PC-3细胞表达PTEN基因,可以明显抑制细胞的增殖,并可降低cyclin D1表达,同时增加p21的表达。
OBJECTIVE: To construct the adenovirus vector carrying PTEN gene and investigate the effect of PTEN gene on the proliferation and the expression of cyclin D1 and p21 in prostate cancer cell line PC-3. Methods: The target gene PTEN was amplified from rat hippocampal neurons by RT-PCR and inserted into pENTR2A vector. The recombinant adenovirus was packaged and amplified in 293A cells. In vitro cultured prostate cancer cell line PC-3 was infected with the adenovirus vector carrying the PTEN gene. Indirect immunofluorescence was used to detect PTEN overexpression. The expression of PTEN, cyclin D1 and p21 were detected by Western blot. The effect of PTEN on the proliferation of PC-3 cells was detected by cell growth assay and plate clone assay. Results: The recombinant adenovirus Ad-PTEN was successfully constructed. The expression of PTEN protein in PC-3 cells was significantly increased after Ad-PTEN infection. The ratio of PTEN protein expression in the Ad-PTEN infected group (0.215 ± 0.065) was significantly higher than that in the control group [(0.052 ± 0.009), t = 4.30 , P <0.05] and Ad-LacZ group [(0.056 ± 0.008), t = 4.21, P <0.05]. The expression of cyclin D1 protein in Ad-PTEN infected group (0.256 ± 0.072) was significantly lower than that in control group [(0.502 ± 0.087, t = 3.77, P <0.05) 3.87, P <0.05)]. The expression of p21 protein in Ad-PTEN infected group (0.589 ± 0.076) was significantly higher than that in control group [(0.146 ± 0.026, t = 9.55, P <0.01)] and Ad-LacZ group [(0.163 ± 0.024, , P <0.01)]. MTT assay showed that the inhibitory effect of Ad-PTEN on PC-3 cells after 48h (21.98%) was significant (t = 6.80, P <0.01). The clone formation rate in Ad-PTEN group [(37.4 ± 4.18)%] was significantly lower than that in control group [(54.9 ± 4.81)%, t = 4.76, P <0.01] and [56.5 ± 5.42 )%, t = 4.83, P <0.01]. CONCLUSION: The expression of PTEN gene in PC-3 cells by adenovirus vector Ad-PTEN can significantly inhibit cell proliferation, decrease cyclin D1 expression and increase p21 expression.