Research On The Cloning, Expression & Function Of HOSM

Objective: To clone hOSM gene and express it in COS7 and WI-38 cell, the biological activity of recombinant is studied and the effect of inhibiting tumor cell and sustaining hematopoiesis of OSM is primarily explained ,which can pave the way of basic and applicative research.Methods: Taking RNA from U937 cell which have been stimulated by PMA, cDNA is amplified by RT-PCR and ligate with plasmid pUCmT forming recombinate pUCmT- hOSM, then the throughout DNA is obtained by PCR , ligating them with plasmid pcDNA3.1/myc-his(-)A and forming recombinate pcDNA3.1A-hOSM. The hOSM sequence is identified with PCR,restriction endonucleases analysis and DNA sequencing , transiently expressing in COS-7 cell.The hOSM gene transcription is tested by reverse-transcribing-polymerase chain reaction, the effect of inhibiting A375 cell of expressed product is investigated with MTT assay.To transfect pcDNA3.1A-hOSM eukaryotic expression vector to WI-38 cell and to obtain cell which steadily express human oncostatin M protein using G418. The human oncostatin M gene tanscription is defined by RT-PCR ,the effect of inhibiting A375 cell and A549 cell of expressed product is investigated with MTT assay and the cell cycle is tested by FCM. human oncostatin M sustainhematopoiesis tested by co-culturing with the fourth marrow stromal cell. Results: The hOSM sequence is defined not only correct oriently but also identical with sequence reported in GeneBank,which transiently express in COS-7 cell. The rhOSM protein that has characteristic of inhibiting A375 cell. Obtaining cell which steadily express human oncostatin M, recombinate human oncostatin M can inhibit A3 75 cell by blocking the cycle of Gl to S period and also inhibit A549 cell .It has the ability to promote the development of marrow stromal cell.Conclusion: This study successfully clone hOSM gene,which express in COS7 cell, construct transfected gene WI-38 cell steadily expressing hOSM. it is proved that the recombined protein have the effect of inhibiting tumor and promoting the development of marrow stromal cell, which paved the way for further research of OSM inhibiting tumor cell and sustaining hematopoiesis.