The Building of Letrozole Induced PCOS Rat Model and the Screening and Validating of Relevant microRNAs
|School||Huazhong University of Science and Technology|
|Course||Obstetrics and Gynaecology|
|Keywords||microRNAs polycystic ovary syndrome rat animal model gene chip|
Objective:1. To establish the polycystic ovary syndrome (PCOS) rat model using the Letrozole. To analyze and compare the changes of serum sex hormone T, E2, LH, the character of insulin resistance, ovarian morphology.2. To screening the different expression of microRNAs between the two groups, and to explore the possible molecular mechanism of microRNAs in PCOS patiants’pathologic physiology.Methods:1. Female Wistar rats of 6W old were divided randomly into two group with 20 numbers each randomly, establishing the PCOS rat model by irrigating Letrozole 1mg/(kg.d) which was dissolved in the 1% carboxymethylcellulose (CMC) into rat’s stomach once a day for 21 days, the control group also were raised normally. Prove the model with the methods as follow: We began to give the rats vaginal smear to detect their oestrous cycle .We tried our best to control the rats in the anoestrum period when the medication period was over .Using the chloral hydrate to anaesthetize the rat, then we cut off their two ovaries, drew blood from their heart, and centrifugalzed the blood for serum. Detect Testosterone, Estradiol, Luteinizing hormone levels, fasting blood glucose(FBG)by Automatic chemiluminescence detector and detect fasting insulin (FINS) levers by radioimmunoassay (RIA) and calculate the insulin resistant index (HOMA1-IR), measure the ovarian weights and body weights and view ovarian morphological changes by HE stain,.2. We selected randomly 3 rats from the model group and control group, used the intraperitoneal injection method with 10% chloralic hydras to anesthetize the rat, then drew blood from heart and quickly took out their bilateral ovaries, weighed them and chose one of each rat’s two ovaries to use liquid nitrogen freezing fixed preservation. Then we sent the samples to CapitalBio Corporation who used the latest version 15.0 LC-science microRNAs gene expression profile microarray chip to screen.Results:1. The vaginal smear showed that the model group had no significant oestrous cycle on the contrary, the controls showed normally. The HE stain of each group’s ovarian slices showed that in the PCOS models, the number of follicular cysts were increased, while the developing follicles and corpus lutea (CL) were significantly reduced, the oocytes and corona radiata in the follicles were diminished, the granule cell (GC) layers were reduced or diminished. There are many CLs and developing follicles in different stages in the control group,also there were many granule cell layers, mostly were 8-9 layers.2. Compared with the control group, the serum level of testosterone (T) was significantly higher in model group (P<0.01), while the fasting insulin, fasting blood glucose concentration and insulin resistance index were had no significant differences; The body weight in two groups showed no significant differences (P>0.05). In consideration of body weight influence, the ovarian weight in model group were higher than the control group (P<0.05).3. We had screened out a lot of microRNAs expressed apparently and differently by comparing the two groups. There were 57 microRNAs expressed higher in the model group than the controls(23 had high signals and 34 had low signals (signals<500)also expressed significantly); while there were 43 microRNAs expressed lower in the model groups than the controls(20 had high signals and 23 had low signals (signals<500) also expressed significantly) .Among them , there were some related with cell proliferation and apoptosis such as mir-16, mir-21, mir-24, mir-31, mir-98, mir-125b, mir-143, mir-145, mir-150 et.Conclusion:1、The changes of the ovarian morphology and serum sex hormone in the PCOS rat models induced by Letrozole were proved to be similar with that of the PCOS patients as well as their vaginal cytology examination. Meanwhile this model had no character of insulin resistance.2、We had screened out a mass of differently expressed microRNAs by the gene chips between the groups of PCOS models and the controls ,so we could say microRNAs were probably have had a close association with the happening and developing of PCOS disease . These results may be provide us new clues to study the pathogenesis of PCOS disease. Also they would play important reference value to carry on the appraisal of microRNAs.Part II The test and verify of mir-21 and mir-320 in the rat PCOS modelObjective:We combined the result of gene chip with reports in the literature and selected mir-21 and mir-320 to test and verify and observed their different expression in the both groups and Then discussing the probable association between the two microRNAs and the PCOS disease. Methods:We used the method of real-time PCR to detect the expression levels of mir-21 and mir-320 in the model group and control group, at the same time we used the snRNA U6 as the internal reference and set 2 parallel after hole and 1 negative control for each sample.Results:The result of real-time PCR showed that compared the control group the content of mir-21 in the model group was about 0.48, while the content of mir-320 in the two groups had no significant differences.Conclusion:The results confirmed that the content changes of the two microRNAs were in accord with our gene chips. On the basis of reports in the literature we knew that mir-21 had an important effect on the anti-apoptosis function in the cell proliferation and apoptosis, so we speculated the reduction of mir-21 may play an important role in the follicular development of PCOS. Mir-320 was proved to had important function in insulin resistence, at the same time, the gene chip showed the two group have nearly the same content, this result further testified the model constructed by letrozole had no insulin resistance, this point was different from the method of using DHEA.