| Part Ⅰ The correlation of ABCA1 gene R219K polymorphism and blood lipid level with abdominal aortic aneurysm[Objective]To investigate the correlation of ABCA1 gene R219K polymorphism and blood lipid level with healthy volunteer and abdominal aortic aneurysm patients.[Method]PCR of 126 cases of abdominal aortic aneurysm patients and 119 healthy volunteer are used to detect SNP patterns in rs2230806 loci of ABCA1 gene and analyze its distribution pattern and the correlation of blood lipid level.To investigate the correlation of ABCA1 gene R219K polymorphism and blood lipid level with healthy volunteer and abdominal aortic aneurysm patients.[Result]①Disease group compared with control group,TG and LDL-C level obviously is higher,TC and HDL-C level obviously is lower.②The result of ABCA1 gene frequency of R219K polymorphism distribution between the two groups,the distribution of the RR and KK genotype,the distribution of R and K allele frequency are different statistical.There was no statistically significant difference on RK genotype distribution.③In disease group,KK and RK genotype carriers have higher level of HDL-C and apoA-I than RR genotype carriers.But there was no statistically significant difference on TQTC and LDL-C.④Logistic analysis showed that R allele may be associated with the onset of AAA.[Conclusion]ABCA1 gene R219K polymorphism KK and RK genotype are related to the HDL-C and apoA-I level.ABCA1 gene R219K polymorphism is related to AAA,and the R allele may be a risk factor of AAA.Part Ⅱ The influence of ABCA1 gene on aortic smooth muscle cell apoptosis and The correlation of ABC A1 with Long noncoding RNA related aneurysmIObjective]To investigate the effect of apoptosis in the human aortic smooth muscle cells with overexpression and knockdown the ABCA1 gene.At the same time,we detect the LncRNA ANRIL,HIF1A-AS1 and AK056155 levels to explore the relationship between them.[Method]ABCA1 gene was harvested in human aortic VSMCs by RNA extraction,reverse transcription and PCR,and the human aortic VSMCs transfected by overexpression vector was constructed through pEGFP-N3.The expression of ABCA1 was verified by qRT-PCR.The apoptotic in human aortic VSMCs with ABCA1-pEGFP-N3 transfected was verified by Annexin V-FITC.The levels of LncRNA ANRIL,HIF1A-AS1 and AK056155 in human aortic VSMCs with ABCA1－pEGFP-N3 transfected were verified by qRT-PCR.Using siRNA technique to knock down ABCA1 in human aortic VSMCs.The efficiency of siRNA knockdown ABCA1 was determined by qRT-PCR technique,and the siRNA with high knockdown efficiency was used for follow-up experiment.The apoptotic in human aortic VSMCs with ABCA1-siRNA transfected was verified by Annexin V-FITC.The levels of LncRNA ANRIL,HIF1A-AS1 and AK056155 in human aortic VSMCs with ABCA1-siRNA transfected were verified by qRT-PCR.[Result]The ABCAl-pEGFP-N3 carrier was successfully constructed.24 hours after transfection of human aortic VSMCs with this carrier,the results of the transfection group were detected:①The expression of ABCA1 in the transfection group was significantly lower than that in the control group(p<0.05).②The apoptotic rate of the aortic VSMC was higher than that of the control group(p<0.05)③The levels of LncRNA ANRIL,HIF1A-AS1 and AK056155 are lower than that of the control group(all p<0.05).The most significant change is LncRNA ANRIL.After transfection of three ABCA1-siRNA into human aortic VSMCs,qRT-PCR confirmed that ABCA1-siRNA-1 had the best interference efficiency,and subsequent experiments were carried out with it.The results of ABCA1-siRNA-1 transfection group were detected:①The expression of ABCA1 in the transfection group was significantly lower than that in the control group(p<0.05);②The apoptotic rate of aortic VSMCs was higher than that of the control group(p<0.05);③The levels of LncRNA ANRIL,HIF1A-AS1 and AK056155 are higher than that of the control group(all p<0.05).The most significant change is LncRNA ANRIL.[Conclusion]① The aortic VSMC cell model of overexpression and knockdown of ABCA1 was successfully constructed.②The overexpression of ABCA1 in vitro cell experiments could decrease the apoptosis of aortic VSMCs,and knockdown ABCA1 could reduce the apoptosis of aortic VSMCs;③At the same time,the overexpression and knockdown of ABCA1 in vitro cell experiments could change the levels of LncRNA ANRIL,HIF1A-AS1 and AK056155,The most significant change is LncRNA ANRIL.④This may indicate that ABCA1 gene involved in the pathophysiological process of aortic VSMCs,and ABCA1 may regulate LncRNA ANRIL,HIF1A-AS1 and AK056155,especially LncRNA ANRIL.Part Ⅲ Effects of ABC A1 Gene-related Long noncoding RNA on Apoptosis and Proliferation of Aortic Smooth Muscle Cells[Objective]To investigate the effect of the overexpression and knockdown LncRNA ANRIL on the apoptosis and proliferation in aortic human smooth muscle cells(VSMCs).[Method]ANRIL gene was harvested in human aortic VSMCs by RNA extraction,reverse transcription and PCR,and the human aortic VSMCs transfected by overexpression vector was constructed through pEGFP-N3.The expression of LncRNA ANRIL was verified by qRT-PCR.The apoptotic,proliferative and Caspase3 and Bcl-2 expression levels of apoptosis-related proteins of aortic VSMCs were detected by Annexin V-FITC staining combined with flow cytometry,MTT colorimetric assay and Western blot technique in aortic VSMC of overexpression LncRNA ANRIL.Using siRNA technique to knock down LncRNA ANRIL in human aortic VSMCs.The efficiency of siRNA knockdown ABCA1 was determined by qRT-PCR technique,and the siRNA with high knockdown efficiency was used for follow-up experiment.Apoptosis,proliferation and Caspase3 and Bcl-2 expression levels of apoptosis-related proteins of aortic VSMC were detected by Annexin V-FITC staining combined with flow cytometry,MTT colorimetric assay and Western blot technique in aortic VSMC of knockdown ABCA1 gene.[Result]The ANRIL-pEGFP-N3 carrier was successfully constructed.24hours after transfection of human aortic VSMCs with this carrier,the results of the transfection group were detected:①The expression of ANRIL in the transfection group was significantly higher than that in the control group(p<0.05);②The apoptotic rate of the aortic VSMCs was higher than that of the control group(p<0.05);③The OD value of cell solution at A = 490nm was lower than that of control group(p<0.05);④The level of Caspase3 was higher than that of the control group,and the level of Bcl-2 was lower than that of the control group(all p<0.05).After transfection of three ANRIL-siRNA into human aortic VSMCs,qRT-PCR confirmed that siRNA-ANRIL-3 had the best interference efficiency,and subsequent experiments were carried out with it.The results of siRNA-ANRIL-3 transfection group were detected:①The expression of ANRIL in the transfection group was significantly lower than that in the control group(p<0.05);(2)The apoptotic rate of aortic VSMCs was lower than that of the control group(p<0.05).③The OD value of cell solution at A = 490nm was higher than that of control group(p<0.05).④The level of Caspase3 was lower than that of the control group,and the level of Bcl-2 was higher than that of the control group(all p<0.05).[Conclusion]①The human aortic VSMCs cell model of overexpression and knockdown of LncRNA ANRIL was successfully constructed.②The overexpression of LncRNA ANRIL in vitro cell experiments could Increase the apoptosis of aortic VSMCs,inhibiting the proliferation,and knowdown LncRNA ANRIL could reduce the apoptosis of aortic VSMCs,promoting the proliferation;③In view of the role of ABCA1 and LncRNA ANRIL in the apoptosis and proliferation of aortic VSMCs,we speculated that the interaction between ABCA1 and LncRNA ANRIL in the pathophysiology of aortic VSMCs play an important role,and thus affect the development of AAA. |
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