左归丸对肾虚仔鼠胸腺内CD4-CD8-T细胞分化的影响

目的:研究左归丸对肾虚仔鼠发育过程中CD4-CD8-T细胞在胸腺内分化的影响以及补肾填精法的应用价值,探索“肾为先天之本”的理论内涵。方法:采用大鼠2∶1进行雌雄配对,3种应激法复合构建肾虚模型,将孕鼠分为正常组,模型组,胸腺肽组(0.003 g·kg^-1),左归丸低、高剂量组(2,8 g·kg^-1)。正常组不造模给予生理盐水灌胃;模型组造模给予生理盐水灌胃,胸腺肽组造模给予胸腺肽胶囊溶液灌胃,左归丸低、高剂量组造模给予左归丸悬浊液灌胃,选取胚胎16,19 d及出生第1天(E16,E19,P1)3个时期,计算仔鼠胸腺指数,电镜观察仔鼠胸腺上皮细胞超微结构形态,酶联免疫吸附测定(ELISA)法检测仔鼠胸腺内胸腺肽β4(TMSβ4)和胸腺肽α1(Tα1)的含量,流式细胞学检测仔鼠胸腺细胞表面CD4,CD8,CD25,CD44表达以鉴定T细胞类型。结果:电镜下模型组胸腺上皮细胞形态不规则,细胞核变形,核染色质明显聚集增加,胸腺肽组和左归丸低、高剂量组胸腺上皮细胞变形不明显;ELISA法检测结果显示,与正常组比较,模型组不同时期的TMSβ4,Tα1含量明显降低(P<0.05);与模型组比较,胸腺肽组和左归丸低、高剂量组胸腺内TMSβ4和Tα1的含量明显升高(P<0.05);流式细胞检测显示,与正常组比较,模型组在E16,E19,P13个时期CD4^-CD8^-T细胞均明显增加(P<0.05),CD4^-CD8^-CD25^-CD44^-T(DN4)明显增加(P<0.05);与模型组比较,胸腺肽组和左归丸低、高剂量组在E16,E19,P1时期CD4-CD8^-T细胞均明显减少(P<0.05),同时DN4也明显减少(P<0.05)。结论:左归丸可以改善肾虚仔鼠胸腺上皮细胞的结构,并通过增加仔鼠胸腺内TMSβ4和Tα1的分泌诱导胚胎期胸腺内T细胞从DN4至DP阶段的分化,说明左归丸通过补肾填精,促进T细胞发育成熟,调节仔鼠肾虚免疫失调状态。

Effect of Zuoguiwan on Differentiation of CD4-CD8-T Cells in Thymus of Kidney-deficient Offspring Rats

Objective:To study the effect of Zuoguiwan on the differentiation of CD4-CD8-T cells in thymus during the development of kidney-deficient fetal rats and the application value of kidney and essence-tonifying method,in order to explore the theoretical connotation of"kidney being the origin of congenital constitution".Method:Male and female rats were paired at a ratio of 2∶1,and three stress methods were combined to construct the kidney-deficient model.Pregnant rats were divided into control group,model group,thymosin group(0.003 g·kg^-1),and low-dose Zuoguiwan group and high-dose Zuoguiwan group(2,8 g·kg^-1).Normal saline was given to the control group,model group was used for modeling and given normal saline by gavage,thymosin group was given thymosin capsule solution by gavage,and low-dose Zuoguiwan group and high-dose Zuoguiwan group were given Zuoguiwan suspension by gavage.Embryos that were 16 days,19 days and born on the first day(E16,E19,P1)were selected to calculate fetal rat thymus index.Electron microscope was used to observe thymic epithelial cell ultrastructure of fetal rats.Enzyme-linked immunosorbent assay(ELISA)method was used to detect thymus thymosinβ4(TMSβ4)and thymosinα1(Tα1)content,fluid cytology detection of fetal rats on the surface of CD4,CD8,CD25,CD44 expression for identifying T cell type.Result:The morphology of thymus epithelial cells in the model group was irregular,the nucleus was deformed,and the chromatin of the nucleus was significantly increased.The results of ELISA showed that the content of TMSβ4 and Tα1 in thymus increased in the model group and the low-dose and highdose Zuoguiwan groups.Flow cytometry showed that,compared with the control group,CD4^-CD8^-T cells were significantly increased in the model group at E16,E19 and P1 stages(P<0.05),suggesting that T cells were blocked during the development from DN to DP.CD4^-CD8^-CD25^-CD44^-T(DN4)was significantly increased(P<0.05),suggesting that T cell development was inhibited during DN4-DP transition.Compared with the model group,CD4^-CD8^-T cells were significantly reduced in low and high-dose groups(P<0.05)at E16,E19 and P1 stages.At the same time,DN4 was significantly reduced(P<0.05),suggesting that Zuoguiwan could promote the development of T cells from DN4 stage to DP stage as thymosin.Conclusion:Zuoguiwan can improve the structure of thymus epithelial cells in rats with kidney deficiency,and induce the differentiation of T cells in thymus from DN4 stage to DP stage by increasing the secretion of TMSβ4 and Tα1 in thymus of embryonic rats,indicating that Zuoguiwan can promote the development and maturation of T cells and regulate the immune status of fetal rats with kidney deficiency by tonifying kidney and spermatogenesis.