中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
10期
1886-1888
,共3页
张萦斐%何传超%毛凯%黄拼搏%金少文%肖治宇
張縈斐%何傳超%毛凱%黃拼搏%金少文%肖治宇
장영비%하전초%모개%황병박%금소문%초치우
肝癌细胞HCCLM3%ATP6L%侵袭%基质金属蛋白酶-2%组织蛋白酶B
肝癌細胞HCCLM3%ATP6L%侵襲%基質金屬蛋白酶-2%組織蛋白酶B
간암세포HCCLM3%ATP6L%침습%기질금속단백매-2%조직단백매B
Hepatocellular carcinoma HCCLM3 cells%ATP6L%Invasion%Matrix metalloproteinase-2%Cathepsin B
目的 观察ATP6L表达变化对人肝癌细胞HCCLM3侵袭能力等肿瘤生物学特性的影响,并探讨其作用机制.方法 构建ATP6L-RNAi真核表达载体ATP6L-sh与阴性对照载体Con-sh,并分别转染至HCCLM3细胞,建立稳定干扰ATP6L的细胞株和阴性对照细胞株,通过细胞侵袭实验(Transwell)检测人肝癌细胞HCCLM3的体外侵袭能力变化,通过Western blot实验、明胶酶谱测定、原位荧光酶谱测定等实验技术,检测ATP6L-sh和Con-sh两株细胞ATP6L蛋白表达水平、上清液基质金属蛋白酶(MMP)-2的活性以及胞内组织蛋白酶B(Cathepsin B)活性的变化.结果 与阴性对照细胞株比较,稳定干扰ATP6L表达的ATP6L-sh细胞株ATP6L蛋白表达水平明显下调,体外侵袭能力明显减弱.ATP6L-sh细胞与对照细胞分泌上清液中MMP-2活性分别为1.81±0.18、18.40±1.26(P ＜0.05)；细胞内Cathepsin B活性分别为228.6±21.1、471.7±21.4(P ＜0.05),活性均明显降低.结论 ATP6L在肿瘤侵袭过程中发挥重要作用,抑制ATP6L蛋白表达可有效降低肝癌细胞HCCLM3的侵袭能力,ATP6L蛋白对肿瘤细胞侵袭能力的影响可能与调控MMP-2和Cathepsin B活性有关.
目的 觀察ATP6L錶達變化對人肝癌細胞HCCLM3侵襲能力等腫瘤生物學特性的影響,併探討其作用機製.方法 構建ATP6L-RNAi真覈錶達載體ATP6L-sh與陰性對照載體Con-sh,併分彆轉染至HCCLM3細胞,建立穩定榦擾ATP6L的細胞株和陰性對照細胞株,通過細胞侵襲實驗(Transwell)檢測人肝癌細胞HCCLM3的體外侵襲能力變化,通過Western blot實驗、明膠酶譜測定、原位熒光酶譜測定等實驗技術,檢測ATP6L-sh和Con-sh兩株細胞ATP6L蛋白錶達水平、上清液基質金屬蛋白酶(MMP)-2的活性以及胞內組織蛋白酶B(Cathepsin B)活性的變化.結果 與陰性對照細胞株比較,穩定榦擾ATP6L錶達的ATP6L-sh細胞株ATP6L蛋白錶達水平明顯下調,體外侵襲能力明顯減弱.ATP6L-sh細胞與對照細胞分泌上清液中MMP-2活性分彆為1.81±0.18、18.40±1.26(P ＜0.05)；細胞內Cathepsin B活性分彆為228.6±21.1、471.7±21.4(P ＜0.05),活性均明顯降低.結論 ATP6L在腫瘤侵襲過程中髮揮重要作用,抑製ATP6L蛋白錶達可有效降低肝癌細胞HCCLM3的侵襲能力,ATP6L蛋白對腫瘤細胞侵襲能力的影響可能與調控MMP-2和Cathepsin B活性有關.
목적 관찰ATP6L표체변화대인간암세포HCCLM3침습능력등종류생물학특성적영향,병탐토기작용궤제.방법 구건ATP6L-RNAi진핵표체재체ATP6L-sh여음성대조재체Con-sh,병분별전염지HCCLM3세포,건립은정간우ATP6L적세포주화음성대조세포주,통과세포침습실험(Transwell)검측인간암세포HCCLM3적체외침습능력변화,통과Western blot실험、명효매보측정、원위형광매보측정등실험기술,검측ATP6L-sh화Con-sh량주세포ATP6L단백표체수평、상청액기질금속단백매(MMP)-2적활성이급포내조직단백매B(Cathepsin B)활성적변화.결과 여음성대조세포주비교,은정간우ATP6L표체적ATP6L-sh세포주ATP6L단백표체수평명현하조,체외침습능력명현감약.ATP6L-sh세포여대조세포분비상청액중MMP-2활성분별위1.81±0.18、18.40±1.26(P ＜0.05)；세포내Cathepsin B활성분별위228.6±21.1、471.7±21.4(P ＜0.05),활성균명현강저.결론 ATP6L재종류침습과정중발휘중요작용,억제ATP6L단백표체가유효강저간암세포HCCLM3적침습능력,ATP6L단백대종류세포침습능력적영향가능여조공MMP-2화Cathepsin B활성유관.
Objective To explore the effect of ATP6L on invasion in human hepatocellular carcinoma cell line HCCLM3 and its mechanism.Methods Stablely ATP6L-shRNA transfected HCCLM3 cell line was established and the expression of ATP6L was evaluated by Western blotting.The invasion capability change was tested by Transwell assay in vitro.The activities of matrix metalloproteinase (MMP)-2 in gelatinase were determinated by zymography.The expression of Cathepsin B was measured by flourescent in situ zymography.Results The expression of ATP6L was markedly decreased in the stable transfected cells compared with the control cells.The invasive ability of HCCLM3 cells were inhibited by ATP6L knockdown.In the supernatant the MMP-2 activity was decreased in stably ATP6L shRNA transfected HCCLM3 cells compared with control group (1.81 ±0.18 vs 18.40 ± 1.26,P ＜0.05).Cathepsin B acitivity was reduced in ATP6L shRNA transfected compared with control (228.6 ± 21.1 vs 471.7 ± 21.4,P ＜ 0.05).Conclusion ATP6L is essential for HCCLM3 cell invasion.Silencing ATP6L expression can inhibit the invasion of HCCLM3 passably by activity reduction of MMP-2 and Cathepsin B.
