( ) Journal of Nantong University (Medical Sciences) 05 35() 3 * OVCAR-3 高赛楠 **, 张玉泉 ***, 苏敏, 黄华 (,, 600) [ ] 目的 : β 5 (chorionic gonadotropin beta polpeptide 5, ) OVCAR-3 方法 :, OVCAR-3, OVCAR-3, BeWo RT-PCR 5 ( OVCAR-3,,,,BeWo ), 6, 5 5, Ki67 结果 :() BeWo 00%(6/6), OVCAR-3 83.3%(5/6), 33.3%(/6) () BeWo, 结论 : OVCAR-3, [ ] ;β 5 ; [ ] R737.3 [ ] A [ ] 674-7887(05)0-003-05 Effect of on human epithelial ovarian cancer cell line OVCAR-3 in the tumor formation rates and growth speed in vivo * GAO Sainan **, ZHANG Yuquan ***, SU Min, HUANG Hua ( Department of Obstetrics and Gynecology, Department of Pathology, the Affiliated Hospital of Nantong University, Nantong 600) [Abstract] Objective: To explore the effect of chorionic gonadotropin beta polpeptide 5() on human epithelial ovarian cancer cell line OVCAR-3 in the tumor formation rates and growth speed in vivo. Methods: The overexpressed vector, targeted sirna vector were constructed and transfected into OVCAR - 3 ( empty vector as control). The human choriocarcinoma BeWo cell line with high expressed was used as a positive control. After sorting by flow cytometry, and selected by Blasticidin, the stable vector expression cell lines were obtained. These cells (five groups: untreated OVCAR-3 cells, targeted sirna transfected OVCAR-3 cells, target-off sirna transfected OVCAR-3 cells, overexpressed OVCAR-3 cells and BeWo cells) were injected subcutaneously into the nude mice (6 mice/group). Then the tumor formation rates and growth speed were examined. The expression of Ki67 was detected by the immunohistochemistry. Results: The tumor formation rates of nude mouse xenografts were 00%(6/6) for overexpressed OVCAR-3 and BeWo cells; 83.3%(5/6) for untreated or target-off sirna transfected OVCAR-3 cells group; and 33.3%(/6) for knockdown OVCAR-3 cells. And the speeds of tumor growth were also different, BeWo and overexpressed OVCAR-3 cells were the growth fastest, but the knockdown OVCAR -3 cells were the slowest. Conclusions: may increase the tumor formation rates and growth speed, which may support a new way for ovarian cancer therapy. [Key words] ovarian cancer; chorionic gonadotropin beta polpeptide 5; in vivo DOI:0.644/j.cnki.cn3-807/r.05.0.004 (human chorionic gonadotropin HCG) HCG [] HCG * [ ] (30806) ** [ ],,, 987 0,,, : *** [ ], :370699363,E-mail:jsnt_zhangyuquan@63.com
4 ( ) 05 35() [] HCG A ( 50 μl) B ( 50 μl) 37 5 min ( β-hcg β-hcg 50 μl) 450 nm 5 (chorionic gonadotropin beta polypertide 5 ).4 RT-PCR 检测 mrna 表达变化 RNAiso OVCAR-3 Plus PrimeScript TM RT Reagent Kit RNA cdna β-hcg 5 -GCTACTGCCCCACCATGACC- 3 5 -ATGGACTCGAAGCGCACATC -3. 材料 OVCAR-3 BeWo β-actin 5 -TGACGTG- SPF 4~6 GACATCCGCAAAG-3 5 -CTGGAAGGTG- BALB/c GACAGCGAGG-3 cdna SPF PCR PCR Invitrogen RP- RT-PCR PCR 94 MI640 Gibco RNA RNAiso Plus PrimeScript TM RT Reagent Kit TaKaRa PCR ( ) (enzyme linked immunosorbent assay, ELISA) %.5 裸鼠皮下移植瘤模型的建立 Ki67 OVCAR-3 OVCAR-3 BeWo 30 5 (. 慢病毒载体转染细胞与细胞分组 OVCAR-3 d BeWo ) 6 0.5% 5 00 μl( 0 6 5 0 5 6 ) 4 6 polybrene( 8 μg/ml) 6 6 ml/ MOI 3 ( ) 6 h 6.6 免疫组化 37 polybrene 3 5 min 5%CO 4 h 0 min 95% 0 min 80% 0 min 0 μg/ml Blasticidin 0 min 3%H O 0 min d PBS 5 min 3 RNA 60 5 min PBS 5 min 5 OVCAR-3 ( ) 3.5% 4 PBS 5 min 3 h BeWo PBS 5 min 3 DAB.3 ELISA 检测各组细胞上清液中 β-hcg 水平 50 μl (.7 统计学方法 SPSS 8.0 ) 50 μl( ) 37 t P<0.05 30 min 0 s 3~5 OD 3 5 min 94 45 s 58 45 s 7 45 s 40 7 0 min PCR
,. OVCAR-3 5. 荧光测定慢病毒载体转染细胞效果 OVCAR-3 ( ). RT-PCR 测定各组细胞中 mrna 的表达 RT-PCR mrna ( ) 3 mrna mrna GAPDH OVCAR-3 mrna.7 免疫组化测定各组中肿瘤细胞增殖指标 Ki67.3 ELISA 测定各组细胞分泌上清液中 的表的表达情况 BeWo 达 ELISA Ki67 Ki67 ( 6 ) ( 3) 3.4 裸鼠皮下移植瘤模型的建立 6 HCG [3-0] ( 4) HCG.5 裸鼠皮下移植瘤的成瘤率比较 BeWo 00%(6/6) OVCAR-3 HCG [3-0] 83.3%(5/6) HCG HCG 33.3%(/6) HCG.6 裸鼠皮下移植瘤的生长速度比较 BeWo HCG ( 5) HCG 3 β-hcg / Unit ml - 4 3 0 OVCAR-3 BeWo OVCAR-3 * P<0.05 ELISA /cm 3 4 4 5 OVCAR-3 BeWo 0 4 6 / α β α α β-hcg 6 β β β3 β5 β7 β8 [-3] 99.7% β5 β3 β8 β5 [3-4] HCG
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(400 ) A B C D E 6 Ki67 (00 ) A 0 μm B 0 μm C 0 μm A FITC-BAFF-R B Hoechst C A B BAFF-R