The Influence of Protein Kinase C-α on actin Cytoskeleton of Internal Medullary Collecting Duct Epithelial Cell Line MIMCD3
|School||Huazhong University of Science and Technology|
|Keywords||protein kinase C-α actin internal medullary collecting duct epithelial cells(mIMCD3)|
Objective:To explore the influence of protein kinase C-αon actin cytoskeleton of internal medullary collecting duct epithelial cell line mIMCD3 and the possible mechanisms of protein kinase C-αin the material transportation.Method:Culture the mIMCD3 cells with routine method in vitro, extraction of four different PKC-αgene expression vector and then transfected into mIMCD3 by lipfectamine2000. The transfected cells were randomly divided into four groups①the cells were transfected empty vector (K group)②the cells were transfected wild-type plasmid vector (W group)③the cells were transfected dominant activating plasmid vector ( A group)④the cells were transfected dominant inactivating plasmid vector (R group) . We get the stable expression groups by give resistance drug of plasmid to the transfected cells. Real-time fluorescent quantization polymerase chain reaction was used to detect the messenger RNA of PKC-αgene in transfected cells, western blot detected the expression and activation of PKC-αgene in transfected cells, measured the structure change of actin cytoskeleton by immunohistochemical way. Results:⑴t he stable expression PKC-αgroups were construct succeed⑵when the time of the cells were transfected was 24h,observed the ratio of fluresence cells in full cells under the fluorescence microscope, the ratio was about36±2 %.⑶the results measured by real-time fluorescent quantization PCR: Compared with the K group, the PKC-αmessenger RNA expressions in other groups obviously increased(p<0.05), but between the other three groups,it dose not show obviously differce(p>0.05); the results measured by western blot: Compared with the K group, the PKC-αprotein expression in other groups obvious increased(p<0.05),and the activation of PKC-αin A group showed obvious increased than other groups(p<0.05),while in R group showed obvious decreased than other groups(p<0.05).⑷the results measured by Immunohistochemistry: the K group and W group showed no significant difference, A group showed the actin more main assemble to the cell apical membrane less distributed in the cytoplasm than the other groups; while R group showed the actin distributed in the cytoplasm, and actin less assemble to the cell apical membrane than the other gourps.Conclusions:The PKC-αaffects the actin cytoskeleton of internal medullary collecting duct epithelial cell line mIMCD3, which may mediate the material transportation, then influence the urine concentration.