激活的富血小板血浆对卵巢储备功能降低患者卵丘颗粒细胞凋亡和增殖的影响  

Effects of activated platelet rich plasma on apoptosis and proliferation of cumulus granulosa cells in patients with diminished ovarian reserve

作　　者：朱文倩 张慧敏[2] 李磊[2] 赵明子 郝翠芳[2] ZHU Wen-qian;ZHANG Hui-min;LI Lei;ZHAO Ming-zi;HAO Cui-fang(Binzhou Medical University,Yantai 264003;Women and Children’s Hospital,Qingdao University,Qingdao 266000;Medical College of Qingdao University,Qingdao 266000)

机构地区：[1]滨州医学院,烟台264003 [2]青岛大学附属妇女儿童医院,青岛266000 [3]青岛大学医学部,青岛266000

出　　处：《生殖医学杂志》2022年第5期652-660,共9页Journal of Reproductive Medicine

摘　　要：目的 探讨激活的富血小板血浆(PRP)对卵巢储备功能降低(DOR)患者卵丘颗粒细胞凋亡和增殖的影响。方法 收集2020年8月至2021年9月在青岛妇女儿童医院生殖医学中心行常规IVF/ICSI-ET治疗的82例DOR不孕症患者的卵丘颗粒细胞,将所获细胞均分为PRP组(使用激活的PRP+培养液培养)和对照组(仅使用培养液培养)。采用细胞增殖及毒性检测法(CCK-8法)测定不同浓度PRP时人卵巢颗粒细胞(KGN细胞)的生长曲线,并选择最适PRP浓度。使用Tunel法检测PRP组和对照组卵丘颗粒细胞的凋亡情况;通过实时荧光定量PCR(qRT-PCR)检测凋亡基因Bax、抗凋亡基因Bcl-2的表达,采用细胞免疫荧光染色法检测凋亡蛋白Bax、抗凋亡蛋白Bcl-2及增殖因子Ki-67的表达;同时,使用罗氏全自动发光仪检测两组卵丘颗粒细胞培养基上清液中雌二醇(E;)浓度,并对上述结果进行统计分析。结果 不同浓度PRP均可促进KGN细胞增殖,和10%胎牛血清联合使用时最适的PRP浓度为1%。PRP组卵丘颗粒细胞凋亡率显著低于对照组(P<0.001),凋亡因子Bax的mRNA和蛋白表达水平均较对照组显著降低(P<0.001),抗凋亡因子Bcl-2的mRNA和蛋白表达水平均较对照组显著升高(P<0.01),增殖因子Ki-67的蛋白表达亦显著高于对照组(P<0.001)。PRP组卵丘颗粒细胞培养基上清液中E;浓度显著高于对照组(P<0.001)。结论 体外培养人卵丘颗粒细胞时,给予激活的PRP能有效抑制颗粒细胞凋亡、促进其增殖,同时增强其分泌功能。Objective:To investigate the effect of activated platelet-rich plasma(PRP) on apoptosis and proliferation of cumulus granulosa cells in patients with diminished ovarian reserve(DOR).Methods:The cumulus granulosa cells were collected from 82 infertile patients with DOR who underwent routine IVF/ICSI-ET in the reproductive medicine center of Qingdao Women and Children’s Hospital from August 2020 to September 2021. The obtained cells were divided into PRP group(cultured with activated PRP+medium) and control group(cultured with medium only). The growth curves of KGN cells at different concentrations of PRP were measured by cell proliferation and toxicity assay(CCK-8 method),and the optimal concentration of PRP was selected. Tunel staining was used to detect apoptosis of cumulus granulosa cells in PRP and control groups. The expression of apoptosis protein Bax and anti-apoptotic protein Bcl-2 in cumulus granulosa cells was detected by using quantitative real-time fluorescence PCR(qRT-PCR). The expression of Bax, Bcl-2 and Ki-67 in cumulus granulosa cells was detected by using cellular immunofluorescence staining. The concentration of estradiol(E;) in the supernatant of cumulus granulosa cells was measured by using a fully automated luminometer.Results:Different concentrations of PRP promoted the proliferation of KGN cells. When combined with 10% fetal bovine serum, the optimal PRP concentration was 1%. The apoptosis of cumulus granulosa cells in PRP group was significantly lower than that in control group(P<0.001). In PRP group, the mRNA and protein expressions of apoptotic factor Bax in PRP group was significantly lower than control group(P<0.001),the mRNA and protein expressions of anti-apoptotic factor Bcl-2 was significantly higher than control group(P<0.01),and the protein expression of proliferative factor Ki-67 was significantly higher than control group(P<0.001). The concentration of estradiol(E;) in the supernatant of cumulus cells culture medium in PRP group was also significantly higher than control grou

关 键 词：卵丘颗粒细胞 富血小板血浆 卵巢储备功能降低 增殖 凋亡 

分 类 号：R711.75[医药卫生—妇产科学]