摘要
目的研究肿瘤转移抑制基因KAI1对肝癌细胞侵袭、转移的影响及其分子机制,为肝癌的基因治疗提供理论和实验依据。方法用亚克隆技术构建KAI1基因正、反义真核表达质粒,DOTAP脂质体法将其分别转入高转移潜能的人MHCC97-H肝癌细胞,以限制性酶切、PCR、Westernblot及免疫组化鉴定基因重组及转染是否成功。电镜观察细胞超微形态变化,流式细胞仪检测细胞周期分布,细胞生长曲线和平板法集落形成试验观察细胞生长和集落形成能力,BoydenChamber试验观察细胞侵袭能力,微管吸吮技术测定细胞的粘弹性和粘附力。BALBCA4～6周龄雄性裸鼠80只,随机分正义组、反义组、亲本细胞组及空载体组进行皮下(n=40)和原位肝(n=40)种植试验,观察体内的成瘤性、侵袭性及远处转移能力,免疫组化研究癌转移灶AFP和肿瘤组织细胞外基质(ECM)及粘附分子的表达状况。结果(1)成功构建了KAI1正、反义基因真核表达质粒,并将其分别转入MHCC97-H人肝癌细胞;(2)发现转入KAI1基因后可导致肝癌MHCC97-H细胞的一些细胞器数量和形态的变化;(3)KAI1基因对肝癌MHCC97-H细胞的体内外生长、细胞周期及体内成瘤性无明显影响;(4)反义KAI1基因可使MHCC97-H细胞的克隆形成能力增强、对体内外肝癌细胞的侵袭和转移有促进作用;(5)KAI1基因表达上调能使MHCC97-H细胞的体内外侵袭能力降低;(6)KAI1基因抑制肝癌侵袭和转移的机制可能与细胞粘弹性增加、ECM表达上调、粘附分子表达下调及粘附力下降等变化有关。结论KAI1基因在肝癌的侵袭转移过程中发挥着重要作用,上调KAI1基因表达能明显降低肝癌细胞的侵袭和转移能力。
Objective To explore the effects and mechanisms of KAI1 gene on invasion and metastasis of human hepatocellular carcinoma（HCC） MHCC97-H cells. Methods KAI1 sense and antisense eukaryotic expression plasmids were constructed using subclone technique and transfected into MHCC97-H cells respectively by DOTAP liposome. After successful transfection was confirmed, in vitro growth curve, cell cycles, plate clone formation efficiency, invasive ability in Boyden Chamber assay, ultrastructural morphology, viscoelastic properties, cell adhesion forces,subcutaneous tumorigenicity and metastasis after implanted orthotopicly into nude mice liver, and the tissue expression of AFP, ECM and intercellular adhesion molecules were studied. Results KAI1 sense and antisense gene had no significant effects on the cell growth curve and cell cycles. After transfection with sense KAI1 gene, decreased invasive ability in Boyden Chamber assay and decreased amount of mitochondrion, but no significant changes of plate clone formation efficiency were observed in MHCC97-H cells; while after transfection with antisense KAI1 gene, the plate clone formation efficiency and invasive ability in Boyden Chamber assay were significantly increased. Furthermore, increased amount of mitochondrion, rough endoplasmic reticulum, Golgi apparatus and expanded endoplasmic reticulum were also noted in the antisense group. The cell elastic coefficients K1, K2 and μ were significantly higher after transfected with sense KAI1 （P〈0.05）, lower after transfected with antisense KAI1 （P〈0.01）,and no significantly different after transfected with its control vector pCI-neo without KAI1 gene （P〉0.05）, as compared with their paternal MHCC97-H cells. The different kind cells inoculated subcutaneously all have tumorigenicity. There were no significant differences in the tumor growth rate among different groups, but the antisense group shows stronger invasion ability than others. The spontaneous metastasis to lung occurred in latency period of six weeks via orthotopie implantation of tumor tissue. The number of lung metastatic lesions increased dramatically in antisense gioup（P〈0.01）, decreased significantly in sense group （P〈0.01） and no significantly dififferenee in vector pCI-nee group （P〉0.05）, as compared with their paternal MHCC97-H cells. There were diferences in expression of ECM, intercellular adhesion molecules and cell adhesion forces after transfection with the antisense or sense gene. Conclusion KAI1 gene may play an important role in the invasion and metastasis of human HCC.Upregulated expression of KAI1 gene in HCC cells may suppress their invasive and metastasis ahility of the tumor.
出处
《浙江医学》
CAS
2005年第12期891-895,共5页
Zhejiang Medical Journal
基金
国家自然科学基金项目(No.30070348)
关键词
肿瘤转移抑制基因
肝细胞癌
肿瘤转移
KAI1基因
基因转染
Metastasis-suppressor gene Hepalneellular carcinoma Tumor metastasis KAII gene Gene transfection
