CAJ | 학술논문

目的观察RhoC高表达诱导人正常肝细胞迁移和侵袭恶性转化.方法 pcDNA3-RhoC转染HL7702细胞,划痕实验和Transwell小室检测细胞迁移和侵袭能力;明胶酶谱分析检测基质金属蛋白酶(MMPs)活性;逆转录-聚合酶链反应(RT-PCR)检测细胞迁移和侵袭相关基因表达;Western blot检测相关蛋白水平;接种裸鼠检测活体成瘤率.结果转染RhoC细胞组与HL7702细胞组和空质粒转染细胞组比较获得了迁移和侵袭能力,相对迁移距离显著增大,分别为(63.33±10.07)％、(28.67±7.02)％和(28.33 ±6.66)％(P＜0.01);穿过Transwell微孔滤膜细胞显著增多,分别为(65.33±6.80)％、(24.33±5.79)％和(23.33±5.73)％(P＜0.01),侵袭能力显著增强,分别为43.67±7.59、13.33±3.40和15.33 ±4.11 (P ＜0.01);MMPs活力增强;迁移和侵袭相关基因MMP-2、MMP-9、血管内皮生长因子(VEGF)、P27RF-Rho和Survivin表达上调,基质金属蛋白酶组织抑制剂1(TIMP1)、TIMP2、p53和人第10号染色体缺失的磷酸酶及张力蛋白同源的基因(PTEN)表达水平下调;接种裸鼠成瘤率为40％.结论 RhoC高表达诱导人正常肝细胞迁移和侵袭的恶性转化.
목적 관찰RhoC고표체유도인정상간세포천이화침습악성전화.방법 pcDNA3-RhoC전염HL7702세포,화흔실험화Transwell소실검측세포천이화침습능력;명효매보분석검측기질금속단백매(MMPs)활성;역전록-취합매련반응(RT-PCR)검측세포천이화침습상관기인표체;Western blot검측상관단백수평;접충라서검측활체성류솔.결과 전염RhoC세포조여HL7702세포조화공질립전염세포조비교획득료천이화침습능력,상대천이거리현저증대,분별위(63.33±10.07)％、(28.67±7.02)％화(28.33 ±6.66)％(P＜0.01);천과Transwell미공려막세포현저증다,분별위(65.33±6.80)％、(24.33±5.79)％화(23.33±5.73)％(P＜0.01),침습능력현저증강,분별위43.67±7.59、13.33±3.40화15.33 ±4.11 (P ＜0.01);MMPs활력증강;천이화침습상관기인MMP-2、MMP-9、혈관내피생장인자(VEGF)、P27RF-Rho화Survivin표체상조,기질금속단백매조직억제제1(TIMP1)、TIMP2、p53화인제10호염색체결실적린산매급장력단백동원적기인(PTEN)표체수평하조;접충라서성류솔위40％.결론 RhoC고표체유도인정상간세포천이화침습적악성전화.
Objective To investigate the role of Ras-homologous GTPase C (RhoC) overexpres-sion in inducing migration and invasion for malignant transformation in normal human hepatocytes.Methods pcDNA3-RhoC vector was transfected into human normal liver cell HL7702.Scratch-healing test combined with Transwell was used to examine cell migration and invasion,and matrix metalloproteinases (MMPs)gelatinase activities were assayed through zymography.Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the migration and invasion associated genes.RhoC transfected HL7702 cells were inoculated into the nude mice to test tumor formation.Results Cells transfected with pcDNA3-RhoC acquired migration capacity,and the migration distance was notably increased as compared with parental cells and negative control groups [(63.33 ± 10.07)％ vs.(28.67 ± 7.02)％ and (28.33 ±6.66)％,P ＜0.01].The number of pcDNA3-RhoC cells through the Transwell was increased significantly (65.33 ± 6.80 vs.24.33 ± 5.79 and 23.33 ± 5.73,P ＜ 0.01),and the invasion capacity was significantly enhanced in pcDNA3-RhoC-transfected group as compared with control groups (43.67 ±7.59 vs.13.33 ± 3.40 and 15.33 ± 4.11,P ＜ 0.01).MMPs gelatinase activities were increased,the expression of migration and invasion associated genes and proteins such as MMP-2,MMP-9,vascular endothelial growth factor (VEGF),P27RF-Rho and Survivin was significantly up-regulated,and the expression of tissue inhibitor of tissue inhibitorof metalloproteinase 1 (TIMP1),TIMP2,p53 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) was down-regulated in pcDNA3-RhoC-transfected group as compared with control group.The tumor formation ratio in nude mice was 40％.Conclusion RhoC overexpression can induce migration and invasion for malignant transformation in human normal hepatocytes.