The Construction Ad5/f35 Adenovirus Vectors Of Rat Glucocorticoid Receptor (gr) Gene And The Role Of Gr Gene In Curing Adriamycin-nephropathy Model With Glucocorticoid

【Abstract】Objective: To construct recombinant replication-defective human adenovirus serotype 35 vector carrying glucocorticoid receptor. Inject the recombinant adenovirus into adriamycin-nephropathy rat model's abdominal aorta while the distal end and proximal end of abdominal aorta was obstructed,and to explore the role of GR in curing adriamycin-nephropathy model with dexamethasone.Methods: 1. The Rat GR cDNA was obtained after rat glucocorticoid receptor gene from pcDNA1 Neo-Rat GR was digested. The Rat GR cDNA and vector plasmid pDC316 were contransformed into DH-5a bacteria to obtain the recombinant of pDC316-GR. After confirmed by restriction enzyme digesting, PCR and plasmid sequencing, the recombinant of pDC316-GR and the skeleton plasmid Ad5/F35 were contransformed into 293 cells to obtain the toxic species of Ad35-GR, and PCR was used to confirm the product. 2. SD rats were administered 6 mg/kg body wt adriamycin via a single tail-vein injection. The nephropathy model was proved by measuring the total proteinuria of 24 hours, and the serum albumin, cholesterol, creatinine, urea nitrogen and the pathological changes. The semiquantitative RT - PCR was used for detecting the levels of GR mRNA and the Western Blot was used to verify the protein expression of rat GR in kidney. The rats were divided into 4 groups: Group A was nephropathy models and was transfected with Ad35-GR; Group B was nephropathy model and was treated with sham operation; Group C was normal controls and was transfected with Ad35-GR; Group D was normal controls and was treated with sham operation. The recombinant adenovirus was injected into the 2-group-rats'abdominal aorta while the distal end and proximal end of abdominal aorta was obstructed. At the end of 1st, 3rd, 7th, 10th, 14th, 21st, 28th day after GR injection, the rats were sacrificed and the renal cortex was acquired. 3. Rats were divided into 5 groups: GroupⅠwas nephropathy models and was transfected with Ad35/GR and was treated with dexamethasone; GroupⅡwas nephropathy models with sham operation and was treated with dexamethasone; GroupⅢwas nephropathy models and was transfected with Ad35/GR without any GC treatment; GroupⅣwas nephropathy models with sham operation without any treatment; GroupⅤwas normal controls with sham operation. 3 days after the recombinant adenovirus was injected into the 5-group-rats'abdominal aorta, dexamethasone was given in the groupⅠand groupⅡ. The rats'24-hour-urine protein, serum albumin, total cholesterol and the pathological changes was detected and compared, and the effect of dexamethasone was judged by these results. Results: 1. After confirmed by restriction enzyme digesting and PCR, recombinant adenoviral vector carrying GR was successfully constructed,and 2.8×109 PFU / ml titer of Ad35-rat GR was obtained by CsCl gradient purification. 2. The 24-hour-urine protein and serum total cholesterol started to increase 1 week after adriamycin was injected. The 24-hour-urine protein increased a lot 4 weeks after injection. And the pathological changes a lot 6weeks after injection. The expression of GR gene started to increased 3 days after the Ad35/GR was transfected into rats. 7 days after the tranfection, the expression increased to the most and lasted to the 14th day. Then the expression began to decrease. During the 28th day, the expressions of GR gene in these transfected groups have no difference with these notransfection groups. The expression of GR in Group A and Group C have no difference in every time points, and it is the same between Group C and Group D. 3. The level of 24-hour-urine protein, serum total cholesterol in GroupⅣincreased a lot, the serum albumin decreased very much. The 24-hour-urine protein level and serum total cholesterol level in GroupⅠandⅡwere much less than the level in GroupⅣ, but these levels were higher than GroupⅤ. The 24-hour-urine protein level and serum total cholesterol level of GroupⅠdecreased the least; the GroupⅡlevel was a little more than GroupⅠ. The pathology changed the worst in GroupⅣ, and the GroupⅠchanged the least. Conclusion: 1. The results indicated that high titer adenovirus vectors of rat glucocorticoid receptor gene can be successfully constructed. 2. The typical adriamycin nephropathy model was successfully constructed by injecting adriamycin via single tail-vein injection. 3. The GR gene was successfully transfected into rats of adriamycin nephropathy model and normal controls by injecting Ad35-GR into rats'abdominal aorta while the distal end and proximal end of abdominal aorta was obstructed, and the expression of GR have no difference in nephropathy models and normal controls. 4. After Ad35-GR was transfected into kidneys of nephropathy models, the effect of treating nephropathy model with glucocorticoid was much better.