Study On The Mechanism Of Ovarian Granulosa Cell Apoptosis Induced By Hyperprolactinemia Model With Liver And Spleen As The Core

Objective:To investigate apoptosis mechanism of ovarian granular cells in vitro animal pattern with hyperprolactinemia, based on liver and spleen ntervention by the method of establishing ovarian granular cells in vitro animal pattern with hyperprolactinemia.Methods:The rat model made with hyperprolactinemia by intraperitoneal injection of metoclopramide. The rat model divided into6groups randomly:blank control group, negative control group, bromocriptine group, the high-dosage Chinese medicine group, the medium-dosage chinese medicine group and the low-dosage chinese medicine group. We would successive medicationon bromocriptine groupthe high-dosage Chinese medicine group, the medium-dosage chinese medicine group and the low-dosage chinese medicine group for30days after successful modeling. We mearured serum prolactin by euzymelinked immunosorbent assay(ELISA), extracted follicular cell of ovary for in vitro primary culture by mechanical separation methods, tested cells with HE stain and FSHR cell immunofluorescence stain, detected cell viability and the condition of24h,48h,72h,96h cell multiplication of each group with MTT, and analyzed phases of cell cycle and apoptotic index with flow cytometry.Resul ts:It showed that it can be identified as rat ovarian granulosa cells through HE stain and FSHR cell immunofluorescence stain, and cell purity>90%. According to the results of cell survival rate by MTT test, negative control group was significantly decreased(P<0.01), bromocriptine group and the low-dosage chinese medicine group was decreased(P<0.05), which were compared with blank control group. Compared with negative control group, high dose group was increased(P<0.05). The result of MTT test showed that there’s no obvious difference between OD value of each cell on24h and48h(P>0.05), then, there’s significant difference between OD value of each cell on72h and96h(P<0.01). All of those can state there’s obvious dose-effect and time-effect relationship on the drug groups, for instance, the high-dosage Chinese medicine group made cell proliferation of S phase significantly increased, G0/G1phase decreased(P<0.05), and there’s no significant difference between the high-dosage Chinese medicine group with Xiaoyao Powde addition and subtraction and bromocriptine group(P>0.05). The results of cell apoptosis index from flow cytometry showed that negative control group and the low-dosage chinese medicine increased and had significant difference compared with blank control group(P<0.01), then, bromocriptine group had differenc(P<0.05). Compared with blank control group, the high-dosage hinese cine group decreased(.P<0.05); Compared with the high-dosage Chinese medicine group, the low-dosage chinese medicine increased(P<0.05), which suggestted that there is a dose-effect relationship within groups.Conclusion:It is concluded that ovarian granular cells of the HPRL rat model were full of vigour which obtained by the methods as mechanical separation, differential attachment HE stain and FSHR cell immunofluorescence tain and builded the original generation of cultivation system of the HPRL rat model. The therapeutic method based on liver and spleen can improve the survival rate of ovarian granular cell from model animals and have quite different effect on cell multiplication to each group(24h、48h:not obvious;72h:significantly ncreased.96h:decreaed) which can show that there is a dose-effect and time-effect relationship within groups. The result of cell cycle detected from fow cytometry shows:Xiaoyao Powder addition and subtraction make cell proliferation of S phase significantly increased which explain Xiaoyao Powder addition and subtraction can promote cells from the stationary phase to S phase transformation, accelerate the process of cell cycle and increase the proportion of S phase cells by reducing cell arrest of Go/G1phase cells to promote cell proliferation and DNA synthesis of ovarian granular cells and recover the function of ovary. The analysis of cell apoptosis index from granule cells shows the method can reduce cell apoptosis index of ovarian granulosa cell from animal pattern which can further demonstrate that the method improve the function of ovary is by the means of inhibiting cell apoptosis of ovarian granular cells and reducing follicular atresia.