| Background and purpose: The chief deathy cause of Type2diabetes mellitus(Type2diabetes mellitus, T2DM) is associated with macrovascular complications,atherosclerosis (Atherosclerosis, AS) is the pathological basis of large vascular disease,oxidative stress is one of the main causes of triggering AS. Selenoprotein S(Selenoprotein S, SelS) may be involved in scavenging oxygen free radical, resisttingoxidative stress-induced endothelial cell dysfunction. The study main purpose is toapply Microbubble ultrasound contrast agent (MCA) and ultrasonic irradiation (UE)combined(MCA-UE), mediate the SelS gene to transfect to T2DM-AS rat model.Observe the SelS expression of vascular endothelial cells in the rat model of T2DM-AS;and the change of TC(Total cholesterol), TG(triglyceride), LDL-C(low densitylipoprotein cholesterol), SAA(Plasma amyloid A), CRP(C-reactive protein), NO(nitricoxide) in serum, ET-1(endothelin-1) in Plasma. Explore the relationship of SelS kurtosisexpression of endothelial cells and gene transfection dose, time after gene transfection,and provide some experimental evidence for the in-depth study of SelS function, and toexplore new strategy for prevention and treatment of T2DM and macrovascular disease.Method：1. Microbubble complex preparation: binding avidin streptavidin and VCAM-1monoclonal antibody to the microbubble shell step by step, through the multi-stageavidin-biotin multi-level bridge chemical method, preparation of targeting ultrasonicmicrobubbles complex with the target gene SelS.2. Purchase140numbers healthy male SD rats of8weeks old (180-200g), thenrandomly divided into normal control group (10numbers, common feed, NC group),T2DM-high fat feeding transfected group (120numbers, TR group), T2DM-high fat feeding untransfected group (10numbers, NTR group). According to each rat is givendifferent dose of plasmids (50μg/kg,100μg/kg,200μg/kg), and different days fromtransferred to sacrificed(1day,3days,5days and7days), TR group divided into12subgroups (each subgroup of10numbers), are set to TR50-1, TR50-3, TR50-5, TR50-7,TR100-1, TR100-3, TR100-5, TR100-7, TR200-1, TR200-3, TR200-5, TR200-7group.3. After high-sugar and high-fat feed for4weeks, intraperitoneal injection ofstreptozotocin (STZ)30mg/kg, buid type2diabetes model. Subsequent application ofvitamin D3intraperitoneal injection of600000U/kg (the total dose average threeinjections weekly for the first three weeks after modeling), to high-sugar and high-fatfeed for16weeks. When25weeks of age, transfect the TR group, inject the preparedtargeting ultrasound microbubble complexes mixed through tail vein (NC group andNTR group as negative control, injection of an equal volume of saline and MMV mixedliquid by tail vein), irradiate the area of thoracicaorta surface projection by the genetransfection therapeutic apparatus. Respectively to death TR group after transfection of1day,3days,5days and7days (NC group and NTR group after transfection of1day),specimens of serum and thoracicaorta, determinate the blood lipids, the serum SAA,CRP, NO and plasma ET-1content; extraction of total RNA and total protein in ratthoracicaorta, detection of SelS mRNA level expression with Real-time PCR method,detection of SelS protein expression by western blot method; fix the rat thoracicaortatissue of0.5cm in10%neutral formalin solution, routine HE staining, to evaluate theT2DM-AS model.Result：1. Successful preparation of microbubble complex, as the static state, the upperlayer is white opacity, lower the clear liquid. The ordinary microscope showed circularbubble structure, the qualitative identification of microbubbles complex is by thefluorescence microscope, the microbubbles can appear around the green fluorescence.2. The successful establishment of T2DM-AS model: weight, blood glucose inNTR group is higher than the NC group (P<0.05). Microscope showed that in the NCgroup, aorticendothelial smooth, no fibrous hyperplasia and fatty streak formation; inNTR and TR group, smooth muscle cells grow larger, round, cell shorter, migrate to theendothelial, endothelial cell aggregation deformation, formation of the fibrous cap;endometrial visible tip size of cracks, loose film structure, elastic fiber hyperplasia, ashyalinization, form fatty streak. The above pathological changes prompted that the NTRgroup and TR group rats have the formation of atherosclerosis. 3. SelS expression abundance and aging study: every groups thoracicaorta haveSelS mRNA expression, TR50-5, TR100-5, TR200-3group corresponding to eachplasmid dose group transfected are the highest expression, TR50-5>TR100-5>TR200-3group; TR100-5group SelS protein expression is higher than NC group and NTR group(P<0.05), the rest TR group SelS protein expression has no statistically significant.TR100-5group SelS mRNA and protein expression is the highest abundance.4. Determination result of blood lipids, serum SAA, CRP, NO, and plasma ET-1:Compared with NC group, blood lipids (TC, TG, LDL-C) and serum NO of NTR groupand TR100-5group rats were significantly higher (P<0.05), but no significantdifference between NTR group and TR100-5group (P>0.05). Compared with NC group,the serum SAA, CRP and plasma ET-1levels in NTR group and TR100-5group wassignificantly increased (P<0.05), transfection SelS by a single tail vein injection SMMVcombined with ultrasound irradiation, can significantly reduced the levels of serumSAA, CRP and plasma ET-1(TR100-5group is lower than NTR group, P<0.05).Conclusion:1. Ultrasound microbubble can improve SelS gene targeting transfection efficiencyof type2diabetes rat thoracicaorta. Its role is related with the plasmid dose transfectedand the time. The best SelS expression capacity appeared when gene dose of100μg andtime of5days, aging for one week.2. After plasmid dose100μg/kg transfected5days, the level of ET-1reduced,endothelial function may be improved, with the level reduction of inflammatory factors.It is prompted that SelS gene transfection could play a certain extent role of endothelialprotection, which may be related with lower levels of inflammatory factors. |
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