Study On The Biological Mechanisms Of Twin Cerebral Palsy With Spleen And Kidney Deficiency Syndrome

ObjectivesO To analyze the risk factors for cerebral palsy, and probe the distribution characteristics of spleen deficiency syndrome and kidney deficiency syndrome through the disease and syndrome survey method for cerebral palsy twins.②To reveal the relationships between the cellular immunity, humoral immunity, serum elements with CP of spleen and kidney deficiency syndrome through checking biochemical indexes testing, and to explore the relationships between the immune function and spleen and kidney of TCM.③To explore the molecular mechanism of CP with spleen and kidney deficiency syndrome based on applying cDNA microarray technology to four "CP-Normal" monozygotic twins.MethodsO Investigation on combination of disease and syndrome of CP43 CP twins were studied by the combination of disease and syndrome method based on spleen and kidney deficiency syndrome questionnaire which was made by our investigation team, and the etiology of CP and distribution characteristics of spleen deficiency syndrome and kidney deficiency syndrome were explored.②Investigation on levels of physical and chemical indexesBlood samples were analyzed for determining related indexes. Immunoglobulin (IgG, IgA, and IgM), C3 and C4 were analyzed using immune turbidity technology. The proportion of CD3+ T cells, CD4+ T cells and CD8+ T cells were compared by flow cytometry. The serum level of Zn, Fe, Ca, Mg and Cu were determined using atomic absorption spectroscopy method.③Screening key genes of CP with spleen and kidney deficiency syndrome through cDNA microarray techniques Four "CP-normal" MZ with spleen and kidney deficiency syndrome were selected as subjects of cDNA microarray test. The gene expression data were functionally annotated through several databases such as GO, KEGG, FatiGO+, etc.④Clinical verification of cDNA microarray resultsIn order to exclude error and false positive results, part of differentially expressed genes key genes screened by cDNA microarray test were further verified through RT-PCR.ResultsO Investigation on combination of disease and syndrome of 43 twins cases of CP patients with spleen and kidney deficiency syndromeThe risk factors for CP with spleen and kidney deficiency syndrome were low birth weight, hypoxia asphyxia, premature birth, eat less irritability after birth, etc. 79.59% of the investigated patients had two or more risk factors for CP. Syndromes survey indicated that most of patients with spleen qi and kidney essence deficiency syndrome. There was consistency of suffering CP and spleen and kidney deficiency syndrome between MZ twins, although the differences in severity of illness were obvious.②Investigative results of physical and chemical test in 51 cases of CPCD3+T cells and CD8+T cell percentage was found to be significantly lower in peripheral blood of CP with spleen and kidney deficiency syndrome compared to controls (P<0.01), and CD4+/CD8+T cell ratio was significantly higher (P<0.01) in CP patients than the controls. The serum concentrations of IgG were found to be significantly lower (P<0.01), IgM significantly higher (P<0.05), and C3 significantly lower (P<0.01) in CP patients than the controls. The serum levels of elements Zn, Fe, Ca and Cu were significantly lower than the control group (P<0.01). For serum level of Mg, the differences between the groups were not significant (P>0.05).③Gene expression profile results of 4 "CP-Normal" MZThere were 2703,20,130 and 350 differentially expressed genes screened from four "CP-normal" MZ, respectively.GO and PATHWAY annotation results of these genes showed that immune-related genes and pathways significantly expressed in all 4 groups of cDNA microarray results, and cytokine-cytokine receptor interaction pathway was the common annotation pathway of differentially expressed genes screened from 4 chips.④RT-PCR verification of 15 differentially expressed genesUsing RT-PCR to verify the screened differentially expressed gene, and the results preliminarily indicating that FIGN gene was significantly lower expressed in all CP patients with spleen and kidney deficiency syndrome than normal, and PTGER2 gene was significantly higher expressed in all CP patients with spleen and kidney deficiency syndrome than normal. The other 13 genes showed no expressed significance between the two groups (P>0.05).ConclusionO Spleen-qi deficiency syndrome and kidney essence deficiency syndrome are the basic syndromes of CP.②There are multiple risk factors for CP, and most CP patients have two or more risk factors.③CP patients with spleen and kidney deficiency syndrome show abnormal cellular immune function and abnormal humoral immune function. The serum levels of elements Zn, Fe, Ca and Cu were significantly lower than the normal children.④The occurrence of CP with spleen and kidney deficiency syndrome is related to multiple differentially expressed genes, and especially the immune genes.It can be inferred that the key pathogenesis of CP with spleen and kidney deficiency syndrome is related to immune-genetic disorders.⑤Significant expression of FIGN and PTGER2 genes according to results of RT-PCR suggests that there is a close relationship between FIGN, PTGER2 gene and CP with spleen and kidney deficiency syndrome. It will provide the basis for the disease and syndrome's further study.