A Study Of The Mechanism Of GH/IGFs On The Growth Of Human Uterine Leiomyoma Cells In Vitro

Objective:GH/IGFs are correlated with the genesis and growth of tumor and affect the biological behavior of neoplasm.Uterine leiomyoma also expresses GH/IGFs.Recent study indicates that IGF-1 and IGF-2 are important mediators of the growth of leiomyoma.Therefore,we speculated GH/IGFs may be involved in the development of leiomyoma.The aim of this study was to demonstrate the effects of different doses of rhGH,rhIGF-1 and rhIGF-2 on the growth of leiomyoma cells and explain the mechanism of their actions on leiomyoma cells in vitro.Methods:26 of Uterine leiomyoma tissues were enzymatically digested for use in primary cell cultures.11 of samples were cultured successfully,5 of them were used for the experiments.The confluent cells about 60%were treated with various concentrations(blank,1ng/ml,10ng/ml,100ng/ml) of rhGH,rhIGF-1 and rhIGF-2. After 48 hours,we underwent the following experiments:(1) The morphological changes were observed with contrast microscopy;(2) The methods of in situ hybridization and image analysis technique were utilized to detect the expression of GHR mRNA and IGF-1R mRNA before and after adding hormone;(3) Flow cytometry(FCM) was used to analyzing the cells cycle.Results:1.The effect of cell proliferation:the growth of cells was observed by the contrast microscopy after 48 hours adding hormone,and we found that the cells of GH 10ng/ml,100ng/ml,IGF-1 10ng/ml,100ng/ml and IGF-2 100ng/ml groups were denser than the control group.The changes of GH groups were more obviously. The same as above,the cells of S stage increased gradually with the upgrade of the concentration of GH,IGF-1,according to the results of flow cytometry;however,in the groups of IGF-2,the cells of G2 stage increased more obviously.These results suggested that GH,IGF-1 and IGF-2 could promote the protein synthesis and mitosis of leimyoma cells in vitro.2.The results obtained in situ hybridization were listed briefly as following:(1) In 10ng/ml and 100ng/ml groups,the expression of GHR mRNA was significantly higher than that of the control group(P＜0.01).With the raising of GH concentration,the expression of GHR mRNA was increased significantly.It suggested that GH could up-regulate the expression of GHR mRNA in a dose-dependent manner when the concentration was higher than 10ng/ml(2) The effect of different IGF-1 concentrations on the expression of the IGF-1R mRNA in leiomyoma cells:the IGF-1R mRNA levels in the control group was remarkable higher than that of the 10ng/ml and 100ng/ml groups(P＜0.01).The higher concentration of IGF-1 was treated,the lower expression of the IGF-1R mRNA was obtained.(3) The effect of different concentrations of IGF-2 on the expression of the IGF-1R mRNA in leiomyoma cells:There was no significant difference in the expression of the IGF-1R mRNA among 1ng/ml,10ng/ml and the control groups (P＞0.05),but compared to the control group,the expression of the 100ng/ml group was decreased greatly(P＜0.01).So IGF-2 has no effect on the expression of IGF-1R mRNA when the concentration was lower than 10ng/ml,and high concentration of IGF-2(100ng/ml) could down-regulate the expression of IGF-1R mRNA.Conclusions:(1) GH,IGF-1 and IGF-2 can promote the proliferation of leiomyoma cells in vitro.With the increasing concentration gradient of GH and IGF-1,the number of the leiomyoma cells of S stage raised gradually;IGF-2 can increase the cell number of G2 stage more obviously;(3) GH can up-regulate the expression of GHR mRNA when the concentration is higher than 10ng/ml;IGF-1 can down-regulate the expression of IGF-1R mRNA,and the expression depended on the dose of IGF-1;High concentrations of IGF-2(100ng/ml) can decrease the expression of IGF-1R mRNA.