慢病毒转染胸苷磷酸化酶基因增强5'-脱氧氟尿苷抗结肠癌细胞活性
만병독전염흉감린산화매기인증강5'-탈양불뇨감항결장암세포활성
Transfection of thymidine phosphorylase cDNA with lentiviral vector enhances the anticancer effect of 5'-deoxy-5-fluorouridine on colorectal cancer cell lines HT29 and LS174T
  • 万方数据
    中华肿瘤杂志 중화종류잡지
    CHINESE JOURNAL OF ONCOLOGY
    2015年 1期 18-24 ,共7页

    叶钿均%王绮雯%张继民%刘奇

    협전균%왕기문%장계민%류기

    结直肠肿瘤%胸苷磷酸化酶%转染%5'-脱氧氟尿苷%氟尿嘧啶 結直腸腫瘤%胸苷燐痠化酶%轉染%5'-脫氧氟尿苷%氟尿嘧啶
    결직장종류%흉감린산화매%전염%5'-탈양불뇨감%불뇨밀정
    Colorectal neoplasms%Thymidine phosphorylase%Transfection%5'-deoxy-5-fluorouridine%Fluorouracil
    目的 探讨5’-脱氧氟尿苷(5’-DFUR)和氟尿嘧啶(5-Fu)对人结肠癌细胞株HT29和LS174T转染胸苷磷酸化酶(TP)基因前后抗癌效应的变化.方法 构建包含TP基因的慢病毒载体,转染结肠癌细胞株HT29和LS174T.传代5代后,以流式细胞术检测转染效率.免疫组织化学染色和Western blot检测转染TP基因前后HT29和LS174T细胞中TP蛋白的表达,逆转录聚合酶链反应(RT-PCR)法检测细胞中TP mRNA的表达水平,四唑氮化合物法检测5’-DFUR和5-Fu对转染TP基因前后细胞株的半数抑制浓度(IC50),高效液相色谱(HPLC)法检测HT29和LS174T在细胞转染TP基因前后转化5'-DFUR为5-Fu的水平.结果 转染TP基因的HT29和LS174T细胞传代5代后,转染效率稳定在约95.0％.转染TP基因的HT29和LS174T细胞中TP表达阳性,而野生型细胞和仅转染病毒载体细胞中TP表达阴性;Westem blot结果显示,HT29和LS174T细胞中TP蛋白的表达明显增强.RT-PCR结果显示,转染TP基因的HT29和LS174T细胞中TP mRNA的相对表达水平分别为8.45 ±0.15和2 615.02±253.97,与野生型细胞比较,差异均有统计学意义(均P＜0.01).5 '-DFUR对转染TP基因的HT29-TP细胞和野生型细胞的IC50分别为(14.33±0.74) μmol/L和(707.66±5.66) μmol/L(P ＜0.05);对转染TP基因的LS174T-TP细胞和野生型细胞的IC50分别为(0.59±0.11)μmol/L和(239.20 ±21.83) μmol/L(P＜0.05).5-Fu对转染TP基因的HT29-TP细胞和野生型细胞的IC5o分别为(5.42±0.75) μmol/L和(14.19 ±0.97) μmol/L(P ＜0.05);对转染TP基因的LS 174T-TP细胞和野生型细胞的IC50分别为(4.41±0.96) μmol/L和(16.42±2.12) μmol/L(P＜0.05).转染TP基因后的HT29和LS174T细胞培养基中,则检出大量转化的5-Fu,较转染前分别增加了12.2 ～23.6倍,差异均有统计学意义(均P＜0.01).在细胞裂解液中也检出有少量5-Fu,但其水平仅相当于培养基中的0.9％ ～4.2％.结论 以慢病毒为载体将TP基因转染至人结肠癌细胞HT29和LS714T,能得到转染效率高和稳定传代的细胞株,并且2株细胞的TP蛋白和TP mRNA的表达水平明显增高.转染后在培养基中转化的5-Fu水平明显增高,使5-Fu和5’-DFUR对HT29和LS714T细胞的抗癌作用明显增强,但5’-DFUR增强效果更加明显.
    목적 탐토5’-탈양불뇨감(5’-DFUR)화불뇨밀정(5-Fu)대인결장암세포주HT29화LS174T전염흉감린산화매(TP)기인전후항암효응적변화.방법 구건포함TP기인적만병독재체,전염결장암세포주HT29화LS174T.전대5대후,이류식세포술검측전염효솔.면역조직화학염색화Western blot검측전염TP기인전후HT29화LS174T세포중TP단백적표체,역전록취합매련반응(RT-PCR)법검측세포중TP mRNA적표체수평,사서담화합물법검측5’-DFUR화5-Fu대전염TP기인전후세포주적반수억제농도(IC50),고효액상색보(HPLC)법검측HT29화LS174T재세포전염TP기인전후전화5'-DFUR위5-Fu적수평.결과 전염TP기인적HT29화LS174T세포전대5대후,전염효솔은정재약95.0％.전염TP기인적HT29화LS174T세포중TP표체양성,이야생형세포화부전염병독재체세포중TP표체음성;Westem blot결과현시,HT29화LS174T세포중TP단백적표체명현증강.RT-PCR결과현시,전염TP기인적HT29화LS174T세포중TP mRNA적상대표체수평분별위8.45 ±0.15화2 615.02±253.97,여야생형세포비교,차이균유통계학의의(균P＜0.01).5 '-DFUR대전염TP기인적HT29-TP세포화야생형세포적IC50분별위(14.33±0.74) μmol/L화(707.66±5.66) μmol/L(P ＜0.05);대전염TP기인적LS174T-TP세포화야생형세포적IC50분별위(0.59±0.11)μmol/L화(239.20 ±21.83) μmol/L(P＜0.05).5-Fu대전염TP기인적HT29-TP세포화야생형세포적IC5o분별위(5.42±0.75) μmol/L화(14.19 ±0.97) μmol/L(P ＜0.05);대전염TP기인적LS 174T-TP세포화야생형세포적IC50분별위(4.41±0.96) μmol/L화(16.42±2.12) μmol/L(P＜0.05).전염TP기인후적HT29화LS174T세포배양기중,칙검출대량전화적5-Fu,교전염전분별증가료12.2 ～23.6배,차이균유통계학의의(균P＜0.01).재세포렬해액중야검출유소량5-Fu,단기수평부상당우배양기중적0.9％ ～4.2％.결론 이만병독위재체장TP기인전염지인결장암세포HT29화LS714T,능득도전염효솔고화은정전대적세포주,병차2주세포적TP단백화TP mRNA적표체수평명현증고.전염후재배양기중전화적5-Fu수평명현증고,사5-Fu화5’-DFUR대HT29화LS714T세포적항암작용명현증강,단5’-DFUR증강효과경가명현.
    Objective To explore the changes of anticancer efficiency of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-Fu) in colorectal cancer cell line HT29 and LS174T cells after transfection of thymidine phosphorylase (TP) cDNA with a lentiviral vector.Methods TP cDNA was transfected into human colorectal cancer cell lines HT29 and LS174T with the lentiviral vector pLenti6.3-MCS-IRES2-EGFP,and the transfection efficiency of the two cell lines passed 5 generations was analyzed by flow cytometry.The expression of TP protein and the relative quantitative expression of TP mRNA in these 2 cell lines were detected by Western blot and RT-PCR,respectively.The 50％ inhibitory concentration (IC50) of 5'-DFUR and 5-Fu in both HT29 and LS174T parent cells and TP-transfected cells were assessed by MTS assay.Finally,the concentration of converted 5-Fu was detected by high performance liquid chromatography (HPLC) either in the medium containing a series of concentrations of 5'-DFUR,in which HT29/HT29-TP or LS174T/LS174T-TP cells were cultured,or in the cell culture lysates.Results The HT29 and LS174T cells transfected with human TP cDNA were monitored for 5 generations,and their transfection efficiency was about 95.0％.Immunohistochemical staining showed that both the parent cells and TP-transfected HT29 and LS174T cells were TP-positive,while vector-transfected cells were TPnegative.Western blotting showed that the TP protein expression in HT29-TP and LS174T-TP cells were significantly increased compared with that in their parents cells.The relative quantity (RQ) values of TP mRNA in HT29-TP and LS174T-TP cells were 8.45 ± 0.15 and 2 615.02 ± 253.97,respectively,which were significantly higher than that in their parents cells (P ＜0.01).The IC50 values of 5'-DFUR on HT29-TP cells and its parents cell were (14.33 ± 0.74) μmol/L and (707.66 ± 5.66) μmol/L,respectively (P＜0.05),while (0.59 ±0.11) μmol/L in LS174T-TP cells and (239.20 ±21.83) μmol/L in its parent cells,respectively (P ＜0.05).The IC50 values of 5-Fu of HT29-TP cells and its parents cells were (5.42 ± 0.75) μmol/L and (14.19 ± 0.97) μmol/L,respectively (P ＜ 0.05),while (4.41 ± 0.96) μmol/L in LS174T-TP cells and (16.42 ± 2.12) μmol/L in its parents cells,respectively (P ＜ 0.05).The HPLC results showed that the 5-Fu concentration detected from media contained a series of concentrations of 5'-DFUR for culturing HT29-TP and LS174T-TP cells were 12.2 to 28.7-folds and 13.1 to 23.6-folds,respectively,higher than that in their parents cells,(P ＜0.01 for all).Otherwise,just a little of 5-Fu was detected in the two TP-transfected cells lysate,about 0.9％ to 4.2％ of 5-Fu detected in the media of the same cultured cells,whereas no 5-Fu was detected in the two parent cell lysates.Conclusions Transfection of TP cDNA into colorectal cancer cell lines HT29 and LS174T with lentiviral vector can improve the expression of both TP mRNA and TP protein levels in passaged cells,enhance the conversion of 5-Fu from 5'-DFUR in the medium,and result in an enhanced anticancer effect on these two cell lines.
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