CAJ | 학술논문

目的研究抑癌基因WWOX对卵巢上皮性癌(卵巢癌)细胞生长的影响,寻求卵巢癌基因治疗的新途径.方法将携有WWOX基因的真核表达载体体外转染卵巢癌细胞系HO8910细胞(重组质粒组),筛选稳定转染的细胞并扩增培养,以转染空载质粒(空质粒组)及未经转染(空白对照组)的HO8910细胞作为对照,用蛋白印迹法检测重组质粒组细胞中WWOX蛋白的表达情况.体外实验:分别用四甲基偶氮唑蓝(MTT)比色法、体外侵袭实验、琼脂克隆形成实验及流式细胞仪等分析WWOX基因对HO8910细胞生物学活性的影响.体内实验:将转染细胞接种于BALB/c裸鼠腹腔,观察裸鼠生存时间和肿瘤生长情况.结果 (1)重组质粒组细胞中WWOX蛋白能稳定表达,空质粒组及空白对照组细胞未检测到WWOX蛋白的表达.(2)MTT比色法检测显示,重组质粒组各时间点的细胞增殖能力较空质粒组及空白对照组明显下降,差异均有统计学意义(P＜0.05).(3)重组质粒组细胞的琼脂克隆形成率(19.8%)明显低于空质粒组(54.5%)及空白对照组(56.0%),差异有统计学意义(P＜0.05).(4)流式细胞仪检测显示,重组质粒组中(72.08±0.39)%的细胞被阻滞于G0/G1期,分别与空质粒组[(41.02±1.08)%]及空白对照组[(39.31±0.67)%]比较,差异均有统计学意义(P＜0.05).(5)体外侵袭实验显示,重组质粒组穿膜细胞数为(89.7±3.1)个,分别与空质粒组[(91.2±1.3)个]及空白对照组[(91.4±1.3)个]比较,差异均无统计学意义(P＞0.05).(6)裸鼠体内实验表明,重组质粒组裸鼠体内的成瘤能力(包括转移灶数目、转移灶重量及移植瘤最大直径、腹水量)明显低于空质粒组及空白对照组,差异均有统计学意义(P＜0.05).结论抑癌基因WWOX可干扰卵巢癌细胞的细胞周期并抑制其生长增殖,可为卵巢癌的基因治疗提供一种新方法.
목적 연구억암기인WWOX대란소상피성암(란소암)세포생장적영향,심구란소암기인치료적신도경.방법 장휴유WWOX기인적진핵표체재체체외전염란소암세포계HO8910세포(중조질립조),사선은정전염적세포병확증배양,이전염공재질립(공질립조)급미경전염(공백대조조)적HO8910세포작위대조,용단백인적법검측중조질립조세포중WWOX단백적표체정황.체외실험:분별용사갑기우담서람(MTT)비색법、체외침습실험、경지극륭형성실험급류식세포의등분석WWOX기인대HO8910세포생물학활성적영향.체내실험:장전염세포접충우BALB/c라서복강,관찰라서생존시간화종류생장정황.결과 (1)중조질립조세포중WWOX단백능은정표체,공질립조급공백대조조세포미검측도WWOX단백적표체.(2)MTT비색법검측현시,중조질립조각시간점적세포증식능력교공질립조급공백대조조명현하강,차이균유통계학의의(P＜0.05).(3)중조질립조세포적경지극륭형성솔(19.8%)명현저우공질립조(54.5%)급공백대조조(56.0%),차이유통계학의의(P＜0.05).(4)류식세포의검측현시,중조질립조중(72.08±0.39)%적세포피조체우G0/G1기,분별여공질립조[(41.02±1.08)%]급공백대조조[(39.31±0.67)%]비교,차이균유통계학의의(P＜0.05).(5)체외침습실험현시,중조질립조천막세포수위(89.7±3.1)개,분별여공질립조[(91.2±1.3)개]급공백대조조[(91.4±1.3)개]비교,차이균무통계학의의(P＞0.05).(6)라서체내실험표명,중조질립조라서체내적성류능력(포괄전이조수목、전이조중량급이식류최대직경、복수량)명현저우공질립조급공백대조조,차이균유통계학의의(P＜0.05).결론 억암기인WWOX가간우란소암세포적세포주기병억제기생장증식,가위란소암적기인치료제공일충신방법.
Objective To study the effects of anti-oncogene WWOX on cell growth of epithelial ovarian cancer,in order to find a new approach of gene therapy for ovarian cancer.Methods A eukaryotic expression vector containing WWOX was transfected into ovarian cancer cell line HO8910 in vitro (recombinant plasmid group),and positive cell clones were selected and amplified.Expression of WWOX protein was detected by western blot. Untransfected cell(blank contrast group) and transfected empty plasmid cell(empty plasmid group)were served as control groups.In vitro,the biology effect of WWOX on HO8910 cell was analyzed throush the methyl thiazolyl tetrazolium test,transwell chamber cell invasion assay in vitro,agarose clony-formation and flow cytometry.In vivo,the cell of transfection was transplanted intraperitoneally in to BALB/c nude mice.The survival time and growth ability of nude mice were observed.Results (1)Recombinant plasmid group cell could steadily express WWOX protein,while in empty plasmid group and blank control group the expression of WWOX protein were not detected.(2)The growth rate of recombinant plasmid group cell was inhibited.(3)The agnrose clony-formation rate of recombinant plasmid group(19.8%)was significantly lower than that of the empty plasmid group(54.5%)and blank control group(56.0%,P＜0.05).(4)Flow cytometry showed that(72.08±0.39)% of cells was arrested at G0/G1 stage in recombinant plasmid group, while in empty plasmid group and blank control group G0/G1 stage cells were at (41.02±1.08)% and (39.31±0.67)% (P＜0.05). (5) In vitro invasion assay showed that invasion cell number in recombinant plasmid group (89.7±3. 1 ) was not significantly different from that of empty plasmid group(91.2±1.3) and blank control group(91.4±1.3, P ＞0. 05). (6) In vivo test in nude mice showed that WWOX gene could inhibit tumor growth of the HO8910 cells. Conclusions Tumor suppressor gene WWOX could interfere with the cell cycles of ovarian cancer cell and inhibit cell proliferation. As a new valuable tool,it premises to have application in the gene therapy of ovarian cancer.