顺铂对FOLR1基因表达上调的卵巢上皮性癌细胞生物学功能的影响
순박대FOLR1기인표체상조적란소상피성암세포생물학공능적영향
Biological effects of upregulated expression of transfected FOLR1 gene on SKOV3 cell lines acted by cisplatin
  • 万方数据
    中华妇产科杂志 중화부산과잡지
    CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
    2013年 9期 676-682 ,共7页

    黄明钜%张玮%王琪%蔡祖艾%李力

    황명거%장위%왕기%채조애%리력

    卵巢肿瘤%叶酸受体1%顺铂%细胞周期%细胞凋亡 卵巢腫瘤%葉痠受體1%順鉑%細胞週期%細胞凋亡
    란소종류%협산수체1%순박%세포주기%세포조망
    Ovarian neoplasms%Folate receptor 1%Cisplatin%Cell cycle%Apoptosis
    目的 探讨顺铂作用于叶酸结合蛋白(FOLRl)基因表达上调的卵巢上皮性癌(卵巢癌)细胞系SKOV3细胞后对其生物学功能的影响.方法 将FOLR1基因与pWPI质粒进行慢病毒包装,构建重组质粒pWPI-FOLR1并转染入SKOV3后得到pWPI-FOLR1-SKOV3细胞,作为重组质粒转染组；以同样方法将pWPI质粒转染入SKOV3后得到pWPI-SKOV3细胞,作为空载体转染组；并设立未转染的SKOV3细胞为空白对照组,采用逆转录(RT)-PCR技术和蛋白印迹法分别检测3组细胞中FOLR1 mRNA和蛋白的表达.采用四甲基偶氮唑蓝(MTT)比色法,检测并绘制3组细胞的生长曲线,检测3组细胞对顺铂的敏感性[以50％抑制浓度(IC50)表示,选择重组质粒转染组的IC50作为下一步实验中顺铂浓度的基准],并检测不同浓度(分别为0.5×IC50、1×IC50、2×IC50,即分别为1.8、3.6、7.2 μg/ml；下同)顺铂作用不同时间(分别为24、48、72 h,下同)后3组细胞生长的抑制率；采用流式细胞仪检测不同浓度顺铂作用不同时间后3组细胞的凋亡率,及不同浓度顺铂作用48 h后3组细胞的细胞周期比例；高效液相色谱法检测同一浓度(3.6 μg/ml)顺铂作用48 h后3组细胞内顺铂的浓度.结果 RT-PCR技术和蛋白印迹法分别检测显示,重组质粒转染组细胞中有FOLR1 mRNA和蛋白的表达,而空载体转染组、空白对照组细胞中无FOLR1 mRNA和蛋白的表达.MTT比色法检测显示,重组质粒转染组细胞生长速度最快、对顺铂最敏感(其IC50最低,为3.6 μg/ml)、相同作用条件(包括顺铂的浓度和作用时间)下其细胞生长的抑制率最高,分别与空载体转染组、空白对照组比较,差异均有统计学意义(P＜0.05)；而空载体转染组与空白对照组比较,差异无统计学意义(P＞0.05).流式细胞仪检测显示,3组细胞的凋亡率随着顺铂浓度(分别为1.8、3.6、7.2 μg/ml)的升高及作用时间(分别为24、48及72 h)的延长而升高,呈明显的剂量-时间依赖关系(P＜0.05),S期细胞比例随着顺铂浓度的升高而升高,呈明显的剂量依赖关系(P＜0.05)；在相同的作用条件下,重组质粒转染组细胞的凋亡率、S期细胞比例分别与空载体转染组、空白对照组比较,差异均有统计学意义(P＜0.05),而空载体转染组与空白对照组比较,差异无统计学意义(P＞0.05).高效液相色谱法检测显示,同一浓度(3.6μg/ml)的顺铂作用48 h后,重组质粒转染组细胞内顺铂浓度为(2.60±0.21) μg/106个细胞,高于空载体转染组的(1.49 ±0.12) μg/106个细胞和空白对照组的(1.54±0.11) μg/106个细胞,差异均有统计学意义(P＜0.05)；而空载体转染组与空白对照组比较,差异无统计学意义(P＞0.05).结论 卵巢癌SKOV3细胞中FOLR1基因表达上调后,SKOV3细胞的生物学功能发生改变,表现为对顺铂的敏感性增加、细胞内顺铂浓度升高、细胞周期中S期比例增加、抑制细胞增殖、诱导细胞凋亡.
    목적 탐토순박작용우협산결합단백(FOLRl)기인표체상조적란소상피성암(란소암)세포계SKOV3세포후대기생물학공능적영향.방법 장FOLR1기인여pWPI질립진행만병독포장,구건중조질립pWPI-FOLR1병전염입SKOV3후득도pWPI-FOLR1-SKOV3세포,작위중조질립전염조；이동양방법장pWPI질립전염입SKOV3후득도pWPI-SKOV3세포,작위공재체전염조；병설립미전염적SKOV3세포위공백대조조,채용역전록(RT)-PCR기술화단백인적법분별검측3조세포중FOLR1 mRNA화단백적표체.채용사갑기우담서람(MTT)비색법,검측병회제3조세포적생장곡선,검측3조세포대순박적민감성[이50％억제농도(IC50)표시,선택중조질립전염조적IC50작위하일보실험중순박농도적기준],병검측불동농도(분별위0.5×IC50、1×IC50、2×IC50,즉분별위1.8、3.6、7.2 μg/ml；하동)순박작용불동시간(분별위24、48、72 h,하동)후3조세포생장적억제솔；채용류식세포의검측불동농도순박작용불동시간후3조세포적조망솔,급불동농도순박작용48 h후3조세포적세포주기비례；고효액상색보법검측동일농도(3.6 μg/ml)순박작용48 h후3조세포내순박적농도.결과 RT-PCR기술화단백인적법분별검측현시,중조질립전염조세포중유FOLR1 mRNA화단백적표체,이공재체전염조、공백대조조세포중무FOLR1 mRNA화단백적표체.MTT비색법검측현시,중조질립전염조세포생장속도최쾌、대순박최민감(기IC50최저,위3.6 μg/ml)、상동작용조건(포괄순박적농도화작용시간)하기세포생장적억제솔최고,분별여공재체전염조、공백대조조비교,차이균유통계학의의(P＜0.05)；이공재체전염조여공백대조조비교,차이무통계학의의(P＞0.05).류식세포의검측현시,3조세포적조망솔수착순박농도(분별위1.8、3.6、7.2 μg/ml)적승고급작용시간(분별위24、48급72 h)적연장이승고,정명현적제량-시간의뢰관계(P＜0.05),S기세포비례수착순박농도적승고이승고,정명현적제량의뢰관계(P＜0.05)；재상동적작용조건하,중조질립전염조세포적조망솔、S기세포비례분별여공재체전염조、공백대조조비교,차이균유통계학의의(P＜0.05),이공재체전염조여공백대조조비교,차이무통계학의의(P＞0.05).고효액상색보법검측현시,동일농도(3.6μg/ml)적순박작용48 h후,중조질립전염조세포내순박농도위(2.60±0.21) μg/106개세포,고우공재체전염조적(1.49 ±0.12) μg/106개세포화공백대조조적(1.54±0.11) μg/106개세포,차이균유통계학의의(P＜0.05)；이공재체전염조여공백대조조비교,차이무통계학의의(P＞0.05).결론 란소암SKOV3세포중FOLR1기인표체상조후,SKOV3세포적생물학공능발생개변,표현위대순박적민감성증가、세포내순박농도승고、세포주기중S기비례증가、억제세포증식、유도세포조망.
    Objective To explore biological effects of up-regulated expression of transfected FOLR1 gene on SKOV3 cell lines following action by cisplatin(DDP).Methods Three groups of cells originated from the same SKOV3 cell line were used in this research,including the SKOV3 cell line (blank control),the cell line transfected with lentiviral pWPI plasmid (no-load control) and the cell line transfected with FOLR1 gene via lentiviral pWPI plasmid (experimental group).Next,the mRNA and protein expression of FOLR1 gene in the three groups were detected by reverse transcription (RT)-PCR and western blot,respectively.Methyl thiazolyl tetrazolium (MTT) assay was used to analysis cells growth curve and identify their sensitivity to cisplatin,and their half inhibition concentration (IC5o) values were calculated.Based on the IC50 value (3.6 μg/ml) in the experimental group,different levels of cisplatin concentration (0.5 × IC50,1 × IC50,2 × IC50,respectively) were administered to the three groups of cells,and the inhibition rates,apoptosis rates as well as apoptosis proportion of each group after 24,48,72 hours were further recorded.Finally,the residual cisplatin concentrations in the three group cells acted successively by 1 × IC50 cisplatin for 48 hours were measured by high performance liquid chromatography(HPLC).P value less than 0.05 were defined as statistically significant.Results RT-PCR and western blot detection showed that stable mRNA and protein expression of the FOLR1 gene in the experimental group while the other two groups were not.MTT assay demonstrated that higher cell growth rate,sensitivity to cisplatin(IC50 =3.6 μg/ml) and inhibition rate in the experimental group compared with those in the other two groups (P ＜ 0.05),which showed no significance in intergroup comparison(P ＞ 0.05).Flow cytometry showed apoptosis rates among three groups increased with higher cisplatin concentrations and longer action duration in dosage-time dependent manner (P ＜ 0.05),and the proportion of S phase cells increased with higher cisplatin concentration in dosage-dependent manner (P ＜ 0.05) ; for the same concentration and duration,the experimental group showed significantly different apoptosis rates and S phase cells compared with the other two groups,which demonstrated no significance in intergroup comparison (P ＞ 0.05).After action by cisplatin(3.6 μg/ml) for 48 hours,HPLC showed significantly higher residual cisplatin concentration (2.60±0.21) μg/106 cell counts in experimental group than those in no-load control group (1.49 ±0.12) μg/106 cell counts and blank control group (1.54 ± 0.11)xg/106 cell counts,respectively (P ＜0.05),and the comparison within the latter two groups showed no significance (P ＞ 0.05).Conclusion Up-regulated expression of the transfected FOLR1 gene in SKOV3 cells may be associated with higher sensitivity to cisplatin,residual cisplatin concentration and higher proportion of S phase cells,and tended to inhibit cancer cells growth and induce apoptosis.
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