Inhibitory effect of cyclosporin A on the immunological rejection of rats with cerebral hemorrhage following transplantation of nerve growth factors transfected glial cells

Rufei Dai; Chao Yan; Lei Wang; Jun Cai; Xiaoming Li; Ning Liu; Fengyi Zhu

摘要

BACKGROUND: At present, it has been confirmed that immunological rejection exists in the cell transplantation in brain tissue, the effects of immunosuppressant on the immunological rejection and the survival of grafts in brain cell transplantation are worthy being investigated further. OBJECTIVE: To observe the immunological rejection after transgeneic cell transplantation in treating cerebral hemorrhage in rats, and investigate the interventional effect of cyclosprin. DESIGN: A randomized controlled study. SETTINGS: Second Affiliated Hospital of Xuzhou Medical College; First Affiliated Hospital of Nanjing Medical University. MATERIALS: Thirty-five healthy clean-degree SD rats of 6-8 weeks old were used, weighing 200-250 g, either male or female; The FACSort flow cytometer (American BD Company) and NYD-1000 image analytical system were used. The rat-anti-rat CD4 monoclonal antibody, rat-anti-rat CD8 monoclonal antibody, and rat-anti-rat MHC Ⅱ antigen monoclonal antibody were purchased from Santa Cruz Company; SP and DAB kits were purchased from Beijing Zhongshan Bio-engineering Company. XSP-8C2 light microscope was the product of Shanghai Zousun Optical Instrument, Co.,Ltd, and KYKY-3800B electron microscope was the product of China KYKY Technology Development Co.,Ltd. METHODS: The experiments were carried out in the animal experimental center of Nanjing Medical University from April to July in 2003. ① Model establishment: The rats were anesthetized, and then the coordinates of left internal capsule were identified, and the needle was withdrawn after 120 μL blood was injected into the internal capsule. Adenoviruses were taken as the carriers, after the astrocytes were successfully transfected by nerve growth factor(NGF) gene, 0.2 mL cell suspension was injected into the sites of cerebral hemorrhage. Thirty successfully established rat models were randomly divided into cyclosporin A group (n=18) and control group, the rats were treated with intraperitoneal injection of cyclosporin A (10 mg/kg per day) intraperitoneal injection of saline of the same dosage from the 1st day after transplantation, once a day for 7 days continuously. ② CD4+ and CD8+ detection: The CD4+ and CD8+ T lymphocytes in caudal vein were counted with flow cytometer at 15 days after treatment. ③ Morphological observation in the transplanted sites: The rats were killed and then brain tissues were taken out, the transplanted sites and the structure of the normal brain tissue around the transplanted sites were observed with light and electron microscopes. ④ Detections of the infiltration of T lymphocyte subsets and expression of major histocompatibility complex (MHC) Ⅱ antigen in the transplanted sites: The image analysis of immunohistochemical sections was performed with the image analytical system, and the integral optical density (IOD) was taken as the statistical value to observe the infiltration of T lymphocyte subsets and expression of MHCⅡ antigen in the transplanted sites, and the normal brain tissue around the transplanted sites were taken as controls. MAIN OUTCOME MEASURES: ① Countings of CD4+ and CD8+ in peripheral blood; ②Results of the morphological observation in the transplanted sites; ③ Infiltration of T lymphocyte subsets and expression of MHC Ⅱ antigen in the transplanted sites. RESULTS: Totally 35 rats were used, and 30 were successfully made into models, 5 died during the treatment, the other 25 were involved in the analysis of results. ① Results of CD4+ and CD8+ T lymphocytes in peripheral blood: The percentages of CD4+ and CD8+ T lymphocytes in the cyclosporin A group were (29.20±3.97)% and (20.65±2.02)%, respectively, which were obviously lower than those in the control group [(47.39±3.01)%, (28.30±2.36)%, t=4.983, 4.012, P < 0.05], and the CD4+/CD8+ ratio was obviously lower than that in the control group (1.41±0.86, 1.68±0.69, t=3. 871, P < 0.05). ② Morphological results in the transplanted sites: Under optical and electron microscopes, the survival region of the transplant was round, and it had an unobvious migration region with the normal brain tissues, the grafts had normal cellular form. Infiltrations of lymphocytes and monocytes were observed in both groups, and mainly located in the transplanted sites, and the expression of lymphocytes in the cyclosporin A group was markedly lower than that in the control group, and no above-mentioned changes were observed in the normal brain tissue around the transplanted sites. ③ Results of CD4+ and CD8+ T lymphocytes and expression of MHC Ⅱ antigen in the transplanted sites: The CD4+ and CD8+ T lymphocytes and expression of MHC Ⅱ antigen in the transplanted sites were observed in both groups. The IOD of CD4+ and CD8+ antigen positive cells in the cyclosporin A group were obviously lower than those in the control group (1.85±0.38, 1.44±0.33; 3.33±0.37, 2.64±0.56, t=4.122, 4.434, P < 0.05), and the IOD of MHC Ⅱ antigen positive cells was markedly lower than that in the control group (0.76±0.22, 0.98±0.24, t=3.885, P < 0.05). CONCLUSION: ① There is immunological rejection in brain tissue after the transplantation of NSC transgeneic glial cells. ② The immunosuppressant of cyclosporin A can reduce the immunological rejection after the cell transplantation.

Inhibitory effect of cyclosporin A on the immunological rejection of rats with cerebral hemorrhage following transplantation of nerve growth factors transfected glial cells

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