The Study On Relationship Between The Expression Of Immune Genes In Rat With The Constitution Of Deficient Kidney And Kidney Zangxiang Theory

Object: According to the physiological and pathological characteristicsof the constitution of deficient kidney, on the basis of our preliminarystudies, this study aims to improve the present method inducing the animalmodel of constitution of deficient kidney and look for a better method whichaccords with the TCM theory. Furthermore, by screening some specific immunemolecular markers in serum and spleen lymphocytes, and assaying thedifferential expression of immune genes in mRNA, to explore the relationshipbetween the constitution of deficient kidney and immunity and the effectiveChinese medicine to improve the constitution of deficient kidney. This methodbased on difference analysis studying the kidney Zangxiang theory will providea new paradigm for modern research about the kidney Zangxiang theory, enrichand develop the kidney Zangxiang theory.Method: Guided by the principle of “fear impairing kidney”, the methodof “cat frightened rat” was used to induce the mother rat with impairedkidney essence and its baby rats with innate deficient kidney. Then, the babyrats were intimidated to induce a complicated constitution of deficient kidneymodel which represents the combination of innate and acquired deficiencykidney. After the model was used, the rats were given Guifudihuang pills（0.9375g/kg）. The serum of rats were taken to measure IL-2,IL-6,IL-10,IFN-γ and TNF-αby ELISA. The T lymphocytes in spleen were isolated to assay thepositive rate of CD3, CD4and CD8by flow cytometry. The mRNA of lymphocytesin spleen was abstracted to perform gene chip technology. The differentialexpression of immune genes were compared and analyzed among the rats in modelgroup, normal control group and Chinese medicine group.Results:1The results about level of cytokines in serum: Compared with the rats in blank control group, the level of IL-2, IL-10and IFN-γ in modelgroup was lower, the level of IL-6and TNF-α in model group was higher. Thedifference of IL-2, IFN-γand TNF-αwas significant (p<0.05). Compared withthe rats in model group, the level of IL-2, IL-10and IFN-γ in Chinesemedicine group was higher, the level of IL-6and TNF-α in Chinese medicinegroup was lower.2The results about the change of T lymphocyte subgroup inspleen: Compared with the rats in blank group, the positive rate of CD3andCD4in model group was lower, the positive rate of CD8in model group was higher,the ratio of CD4+/CD8+was lower than1.0. The difference of CD4+and CD4+/CD8+ratio was significant (p<0.05). Compared with the rats in model group, thepositive rate of CD3and CD4in Chinese medicine group was higher, the positiverate of CD8in Chinese medicine group was lower, the ratio of CD4+/CD8+washigher than1.0.3The result about differential expression of immune genesin: There are149differential expression genes in spleen lymphocytes betweenmodel and blank control group by the mRNA hybridization.77genes among themare related to immunity. There are45differential expression genes in spleenlymphocytes between Chinese medicine and model group.26genes among them arerelated to immunity. By statistical analysis of the above immune genes,12different expression genes were found in twice hybridization. Among them,5genes were up-regulated and7genes were down-regulated in model group. Afterthe treatment of Chinese medicine, above5up-regulated genes weredown-regulated and7down-regulated genes were up-regulated.Conclusion: The complicated model which represents the combination ofinnate and acquired deficient kidney is feasible to be reproduced. This modelin line with the physiological and pathological mechanism of the constitutionof deficient kidney, reflects the characteristics of TCM constitution theory.The imbalance of immune homeostasis can be found in the rat model of theconstitution of deficient kidney. Some parameters, such as the level of somecytokines are related to the development of the constitution of deficientkidney. The positive rate of CD3, CD4and CD8in T lymphocyte subgroup and CD4+/CD8+ratio can be regarded as an objective index diagnosing theconstitution of deficient kidney. The12differential expression of immunegenes are screened as the important and direct index related to theconstitution of deficient kidney. The CD4+/CD8+ratio and12differentialexpression of immune genes are targets of Guifudihuang pills improving theimmune function in rats with the constitution of deficient kidney.