| Objective: To study the effects of AAV transfected newborn porcine islets (NPI) expressed CTLa4-Ig and immunologic features of xenotransplantation of NPI. Methods: Isolated and purified newborn porcine islets cell were divided into group A (transfected with AAV-CTLa4-Ig) and B (controlled group, untransfected group). Glucose stimulating test (GST) were performed to assay the function of islets cell; RT-PCR and immunohistochemistry were individually to assay the expressing of CTLa4-Ig on mRNA and protein level. 30 drug-induced diabetes Wistar rats were divided into group A, B, C at random. Group A and group B islets were transplanted to left subcapsular of kidney of A and B DM Wistar rats individually, group C as the blank controlled group, (no graft group DM rat). Observe the clinical symptom change of DM Wistar rat more than 90 days on condition that insulin is not administrated the group A, B and C. 5, 10, 15, 30, 60, 90 days after the transplantation of the rats, blood plasma were obtained to assay IL-2, IFN-γ, α-TNF by EL1SA so as to evaluate the rat immune status. 15, 30, 90 days after the transplantation, the subcapsular grafts of left kidney were performed with HE Stain and insulin immunohistochemistry to evaluate the morphology change of the islets cell grafts andthe inflammatory cell infiltrating status around the transplantation situs. Results: Group A and B DM rats show descent of urine sugar and blood sugar level 1 week after the transplantation. The average blood glucose of group A, B and C is 11.36 ±2.69mmol/K 16.11 ±4.16mmol/l, 19.29 ±1.73mmol/l individually, the grafts of group A survive for 38.5 ±24.0 days in average, and for 3 months at most; the group B is 10.7 ± 3.3 days, significant difference existence between them. The survival of DM Wistar rats of group A is 58.7 ± 30.6 days, group B is 28.4 ±6.9 days, group C is 20.4 ±5.8 days. Compare the cytokine expression in the blood plasma of group A and B, group A is obviously lower than group B, and significant difference between them. Electrophoresis (EP) of RT-PCR products in group A islets shows 200bp positive strap representing CTLa4-Ig-mRNA, but no positive strap in group B. Immunohistochemistry staining of group A show large number of CTLa4-Ig positive islet cell in vitro, but no in group B. HE staining and insulin immunohistochemistry of the grafts show that the structure of the islets are intact and no inflammatory cell infiltrate 15, 30, 90days after transplantation of group A, and islets cell are destroyed and disappeared with inflammatory cell infiltrating in group B.Conclusion: AAV-CTLa4-Ig can transfect NPI and express CTLa4-Ig efficiently in vitro, which can keep the intact of islets, inhibit T cell-dependant immunological rejection, and prolong the survival of xenotransplantation islets, which provide experimental data for the study of anti-rejection therapy of cell transplantation with gene modification in clinic.
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