CAJ | 학술논문

目的：研究不同浓度的氯化钴（CoCl2）对大鼠肝癌细胞株（RH35）的缺氧诱导因子-1α（HIF-1α）及血管内皮生长因子（VEGF）表达的影响，及对RH35增殖及侵袭能力影响。方法利用不同浓度的（0、50、100、150、200μmol／L）CoCl2建立RH35缺氧模型。通过Cell Counting Kit-8（CCK-8）检测CoCl2对RH35的体外生长增殖活性的影响。定量PCR （Q-PCR）、蛋白免疫印迹法及ELISA检测RH35中HIF-1α及VEGF的mRNA及蛋白表达情况。体外侵袭实验（Transwell）检测细胞的体外侵袭迁移力。结果 CCK-8显示在CoCl2浓度为50、100、150μmol／L时，RH35细胞体外生长增殖活性与0μmol／L 无差异（P＞0．05），在200μmol／L 时，RH35增殖活性降为（62．90±1．57）％，与0μmol／L时比较差异有统计学意义（P #0．01）。Q-PCR 显示 RH35细胞 HIF-1α及VEGF的mRNA表达随CoCl2浓度升高而增加，并且HIF-1α与VEGF呈显著正相关（r＝0．980，P＝0．020）。蛋白免疫印迹法及ELISA显示HIF-1α及VEGF的蛋白表达均随CoCl2浓度升高而增加。Tr-answell显示RH35随CoCl2浓度增高，侵袭能力增加。结论 CoCl2化学缺氧模型可较好的模拟缺氧引起的肝癌细胞HIF-1α上调，使VEGF表达增加，并且两者具有相关性；通过上调HIF-1α可增加肝癌细胞的侵袭能力，为下一步实验奠定基础。
목적：연구불동농도적록화고（CoCl2）대대서간암세포주（RH35）적결양유도인자-1α（HIF-1α）급혈관내피생장인자（VEGF）표체적영향，급대RH35증식급침습능력영향。방법이용불동농도적（0、50、100、150、200μmol／L）CoCl2건립RH35결양모형。통과Cell Counting Kit-8（CCK-8）검측CoCl2대RH35적체외생장증식활성적영향。정량PCR （Q-PCR）、단백면역인적법급ELISA검측RH35중HIF-1α급VEGF적mRNA급단백표체정황。체외침습실험（Transwell）검측세포적체외침습천이력。결과 CCK-8현시재CoCl2농도위50、100、150μmol／L시，RH35세포체외생장증식활성여0μmol／L 무차이（P＞0．05），재200μmol／L 시，RH35증식활성강위（62．90±1．57）％，여0μmol／L시비교차이유통계학의의（P #0．01）。Q-PCR 현시 RH35세포 HIF-1α급VEGF적mRNA표체수CoCl2농도승고이증가，병차HIF-1α여VEGF정현저정상관（r＝0．980，P＝0．020）。단백면역인적법급ELISA현시HIF-1α급VEGF적단백표체균수CoCl2농도승고이증가。Tr-answell현시RH35수CoCl2농도증고，침습능력증가。결론 CoCl2화학결양모형가교호적모의결양인기적간암세포HIF-1α상조，사VEGF표체증가，병차량자구유상관성；통과상조HIF-1α가증가간암세포적침습능력，위하일보실험전정기출。
Objective To investigate the effect of different concentration of Cobalt chloride (CoCl2 ) on the expression levels of hypoxia inducible factor-1α(HIF-1α) and vascular endothelial growth factor (VEGF)in rat RH35 hepatoma cell line,and the influence of HIF-1αon the tumor invasion. Methods Hy-poxic cell models were established by CoCl2 with different concentration (0,50,100,150 and 200 μmol/L). Cell counting kit-8 (CCK-8)was used to detect the proliferation of RH35. Quantitative polymerase chain reac-tion (Q-PCR)was employed to detect the expression levels of HIF-1αand VEGF mRNA. Western blot was adopted to detect the levels of HIF-1αprotein and enzyme-linked immunosorbent assay (ELISA)was conduc-ted to detect the level of VEGF protein. Invasion assay in vitro (Transwell)was applied to evaluate the inva-sion and migration of RH35. Results In the hypoxic microenvironment (50,100 and 150μmol/L of CoCl2 ), the proliferation of RH35 did not significantly differ from that in the control group (0 μmol/L of CoCl2 ). At 200 μmol/L of CoCl2,the proliferation of RH35 decreased by (62. 90 ±1. 57)%. Q-PCR revealed that the expression of HIF-1αand VEGF increased along with elevated concentration of CoCl2 and HIF-1αwas positive-ly correlated with VEGF (r=0. 980,P=0. 020). Western blot and ELISA demonstrated that the expression levels of HIF-1αand VEGF proteins were increased along with the rising concentration of CoCl2. Transwell method showed that the invasion and migration of RH35 were enhanced over the rising concentration of CoCl2. Conclusions CoCl2-induced RH35 cell model could simulate the up-regulated expression of HIF-1αby hy-poxia in hepatocellular carcinoma and then enhance the expression of VEGF. And it also could improve the in-vasion and migration of RH35 by up-regulating the expression of HIF-1α,offering the basis for subsequent in-vestigations related to HIF-1αin hepatocellular carcinoma.