复旦学报(医学版)
複旦學報(醫學版)
복단학보(의학판)
JOURNAL OF FUDAN UNIVERSITY
2010年
2期
146-152
,共7页
江颖%周信达%刘银坤%黄晓武%孙瑞霞%张春晖%陆维祺%朱隽%罗文杰%张华
江穎%週信達%劉銀坤%黃曉武%孫瑞霞%張春暉%陸維祺%硃雋%囉文傑%張華
강영%주신체%류은곤%황효무%손서하%장춘휘%륙유기%주준%라문걸%장화
T淋巴细胞%肝细胞癌%反义核糖核酸%转染
T淋巴細胞%肝細胞癌%反義覈糖覈痠%轉染
T림파세포%간세포암%반의핵당핵산%전염
T-lymphocytes%hepatocellular carcinoma%antisense RNA%transfection
目的 构建T细胞转录因子(Tcf)反义RNA真核表达载体以阻断异常Wnt信号通路,探讨其对肝癌细胞生物学特性的影响.方法 利用GeneJammer将反义基因转染人肝癌细胞系SMMC-7721,应用RT-PCR及Western blot方法分别检测转染前后细胞mRNA及蛋白表达差异,通过生长曲线、Transwell小室实验比较转染细胞生长增殖及运动侵袭能力,并进一步用流式细胞仪检测细胞凋亡及生长周期的改变.结果 反义RNA转染降低了Tcf表达,减慢了肝癌细胞的生长速度并抑止其运动侵袭能力.转染反义RNA的肝癌细胞7721-pTas凋亡比例增加[(26.34±2.07)%],明显高于空载体转染细胞7721-vector[(6.53±1.02)%]和亲本SMMC-7721细胞[(4.33±0.68)%] (P<0.001).同时,处于G0-G1期的反义转染细胞比相应亲本SMMC-7721细胞和转染空载体的细胞7721-vector分别高20.24%和20.95%,而S期细胞比亲本细胞SMMC-7721和7721-vector细胞分别低11.8%和11.38%.结论 反义Tcf RNA转染可通过诱导细胞凋亡和阻止细胞周期的进程而抑制肝癌细胞的恶性增殖, 提示选择性阻断异常Wnt信号通路有望成为肝癌基因治疗的新途径.
目的 構建T細胞轉錄因子(Tcf)反義RNA真覈錶達載體以阻斷異常Wnt信號通路,探討其對肝癌細胞生物學特性的影響.方法 利用GeneJammer將反義基因轉染人肝癌細胞繫SMMC-7721,應用RT-PCR及Western blot方法分彆檢測轉染前後細胞mRNA及蛋白錶達差異,通過生長麯線、Transwell小室實驗比較轉染細胞生長增殖及運動侵襲能力,併進一步用流式細胞儀檢測細胞凋亡及生長週期的改變.結果 反義RNA轉染降低瞭Tcf錶達,減慢瞭肝癌細胞的生長速度併抑止其運動侵襲能力.轉染反義RNA的肝癌細胞7721-pTas凋亡比例增加[(26.34±2.07)%],明顯高于空載體轉染細胞7721-vector[(6.53±1.02)%]和親本SMMC-7721細胞[(4.33±0.68)%] (P<0.001).同時,處于G0-G1期的反義轉染細胞比相應親本SMMC-7721細胞和轉染空載體的細胞7721-vector分彆高20.24%和20.95%,而S期細胞比親本細胞SMMC-7721和7721-vector細胞分彆低11.8%和11.38%.結論 反義Tcf RNA轉染可通過誘導細胞凋亡和阻止細胞週期的進程而抑製肝癌細胞的噁性增殖, 提示選擇性阻斷異常Wnt信號通路有望成為肝癌基因治療的新途徑.
목적 구건T세포전록인자(Tcf)반의RNA진핵표체재체이조단이상Wnt신호통로,탐토기대간암세포생물학특성적영향.방법 이용GeneJammer장반의기인전염인간암세포계SMMC-7721,응용RT-PCR급Western blot방법분별검측전염전후세포mRNA급단백표체차이,통과생장곡선、Transwell소실실험비교전염세포생장증식급운동침습능력,병진일보용류식세포의검측세포조망급생장주기적개변.결과 반의RNA전염강저료Tcf표체,감만료간암세포적생장속도병억지기운동침습능력.전염반의RNA적간암세포7721-pTas조망비례증가[(26.34±2.07)%],명현고우공재체전염세포7721-vector[(6.53±1.02)%]화친본SMMC-7721세포[(4.33±0.68)%] (P<0.001).동시,처우G0-G1기적반의전염세포비상응친본SMMC-7721세포화전염공재체적세포7721-vector분별고20.24%화20.95%,이S기세포비친본세포SMMC-7721화7721-vector세포분별저11.8%화11.38%.결론 반의Tcf RNA전염가통과유도세포조망화조지세포주기적진정이억제간암세포적악성증식, 제시선택성조단이상Wnt신호통로유망성위간암기인치료적신도경.
Objective To construct the recombinant expression vector encoding antisense Tcf fragment for the blockage of abnormal Wnt pathway, and to investigate its effect on the biological behaviors of human hepatocarcinoma cells. Methods Antisense expression vector was transfected into hepatocarcinoma cells SMMC-7721 with GeneJammer. RT-PCR and Western blot were used to detect Tcf expression. Cell proliferation and motility were compared by growth curves and Transwell plate assay. Cell apoptosis was determined by Annexin V and cell cycle was examined by fluorescent staining. Results The stable transfection of antisense Tcf in SMMC-7721 cells significantly reduced Tcf expression at both mRNA and protein levels. Compared with parental and mock-transfected 7721 (7721-vector) cells, antisense Tcf RNA transfected cells 7721-pTas showed much decreased activities of proliferation, migration and invasion in vitro. Furthermore, the apoptosis rate of 7721-pTas cells [(26.34±2.07)%] was significantly higher than that of 7721-vector cells [(6.53±1.02)%] and parental SMMC-7721 cells [(4.33±0.68)%] (P<0.001). The percentages of G0-G1 phase antisense transfected cells were 20.24% and 20.95%, higher than parental SMMC-7721 and 7721-vector cells, and percentages of S phase antisense transfected cells were 11.8% and 11.38%, lower than parental SMMC-7721 and 7721-vector cells, respectively. Conclusions Antisense RNA suppress the growth ability of liver cancer cells by inducing cell apoptosis and impeding the progress of cell cycle, which suggests that selective blockage of abnormal Wnt signal pathway by antisense Tcf RNA may be a potential new gene therapy for liver cancer.
