Research On Effects And Mechanism Of RAAV2/1-Acrp30 On GK Rats With Macroangiopathy

Part 1 Gaining and Clearing of the Recombinant Adeno-associated virus Vector with AdiponectinObjective Adiponectin(Acrp30)was cloned to the recombinant adeno-associated virus vector,the recombinant adeno-associated virus vector with adiponectin(AAV2/1-Acrp30)was gained by packaging and purification.Methods The Acrp30 gene primer was designed based on the corresponding gene sequence GenBank NM₁44744.EcoR I site was introduced into the upstream of the primer and Sal I site into downstream. The total RNA was drawed from adipose tissue,complementary deoxyribonucleic acid(cDNA)was gained by reverse transcription with reverse transcription kit,Aerp30 gene was amplified with the template of cDNA by reverse transcription-polymerase chain raction,then it was cloned into pUC19 vector to construct recombinant plasmid pUC19-Acrp30.The plasmid spUC19-Acrp30 and plasmid pSNAV were sliced up by EcoR I and Sal I enzyme,the fragments were collected and linked with T4DNA ligase at 16℃overnight,recombinant plasmid pSNAV-Aerp30 was obtained.The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM 2000.The G418 resistant cells were obtained consequently.These cells were infected with HSVI-rc/â–³M L2 which has the function of packaging and copying combinant AAV.After purification,the construction of recombinant rAAV2/1-Acrp30 was collected.Results The construction of combinant pSNAV-Acrp30 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequencing showed that the recombinant pSNAV-Acrp30 was correct.The virus titer was about 1.0×10¹²Î¼g/ml.The purity of the the Recombinant AAV2/1 was fairly high using the SDS-PAGE method.Conclusion With this method,rAAV2/1-Acrp30 with high virus titers and purity can be acquired successfully and it could meet the demands of the experimental study of Acrp30 gene therapy of GK rats. Part 2 Comparative study of rAAV2/1-Acrp30 internal expression in RatsObjective To observe the differences of adiponectin expression efficiency in rats by injecting recombinant adeno-associated virus encoding adiponectin with different pathway and dosageMethods Recombinant adeno-associated virus encoding adiponectin gene were injected to two-month-old Wistar rats by hypodermic, intraperitoneal injection and intramuscular injection in 3 different dosages for 21 days.Aorta,muscle,liver and blood serum were obtained after rats were sacrificed.Serum adiponectin and adiponectin expression in aorta, muscle liver tissue were detected by enzyme linked immunosorbent assay and in situ hybridization analysis respectively.The best pathway and dosage of gene therapy was also determined.Results Compared with intramuscular injection of rAAV2/1-Acrp30, adiponectin showed significantly decrease in groups with hypodermic and intraperitoneal injection rAAV2/1-Acrp30 in rats(P＜0.05),as well as concentration dependence were accompanied by dosage rAAV2/1-Acrp30, but serum adiponectin were not change(P＞0.05).Conclusion Intramuscular injection may be the best pathway for gene therapy of adiponectin,the best dosage may be 1×10¹¹v.g/ml or above. Part 3 Establishing Rat Models with Diabetic MacroangiopathyObjective To establish animal model with macroangiopathy in spontaneous diabetic rats.Methods Rat models were set up by feeding Goto-Kakizaki rats with high fat diet and L-NAME.Urine volume,body weight,fasting blood glucose,two-hour postprandial blood sugar,glycosylated hemoglobin,serum adiponectin,serum insulin,cholesterol,triglyceride, low-density lipoprotein and high-density lipoprotein cholesterol in plasma were detected after 8 weeks.Ultramicrostructure and pathology in random aorta were also observed by light microscope and electron microscope.Results Compared with GK control group,body weight gain slowly in model group,urine volume,fasting blood glucose,two hour postprandial blood sugar,glycosylated hemoglobin,cholesterol, triglyceride and low density lipoprotein cholesterol were increased (P＜0.05),decreased serum adiponectin and high density lipoprotein cholesterol in model group compared with that in control group(P＜0.05). The results observed by electron microscope showed that the aortosclerosis in the GK control group was lighter than model group.And it was found that there were not lipid droplet vacuoles in cytoplasm of the endothelial cells,and various cell organs and elastic membrane were existed,but no lipid droplet vacuoles in cytoplasm of the medial smooth muscle cells aorta hypertrophia were found,lipid droplet vacuoles were deposited in endothelial cells,Foam cells were emerged in endarterium and tunica media of aorta,and there were a lot of pathology for aorta and endarterium impaired in model group.Conclusion Animal models for aortosclerosis in spontaneous diabetic rats were established by nicotin based on high fat diet which could be animal models for macroangiopathy in diabetes mellitus. Part 4 Effects and Mechanism of rAAV2/1-Acrp30 on GK Rats with MacroangiopathyChapter 1Effects of rAAV2/1-Acrp30 on Metabolism of Lipid and Glucose and Ultrastructure in Diabetic Rats with AortosclerosisObjective To study the effects of rAAV2/1-Acrp30 on metabolism of lipid and glucose and aorta ultrastructure in GK rats with diabetic aortoselerosis.Methods GK rats with diabetic aortosclerosis were used to be tested models,Thirty GK rats with diabetic aortosclerosis group randomly were divided into three groups,saline,vacuity virus and rAAV2/1-Aerp30 were administered to the rats in model 1 group,model 2 group and rAAV2/1-Aerp30-treated group at a daily respectively.Blood was collected by orbital vein in four weeks,At the end of the 8th week,the levels of fasting blood glucose,two-hour postprandial blood sugar, glycosylated hemoglobin,serum insulin,serum total cholesterol(TC), triglycerides(TG),high-density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)were tested in each group,and the ultrastructures of the aorta were also observed by light microscope and electron microscopeResults Compared with model 1,2 group,rAAV2/1-Acrp30 could reduce the levels of urine volume,fasting blood glucose,two-hour postprandial blood sugar,glycosylated hemoglobin,TC,TG,and LDL-C. increase the levels of body weight and high-density lipoprotein cholesterol(P＜0.05),but serum insulin were not change(P＞0.05).The results observed by electron microscope showed that,as compared with model 1,2 group,the aortosclerosis in the rAAV2/1-Acrp30-treated group was lighter.And it was found that there were few lipid droplet vacuoles in cytoplasm of the blood lipid,various kinds of cell organs and internal elastic lamina were existed,there were no formation of lipid droplet and foam cells in cytoplasm of media smooth muscle.Conclusion rAAV2/1-Acrp30 can regulate lipid metabolism, decrease blood glucose level and has recovering effects on endothelium ultrastructure in aorta. Chapter 2Regulation of rAAV2/1-Acrp30 on Blood Adherence Factor and Peroxsome Proliferator-Activated Receptor in Rats with Diabetic AortosclerosisObjective To study effects of rAAV2/1-Acrp30 on blood adherence factor and peroxisome proliferator-activated receptor in rats with diabetic aortosclerosis.Methods GK rats with diabetic aortosclerosis were tested models. Thirty GK rats with diabetic aortoselerosis group randomly were divided into three groups,model 1 group,model 2 group and rAAV2/1-Acrp30-treated group,while saline,vacuity virus and rAAV2/1-Acrp30 were administered to the rats in model 1 group,model 2 group and rAAV2/1-Acrp30-treated group at a daily oral dose respectively.Blood was collected by orbital vein in four weeks,At the end of the 8th week, the rats were sacrificed,the levels of serum C-reactive protein, E-selectin,serum soluble vascular cell adhesion molecule(sVCAM)and soluble intercellular adhesion molecular(sICAM)were tested in each group.Peroxisome proliferator-aetivated receptorα(PPARα)mRNA, Peroxisome proliferator-activated receptorγ(PPARγ)mRNA and nuclear factor(NF-kB)p65mRNA in aorta were detected by RT-PCR after the aorta was taken out from the rats executed by narcosis.Results Compared with model 1,2 group,the levels of serum C-reactive protein,E-selectin,serum soluble vascular cell adhesion molecule and soluble intercellular adhesion molecular decreased in rAAV2/1-Acrp30 group(P＜0.05),as well as NF-kB p65mRNA in the rAAV2/1-Aerp30-treated group were inhibited(P＜0.05)in rAAV2/1-Acrp30-treated group.PPARγmRNA and PPARαmRNA were upregulation in aorta of rAAV2/1-Acrp30-treated group rats but serum adiponectin was not changed.(P＞0.05)Conclusion rAAV2/1-Acrp30 could play a role in the recovering diabetic aortosclerosis by up-regulating peroxisome proliferator-activated receptor family gene,and also the nuclear factor p65 of aorta and adherence factor with C-reactive protein were suppressed.