切换至 "中华医学电子期刊资源库"
高级检索
中华移植杂志(电子版)

中华移植杂志(电子版) ›› 2020, Vol. 14 ›› Issue (04) : 229 -233. doi: 10.3877/cma.j.issn.1674-3903.2020.04.007

所属专题： 文献；

论著

大鼠卵巢体外低温机械灌注模型的建立
张诗辰1, 姚翰林1, 杨志坚1, 叶啟发2, 王彦峰1,†()   
  1. 1. 430071　武汉大学中南医院，武汉大学肝胆疾病研究院，武汉大学移植医学中心，移植医学技术湖北省重点实验室
    2. 430071　武汉大学中南医院，武汉大学肝胆疾病研究院，武汉大学移植医学中心，移植医学技术湖北省重点实验室；410013　长沙，中南大学湘雅三医院，卫生部移植医学工程技术研究中心
  • 收稿日期:2020-03-06 出版日期:2020-08-25
  • 通信作者: 王彦峰
  • 基金资助:
    国家自然科学基金面上项目(81570079)

The establishment of hypothermic mechanical perfusion model of rat ovary in vitro

Shichen Zhang1, Hanlin Yao1, Zhijian Yang1, Qifa Ye2, Yanfeng Wang1,†()   

  1. 1. Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of Wuhan University, Transplant Center of Wuhan University, Hubei Key Laboratory of Medical Technology on Transplantation, Wuhan, 430071, China
    2. Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of Wuhan University, Transplant Center of Wuhan University, Hubei Key Laboratory of Medical Technology on Transplantation, Wuhan, 430071, China; The 3rd Xiangya Hospital of Central South University, Research Center of National Health Ministry on Transplantation Medicine Engineering and Technology, Changsha 410013, China
  • Received:2020-03-06 Published:2020-08-25
  • Corresponding author: Yanfeng Wang
  • About author:
    Corresponding author: Wang Yanfeng, Email:
全文 ( ) 下载PDF ( ) 导出引用
引用本文：

张诗辰, 姚翰林, 杨志坚, 叶啟发, 王彦峰. 大鼠卵巢体外低温机械灌注模型的建立[J/OL]. 中华移植杂志(电子版), 2020, 14(04): 229-233.

Shichen Zhang, Hanlin Yao, Zhijian Yang, Qifa Ye, Yanfeng Wang. The establishment of hypothermic mechanical perfusion model of rat ovary in vitro[J/OL]. Chinese Journal of Transplantation(Electronic Edition), 2020, 14(04): 229-233.

目的

建立大鼠卵巢体外低温机械灌注(HMP)模型，并验证其有效性。

方法

26只雌性8～10周龄SD大鼠，供体组、受体组、正常对照组和卵巢摘除组各6只，另有2只大鼠HMP结束后留取卵巢标本以备检测。供体组和取材大鼠获取卵巢和肾脏后置于0～4 ℃林格氏液中保存2 h，再行HMP 2 h。供体组大鼠卵巢组织HMP结束后移植至受体组大鼠左肾包膜下，移植7 d后检测血清抗苗勒管激素(AMH)水平，同时获取移植卵巢进行组织病理检查以及Ki67和CD31免疫组织化学染色。采用单因素方差分析比较正常对照组、卵巢摘除组和受体组大鼠血清AMH水平，采用t检验比较正常对照组和受体组卵泡计数以及Ki67和CD31阳性细胞面积百分比。P<0.05为差异有统计学意义。

结果

HMP后的卵巢组织血管未见明显扩张，受体组大鼠移植7 d后的卵巢组织卵泡结构完整，成熟卵泡数为(7.0±0.6)个，低于正常对照组(26.3±3.4)个，差异有统计学意义(t＝5.633，P<0.05)。受体组血清AMH为(4.63±0.87)ng/mL，高于卵巢摘除组(2.55±0.16)ng/mL，低于正常对照组(7.39±1.04)ng/mL，但差异均无统计学意义(P均>0.05)。移植7 d后受体组大鼠卵巢组织Ki67和CD31阳性细胞面积百分比分别为(29.0±2.4)%和(12.3±1.0)%，正常对照组分别为(21.9±3.8)%和(12.6±1.2)%，差异均无统计学意义(t＝1.634和0.178，P均>0.05)。

结论

本研究构建的大鼠卵巢体外HMP模型，方法简单，可短时间维持卵巢的正常功能，为卵巢器官保存提供了一个新的方式。

关键词: 大鼠, 卵巢移植, 低温机械灌注, 卵巢
Objective

To establish a hypothermic mechanical perfusion (HMP) model of rat ovary in vitro and test its effectiveness.

Methods

26 female SD rats aged 8-10 weeks were divided into donor group, recipient group, normal group and ovariectomy group, with 6 rats in each group. Ovarian tissue of 2 rats was harvested for test after HMP. Ovaries and kidneys from donor group and HMP management only group were placed in Ringer′s solution at 0-4 ℃ for 2 h, and follwed by HMP for 2 h. Ovarian tissue from donor group was transplanted under the recipient′s left kidney capsule after HMP, and the serum anti-Mullerian hormone (AMH) in recipient rat was tested 7 days after transplantation, and the ovaries were harvested for histological analysis, and immunohistochemistry for CD31 and Ki67. ANOVA was used to compare the serum AMH levels in the normal group, the ovariectomy group and the recipient group. The t-test was used to compare the follicle counts and the percentages of Ki67 and CD31 positive cells in the normal group and the recipient group. P<0.05 was considered statistically significant.

Results

No obvious dilatation was observed in the blood vessels of ovarian tissue after HMP. The follicle structure of rats from recipient group was complete 7 days after transplantation, and the number of mature follicles was (7.0±0.6), which was lower than that of the normal group (26.3±3.4), the difference had statistical significance (t=5.633, P<0.05). The serum AMH of recipient group was (4.63±0.87) ng/mL, higher than that of the ovariectomy group [(2.55±0.16) ng/mL], and lower than that of the normal group [(7.39±1.04) ng/mL]. The percentage of Ki67 and CD31 positive cells in ovarian tissue from recipient group 7 days after transplantation were (29.0±2.4)% and (12.3±1.0)%, which had no significant difference with normal group [(21.9±3.8%) and (12.6±1.2%)] (t=1.634 and 0.178, P all>0.05).

Conclusions

The method to construct the model of HMP in vitro of rat ovary in this study is simple and can maintain the normal function of ovary for a short time, providing a new method for organ preservation of ovary.

Key words: Rats, Ovarian transplantation, Hypothermic mechanical perfusion, Ovary
图1 SD大鼠卵巢获取解剖示意图
图2 SD大鼠卵巢体外低温机械灌注前后大体观
图3 SD大鼠卵巢体外低温机械灌注前后和移植7 d后病理学检查结果(HE ×100)
图4 SD大鼠卵巢免疫组织化学染色结果(×200)
1
Mason JB, Cargill SL, Griffey SM, et al. Transplantation of young ovaries restored cardioprotective influence in postreproductive-aged mice[J]. Aging Cell, 2011，10(3): 448-456.
2
Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women′s Health Initiative randomized controlled trial[J]. JAMA, 2002，288(3): 321-333.
3
Westerkamp AC, Karimian N, Matton APM et al. Oxygenated hypothermic machine perfusion after static cold storage improves hepatobiliary function of extended criteria donor livers[J]. Transplantation, 2016，100(4): 825-835.
4
程颖. 机械灌注在肝移植中的应用[J]. 器官移植，2016，7(5): 390-393.
5
胡晓燕，王彦峰，叶殷发，等. 低温机械灌注在离体肝脏中的保存与修复作用[J]. 中华器官移植杂志，2015，36(10): 632-635.
6
Milenkovic M, Diaz-Garcia C, Wallin A, et al. Viability and function of the cryopreserved whole rat ovary: comparison between slow-freezing and vitrification[J]. Fertil Steril, 2012，97(5): 1176-1182.
7
Ding Y, Shao J, Li J, et al. Successful fertility following optimized perfusion and cryopreservation of whole ovary and allotransplantation in a premature ovarian insufficiency rat model[J]. J Ovarian Res, 2018，11(1): 35.
8
Ladanyi C, Mor A, Christianson MS, et al. Recent advances in the field of ovarian tissue cryopreservation and opportunities for research[J]. J Assist Reprod Genet, 2017，34(6): 709-722.
9
Mason JB, Terry BC, Merchant SS, et al. Manipulation of ovarian function significantly influenced trabecular and cortical bone volume, architecture and density in mice at death[J]. PLoS One, 2015，10(12): e0145821.
10
Peterson RL, Parkinson KC, Mason JB. Restoration of immune and renal function in aged females by re-establishment of active ovarian function[J]. Reprod Fertil Dev, 2017，29(10): 2052-2059.
11
Mason JB, Cargill SL, Anderson GB, et al. Transplantation of young ovaries to old mice increased life span in transplant recipients[J]. J Gerontol A Biol Sci Med Sci, 2009，64(12): 1207-1211.
12
Silber S, Pineda J, Lenahan K, et al. Fresh and cryopreserved ovary transplantation and resting follicle recruitment[J]. Reprod Biomed Online, 2015，30(6): 643-650.
13
Xu Z, Wang X, Wu Y, et al. Slow-controlled freezing versus speed-cooling for cryopreservation of whole guinea pig ovaries[J]. Theriogenology, 2012，77(3): 483-491.
14
Kamoshita K, Okamoto N, Nakajima M, et al. Investigation of in vitro parameters and fertility of mouse ovary after storage at an optimal temperature and duration for transportation[J]. Hum Reprod, 2016，31(4): 774-781.
15
贾俊君，李建辉，姜骊，等. 肝移植供肝保存新途径：机械灌注[J/CD]. 中华移植杂志：电子版，2014，8(3): 164-168.
16
Dewailly D, Andersen CY, Balen A, et al. The physiology and clinical utility of anti-Mullerian hormone in women[J]. Hum Reprod Update, 2014，20(3): 370-385.
17
Lee J, Kim EJ, Kong HS, et al. A combination of simvastatin and methylprednisolone improves the quality of vitrified-warmed ovarian tissue after auto-transplantation[J]. Hum Reprod, 2015，30(11): 2627-2638.
18
Li J, Mao Q, He J, et al. Human umbilical cord mesenchymal stem cells improve the reserve function of perimenopausal ovary via a paracrine mechanism[J]. Stem Cell Res Ther, 2017，8(1): 55.
19
Broer SL, Eijkemans MJ, Scheffer GJ, et al. Anti-mullerian hormone predicts menopause: a long-term follow-up study in normoovulatory women[J]. J Clin Endocrinol Metab, 2011，96(8): 2532-2539.
20
Xu J, Long YS, Gozal D, et al. Beta-cell death and proliferation after intermittent hypoxia: role of oxidative stress[J]. Free Radic Biol Med, 2009，46(6): 783-790.
21
Onions VJ, Mitchell MRP, Campbell BK, et al. Ovarian tissue viability following whole ovine ovary cryopreservation: assessing the effects of sphingosine-1-phosphate inclusion[J]. Hum Reprod, 2008，23(3): 606-618.
22
Onions VJ, Webb R, Pincott-Allen C, et al. The effects of whole ovarian perfusion and cryopreservation on endothelial cell-related gene expression in the ovarian medulla and pedicle[J]. Mol Hum Reprod, 2013，19(4): 205-215.
[1] 刘思锐, 赵辰阳, 张睿, 张一休, 杨萌. 多普勒超声对孕鼠子宫动脉不同节段血流动力学参数的评估[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 877-883.
[2] 卢菊, 赵胜, 范建华, 高艳多. 探讨IOTA、GI-RADS及O-RADS在附件肿瘤良恶性鉴别诊断中的价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(05): 484-490.
[3] 张焱, 刘春媚, 姚瑾, 陈苗苗, 徐雯, 黄品同. 超声O-RADS分类和临床特征对不同病理类型卵巢浆液性肿瘤的诊断价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(03): 268-274.
[4] 林琳, 田思萌, 于永华, 徐飞飞, 黄明莉. 干细胞及其外泌体治疗宫腔黏连的研究现状[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(03): 271-275.
[5] 刘锦程, 王斌, 张雯, 张明周, 刘禹, 叶东樊, 黄赞胜, 邱凌霄, 卿斌, 王创业, 王南博, 王苹, 郭宇航, 周培花, 程秋霞, 徐智. 肺泡灌洗液RASSF1A及SHOX2甲基化联合径向超声特征对肺结节性质鉴别诊断的意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(04): 505-511.
[6] 蒋露, 郑莹, 杨帆, 王乔, 王娜, 阳川华, 陈宇, 苟嘉妮, 邓露丝, 杨旭. 经脐单孔腹腔镜联合上腹部开腹行晚期卵巢癌肿瘤细胞减灭术[J/OL]. 中华腔镜外科杂志(电子版), 2024, 17(03): 177-181.
[7] 刘庭秀, 刘新敏, 刘莹, 隋娟, 武宇, 赵瑜敬, 毕红, 孙雪梅, 范秀华. 腹壁整形术后腹腔镜新脐入路治疗卵巢肿物的安全性探讨[J/OL]. 中华腔镜外科杂志(电子版), 2024, 17(03): 189-192.
[8] 陈丽璇, 窦培宁, 肖扬. 干细胞治疗早发性卵巢功能不全的现状及未来展望[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 239-248.
[9] 徐靖亭, 孔璐. PARP抑制剂治疗卵巢癌的耐药机制及应对策略[J/OL]. 中华临床医师杂志(电子版), 2024, 18(06): 584-588.
[10] 王星, 陈园, 热孜万古丽·乌斯曼, 郭艳英. T2DM、Obesity、NASH、PCOS共同致病因素相关的分子机制[J/OL]. 中华临床医师杂志(电子版), 2024, 18(05): 481-490.
[11] 袁捷, 乔钰琪, 李彦冬. 二甲双胍、来曲唑联合地屈孕酮治疗多囊卵巢综合征合并不孕症的效果评价[J/OL]. 中华临床医师杂志(电子版), 2024, 18(04): 343-347.
[12] 胡小靖, 张华. 妊娠期卵巢囊肿蒂扭转的诊断与治疗[J/OL]. 中华产科急救电子杂志, 2024, 13(04): 197-201.
[13] 洪凡, 陈敦金, 傅洋, 梁新月, 吴毅, 王晓怡. 体外受精-胚胎移植妊娠合并前置胎盘临床研究[J/OL]. 中华产科急救电子杂志, 2024, 13(03): 176-182.
[14] 何玉花, 钟欢妹, 王文惠, 沈永棋, 刘映云, 顾国威, 陈丹娜. 不同表型多囊卵巢综合征患者代谢指标及肥胖相关指标对多囊卵巢综合征合并代谢综合征人群的诊断效能分析[J/OL]. 中华临床实验室管理电子杂志, 2024, 12(04): 212-220.
[15] 张允耀, 王静, 范爱娟, 牟海萍. 超声诊断卵巢交界性肿瘤临床价值的荟萃分析[J/OL]. 中华诊断学电子杂志, 2024, 12(02): 85-89.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号