Clone And Identification Of Precocious Puberty Related Differential Expression Genes And Effects Of Chinese Herb Medicine Of Prescription Nourishing "Yin" And Removing "Fire" On Expression Of These Genes

Precocious puberty is one of the most popular endocrine disorders in children. The incidence of this disease is about 0.6% throughout the world. But until now the pathogenesis of precocious puberty is unclear. In this study, we use RNA arbitrarily primed PCR (RAP-PCR), Dot blot or Northern blot and other molecular biological methods to clone and identify the differential expression genes in danazol induced precocious puberty rat model. Then the expression of these genes in rat and children of different age peripheral blood mononuclear cells are detected by means of RT-PCR. Further we probe the effects of Chinese herb medicine for nourishing "Yin" and removing "Fire" (CHM) on the differential genes. The main results are showed as follows,1. The day of vaginal opening and onset of regular estrus cycle (25.97±2.24 days, 32.43±0.75 days, respectively) was significant (p<0.01) advancement compared with normal rats (34.00±0.98 days, 41.26±2.35 days, respectively) after female rats of 5 days age were treated with danazol 300μ.g. The ovary and uterus organ coefficients of model group rats were higher than that of normal and vehicle groups (p<0.05). And there was no difference about cyclic morphology of exfoliative cells in vaginal smear between model rats and normal rats. These suggested that danazol may induce precocious puberty, the model here is kind of true precocious puberty model.2. Five differential displayed cDNA fragments were separated from danazol induced precocious puberty rat's hypothalamus. Two of them were identified, one is the partial fragment of GnRH cDNA (identity=100%). The expression of GnRHmRNA was significantly up-regulated in model group by Dot blot. Anotheridentified cDNA fragments was named IPP-1 (idiopathic precocious puberty-1), which is 84% identity with homo sapiens KIAA1538 cDNA. The expression of IPP-1 mRNA was down-regulated in model rats by Northern blot and Dot blot.3. CHM might significantly delay the day of vaginal opening and the onset of regular estrus cycle in model group (p>0.05, compared with normal group). The expression of IPP-1 mRNA was increased in CHM group. The number of hypothalamic GnRH immunoreactive positive cells increased in CHM group by immunocytochemistry, and the expression of GnRHmRNA in hypothalamus and its receptor mRNA in pituitary were significantly decreased in CHM group by RT-PCR. These suggested that CHM might inhibit the abnormal hyper-function of hypothalamus-pituitary-ovary axis via reducing the synthesis and release of GnRH, and lowering the responsibility of pituitary cells to GnRH, which may be the primary mechanism of CHM in effective treating the true precocious puberty.4. GnRHmRNA was expressed in peripheral blood mononuclear cells of rats and varies age children. It was significantly increased in precocious puberty children (n=2) compared with pre-puberty (4,6,8 years old) and puberty (17 years old) subjects. Also it was expressed higher in model rats' mononuclear cells than in normal and vehicle groups. Though the expression of KIAA1538mRNA was also detected in blood mononuclear cells, there was no difference among various groups.In sum, the expression and regulation of certain genes may be involved in the pathogenesis of precocious puberty, and the targets of CHM that effectively treat this disease may just at the level of gene.