In Vitro Study Of Fibroblast Growth Factor-18in Up-Regulating Expression Of Elastic Fibers In The Artery

【Objective】To construct and package lentivirus carrying the gene of human fibroblast growthfactor-18(FGF-18),and test the effects of this facotor on the synthesis of elastic fibers byinfecting the human umbilical vein endothelial cells(HUVECs) and human arterialsmooth muscle cells(HASMCs) respectively with the well-packaged lentivirus,and toinitially explore the direction of the next in vivo study on FGF-18in regulating theexpression of elastic fibers in the artery.【Methods】1.Letivirus carrying the gene of FGF-18tagged with flag and GFP were packagedwith293T cells and validated by infecting293T cells and observing the express of GFPas well as detecting the express of FGF-18.2.The expression of FGFR-2both in HUVECs and HASMCs were identified byimmunocytochemical stain.3. The best MOI was tested by infecting HUVECs and HASMCs respectively withthe lentivirus of different MOI,and the poorly infected cells were selected with the flowcytometer.4. Experimental group,control group of zero-load vector and blank grop wereestablished for HUVECs and HASMCs respectively.The expression of the mRNA aswell as the protein of five elements,including FGF-18,FGFR-2,elastin,fibrillin-1andfibulin-5,were detected by real-time PCR,ELISA and Western-Blot methods.【Results】1.The lentivirus carrier palsmid with FGF-18were successfully constructed andidentified by PCR and sequencing.The final virus titer of the well-packagedlentivirus-FGF-18was3.45×108IU/ml.2.GFP were observed obviously in293T cells which were infected with lentivirus-FGF-18,and FGF-18was detected to be highly expressed in the goup which293T cells were infected with MOI=10.3.The stainning of FGFR-2was positive in the membrane as well as the nucelus ofHUVECs,and the membrane of HASMCs.4.The best MOI for HUVECs was20.The infectious rate for HASMCs was lowerthan30%when MOI=160,so the infected HASMCs were selected by the flowcytometer,and the selected cells showed flourescence positive rate were higher than90%.5.With real-time PCR,the mRNAs of three elements(FGF-18、FGFR-2and elastin)were detected to be higher expressed in the experimental group of HUVEC comparingwith the other goups(P<0.05),while mRNAs of fibulin-5and fibrillin-1showed nostatistical difference(P>0.05);As to HASMC,mRNAs of FGF-18and FGFR-2werefound to be higher expressed in experimental group(P<0.05),while the mRNA of theother three elements showed no statistical difference(P>0.05).6.With ELISA method,FGF-18was detected to be higher expressed in theexperimental group of HUVEC than the other groups,while HASMCs didn’t show thesame result.7.With Western Blot method,fibulin-5was found to be higher expressed inexperimental group in HUVEC,while the elastin of the three groups showed no obviousdifference.As to HASMC,the expressions of both the elastin and fibulin-5were lower inexperimental group.8. The confocal laser scanning microscope observed more positive stained elastin inthe experimental group of HUVEC,while the experimental group of HASMC did notshow any positive stainned elastin.9.With MTT method,the experimental goup of both HUVEC and HASMC showeda more OD value than the control goup with zero-load vector（P<0.05）.【Conclusion】1.Lentivirus-FGF-18can be successfully packaged with the three plasmid packagingsystem;2.FGFR-2exists at least in the surface of membranes of HUVEC and HASMC. 3.The mRNA of FGF-18can be highly expressed when the HUVECs and HASMCsare infected with lentivirus-FGF-18.4.FGF-18can induce HUVEC to express elastin and fibulin-5,and can enhance theproliferation of HUVEC.5.Whether FGF-18can induce HASMC to express elastic fibers is still need to bestudied.