鞘氨醇-1-磷酸干预对小鼠玻璃化冻融卵巢移植后凋亡的影响

Effect of S1P Intervention on Ovarian Apoptosis after Vitrified Freeze-thaw Transplantation in Mice

目的探讨鞘氨醇-1-磷酸(sphingosine-1-phosphate,S1P)干预对小鼠玻璃化冻融卵巢移植后卵泡形态和凋亡的影响。方法取21日龄ICR雌性小鼠卵巢分为新鲜对照组、冻存对照组和2μmol·L-1 S1P干预组,新鲜卵巢及两组冻存卵巢解冻后移植于6~8周龄ICR雌性去势小鼠肾被膜下2 d、14 d后,通过HE染色观察移植后卵巢的卵泡形态,TUNEL检测移植后卵巢的凋亡率;实时荧光定量PCR检测卵巢移植后Caspase3、Bax、Bcl-2的基因表达情况。结果移植2 d和14 d后,与新鲜对照组相比,冻存对照组和S1P干预组卵泡形态的完整性较差,闭锁卵泡多,正常卵泡明显减少,卵泡凋亡率较高(P均<0.05)。与冻存对照组相比,S1P干预组卵泡形态较完整,闭锁较少,卵泡发育良好,原始卵泡较多,卵泡凋亡率降低(P均<0.05),促凋亡基因Bax和凋亡基因Caspase3表达降低(P均<0.05);抗凋亡基因Bcl-2表达增高(P<0.05)。结论小鼠玻璃化冻融卵巢移植后S1P的干预可维持较好的卵泡发育,抑制卵泡闭锁,降低移植后卵巢凋亡,抵抗玻璃化冷冻移植对卵巢的损伤。

Objective To study the effect of sphingosine-1-phosphate(S1P)intervention on the morphology and apoptosis of follicles after vitrification and freeze-thaw transplantation of mouse ovaries.Methods The ovaries of 21-day-old ICR female mice were divided into a fresh control group,a vitrification cryopreservation control group,and an S1P intervention vitrification cryopreservation group.The fresh ovaries and the two cryopreserved ovaries were thawed and transplanted into 6-8 weeks old ICR female mice.After 2 days and 14 days under the renal capsule,the follicular morphology of the transplanted ovary was observed by HE staining,the apoptosis rate of the transplanted ovary was measured by TUNEL;the gene expression of Caspase3,Bax,and Bcl-2 after ovarian transplantation was measured by real-time fluorescent quantitative PCR.Results Two days and 14 days after transplantation,compared with the fresh control group,the frozen-preserved control group and the S1P intervention group had poorer follicular morphology,with more atresia follicles,significantly reduced normal follicles,and higher follicular apoptosis(P all<0.05).Compared with the cryopreserved control group,the S1P intervention group had more complete follicles,less atresia,well-developed follicles,more primitive follicles,and decreased follicular apoptosis rate(P all<0.05).The expression of pro-apoptotic gene Bax and apoptosis gene Caspase3 decreased(P all<0.05);the expression of anti-apoptotic gene Bcl-2 increased(P<0.05).Conclusion S1P intervention after vitrification freeze-thaw transplantation of mouse ovaries can maintain good follicular development,inhibit follicular atresia,reduce ovarian apoptosis after transplantation,and resist the damage of vitrification freeze-thaw transplantation to the ovary.