A New Hsp90 And Hsp70 Chaperones The Ttc36 Positioning And Function Of Human Retinal Pigment Epithelial Cells And Cloning Of The Gene Limsa Analysis

Objective : To investigate subcellμlar location and function of TTC36,as a Novel Chaperone Protein for both HSP90 and HSP70 in human retinal pigment epithelial(RPE) cells and further explore the mechanism of TTC36 and HSP90/70 effects on the proliferation and apoptosis of RPE cells and the pathogenesis and development of some RPE cell proliferative diseases such as proliferative vitreoretinopathy(PVR).Methods: First, red fluorescence expressing vector of TTC36 was constructed by using gene recombination technique.Second, plasmids plp-RFP-TTC36 and plp-GFP-HSP90/plp-GFP-HSP70 were transfected into ARPE-19 cells using LipofectamineTM 2000 to visualize co-localization of TTC36 and HSP90/HSP70 by Laser Confocal Microscope.Third, we examined cell cycle of ARPE-19 cells transfected by TTC36 using flow cytometry .At last, real-time fluorescence quantitative PCR was used to study the mRNA expression of TTC36 in ARPE-19 cells which were treated with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG).Results: Confirmed by PCR and DNA sequencing,the fluorescence expressing vector of TTC36 was successfully constructed.We find that the co-localization of HSP90/HSP70 and TTC36 is cytoplasm of ARPE-19 cells and the role of TTC36 in promoting the proliferation of ARPE-19 cells. Treatment of A-RPE19 cells with the 17-AAG results in downregulation of TTC36 expression .Conclusion: TTC36 can interact with HSP90/70 in RPE cells,so TTC36 and HSP90/70 may be involved in the proliferation and apoptosis of RPE cells.It is possible that TTC36 and HSP90/70 contribute to some RPE cell proliferative diseases such as PVR .This study lays a foundation for the functional study of TTC36 and HSP90/70,and provides new pathway for the clinical application of therapies for RPE cell proliferative diseases. Objective: To clone and analyze the different splicing variant of Lims gene in rat. Methods: Splicing variants of Lims gene were amplified by RT-PCR from rat cDNA and inserted into PinPointTM Xa-1T vector, the position clones selected by PCR were sequenced.Results: Lims A, a novel splicing variant with a 1014 bp open reading frame (ORF) , encoding a 338-amino acid (AA) protein was cloned.Conclusion: Comparative genome analysis displayed Lims A, a novel variant of Lims gene was confirmed in rat and established the foundation for further to studying the function of Lims A in proliferative vitreoretinopathy.