四种环磷酰胺诱导免疫抑制小鼠模型的对比研究

Comparison of Four Different Immunosuppression Models Induced by Cyclophosphamide in Mice

目的研究并对比环磷酰胺4种不同造模方式对小鼠免疫功能的影响。方法采用4种不同方式诱导环磷酰胺小鼠免疫低下模型,A组:第1、3、5天模型组小鼠腹腔内注射环磷酰胺80 mg/kg;B组:连续5 d模型组小鼠腹腔内注射环磷酰胺60 mg/kg;C组:第1、3、5、7、9、11、13天模型组小鼠腹腔内注射环磷酰胺60 mg/kg;D组:第1、4、7、10、13天模型组小鼠腹腔内注射环磷酰胺60 mg/kg,观察并比较不同造模方式对小鼠一般免疫学指标(体质量、胸腺指数、脾指数、全血白细胞及淋巴细胞计数)、细胞免疫(迟发型变态反应)和体液免疫(半数溶血值的测定)等相关指标的影响。结果各实验组中模型组的相关检测指标均有不同程度的改变,实验A组模型组较空白对照组第7天小鼠体质量、胸腺指数、脾指数及全血白细胞计数分别下降19.15%、67.69%、67.59%、83.87%,而第14天模型组小鼠体质量下降17.91%,而胸腺指数、脾指数及全血白细胞计数分别升高了7.14%、173.89%、67.76%;实验B组模型组较空白对照组第7天小鼠体质量、胸腺指数、脾指数分别下降了15.64%、64.91%、64.60%,第14天模型组小鼠体质量、胸腺指数分别下降17.86%、48.00%,而脾指数升高了148.80%;实验C组模型组较空白对照组小鼠体质量、胸腺指数、脾指数、全血白细胞计数及淋巴细胞计数分别下降11.74%、82.61%、37.42%、88.40%、91.44%;实验D组模型组较空白对照组小鼠体质量、胸腺指数、脾指数、全血白细胞计数及淋巴细胞计数分别下降10.83%、68.31%、14.34%、66.15%、73.42%,DTH实验中,免疫低下小鼠左右耳重量差异减小66.88%,体液免疫中,免疫低下小鼠血清HC50下降74.52%。对比A、B两组,结果显示实验第14天相关指标与第7天相比均有不同程度的恢复且A组小鼠短期内免疫低下状态较为严重,B组小鼠免疫低下状态维持较持久;延长造模周期时,对比C、D组结果显示,两种方式均能造成小鼠免疫低下,且C组造成的短期内小鼠免疫低下状态较D组严重。结论上述4种方法均可成功建立小鼠免疫抑制模型,免疫低下的程度与注射环磷酰胺的次数、剂量、间隔时间及检测终点均有密切关系,可根据具体的药物评价要求,选择合适的评价模型。

Objective The aim of present study is to compare the effects of four different modeling method of cyclophosphamide on the immune function of mice.Method Four different ways were employed to develop immunocompromised mouse models by cyclophosphamide,Group A:mice were intraperitoneally injected with cyclophosphamide 80 mg/kg on days 1,3,and 5,Group B:mice were intraperitoneally injected with cyclophosphamide 60 mg/ kg for 5 consecutive days, Group C: mice were intraperitoneally injected with cyclophosphamide 60 mg/ kg on days 1, 3, 5, 7, 9, 11 and 13, Group D: mice were intraperitoneally injected with cyclophosphamide 60 mg/ kg on days 1, 4, 7, 10 and 13). The general immunological indexes of mice (body weight, thymus index, spleen index, whole blood leukocyte and lymphocyte count ), cellular immunity ( delayed allergic reaction ) and humoral immunity (determination of half hemolysis value) were observed and compared in the four different models. Result In each experimental group, the relevant detection indexes changed in varying degrees. Compared with the blank control group, the body mass, thymus index, spleen index and WBC count of mice in experimental group A and model group decreased by 19. 15%, 67. 69%, 67. 59% and 83. 87% respectively on the 7th day, while the body mass of mice in model group decreased by 17. 91%, while the thymus index, spleen index and WBC count increased by 7. 14%, 173. 89% and 67. 76% respectively on the 14th day;Compared with the blank control group, the body mass, thymus index and spleen index of mice in experimental group B and model group decreased by 15. 64%, 64. 91% and 64. 60% respectively on the 7th day, and the body mass and thymus index of mice in model group decreased by 17. 86% and 48. 00% respectively on the 14th day, while the spleen index increased by 148. 80%;Compared with the blank control group, the body mass, thymus index, spleen index, WBC count and lymphocyte count of mice in experimental group C and model group decreased by 11. 74%, 82. 61%, 37. 42%, 88. 40% and 91. 44% respectively;Compared with the blank control group, the body mass, thymus index, spleen index, whole blood leukocyte count and lymphocyte count of mice in experimental group D and model group decreased by 10. 83%, 68. 31%, 14. 34%, 66. 15% and 73. 42% respectively. In DTH experiment, the weight difference between left and right ears of immunosuppressive mice decreased by 66. 88%, and in humoral immunity, the serum HC50 of immunosuppressive mice decreased by 74. 52%. In the experiments of group A and B, the relevant detection indexes recovered in varying degrees on the 14th day compared with the 7th day. Meanwhile, the immunosuppressive state of group A mice was more serious in the short term, while the immunosuppressive state of group B mice remained more lasting. Conclusion The above four method could successfully establish immunosuppressive model in mice, and the times, dose, interval days of intraperitoneal injection of cyclophosphamide and the detection endpoint will affect the effect of the immunosuppressive state of mice. So the appropriate model can be selected according to the specific evaluation requirements of drugs.