The Study Of The Effect Of BMSCs On Preantral Follicle Culture In Vitro And 3D Culture System Construction

Part 1 The study of the effect of BMSCs on preantral follicle culture in vitro[Purpose] To observe the effect of BMSCs on the culture of mouse preantral follicles in vitro.[Methods] Mouse preantral follicles co-cultured with BMSCs using transwell chamber was co-culture group.Untreated mouse preantral follicles were cultured separately in vitro under the same conditions,and mature oocytes of untreated young mice in vivo were used as the control group.Observation before preantral follicle growth speed,oocyte maturation rate,by immunofluorescence test phase M Ⅱ spindle abnormal use chemiluminescence immunoassay detection rate and the medium concentration of estrogen.[Results] BMSCs co-culture had no significant effect on the growth rate of preantral follicles in vitro,and the survival rate of preantral follicles after BMSCs co-culture showed no significant change.The antrum formation rate(49.22%)in the co-culture group was higher than in vitro group alone(29.47%)(P<0.05),and the oocyte maturation rate(31.75%)in the co-culture group was higher than in vitro group(16.84%)(P<0.05).The results of immunofluorescence showed that the mallet morphology and microtubule arrangement of oocytes in the in vitro group were abnormal.In the co-culture group and in vivo group,the spindle morphology was intact and microtubules were arranged neatly.Oocyte spindle abnormality rate(34.78%)in the co-culture group was significantly lower than in vitro group(52.17%)(P<0.05),but still higher than that in vivo group(25.53%)(P<0.05).At the beginning of the fourth day of culture,the estrogen in the culture medium in the co-culture group was significantly higher than in vitro group(P<0.05).[Conclusions] After BMSCs co-culture,the rate of antrum formation and maturation increased,the rate of spindle abnormality decreased,and the secretion of estrogen in theculture medium increased,indicating that the BMSCs co-culture can improve the quality of in vitro culture of preantral follicles and the secretion function of granulosa cells.Part 2 Construction of 3D culture system and safety test[Purpose] Establish the best 3D culture system and determine its safety.[Methods] Four 3D culture systems were constructed: collagen system,alginate system,collagen +DNA hydrogel system,alginate +DNA hydrogel system,and 2D culture system as the control.The five systems were co-cultured with preantral follicles to observe the growth and development of follicles.The five systems were co-cultured with GCs and BMSCs for 72 h.Annexin v-egfp apoptosis assay kit was used to detect the apoptosis of granulosa cells and BMSCs in each group,and the expressions of apoptosis-related proteins Bax and bcl-2 were detected by Western blot.[Results] Four 3D culture systems were successfully constructed,and all the preantral follicles could survive and grow in the four 3D culture systems.GCs and BMSCs grew well in the four culture systems,apoptosis flow detection showed that the apoptosis index of each group was less than 1%,and there was no significant difference in the expression level of apoptosis-related proteins between the groups(P>0.05).[Conclusions] The four 3D culture systems had no effect on the proliferation of GCs and BMSCs.In view of the best development of preantral follicles in alginate +DNA hydrogel system,the alginate +DNA hydrogel culture system was preliminarily determined to be the optimal system.