广东医学
廣東醫學
엄동의학
GUNAGDONG MEDICAL JOURNAL
2015年
1期
30-34
,共5页
王绮雯%刘剑%叶钿均%张继民
王綺雯%劉劍%葉鈿均%張繼民
왕기문%류검%협전균%장계민
胸苷磷酸化酶%基因转染%慢病毒%结肠癌细胞株
胸苷燐痠化酶%基因轉染%慢病毒%結腸癌細胞株
흉감린산화매%기인전염%만병독%결장암세포주
thymidine phosphorylase%gene transfection%lentiviral vector%SW480%LOVO%HT29%LS174T
目的:构建表达高度胸苷磷酸化酶(TP)活性并可以稳定传代的结肠癌细胞株。方法合成包含TP cDNA 全长的真核细胞表达载体,用慢病毒包装后转染人结肠癌细胞株 SW480、LOVO、HT29和 LS174T,流式细胞仪检测转染效率,实时荧光定量 RT -PCR 方法检测 TP 的 mRNA 表达,蛋白印迹法(Western blot)检测 TP 蛋白表达。结果转染 TP cDNA 后,SW480、LOVO、HT29与 LS174T 在传代5代后转染效率仍稳定在95%左右。与未转染 TP 基因的亲代细胞相比,SW480-TP、LOVO -TP、HT29-TP 与 LS174T -TP 的 TP mRNA 的表达分别提高了(694.56±171.53)、(282.46±86.85)、(8.45±0.15)和(2615.02±253.97)倍,差异有统计学意义(P <0.05);并且 TP 蛋白的表达水平也显著提高。结论慢病毒载体能够高效地将 TP cDNA 转染至人结肠癌细胞 SW480、LO-VO、HT29和 LS174T 中,所得克隆能够稳定传代,并能显著提高4株结肠癌细胞 TP 的 mRNA 和蛋白表达水平。
目的:構建錶達高度胸苷燐痠化酶(TP)活性併可以穩定傳代的結腸癌細胞株。方法閤成包含TP cDNA 全長的真覈細胞錶達載體,用慢病毒包裝後轉染人結腸癌細胞株 SW480、LOVO、HT29和 LS174T,流式細胞儀檢測轉染效率,實時熒光定量 RT -PCR 方法檢測 TP 的 mRNA 錶達,蛋白印跡法(Western blot)檢測 TP 蛋白錶達。結果轉染 TP cDNA 後,SW480、LOVO、HT29與 LS174T 在傳代5代後轉染效率仍穩定在95%左右。與未轉染 TP 基因的親代細胞相比,SW480-TP、LOVO -TP、HT29-TP 與 LS174T -TP 的 TP mRNA 的錶達分彆提高瞭(694.56±171.53)、(282.46±86.85)、(8.45±0.15)和(2615.02±253.97)倍,差異有統計學意義(P <0.05);併且 TP 蛋白的錶達水平也顯著提高。結論慢病毒載體能夠高效地將 TP cDNA 轉染至人結腸癌細胞 SW480、LO-VO、HT29和 LS174T 中,所得剋隆能夠穩定傳代,併能顯著提高4株結腸癌細胞 TP 的 mRNA 和蛋白錶達水平。
목적:구건표체고도흉감린산화매(TP)활성병가이은정전대적결장암세포주。방법합성포함TP cDNA 전장적진핵세포표체재체,용만병독포장후전염인결장암세포주 SW480、LOVO、HT29화 LS174T,류식세포의검측전염효솔,실시형광정량 RT -PCR 방법검측 TP 적 mRNA 표체,단백인적법(Western blot)검측 TP 단백표체。결과전염 TP cDNA 후,SW480、LOVO、HT29여 LS174T 재전대5대후전염효솔잉은정재95%좌우。여미전염 TP 기인적친대세포상비,SW480-TP、LOVO -TP、HT29-TP 여 LS174T -TP 적 TP mRNA 적표체분별제고료(694.56±171.53)、(282.46±86.85)、(8.45±0.15)화(2615.02±253.97)배,차이유통계학의의(P <0.05);병차 TP 단백적표체수평야현저제고。결론만병독재체능구고효지장 TP cDNA 전염지인결장암세포 SW480、LO-VO、HT29화 LS174T 중,소득극륭능구은정전대,병능현저제고4주결장암세포 TP 적 mRNA 화단백표체수평。
Objective To build the human colorectal cancer cell lines with the high expression of thymidine phos -phorylase (TP) by transfected TP cDNA with lentiviral vector .Methods Human colorectal cancer cell lines SW480, LOVO, HT29 and LS174T were transfected with human lentiviral vector TP cDNA by pLenti 6.3_MCS_IRES2 -EGFP. The transfection efficiency was analyzed by flow cytometer , the mRNA expression of TP was detected by RT -PCR and the TP level was detected by Western blot .Results The stabilized transfection efficiency was about 95% past 5 generations. Comparing with parental SW480, LOVO, HT29 and LS174T, the mRNA expression of SW480 -TP, LOVO -TP, HT29 -TP and LS174T -TP were significantly up -regulated by (694.56 ±171.53), (282.46 ±86.85), (8.45 ±0.15) and (2 615.02 ±253.97) folds, respectively (P <0.05).The Western blot showed that the TP levels of SW 480 -TP, LOVO -TP, HT29 -TP and LS174T -TP were obvious up -regulated.Conclusion Stabilized transfections of 4 cell lines with a high TP expression is successfully constructed by lentiviral vector , with significantly up -regulation of TP in mRNA and protein levels.