| Cardiovascular diseases(CVDs)have become the leading cause of death worldwide.Arteriosclerosis is the main cause of cardiovascular disease.Experts predict that the incidence of cardiovascular diseases will continue to increase in the next 10 ~ 15 years.Cardiovascular diseases are becoming one of the focuses of disease prevention and treatment.Percutaneous transvascular interventional therapy is one of the most widely used method to treat cardiovascular diseases,but the subsequent in-stent restenosis(ISR)is a serious complication after interventional therapy,which has become another urgent problem to be solved.The injury of vascular endothelial cells and the proliferation and migration of vascular smooth muscle cells(VSMCs)are the important links of ISR.In addition,vascular endothelial cell injury and neointimal hyperplasia play an important role in the formation of atherosclerosis.At present,there are many methods to establish animal models of vascular injury,but their consistency is not good.On the other hand,the rapid development of molecular biology and genomics makes gene therapy possible.HGF is a multipotent growth factor,but its role in cardiovascular system is controversial.This study is divided into three parts.Firstly,to establish animal model of vascular injury,the proliferation of neointimal tissue induced by ultrasound-guided percutaneous balloon angioplasty in rabbit abdominal aorta.Secondly,to observe the expression of HGF gene in rabbit abdominal aorta in vivo and to discuss the effect of vascular injury on gene expression.Lastly,to detect the effect of HGF gene transfection on neointimal hyperplasia and endogenous HGF expression after abdominal aortic vascular injury in rabbits.This study provides a theoretical basis forHGF gene transfection to inhibit early neointimal hyperplasia.Part Ⅰ: The establishment of ultrasound guided neointimal hyperplasia model of abdominal aorta in rabbitsObjective: To investigate the difference of neointimal hyperplasia induced by different pressure balloon under ultrasound guidance.Methods:(1)Twenty New Zealand white rabbits were divided into three groups according to balloon dilatation pressure: 1 atm,5 atm and 10 atm groups,5 rabbits in each group,and 5 rabbits in the normal control group.The diameter of abdominal aorta was measured by transabdominal high frequency ultrasound under general anesthesia.The balloon was selected according to the balloon/vessel diameter=1.2.The right femoral artery was separated and the balloon catheter was guided into the abdominal aorta to the renal artery level under ultrasound guidance.The abdominal aorta blood flow was completely blocked by filling balloon under ultrasonic monitoring.According to different groups,the balloon pressure reached to 1 atm,5 atm and 10 atm.Then pull back the balloon to the bifurcation of iliac artery.The operation was repeated three times with an interval of30 seconds.The diameter of the middle abdominal aorta was measured before,immediately and 2 weeks after the injury.The immediate dilatation rate was calculated.(2)Two weeks after operation,the rabbit abdominal aorta was separated and stained with HE to observe the formation of neointimal tissue.Results:(1)Ultrasound imaging showed that the balloons appressed the vessel wall,and there was no blood flow around the balloon when the balloon inflated in groups 1 atm,5atm and 10 atm.(2)The vascular diameters immediately after operation were significantly larger than those before operation intra-group(P<0.05).There was no significant difference in the intravascular diameters among the three groups before operation,immediately and 2weeks after operation.(3)Two weeks after vascular injury,varying neointimal tissue appeared in three groups at 2 weeks after operation.The ratio of neointimal circumference and area ratio in groups 5 atm and 10 atm was significantly higher than that in group 1 atm.The thickness of neointima increased with the increase of balloon pressure,group 10 atm >5atm >1 atm.Conclusion:(1)High frequency transabdominal ultrasonography was used to measurethe diameter of abdominal aorta in rabbits,balloon size was selected according to the diameter,and ultrasonography was used to monitor the process of vascular injury to ensure the consistency of all experimental animals.(2)The degree of neointimal hyperplasia was related to the balloon filling pressure.Part Ⅱ: the expression of human HGF(hHGF)plasmid transfected into rabbit abdominal aorta and influencing factorObjective: To investigate the in vivo expression of hHGF transfected rabbit abdominal aorta in different doses,different time points and different vascular states.Methods: The experiment was divided into four parts:(1)According to the different doses of hHGF,the experiment was divided into four groups: empty plasmid group(Con),low dose group(Low,0.02mg),medium dose group(Medium,0.2mg),high dose group(High,2mg),3 rabbits in each group.The expression of hHGF mRNA and protein in abdominal aorta of rabbits was detected by RT-PCR and ELISA on the third day after transfection.(2)According to the time point of transfection,the experimental animals were divided into the following four groups: immediate transfection(group Op0),one day transfection(group Op1),three days transfection(group Op3)and seven days transfection(group Op7),3 rabbits in each group.The expression of hHGF mRNA and protein in abdominal aorta of rabbits was detected by RT-PCR and ELISA on the third day after transfection.(3)According to the extent of vascular injury,the experimental animals were divided into three groups: normal rabbit abdominal aorta hHGF transfection(group Nor),partial pull(1/2)immediately after operation hHGF transfection(group 1/2Op),complete pull immediately hHGF transfection(group Op0),3 rabbits in each group.The expression of hHGF mRNA and protein in abdominal aorta of rabbits was detected by RT-PCR and ELISA on the third day after transfection.(4)The experimental animals were divided into two groups according to whether there was vascular injury: hHGF transfection without injury(group HGF)and hHGF transfection with injury(group EI-HGF),12 rabbits in each group.The expression of hHGF mRNA and protein in abdominal aorta of rabbits was detected by RT-PCR and ELISA on the 3rd,7th and 14 th days after transfection.Results:(1)In vivo expression of hHGF in normal rabbit abdominal aorta transfectedwith different doses of hHGF gene.hHGF mRNA and protein were detected in group Medium on the 3rd day after transfection.No significant expression of hHGF mRNA and protein was detected in groups Con,Low and High.(2)hHGF gene transfection was performed at different time points after vascular injury.The expression of hHGF mRNA and protein was detected in group Op7 on the 3rd day after transfection.No expression of hHGF mRNA and protein was detected in group Op0,group Op1 and Op3.(3)The expression of hHGF mRNA and protein was detected in groups Nor and 1/2Op and Op0.The expression of hHGF mRNA and protein in group 1/2Op was significantly lower than that in group Nor.HHGF mRNA and protein was not detected in group Op0.(4)The expression of hHGF mRNA and protein was detected in groups HGF and EI-HGF on the 3rd,7th and 14 th day after transfection.The peak appeared on the 3rd day,and lasted for more than 2 weeks.The expression of hHGF mRNA in group EI-HGF was lower than that in group HGF on the 3rd day after transfection(P < 0.05),and the expression of hHGF protein in group EI-HGF was lower than that in group HGF on the 3rd,7th and 14 th day after transfection.CONCLUSION: In this study,we found that the greater the extent of balloon injury,the greater the effect on in vivo expression of gene transfection.Vascular injury reduced the expression of hHGF in vivo.The optimal dose of gene transfection was 0.2 mg,and the optimal time point of transfection was 7 days after balloon injury,which provided a basis for further experiments.Part Ⅲ: Effect of human hepatocyte growth factor on early intimal hyperplasia of rabbit abdominal aorta after endothelial injury and its mechanismObjective: To investigate the effect of exogenous hHGF transfection on neointima formation after endothelial injury and its mechanism.Methods: Fifty-five healthy New Zealand white rabbits were divided into four groups:simple balloon injury group(EI group,20 rabbits),balloon injury + hHGF transfection(EI-HGF group,15 rabbits),balloon injury + empty plasmid transfection(EI-V group,15rabbits)and normal control group(control group,5 rabbits).Balloon pressure was set at 10 atm.The hHGF transfection time was on the 7th day after balloon injury,and the transfection dose was 0.2mg.The rabbit abdominal aorta was separated at 1,2,4 and 8 weeks aftervascular injury.The neointima of rabbit abdominal aorta was observed by HE staining.Immunohistochemical staining were used to observe the expression of rHGF,c-Met,CD31 and α-SMA in neointima in each group.The expression of endogenous rHGF,c-Met,α-SMA and e NOS in abdominal aorta of rabbits was detected by real-time RT-PCR.Results:(1)hHGF inhibits early neointima formation: Neointima formation was observed in EI group,EI-HGF group and EI-V group after vascular injury,gradually thickening with time,and N/M ratio increased significantly.The ratio of N/M in group EI-HGF was significantly lower than that in group EI-V at 2 weeks(P<0.001).(2)hHGF altered the expression of ECs and VSMCs in neointima: At 2 weeks,the proportion of CD31 positive cells on the surface of neointima in EI-HGF group was significantly higher than that in EI-V group(P< 0.01).The expression of e NOS mRNA in group EI-HGF was significantly higher than that in group EI-V(P<0.001)at 2 weeks after operation.At 2 and 4 weeks,the number of α-SMA positive cells in group EI-HGF was significantly lower than that in group EI-V(P < 0.05).The expression of α-SMA mRNA in group EI-HGF increased gradually and reached the peak at 4 weeks.The expression of α-SMA mRNA in group EI-HGF was significantly lower than that in group EI-V at 2 and 4 weeks(P < 0.001 at 2 weeks and P <0.01 at 4 weeks).(3)Expression of endogenous rHGF and c-Met induced by hHGF: The peak expression of endogenous rHGF mRNA in group EI-HGF was earlier than that in group EI.The expression of endogenous rHGF mRNA in group EI-HGF was significantly higher than that in group EI-V at 2 and 4 weeks after balloon injury(2 weeks: P< 0.01;4 weeks: P< 0.05).At 2 weeks after operation,the expression of c-Met mRNA in group EI-HGF was significantly higher than that in group EI-V(P<0.05).CONCLUSION: Exogenous administration of hHGF can significantly inhibit the formation of neointima,promote re-endothelialization,improve endothelial function and inhibit the proliferation of smooth muscle cells at the early stage after balloon injury.The effect of hHGF may be achieved by increasing the expression of endogenous rHGF and c-Met mRNA. |
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