中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
10期
2155-2157
,共3页
吴潇%林泽宇%陈治平%翁杰%杨青壮%万云乐%陈涛%贾长库
吳瀟%林澤宇%陳治平%翁傑%楊青壯%萬雲樂%陳濤%賈長庫
오소%림택우%진치평%옹걸%양청장%만운악%진도%가장고
肝纤维化%细胞增殖抑制基因%瞬时转染%细胞周期%细胞凋亡
肝纖維化%細胞增殖抑製基因%瞬時轉染%細胞週期%細胞凋亡
간섬유화%세포증식억제기인%순시전염%세포주기%세포조망
Hepatic fibrosis%HSG%Transient transfection%Cell cycle%Cell apoptosis
目的 观察细胞增殖抑制基因(HSG)对大鼠肝星状细胞系HSC-T6增殖与凋亡的影响.方法 制备携带HSG基因的重组腺病毒载体,瞬时转染至大鼠肝星状细胞HSC-T6,逆转录-聚合酶链反应(RT-PCR)和Western blot分别检测细胞中HSG信使核糖核酸(mRNA)及蛋白的表达水平,细胞计数试剂盒(CCK-8)法检测细胞增殖活性,流式细胞术分析HSG对细胞周期和凋亡状态的影响,Western blot检测HSG对细胞周期蛋白激酶抑制剂p21、p27及细胞增殖核抗原(PCNA)的作用.结果 与空白对照组和阴性对照组比较,转染组HSG的mRNA(14.93 ±1.10比1.03±0.33,14.93±1.10比1.00 ±0.15)和蛋白表达量明显增加(P＜0.05);转染后细胞生长减缓,细胞增殖活性明显受到抑制(P＜0.05);转染组G0/G1期细胞比例和凋亡率分别为[(66.57±1.29)％、(23.88 ±0.96)％],与空白对照组[(49.32±2.86)％、(3.38±0.16)％]和阴性对照组[(52.46±3.17)％、(3.54±0.24)％]比较明显增高(P＜0.05);转染后细胞周期调控蛋白p21、p27表达升高,PCNA表达降低,差异均有统计学意义(P＜0.05).结论 HSG基因过表达可有效的抑制大鼠肝星状细胞系HSC-T6的增殖,使其阻滞于G0/G1期并诱导其凋亡,并可能是通过参与细胞周期的调节发挥作用.
目的 觀察細胞增殖抑製基因(HSG)對大鼠肝星狀細胞繫HSC-T6增殖與凋亡的影響.方法 製備攜帶HSG基因的重組腺病毒載體,瞬時轉染至大鼠肝星狀細胞HSC-T6,逆轉錄-聚閤酶鏈反應(RT-PCR)和Western blot分彆檢測細胞中HSG信使覈糖覈痠(mRNA)及蛋白的錶達水平,細胞計數試劑盒(CCK-8)法檢測細胞增殖活性,流式細胞術分析HSG對細胞週期和凋亡狀態的影響,Western blot檢測HSG對細胞週期蛋白激酶抑製劑p21、p27及細胞增殖覈抗原(PCNA)的作用.結果 與空白對照組和陰性對照組比較,轉染組HSG的mRNA(14.93 ±1.10比1.03±0.33,14.93±1.10比1.00 ±0.15)和蛋白錶達量明顯增加(P＜0.05);轉染後細胞生長減緩,細胞增殖活性明顯受到抑製(P＜0.05);轉染組G0/G1期細胞比例和凋亡率分彆為[(66.57±1.29)％、(23.88 ±0.96)％],與空白對照組[(49.32±2.86)％、(3.38±0.16)％]和陰性對照組[(52.46±3.17)％、(3.54±0.24)％]比較明顯增高(P＜0.05);轉染後細胞週期調控蛋白p21、p27錶達升高,PCNA錶達降低,差異均有統計學意義(P＜0.05).結論 HSG基因過錶達可有效的抑製大鼠肝星狀細胞繫HSC-T6的增殖,使其阻滯于G0/G1期併誘導其凋亡,併可能是通過參與細胞週期的調節髮揮作用.
목적 관찰세포증식억제기인(HSG)대대서간성상세포계HSC-T6증식여조망적영향.방법 제비휴대HSG기인적중조선병독재체,순시전염지대서간성상세포HSC-T6,역전록-취합매련반응(RT-PCR)화Western blot분별검측세포중HSG신사핵당핵산(mRNA)급단백적표체수평,세포계수시제합(CCK-8)법검측세포증식활성,류식세포술분석HSG대세포주기화조망상태적영향,Western blot검측HSG대세포주기단백격매억제제p21、p27급세포증식핵항원(PCNA)적작용.결과 여공백대조조화음성대조조비교,전염조HSG적mRNA(14.93 ±1.10비1.03±0.33,14.93±1.10비1.00 ±0.15)화단백표체량명현증가(P＜0.05);전염후세포생장감완,세포증식활성명현수도억제(P＜0.05);전염조G0/G1기세포비례화조망솔분별위[(66.57±1.29)％、(23.88 ±0.96)％],여공백대조조[(49.32±2.86)％、(3.38±0.16)％]화음성대조조[(52.46±3.17)％、(3.54±0.24)％]비교명현증고(P＜0.05);전염후세포주기조공단백p21、p27표체승고,PCNA표체강저,차이균유통계학의의(P＜0.05).결론 HSG기인과표체가유효적억제대서간성상세포계HSC-T6적증식,사기조체우G0/G1기병유도기조망,병가능시통과삼여세포주기적조절발휘작용.
Objective To discuss the effects of HSG gene on proliferation and apoptosis of rat hepatic stellate cell line HSC-T6.Methods To construct the recombinant retroviral vector carrying rat HSG gene,transient transfection into rat hepatic stellate cell line HSC-T6,real-time quantitative polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA expression and protein levels of HSG respectively.Cell counting kit-8 (CCK-8) assay was used to investigate cell proliferation activity.Flow cytometry was used to measure the cell cycle progression and cell apoptosis.Effect of HSG on HSC-T6 cells proliferation were investigated by Western blotting for the expression of cyclin dependent kinase inhibitors p21,p27 and proliferating cell nuclear antigen (PCNA).Results Compared with blank control group and negative control group,the protein and mRNA expression (14.93 ± 1.10 vs.1.03 ± 0.33,14.93 ± 1.10 vs.1.00 ± 0.15) of HSG was significantly increased in transfection group (all P ＜ 0.05).Meahwhile,the cell proliferation activity remarkably decreased in transfection group (both P ＜ 0.05).Furthermore,cell cycle arrested in the G0/G1 phase with the increasing apoptosis rate [(23.88 ± 0.96) ％ vs.(3.38±0.16)％,(23.88±0.96)％ vs.(3.54 ±0.24)％] as well as the expression of p21 and p27,and the reducing expression of PCNA (all P ＜ 0.05).Conclusion Overexpression of HSG could inhibit the proliferation of rat hepatic stellate cell line HSC-T6,the cell cycle arrest in the G0/G1 phase and induce its apoptosis.HSG may play a role by participating in the regulation of cell cycle.
