The Research Of Rh-HGF Combined With Rh-VEGF165 In Promoting Vascular Endothelial Cell Proliferation And The Treatment Of Lower Limb Ischemia In Rats

Objective:To construct the model of experimental rats with lower limb ischemia and cultivate human umbilical vein endothelial cells(HUVECs),the use of recombinant human vascular endothelial cells growth factor 165(rh-VEGF165)and recombinant human hepatocyte growth factor(rh-HGF)stimulate HUVECs and ischemic limb,to evaluate the proliferation of HUVECs and formation of capillary-like tubes in vitro,neovascularization in the rat ischemic limb.Methods:1.Evaluate the influence of rh-VEGF165 or rh-HGF to HUVECs' proliferation:HUVECs were cultured in different concentration of rh-VEGF165 or rh-HGF(0ng/ml,5 ng/ml,10 ng/ml,30 ng/ml,50 ng/ml,75 ng/ml,100 ng/ml,120 ng/ml),then evaluated the cell proliferation by CCK-8.2.Evaluate the influence of rh-VEGF165 combined with rh-HGF to HUVECs' proliferation :HUVECs were cultured in different concentration of rh-VEGF165 combined with rh-HGF(0 ng/ml+0ng/ml,5 ng/ml+5ng/ml,10 ng/ml+10ng/ml,30ng/ml+30ng/ml,50ng/ml+50ng/ml,75ng/ml+75ng/ml,100ng/ml+100ng/ml,120ng/ml+ 120ng/ml),then evaluated the cell proliferation by CCK-8.3.Evaluate formation of capillary-like tubes in vitro:HUVECs were stimulated in different factor such as control group,100ng/ml rh-VEGF165,100ng/ml rh-HGF or100ng/ml rh-VEGF165 combined with 100ng/ml rh-HGF,then evaluated the formation of capillary-like tubes by inverted contrast phase microscope.4.Evaluate effection of rh-VEGF165 and rh-HGF in vivo :established the rat model of lower limb ischemia,Models were stimulated in different factor such as control group,100ng/ml rh-VEGF165,100ng/ml rh-HGF or 100ng/ml rh-VEGF165 combined with 100ng/ml rh-HGF,took the gastrocnemius muscle from every groups in the day 28.Then the HE and IHC were used to analysed cell nucleus and capillaries.Results:1.rh-VEGF165 stimulated HUVECs' proliferation : HUVECs' OD value was increased with the addition in rh-VEGF165 concentration.There were signficant differences compared with the control group(P<0.05).When the concentration of rh-VEGF165 was 10ng/ml,the OD value could achieve the highest.But the OD value was no statistically signficant if continued increasing the rh-VEGF165' concentration(P>0.05).2.rh-HGF stimulated HUVECs' proliferation: HUVECs' OD value was increased with the addition in rh-HGF concentration.There were signficant differences compared with the control group(P<0.05).When the concentration of rh-HGF was100ng/ml,the OD value could achieve the highest.But the OD value was no statistically signficant if continued increasing the rh-HGF concentration(P>0.05).3.rh-VEGF165 combined with rh-HGF stimulated HUVECs' proliferation :HUVECs' OD value was increased with the addition in rh-VEGF165 combined with rh-HGF concentration.There were signficant differences compared with the control group(P<0.05).When the concentration of rh-VEGF165 combined with rh-HGF respectively was 100ng/ml+100ng/ml,the OD value could achieve the highest.But the OD value was no statistically signficant if continued increasing the two combined concentration(P>0.05).4.When the concentration of rh-VEGF165,rh-HGF,rh-VEGF165 combined with rh-HGF respectively was 100ng/ml,100ng/ml,100ng/ml+100ng/ml,HUVECs' OD value in combined group was the highest,the second was in rh-HGF group,the least was in rh-VEGF165 group,there were signficant differences in the three groups(P<0.05).5.Formation of capillary-like tubes in vitro :the experiment group could saw obvious capillary-like tubes structure,especially in combined group,while there were no apparent results in control group.6.The result of HE : the rh-VEGF165 group ? rh-VEGF165 combined with rh-HGF group had signficant differences compared with the control group(P<0.05),while rh-HGF group had no statistically signficant compared with the control group(P>0.05).there were signficant differences in between rh-VEGF165 and thecombined group(P<0.05).7.the result of IHC :The rh-VEGF165 group ? rh-VEGF165 combined with rh-HGF group had signficant differences compared with the control group(P<0.05),while rh-HGF group had no statistically signficant compared with the control group(P>0.05).there were signficant differences in between rh-VEGF165 and the combined group(P<0.05).Conclusion:rh-VEGF165 combined with rh-HGF can promote angiogenesis in vivo and vitro,and its effection was stronger than rh-VEGF165 or rh-HGF used alone.The combined application can promote proliferation of vascular endothelial cells and capillaries,and improve the symptoms of lower limb ischemia.