摘要
建立了线粒体DNA缺失SK-Hep1细胞(ρ°SK-Hep1)转线粒体的模型(SK-Hep1Cyb),并探讨了线粒体DNA缺失对肝癌细胞恶性表型的影响。以正常人血小板为外源性线粒体供体,采用聚乙二醇融合方法建立ρ°SK-Hep1细胞转线粒体模型SK-Hep1Cyb,并以PCR、Southern杂交及Western杂交进行鉴定,MTT法测定生长曲线,Transwell实验检测细胞侵袭能力,Western杂交检测凋亡相关蛋白Bcl-2和Bax的表达情况。PCR、Southern杂交及Western杂交证实成功建立了ρ°SK-Hep1细胞转线粒体模型SK-Hep1Cyb。与SK-Hep1和ρ°SK-Hep1细胞相比,SK-Hep1Cyb细胞群体倍增时间明显延长,生长速度减慢,侵袭能力明显下降,抗凋亡能力下降;而ρ°SK-Hep1细胞与母本细胞相比生长速度与侵袭能力明显增强,抗凋亡能力增强。采用细胞融合技术成功建立了ρ°SK-Hep1转线粒体模型。融合细胞肿瘤恶性相关表型如生长速度、侵袭能力、抗凋亡能力明显下降。线粒体DNA损伤对肝癌细胞恶性表型可能有影响,而恢复正常线粒体功能后或可逆转恶性表型。
To establish p°SK-Hepl cells transmitochondrial model (Cybrids) and analyze mitochondrial DNA (mtDNA) depletion on hepatoma cell's malignant phenotypes. Normal human blood platelets were used as mitochondrial donors, and polyethylene glycol was used as fusion promoting reagent to establish transmitochondrial cell mode/. These cybrids were confirmed by PCR, Southern hybridization and Western blot. Cells growth status and their invasion capability were detected by MTT assays and Transwell chamber respectively. Western blot were carried out to analyze the expression of apoptosis related protein Bcl-2 and Bax. PCR, Southern hybridization and Western blot confirmed that cybrids cells had objective fragments of mtDNA and positive COX II activity. Cybrids cells showed lower growth rates and less invasive capability than SK-Hepl and p°SK-Hepl cells. Otherwise, p°SK- Hepl cells had higher growth rates and stronger invasive capability than its parental cells. The ability of apoptotic resistance was highest in p°SK-Hepl cell but reversely in SK-HeplCyb cell. p°SK-Hepl cells transmitochondrial model was successfully established. The cybrid cells showed lower growth rates, less invasive capability and decreased anti-apoptotic ability. Damage to mtDNA might have an effect on malignant phenotypes of human hepatoma cells, and those might be reversed by transferring normal mitochondria to the mtDNA depleted cells.
出处
《细胞生物学杂志》
CSCD
2009年第1期107-112,共6页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30470865)
新桥医院1520基金资助~~
关键词
线粒体DNA
融合细胞
肝癌
恶性表型
mitochondrial DNA
cybrids
hepatocellular carcinoma
malignant phenotypes