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目的经口染毒给予大鼠不同剂量环磷酰胺(CTX)以确定脾脏免疫相关指标在大鼠免疫抑制模型中的变化,为毒性评价免疫抑制模型提供科学的检测指标。方法选用40只28日龄Wistar雌性大鼠称重后随机分为对照组及环磷酰胺染毒组(剂量分别为5、10和20 mg/kg·BW)。经口连续染毒环磷酰胺28 d后,取脾脏、胸腺、淋巴结、肝、肾、心经苏木素-伊红(HE)染色后进行形态学观察;FACS法检测脾脏中免疫细胞类型及比例的变化;IHC染色观察免疫细胞形态,FACS法检测脾脏细胞凋亡情况。结果经口给予环磷酰胺28 d后,CTX染毒组大鼠体重显著下降,与对照组比较有统计学意义(P<0.05、P<0.01);5 mg/kg·BW组大鼠脾脏、胸腺指数降低,20 mg/kg·BW组大鼠脾脏指数升高,与对照组比较有统计学意义(P<0.01);组织病理学观察发现脾、胸腺、淋巴结发生不同程度的坏死、凋亡,随着给药剂量的不同,在免疫组织中发生不同程度的纤维结缔组织增生;肝、肾、心脏也发生不同程度的坏死、心肌断裂。脾脏B细胞百分含量随染毒剂量的增加而降低,与对照组比较有统计学意义(P<0.01);5 mg/kg·BW组脾脏T细胞百分含量升高,10和20 mg/kg·BW组脾脏T细胞百分含量降低,与对照组比较有统计学意义(P<0.01);脾脏NK细胞百分含量随染毒剂量升高而升高,10和20 mg/kg·BW组与对照组比较有统计学意义(P<0.01);5 mg/kg·BW组脾脏Th细胞百分含量略有升高,10和20 mg/kg·BW组Th细胞百分含量降低;染毒组脾脏Tc细胞百分含量升高;脾脏细胞凋亡数量随染毒剂量升高而升高,早期凋亡细胞数量与染毒剂量呈正相关性,各染毒组细胞凋亡数量与对照组比较有统计学意义(P<0.01)。结论大鼠CTX 10 mg/kg·BW经口染毒可作为脾细胞免疫抑制模型。CTX通过调节CD8+细胞分化、上调细胞凋亡造成中枢免疫功能低下。
Objective To investigate the changes of immunosuppression in rats induced by different doses of CTX after oral administration of CTX to provide a scientific indicator of toxicity for immunosuppression model. Methods Forty-eight female Wistar rats aged 28 days were weighed and randomly divided into control group and cyclophosphamide-treated group (5,10 and 20 mg / kg · BW). Spleen, thymus, lymph nodes, liver, kidney and heart were stained with hematoxylin-eosin (HE) for morphological observation after oral continuous exposure to cyclophosphamide for 28 days. The changes of immune cell types and their proportion in spleen were detected by FACS The morphology of immune cells was observed by IHC staining and the apoptosis of spleen cells was detected by FACS. Results After 28 days of oral administration of cyclophosphamide, the body weight of CTX-treated rats decreased significantly compared with that of the control group (P <0.05, P <0.01). In the 5 mg / kg · BW group, The thymus index decreased, the index of spleen increased in 20 mg / kg · BW group, compared with the control group (P <0.01). Histopathological examination showed that necrosis and apoptosis of spleen, thymus and lymph node were different , With different dosage, different degrees of fibrous connective tissue hyperplasia occurred in the immune tissue; liver, kidney and heart also have varying degrees of necrosis and myocardial rupture. The percentage of splenic B cells decreased with the increase of dose, which was significantly higher than that of the control group (P <0.01). The percentage of T cells in 5 mg / kg · BW group was significantly increased (P <0.01). The percentage of splenic NK cells increased with the increase of the dose of 10 and 20 mg / kg · BW (P <0.01). The percentage of Th cells in spleens increased slightly in 5 mg / kg · BW group and the percentage of Th cells in 10 and 20 mg / kg · BW groups was lower than that in control group The percentage of Tc cells in the spleen of the poisoned group increased; the number of apoptotic cells in the spleen increased with the increase of the dose; the number of apoptotic cells in the early stage was positively correlated with the dose of the treated cells; More statistically significant (P <0.01). Conclusion CTX 10 mg / kg · BW can be used as a model of immunosuppression in spleen cells. CTX can cause central immune dysfunction by regulating CD8 + cell differentiation and up-regulating apoptosis.