小鼠卵巢玻璃化冻融过程中FSH联合S1P干预对血管内皮生长因子和缝隙连接蛋白表达的影响  

Effects of FSH Combined with S1P Interpose on the Expression of Vascular Endothelial Growth Factor and Gap Junction Protein During Vitrification of Mouse Ovary

作　　者：田媛 杨延周[1] 秦天 俞晓丽 王燕蓉[1] 裴秀英[1] TIAN Yuan;YANG Yanzhou;QIN Tian;YU Xiaoli;WANG Yanrong;PEI Xiuying(Key Laboratory of Fertility Preservation and Maintenance,Ministry of Education,Key Laboratory of Reproduction in Ningxia,Department of Biological Chemistry and Molecular Biology,School of the Basic Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学生育力保持教育部重点实验室,宁夏回族自治区生殖与遗传重点实验室,宁夏医科大学基础医学院生物化学与分子生物学学系,银川750004

出　　处：《宁夏医科大学学报》2020年第4期325-331,339,共8页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81760286);宁夏回族自治区重点研发项目(2019BFG02007)。

摘　　要：目的研究小鼠卵巢玻璃化冻融过程中重组人促卵泡刺激素(rhFSH)和1-磷酸鞘氨醇(S1P)联合干预对卵泡形态、血管内皮生成相关因子(VEGF)及其受体(VEGFR-2)、缝隙连接蛋白(CX43、CX37)表达的影响。方法将21日龄ICR雌性小鼠卵巢分为新鲜组、冻存组、0.3 IU·mL^-1FSH干预玻璃化冻融组、2μmol·L^-1S1P干预玻璃化冻融组、不同浓度FSH(0.3、1、0.5、0.2、0.1 IU·mL^-1)+2μmol·L^-1S1P干预玻璃化冻融组、不同浓度S1P(4、10、20、40μmol·L^-1)+0.3 IU·mL-1FSH干预玻璃化冻融组。通过TUNEL检测冻融卵巢的卵巢凋亡率、形态学卵泡计数分析各组正常卵泡百分比及免疫组织化学法观察卵巢组织VEGF、VEGFR-2、CX43、CX37蛋白表达情况。结果除0.1 IU·mL^-1FSH+2μmol·L^-1 S1P组及0.3 IU·mL^-1 FSH+4、10μmol·L^-1S1P组,各组闭锁卵泡数均低于冻存组(P均<0.05);添加FSH及S1P后各组卵巢凋亡率均低于冻存组(P均<0.05);除0.5、0.1 IU·mL^-1 FSH+2μmol·L^-1S1P组及0.3 IU·mL^-1FSH+40μmol·L^-1SIP组,各组VEGF表达均高于冻存组(P均<0.05);2μmol·L^-1S1P组及0.3 IU·mL^-1FSH+10、40μmol·L^-1SIP组VEGFR-2表达均高于冻存组(P均<0.05);0.3、0.5、0.2 IU·mL^-1FSH+2μmol·L^-1S1P组及0.3 IU·mL^-1FSH+4、20μmol·L^-1S1P组CX43蛋白表达均高于冻存组(P均<0.05);除0.3 IU·mL^-1FSH+40μmol·L^-1SIP组,各组CX37表达均高于冻存组(P均<0.05)。联合干预结果显示1IU·mL^-1FSH、0.1 IU·mL^-1FSH及40μmol·L^-1SIP不能维护较好的卵泡结果及相关蛋白的表达。结论玻璃化冻存过程FSH联合S1P的干预可抑制卵泡闭锁,但过高的FSH及S1P导致卵泡激活成熟;0.3 IU·mL^-1FSH与2μmol·L^-1S1P干预可维持较高数目的原始卵泡池,该作用与提高VEGF、VEGFR-2、CX43及CX37蛋白表达有关。Objective To study the effects of recombinant human follicle stimulating hormone(rhFSH)and sphingosine 1-phosphate(S1P)during vitrification of mouse ovary on follicular morphology and vascular endothelial growth factor(VEGF),and its receptor(VEGF-2),Gap Connexin(CX43 and CX37)expression.Methods The ovaries of 21-day-old ICR female mice were divided into:Fresh group;Vitrification group;0.3 IU·mL^-1FSH intervention vitrification group;2μmol·L^-1S1P intervention vitrification group;0.3,1,0.5,0.2,0.1 IU·mL^-1FSH and 2μmol·L^-1S1P intervention vitrification group;4,10,20,40μmol·L^-1S1P and 0.3 IU·mL^-1FSH intervention vitrification group.Apoptosis of vitrification ovary was detected by TUNEL.The percentage of normal follicles in each group was analyzed by morphological follicle count.The expression of VEGF,VEGFR-2,CX43,CX37 protein in ovarian tissue was observed by immunohistochemistry.Results Except for 0.1 IU·mL-1FSH+2μmol·L^-1S1P group and 0.3 IU·mL^-1FSH+4,10μmol·L^-1S1P group,the number of atretic follicles in each group was less than the vitrification group(P all<0.05).After adding FSH and S1P,the ovarian apoptosis rate of each group was lower than the vitrification group(P all<0.05).Except for 0.5,0.1 IU·mL^-1FSH+2μmol·L^-1S1P group and 0.3 IU·mL^-1FSH+40μmol·L^-1SIP group,the expression of VEGF in each group was higher than the vitrification group(P all<0.05).The expression of VEGFR-2 in the 2μmol·L-11S1P group and 0.3 IU·mL-1FSH+10,40μmol·L-1SIP group was higher than the vitrification group(P all<0.05).The expression of CX43 protein in the 0.3,0.5,0.2 IU·mL-1FSH+2μmol·L-1S1P group and the 0.3 IU·mL-1FSH+4,20μmol·L-1S1P group was higher than the vitrification group(P all<0.05).Except for the 0.3 IU·mL-1FSH+40μmol·L-1SIP group,the expression of CX37 in each group was higher than the vitrification group(P all<0.05).The combined intervention results showed that 1 IU·mL-1FSH,0.1 IU·mL-1FSH,and 40μmol·L-1SIP could not maintain good follicular results and related protein expres

关 键 词：小鼠卵巢玻璃化冻存 促卵泡刺激素 1-磷酸鞘氨醇 血管生成 

分 类 号：R71[医药卫生—妇产科学]