The Preparation Of VCAM-1Monoclonal Antibody Connected SelS Gene Microbubble Ultrasound Contrast Agent Complex And Its Identification

Background and Objective: Vascular endothelial cell dysfunction is a key link ina variety of vascular diseases. SelS has antioxidant capacity to protect endothelial cells.So far, vascular cell adhesion molecular-1(VCAM-1) monoclonal antibody-microbubble contrast agent (MCA)-the target gene-ultrasound exposure (UE) jointlyapplied to the targeted drug or gene therapy has not been reported.This study was to construct pcDNA3.1-SelS eukaryotic expression vector. In thisstudy, SelS gene-containing microbubble contrast agent linked with the monoclonalantibody against VCAM-1(SMMV) was prepared by the multi-stage avidin-biotinbridging chemistry method. Evaluation of the combination of ultrasound microbubblewith ligands lays the material and technical basis for targeted transfection. Exploringefficiency of SMMV mediated gene transfection of rat thoracic aorta provides anexperimental basis for seeking an effective way of gene therapy for vascular disease.Methods:1. Plasmid preparation: pcDNA3.1-SelS transformed and amplified throughcolibacillus. It is extracted in accordance with endotoxin-free plasmid big mention kitand finally identified by EcoRI/XhoI digestion and sequence.2. Preparation of SMMV: Microbubble contrast agent linked with the monoclonalantibody against VCAM-1(MMV) was prepared by the multi-stage avidin-biotinbridging chemistry method. SMMV was prepared by mixing MMV andpcDNA3.1-SelS.3. Manner of Microbubbles identification:①Measuring the microbubbleconcentration by blood count analyzer.②Observeding in shape, size, distribution andstability by ordinary light microscope.③Fluorescently labeled secondary antibody and MMV mixed, dark, incubated on ice. Observeding fluorescence marker situation byfluorescence microscopy.4. The establishment and group of type2diabetic rat model:50healthy maleSprague-Dawley (SD) rats fed with high sugar, high fat diet. After4weeks, the diabeticrat models were induced by intraperitoneal injection of streptozotocin (STZ)(30mg/kg).The diabetic rats were injected with vitamin D3by intraperitoneal(600000U/kg, inthree injections, injection once every other week); After16weeks of feeding, the40type2diabetic rats were randomly divided into five groups: Blank control group (C,n=8), Ultrasound+Microbubbles group (UE+MB, n=8), pcDNA3.1-SelS group (SelS,n=8), SMMV group (SMMV, n=8), Ultrasound+SMMV group (UE+SMMV, n=8).5. Transfection of SMMV: SMMV infusioned through the tail vein in constantrate in2minutes. Ultrasound irradiated rat precordial for3minutes using gene therapyinstrument in30seconds after the end of the injection. The irradiation energy is2.06W/㎝2, continuous pulse. The irradiation time is180s.72hours after transfection, ratswere killed. Take the thoracic aorta, Real-time PCR method analysis the mRNAexpression of SelS. Western blot analysis the protein expression of SelS.Results:1. Amplification of plasmid: The EcoRI/XhoI digestion and sequence resultsshowed that SelS gene segment had been successfully cloned into eukaryotic expressionvector pcDNA3.1. The plasmid concentration was approximately600~1100ng/μl byUV spectrophotometer.2. Determination of the microbubble concentration: Ordinary microbubbleconcentration was about8.0×10~9cells/ml. The MMV concentration was about9.5×10~8cells/ml.3. The microbubbles appearance and microscopic observation: Ordinarymicrobubbles and MMV showed a milky white suspension in appearance. Microbubblesfloating on the liquid surface in static. Ordinary microbubbles compared with MMVusing ordinary light microscope, there was no significant difference in shape, size,distribution.4. The stability of the microbubbles: Ordinary microbubbles and MMV form stillcomplete rules in the microscope at room temperature for one day. Commonmicrobubble concentration decreases at room temperature for one day. MMV onlyscattered at room temperature for one day. MMV compared with ordinary microbubbles,stability slightly worse. 5. The qualitative identification of the MMV: Observed under fluorescencemicroscope, ordinary microbubbles showed no fluorescence. MMV showed brightgreen fluorescence.6. The SelS mRNA expression of rat thoracic aorta: Real-time PCR results showedthat the SelS mRNA expression of the UE+SMMV group was significantly enhanced,higher than the other four groups (P＜0.05); the SelS mRNA expression of the SMMVgroup was higher than the previous three groups (P＜0.05). The SelS mRNA expressionwas no significant difference between the C group and the UE+MB group(P＞0.05).The SelS mRNA expression was no significant difference between the C group and theSelS group(P＞0.05).7. The SelS protein expression of rat thoracic aorta: Real-time PCR results showedthat the SelS protein expression of the UE+SMMV group was significantly enhanced,higher than the other four groups (P＜0.05); the SelS protein expression of the SMMVgroup was higher than the previous three groups (P＜0.05). The SelS protein expressionwas no significant difference between the C group and the UE+MB group(P＞0.05).The SelS protein expression was no significant difference between the C group and theSelS group(P＞0.05).Conclusions:1. SMMV was prepared by the multi-stage avidin-biotin bridging chemistrymethod. It lays the material and technical basis for targeted transfection.2. SMMV enhanced the efficiency that pcDNA3.1-SelS was transfected into ratthoracic aorta. This study provided the experimental basis for seeking an effective wayof gene therapy for vascular disease.