过继转移过表达Pellin...不全小鼠卵巢功能的修复作用_卓爱萍.pdf

海军军医大学学报2023 年 4 月第 44 卷第 4 期http:/Academic Journal of Naval Medical University,Apr.2023,Vol.44,No.4 409 论 著 收稿日期 2022-04-26 接受日期 2022-07-01基金项目 广东省自然科学基金（2020A1515010205，2021A1515010701）Supported by Natural Science Foundation of Guangdong Province(2020A1515010205,2021A1515010701).作者简介 卓爱萍，硕士生 E-mail:*通信作者（Corresponding author）.Tel:020-62782976,E-mail:过继转移过表达 Pellino-1 的调节性 T 细胞对自身免疫性早发性卵巢功能 不全小鼠卵巢功能的修复作用卓爱萍，王 袁，杨雨涛，谢嘉欣，高 萌，付霞霏*南方医科大学珠江医院妇产科，广州 510220摘要 目的 探讨过继转移过表达Pellino-1（Peli1）的调节性T细胞（Treg细胞）对自身免疫性早发性卵巢功能不全（POI）小鼠的卵巢功能是否有修复作用，以及对其免疫功能是否具有调控作用。方法 采用透明带 3 多肽（pZP3）构建自身免疫性POI小鼠模型，将构建的过表达Peli1 的慢病毒载体（LV-Peli1）转染至Treg细胞，最后将过表达Peli1 的Treg细胞过继转移到模型小鼠体内。将POI模型小鼠随机分为 4 组（n5），分别为 LV-Peli1-POI 组（过继转移过表达 Peli1 的 Treg 细胞）、LV-POI 组（过继转移转染慢病毒空载体的 Treg 细胞）、Treg-POI 组（过继转移未经任何处理的Treg 细胞）、POI 模型组（经尾静脉注射等体积 PBS）。采用H-E染色观察各组卵巢结构并行各级卵泡计数，免疫组织化学染色检测卵巢组织增殖蛋白Ki-67 表达及CD3 T淋巴细胞水平，TUNEL法检测卵巢颗粒细胞凋亡情况，ELISA法检测各组小鼠血清卵巢激素促卵泡激素（FSH）、雌二醇（E2）及细胞免疫因子IL-10、TGF-的表达水平，考察过表达Peli1 对自身免疫性POI的作用。结果 与LV-POI组、Treg-POI组及POI模型组相比，LV-Peli1-POI组中始基卵泡、初级卵泡、次级卵泡、成熟卵泡的数目均增加，Ki-67 表达增高，卵巢颗粒细胞凋亡减少，FSH表达水平下降，E2表达水平升高（P0.05，P0.01）；LV-Peli1-POI组的CD3 T淋巴细胞的数量减少，IL-10 和TGF-表达水平升高（P0.05，P0.01）。结论 过继转移过表达Peli1 的Treg细胞可有效促进POI小鼠卵巢颗粒细胞增殖、抑制其凋亡，同时改善POI小鼠的卵巢内分泌功能和调节免疫功能，对自身免疫性POI有一定的修复作用。关键词 自身免疫性早发性卵巢功能不全；Pellino-1；调节性T淋巴细胞；颗粒细胞；自身免疫性卵巢炎中图分类号 R 711.75文献标志码 A文章编号 2097-1338(2023)04-0409-09Repairing effect of adoptive transfer of regulatory T cells overexpressing Pellino-1 on ovarian function of mice with autoimmune premature ovarian insufficiencyZHUO Ai-ping,WANG Yuan,YANG Yu-tao,XIE Jia-xin,GAO Meng,FU Xia-fei*Department of Obstetrics and Gynaecology,Zhujiang Hospital of Southern Medical University,Guangzhou 510220,Guangdong,China Abstract Objective To investigate whether adoptive transfer of regulatory T cells(Treg cells)overexpressing Pellino-1(Peli1)can repair the ovarian function of mice with autoimmune premature ovarian insufficiency(POI)and regulate its immune function.Methods The autoimmune POI mouse model was constructed using zona pellucida 3 peptide(pZP3),and the lentiviral vector overexpressing Peli1(LV-Peli1)was constructed.Then LV-Peli1 was transfected into Treg cells,and finally the Treg cells overexpressing Peli1 were adoptively transferred into the model mice.The POI model mice were randomly divided into 4 groups(5 in each group),namely,LV-Peli1-POI group(Treg cells overexpressing Peli1 were adoptively transferred to POI mice),LV-POI group(Treg cells transfected with empty-vector lentivirus were adoptively transferred to POI mice),Treg-POI group(Treg cells without any treatment were adoptively transferred to POI mice)and POI model group(an equal volume of phosphate-buffered saline was injected into POI mice via the tail vein).Hematoxylin-eosin staining was used to observe the ovarian structures and follicle counts at various stages of mice in each group.Immunohistochemical staining was used to detect the levels of proliferation protein Ki-67 and CD3 T lymphocytes in ovarian tissue,and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay was used to detect the apoptosis of granulosa cells in ovarian.Enzyme-linked immunosorbent assay was used to detect the expression levels of serum ovarian hormones follicle-stimulating hormone(FSH),estradial(E2),and cellular immune factors interleukin 10(IL-10)and transforming growth factor (TGF-)in mice in each group,so as to explore the effect of Peli1 overexpression on autoimmune POI.Results Compared with the LV-POI group,Treg-POI group and POI model group,the numbers of DOI：10.16781/j.CN31-2187/R.20220346海军军医大学学报 2023 年 4 月，第 44 卷 410 primordial follicles,primary follicles,secondary follicles and mature follicles were significantly increased,the expression of Ki-67 was significantly increased,the apoptosis of ovarian granulosa cells was significantly reduced,the expression level of FSH was decreased,and the expression level of E2 was significanly increased in the LV-Peli1-POI group(P0.05,P0.01).The level of CD3 T lymphocytes in the LV-Peli1-POI group was significantly decreased,and the expression levels of IL-10 and TGF-were significantly increased(P0.05,P0.01).Conclusion Adoptive transfer of Treg cells overexpressing Peli1 can effectively promote the proliferation of POI ovarian granulosa cells of mice and inhibit their apoptosis.At the same time,it can improve the ovarian endocrine function and regulate immune function in POI mice,thereby producing a certain repairing effect on autoimmune POI.Key words autoimmune premature ovarian insufficiency;Pellino-1;regulatory T-lymphocytes;granulosa cell;autoimmune oophoritisAcad J Naval Med Univ,2023,44(4):409-417早 发 性 卵 巢 功 能 不 全（premature ovarian insufficiency，POI）是妇科内分泌领域的常见疾病，在一般人群中的发病率为 1%3%，且近年来呈逐渐上升趋势1。根据欧洲人类生殖与胚 胎学会（European Society for Human Reproduction and Embryology，ESHRE）的标准，当年龄40 岁 的女性闭经或月经稀发至少 4 个月且间隔 4 周以上检测的 2 次促卵泡激素（follicle-stimulating hormone，FSH）水平升高（FSH25 IU/L）可诊断为 POI2。POI 临床表现为女性在 40 岁之前出现闭经、不孕、性欲下降、潮热等一系列卵巢功能衰竭症状，其激素水