【作者】 卢小东；

【导师】 杨胜利； 覃文新； 王文兵；

【作者基本信息】 江苏大学 ， 农产品加工及贮藏工程， 2005， 博士

【摘要】 肿瘤细胞的两大特征是无限增殖和侵袭转移。端粒酶异常活跃是细胞永生化的重要原因,因此成为抑瘤靶点。肿瘤细胞的侵袭转移与细胞外基质的分解密切相关。侵袭性实体瘤细胞周围较正常细胞有较低的pH,这主要与肿瘤细胞膜上氢泵V-ATPase活性通常增高、促氢离子外排有关。酸性条件有利于蛋白水解酶激活、消化细胞外基质,从而利于细胞侵袭。本文用基于DNA质粒的RNAi干扰技术分别抑制高转移潜能的肝癌细胞株HCCLM3端粒酶亚基hTERT和V-ATPase的ATP6L亚基表达,以达到抑制肝癌细胞生长和转移的目的。构建针对hTERT靶基因的siRNA表达质粒载体,转染HCCLM3。转染后,western blot显示hTERT被抑制;TRAP和ELISA方法显示端粒酶活性降低;MTT方法显示细胞增殖减慢。构建针对ATP6L靶基因的siRNA表达质粒载体,转染HCCLM3,经G418筛选的转染细胞命名为si-A-M3。northern blot结果显示si-A-M3的ATP6L被抑制～60%。用pH敏感荧光染料BCECF/BCECF-AM结合实时荧光分光光度计方法分析细胞培养液pH（pHe）和细胞内pH（pHi）的变化,结果为:①si-A-M3培养液培养12小时后细胞泌氢较对照组减少;②si-A-M3细胞内pHi在NH₄Cl诱导酸化后,去除NH₄⁺后恢复速度明显减慢,且不能恢复到静息pHi:③用细胞内缓冲能力校正测得si-A-M3即时排氢量明显降低。上述结果提示转染细胞的V-ATPase功能被抑制。分解细胞外基质的蛋白水解酶受pH调节。V-ATPase功能被抑制影响MMP-2/9的活性。western blot结果显示si-A-M3的MMP-2表达降低,用ELISA方法和酶谱测定细胞培养上清液中MMP-2/9活性以及用FITC-标记的gelatin原位显示移植瘤细胞周围MMP-2/9活性,均发现si-A-M3的MMP-2/9活性下调。体外培养si-A-M3的侵袭实验显示si-A-M3的侵袭力明显下降;在小鼠[BalB/c（nu+/nu+）]肝原位移植瘤模型中发现si-A-M3组肝内转移率和肺转移率明显降低;si-A-M3组肝原位生长的肿瘤体积明显小于对照组。综合上述结果,HCCLM3细胞的ATP6L被抑制后,细胞侵袭转移和生长受抑制,与细胞的V-ATPase功能被抑制及其MMP-2表达和MMP-2/9活性被抑制有关。本文证实hTERT是抑制肿瘤生长的有效标靶,同时首次报道ATP6L的RNA干扰对是肿瘤转移和生长具有明显抑制作用。本文的结果为研究肿瘤发生和转移机制以及探索抑瘤方法提供了参考。更多还原

【Abstract】 The two main characteristics of tumor cells are uncontrolled proliferation （immortalization） and aggressive behavior.Telomerase is a major contributing factor of immortalization of cells and therefore is a potential target to suppress tumor.Extracellular pH is usually low in metastastic solid tumors,mainly resulted from abnormally active hydrogen pump V-ATPase on plasma membrane.In acidic conditions,activity of proteases that digest extracellular matrix（ECM） are up-regulated,benefiting invasion or metastasis.In our experiments,in order to suppress the growth and metastasis of HCCLM3,a human hepatocellular carcinoma cell（HCC） line with highly metastatic potential, DNA vector-based small interfering RNA was used respectively to knock down hTERT,the subunit of telomerase and ATP6L,the subunit of V-ATPase.siRNA-expressing vector targeting hTERT was constructed and transfected into HCCLM3.Western blot demonstrated that hTERT was inhibited.Assayed by TRAP and ELISA,the activity of telomerase was decreased in transformed cells.MTI cell proliferation curve showed that the proliferation of HCCLM3 with hTERT knockdown was inhibited.HCCLM3 was transfected siRNA-expressing vector targeting ATP6L,screened by G418 and designated as si-A-M3.ATP6L in si-A-M3 was knocked down by～60%, assayed by northern blot.The ability of proton extrusion of si-A-M3 was analyzed by determining the alteration of extracellular pH（pH of culture media） and intracellular pH（pHi） using pH-sensitive fluorescent dyes BCECF/BCECF-AM,real-time monitored on a luminescence spectrometer. The results showed:①The extrusion of proton by long-term（12 hr） cultured si-A-M3 was notably reduced.②The pHi of si-A-M3 was only marginally recovered from NH₄Cl-prepulsed acidification,contrasting to the full recovery to the resting pHi of the control.The rate of the pHi recovery of si-A-M3 was significantly dropped.^In si-A-M3,at the initial pHi recovery point,the H⁺ effiux（JH+）,corrected by intrinsic buffering capacity,was significantly decreased.The above results indicate the inhibition of the activity of V-ATPase in si-A-M3.The secretion and activation of ECM-degenerating proteases are pH-regulated.The expression of matrix metalloproteinase-2 and the gelatinase activity were affected,concomitant with the inhibition of V-ATPase.Western blot showed that the expression of MMP-2 was down-regulated in si-A-M3. The activity of gelatinase of the supernatant of cultured si-A-M3 cells was apparently reduced,further confirmed by zymography of the supernatant and in situ zymography of si-A-M3 xenografts on mice by using FITC-labeled gelatin.In vitro,the invasion ratio of si--A-M3 was decreased remarkably.On the animal model in vivo,the sizes of the xenografts of si-A-M3 implantated into the livers in BalB/c（nu+/nu+） mice were apparently decreased. Intrahepatic and pulmonary metastasis of si-A-M3 group were respectively strikingly decreased.Taken together,The results suggest that the inhibition of V-ATPase function via knockdown of ATP6L expression using RNA interfering technology effectively retarded the cancer growth and suppressed the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity.In conclusion,our experiments confirmed hTERT of telomerase was an effective target to inhibit the proliferation of HCC.As for a target to inhibit the metastasis as well as the growth of HCC,ATP6L was a good candidate.So far as we could know,our experiments firstly reported that the knockdown of ATP6L expression using siRNA technology suppressed the growth and metastasis of HCC.Our results implicate possible mechanisms of carcinogenisis and metastasis of tumor,and furthermore,the potential for cancer treatment.更多还原