| Chapter One Research on pretreatment effect of cyclophosphamide on the NOD/SCID miceObjective:Study the effect difference between single high dose and multiple low dose of cyclophosphamide pretreatment on NOD/SCID mice, and discuss the optimum does.Methods:36NOD/SCID mice bred in SPF environment were randomly divided into five groups. Group I:Negative control group without any treatment; Group II to VI were the experimental group, treated by cyclophosphamide single injection in the dose of200mg/kg,250mg/kg,300mg/kg, and multiple injection in the dose of300mg/kg respectively. The survival status and presence of aGVHD reaction of the experimental mice were recorded. Two mice of each group were sacrificed after one, three and five days respectively. The spleen and the body mass of each mouse were weighed to calculate the mice spleen index of each group. The mice femur of each group were sent to HE staining observation.Results:1, The survival rate of each group was100%;2, The aGVHD scores display: Compared to the negative control group, the scores of each experimental group were statistically significant, P<0.05; Compared to other experimental groups, the score of250mg/kg cyclophosphamide single injection group was statistically significant, P<0.05.3, The spleen index of all experimental groups were statistically significant compared to the negative control group, P<0.05; Spleen index of each experimental group after injection in the third and fifth day were significantly different from the first day, P<0.05.4, HE staining result showed that different dose of cyclophosphamide pretreatment to mice caused the femur structure change based on the micrography record, among which three days after injection of the250mg/kg single dose group presented the best pretreatment result.Conclusion:1, Cyclophosphamide intraperitoneal injection in different doses do effect in pretreatment to NOD/SCID mice, furthermore, the250mg/kg cyclophosphamide single injection group gave the best result.2, When given the same cyclophosphamide injection dose, pretreatment result of single high dose are better than multiple low dose.3, The optimum pretreatment effect is relevant to the injection time, single high dose injection show the best result after three days.4, The best myeloablative dose of cyclophosphamide for the NOD/SCID mice was as follows:250mg/kg, three days in advance, a single intraperitoneal injection. Chapter Two Cell immune system reconstruction of the NOD/SCID mice by transplanting the bone marrow CD34+cell of the SD rats of ALCL and assessmentObjective:Reconstructing the immune system of the NOD/SCID mice by transplanting the bone marrow CD34+cell of the SD rats of ALCL and assessing.Methods:1, Establish the human anaplastic large cell lymphoma animal (ALCL) mode in the male SD donor rats with functioning immune system by using ACLC cell line, Karpas-299.2, Sort the bone marrow CD34+cells of these SD rats by the method of1MB.3, Pretreat the female NOD/SCID receptor mice with cyclophosphamide in the optimum dose which derives from the experimental result in chapter one.4, Transplant the sorted bone marrow CD34+cells from SD rats above to the female NOD/SCID mice pretreated by cyclophosphamide through tail vein injection, reconstruct its immune system and assess.Results:1, Establishing the ALCL animal mode of male SD donor rats with functioning immune system was identified to be successfully by HE staining and immunohistochemistry.2, Significant aGVHD reaction was found after transplantation among the experimental group of NOD/SCID mice. The success rate of transplantation is80%.3, Based on the PCR result, the expression of specific Sry gene on Y chromosome can be detected in the peripheral blood and spleen of the female NOD/SCID mice within1-4weeks after transplantation.4, By the method of FCM, expression of T, B lymphocytes in the peripheral blood of the NOD/SCID mice are detected in varying degrees, within1-4weeks after immune reconstitution.5, The expression of CD20of SD rats are detected in the spleen, sternum and femur of the NOD/SCID mice in the fourth week after transplantation by immunohistochemical.Conclusion:1, The ALCL animal mode can be established in the male SD rats with functioning immune system by continuous hige-dose Karpas-299cells transplantation (107/per mice/day).2, High purity and activity of the bone marrow CD34+cells can be sorted by using the technology of1MB.3, The immune system of the NOD/SCID mice pretreated by cyclophosphamide can be reconstructed by transplanting5×106of the bone marrow CD34+cell. |
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