【作者】 丰盛梅；

【导师】 李亦平； 邵健忠；

【作者基本信息】 浙江大学 ， 细胞生物学， 2008， 博士

【摘要】 V-ATPase广泛存在于真核细胞,调控细胞内外环境pH的变化而发挥功能。存在于激活的破骨细胞皱褶缘(Ruffled border,RB)的V-ATPase质子泵的细胞外酸化功能是骨吸收所必需的。然而特异的破骨细胞皱褶缘V-ATPase的组成和结构仍然没有被阐明清楚。本研究发现V-ATPase的C亚基Atp6v1c1(C1)亚型在破骨细胞中高表达,而C亚基的另外两个亚型Atp6v1c2a(C2a)和Atp6v1c2b(C2b)在破骨细胞中几乎不表达;在破骨细胞体外诱导分化过程中RANKL诱导Atp6v1c1高表达;Atp6v1c1与Atp6voa3(a3)相互作用;在体内激活的破骨细胞中Atp6v1c1主要定位在皱褶缘;体外培养的成熟破骨细胞中Atp6v1c1和微管共定位在细胞浆。本研究首次采用慢病毒介导RNA干扰技术在破骨细胞中表达Atp6v1c1特异siRNA,发现Atp6v1c1表达被敲低后严重影响了破骨细胞的细胞外酸化和骨吸收功能而骨髓单核细胞分化成多核破骨细胞并不受影响,与Atp6v0a3表达敲低的破骨细胞表现相似。主要包含微丝的环(F-actin ring)的形成由于C1表达被敲低而被严重破坏,而a3表达敲低或a3敲除并不影响破骨细胞F-actin环的形成;C1与F-actin在抗酒石酸酸性磷酸酶阳性(TRAP+)的单核细胞中共定位在细胞浆,且随着单核细胞分化为成熟的多核破骨细胞C1与F-actin共同定位大部分从细胞浆迁移到了细胞外周并行成环状带。本研究结果表明Atp6v1c1是破骨细胞皱褶缘质子泵的主要成分,且参与调控破骨细胞激活过程中F-actin环的形成。本研究采用逆转录病毒介导C1在破骨细胞中过表达,发现C1过表达不能抑制Src激酶的抑制剂PP2对破骨细胞F-actin环的破坏作用,结果表明C1调控破骨细胞F-actin环的形成过程中需要其它因子的协同作用。在肿瘤细胞中胞浆膜V-ATPase在肿瘤细胞的生长、迁移、凋亡和癌细胞的抗药性中发挥重要功能。虽然C1亚基在人口腔鳞状细胞癌中明显较正常组织高表达,但C1高表达在肿瘤细胞的生长和迁移中的作用仍未阐明。本研究发现V-ATPase的一个亚基Atp6v1c1在肿瘤细胞中广泛表达,且C1敲低后小鼠乳腺癌细胞系4T1和大鼠骨肉瘤细胞系UMR-106的生长明显延缓,且二者的迁移能力明显下降。结果表明ATP6v1c1可能参与调控肿瘤细胞的生长和迁移。更多还原

【Abstract】 V-ATPase exsits in all eukaryotic cells and palys a role by regulating pHcyt and pHex.The extracellular acidification function of vacuolar ATPase（V-ATPase） proton pump（s） present in the activated osteoclasts plasma membrane is necessary to osteoclast bone resorption.The exact configuration of osteoclast-specific ruffled border V-ATPase remains largely unknown.In this study,we found that V-ATPase subunit Atp6v1cl（C1） is highly expressed in osteoclasts but not subunits Atp6v1c2a （C2a） or Atp6v1c2b（C2b）;the expression level of C1 is highly induced by RANKL during osteoclast differentiation;C1 interacts with Atp6v0a3（a3） and is mainly localized on the ruffled border of activated osteoclasts.In vitro,C1 co-localized with tublin just in cytoplasma in mature osteoclast.Our data showed for the first time that C1 silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption while cell differentiation did not appear to be affected,which is similar to a3 silencing;F-actin ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3^-/- osteoclasts;and that C1 is co-localized with F-actin in cytoplasm,however,the co-localization is largely moved to plasma membranes of mature osteoclasts.Our study demonstrated that Atp6v1c1 is an essential component of the proton pump at the osteoclast ruffled border,and may regulate F-actin ring formation in osteoclast activation.In this study, we used retrovirus-mediated C1 overexpression in osteoclast,and found that C1 overexpression didn’t inhibit the distruction of F-actin ring due to PP2（Src inhibitor）, our study demonstrated that C1 needs other factors together to regulate the F-actin formation during osteoclast activation.In tumor cells,plasma memebrane V-ATPase（pmV-ATPase） are critical to cell growth,cell migration,apoptosis and drug resistance in cancer cells.Though subunit C1 was found sample wide over-expression in OSCC tumor cells compared to normal cells,the role of C1 higher expression in tumor cells growth and migration is not yet clear.In this study,we found that Atp6v1c1 ubiquitously expressed in tumor cells and the cell growth was retarded in C1-depleted mouse breast cancer cell line 4T1 and C1-depleted rat osteosarcoma cell line UMR-106,furthermore,the cell migration ability obviously decreased in the two C1-depleted cell lines.Our results suggested that Atp6v1c1 was involved in the regulation of tumor cells growth and migration.更多还原