中华普通外科杂志
中華普通外科雜誌
중화보통외과잡지
CHINESE JOURNAL OF GENERAL SURGERY
2015年
5期
386-390
,共5页
刘奇%王绮雯%张继民%方耿
劉奇%王綺雯%張繼民%方耿
류기%왕기문%장계민%방경
结肠肿瘤%胸苷磷酸化酶%干扰素α%氟尿苷
結腸腫瘤%胸苷燐痠化酶%榦擾素α%氟尿苷
결장종류%흉감린산화매%간우소α%불뇨감
Colonic neoplasms%Thymidine phosphorylase%Interferon-alpha%Fluorouridine
目的 利用慢病毒载体将胸苷磷酸化酶(TP)基因转染人结肠癌细胞株LOVO,然后应用干扰素-α2a(INF-α2a)处理转染前后细胞,分别测定5'-脱氧氟尿苷(5'-DFUR)和5-氟尿嘧啶(5-FU)对LOVO抗肿瘤效应的影响.方法 构建包含TP基因的慢病毒载体,转染结肠癌细胞株LOVO,以流式细胞技术和免疫荧光法检测在第5代LOVO中的转染效率;加INF-α2a培养24h后分别应用RT-PCR和Wester blot检测细胞的TP mRNA和TP蛋白表达;MTS法检测5'-DFUR和5-FU对各组细胞的半数抑制浓度(IC50);高效液相色谱法检测各组细胞转化5'-DFUR为5-FU的量.结果 转染TP基因的效率达95％.LOVO+INF-α2a组的TP mRNA表达是LOVO组的1.87倍,而LOVO-TP组和LOVO-TP+ INF-α2a组TPmRNA表达则比LOVO组分别增加了18.56倍和59.61倍(P＜0.01).LOVO-TP+ INF-α2a组和LOVO-TP组蛋白表达水平比LOVO组分别增加242.23倍和197.69倍(P＜0.01).5'-DFUR对LOVO-TP组细胞的IC50值仅为LOVO组细胞的37.2％,而LOVO-TP+INF-α2a组的IC50则是LOVO组的18.2％.LOVO-TP组在加入100、200、400 μmol/L5'-DFUR的培养基中检测的5-FU浓度比LOVO组分别增高了26.13％、33.23％、32.95％,而LOVO-TP+ INF-α2a组比LOVO组分别增高了49.94％、61.66％、72.14％. 结论 转染TP eDNA能够明显提高LOVO细胞的TP mRNA及TP蛋白的表达水平,而INF-α2a则进一步增强这种上调作用.增高的TP表达使细胞外5'-DFUR转化为5-FU含量增加,明显提高5'-DFUR的细胞毒性.
目的 利用慢病毒載體將胸苷燐痠化酶(TP)基因轉染人結腸癌細胞株LOVO,然後應用榦擾素-α2a(INF-α2a)處理轉染前後細胞,分彆測定5'-脫氧氟尿苷(5'-DFUR)和5-氟尿嘧啶(5-FU)對LOVO抗腫瘤效應的影響.方法 構建包含TP基因的慢病毒載體,轉染結腸癌細胞株LOVO,以流式細胞技術和免疫熒光法檢測在第5代LOVO中的轉染效率;加INF-α2a培養24h後分彆應用RT-PCR和Wester blot檢測細胞的TP mRNA和TP蛋白錶達;MTS法檢測5'-DFUR和5-FU對各組細胞的半數抑製濃度(IC50);高效液相色譜法檢測各組細胞轉化5'-DFUR為5-FU的量.結果 轉染TP基因的效率達95％.LOVO+INF-α2a組的TP mRNA錶達是LOVO組的1.87倍,而LOVO-TP組和LOVO-TP+ INF-α2a組TPmRNA錶達則比LOVO組分彆增加瞭18.56倍和59.61倍(P＜0.01).LOVO-TP+ INF-α2a組和LOVO-TP組蛋白錶達水平比LOVO組分彆增加242.23倍和197.69倍(P＜0.01).5'-DFUR對LOVO-TP組細胞的IC50值僅為LOVO組細胞的37.2％,而LOVO-TP+INF-α2a組的IC50則是LOVO組的18.2％.LOVO-TP組在加入100、200、400 μmol/L5'-DFUR的培養基中檢測的5-FU濃度比LOVO組分彆增高瞭26.13％、33.23％、32.95％,而LOVO-TP+ INF-α2a組比LOVO組分彆增高瞭49.94％、61.66％、72.14％. 結論 轉染TP eDNA能夠明顯提高LOVO細胞的TP mRNA及TP蛋白的錶達水平,而INF-α2a則進一步增彊這種上調作用.增高的TP錶達使細胞外5'-DFUR轉化為5-FU含量增加,明顯提高5'-DFUR的細胞毒性.
목적 이용만병독재체장흉감린산화매(TP)기인전염인결장암세포주LOVO,연후응용간우소-α2a(INF-α2a)처리전염전후세포,분별측정5'-탈양불뇨감(5'-DFUR)화5-불뇨밀정(5-FU)대LOVO항종류효응적영향.방법 구건포함TP기인적만병독재체,전염결장암세포주LOVO,이류식세포기술화면역형광법검측재제5대LOVO중적전염효솔;가INF-α2a배양24h후분별응용RT-PCR화Wester blot검측세포적TP mRNA화TP단백표체;MTS법검측5'-DFUR화5-FU대각조세포적반수억제농도(IC50);고효액상색보법검측각조세포전화5'-DFUR위5-FU적량.결과 전염TP기인적효솔체95％.LOVO+INF-α2a조적TP mRNA표체시LOVO조적1.87배,이LOVO-TP조화LOVO-TP+ INF-α2a조TPmRNA표체칙비LOVO조분별증가료18.56배화59.61배(P＜0.01).LOVO-TP+ INF-α2a조화LOVO-TP조단백표체수평비LOVO조분별증가242.23배화197.69배(P＜0.01).5'-DFUR대LOVO-TP조세포적IC50치부위LOVO조세포적37.2％,이LOVO-TP+INF-α2a조적IC50칙시LOVO조적18.2％.LOVO-TP조재가입100、200、400 μmol/L5'-DFUR적배양기중검측적5-FU농도비LOVO조분별증고료26.13％、33.23％、32.95％,이LOVO-TP+ INF-α2a조비LOVO조분별증고료49.94％、61.66％、72.14％. 결론 전염TP eDNA능구명현제고LOVO세포적TP mRNA급TP단백적표체수평,이INF-α2a칙진일보증강저충상조작용.증고적TP표체사세포외5'-DFUR전화위5-FU함량증가,명현제고5'-DFUR적세포독성.
Objective Thymidine phosphorylase (TP) cDNA was transfected into colorectal cancer cell lines LOVO with the lentiviral vector,the anticancer effeciency of 5'-deoxy-5-fluorouridine (5'-DFUR)and 5-fluorouracil (5-FU) on LOVO cells were evaluated.Methods TP cDNA were transfected into LOVO cell line with the lentiviral vector pLenti6.3_MCS_IRES2-EGFP,and the transfection efficiency was analyzed by flow cytometer and immunohistochemistry.Cells were divided into six groups:LOVO,LOVO-TP,LOVO-control,LOVO + INF-α2a,LOVO-TP + INF-α2a,LOVO-control + INF-α2a.TP protein expression and the relative quantitative analysis for TP mRNA in transfections cells were detected by Western blot and RT-PCR respectively.Volumes of converted 5-FU from either in the medium containing different concentration of 5'-DFUR,in which all cells were cultured,or in cells lysate,were detected by high performance liquid chromatography (HPLC).Results The transfection efficiency of TP cDNA in LOVO cells was 95％.The Mean gray value of TP protein expression in LOVO-TP and LOVO-TP + INF-α2a were 198.15 folds and 243.22 folds higher than LOVO cell,respectively (P ＜ 0.01).The RQ values of TP mRNA expression in LOVO-TP and LOVO-TP + INF-α2a were also 18.56 folds and 59.61 folds higher than wild LOVO cell,respectively (P ＜ 0.01).The IC50 values of 5'-DFUR on LOVO-TP and LOVO-TP + INF-α2a were 1 660 μ mol/L and 813 μ mol/L,respectively,significantly lower than 4 462.59 μ mol/L in wild LOVO (P ＜ O.01).The 5-FU volumes detected from media contained series concentration of 5'-DFUR for culturing LOVO-TP and LOVO-TP + INF-α2a were 2.0-5.3 folds,and 2.9-10.4 folds more than wild LOVO,respectively (P ＜ 0.01).Conclusions Transfected TP cDNA into colorectal cancer cell line LOVO with lentiviral vector increases the expression of TP mRNA and TP protein and the amount of 5-FU converted from 5'-DFUR,enhancing its anticancer effect.
