中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (5): 828-832.doi: 10.3969/j.issn.1673-8225.2010.05.017

• 细胞与组织移植 cell and tissue transplantation • 上一篇    下一篇

大鼠卵巢组织的冷冻移植

鞠晓红1，马爱新1，王  冬2   

  1. 1吉林医药学院病原教研室，吉林省吉林市 132013 ；2吉林医药学院附属医院妇产科，吉林省吉林市  132013
  • 出版日期:2010-01-29 发布日期:2010-01-29
  • 通讯作者: 王 冬，主任医师，吉林医药学院附属医院妇产科，吉林省吉林市 132013
  • 作者简介:鞠晓红★，女，1968年生，吉林省敦化市人，汉族，2005年北华大学毕业，硕士，副教授，主要从事肿瘤免疫和移植免疫研究。 lijin838@126.com
  • 基金资助:

    吉林省科技厅项目(200705415)。

Ovarian tissue autografe after cryopreservation in rats

Ju Xiao-hong1, Ma Ai-xin1, Wang Dong2   

  1. 1 Staff Room of Etiology, Jilin Medical and Pharmacal College, Jilin  132013, Jilin Province, China; 2 Department of Gynaecology and Obstetrics, Affiliated Hospital of Jilin Medical and Pharmacal College, Jilin  132013, Jilin Province, China
  • Online:2010-01-29 Published:2010-01-29
  • Contact: Wang Dong, Chief physician, Department of Gynaecology and Obstetrics, Affiliated Hospital of Jilin Medical and Pharmacal College, Jilin 132013, Jilin Province, China
  • About author:Ju Xiao-hong★, Master, Associate professor, Staff Room of Etiology, Jilin Medical and Pharmacal College, Jilin 132013, Jilin Province, China lijin838@126.com
  • Supported by:

    the grant from Julin Science and Technology Bureau, No. 200705415*

PDF

538

被引次数

6

摘要：

背景：玻璃化冷冻是应用高浓度的冷冻保护液结合极快的制冷速度，使细胞快速冷冻，避免细胞内外冰晶形成对细胞的破坏作用，是一种比较新的冷冻方法，具有操作简单、冷冻效率高等特点。
目的：采用玻璃化冷冻技术冷冻大鼠卵巢组织，比较4种冷冻保护剂对大鼠卵巢组织学和功能的影响。
方法：将大鼠随机数字表法分为6组，每组6只：二甲基亚砜+乙二醇组、二甲基亚砜+乙二醇+蔗糖组、二甲基亚砜+乙二醇+蔗糖+乙酰胺组、乙二醇+蔗糖+乙酰胺组、去势组、正常对照组。4冷冻组摘除卵巢后应用相应的冷冻保护液冻存，去势组仅摘除卵巢不冻存、不移植，正常对照组不作处理。冻存2周后解冻复苏行同体异位移植，将卵巢组织植入大鼠后肢大腿内侧。移植后30 d，观察阴道上皮类型及动情周期；移植后3个月，经腹主动脉采血检测血清雌二醇水平;回收卵巢组织做组织学检查。
结果与结论：4冷冻组均可见卵巢组织结构受损表现，原始卵泡的形态正常率分别为67.9%，71.6%，80.5%，59.4%，初级卵泡形态正常率分别是41.6%，52.3%，55.9%，36.7%。二甲基亚砜+乙二醇+蔗糖+乙酰胺组卵泡正常率较二甲基亚砜+乙二醇组、乙二醇+蔗糖+乙酰胺组高(P < 0.05)；不同发育阶段卵泡构成比差异无显著性意义，均未见典型的次级卵泡。受损的卵细胞主要表现为体积变小、胞浆内出现空泡、核皱缩等。4冷冻组未出现典型动情周期的细胞类型。移植物自周围组织获得可见的血管供应，将卵巢组织块连接形成网络。移植物内毛细血管丰富，皮质中可见成群的原始卵泡，初级卵泡、次级卵泡所占比例低于原始卵泡，形态与正常者相似。4种冷冻方法冻存卵巢组织血清雌二醇水平较正常对照组明显降低(P < 0.01)，二甲基亚砜+乙二醇+蔗糖+乙酰胺组高于其他冷冻组(P < 0.05)。结果提示4种冷冻方法均对大鼠卵巢组织有一定程度破坏，初级卵泡的损伤大于原始卵泡。冻存复苏后，卵巢功能恢复，但并不完全，二甲基亚砜+乙二醇+蔗糖+乙酰胺组效果优于另外3组，但移植后动物没有出现规律的动情周期，提示冷冻方法尚需进一步完善。

关键词: 冷冻保护剂, 大鼠, 卵巢组织, 玻璃化冷冻, 组织移植

Abstract:

BACKGROUND: Vitrification is a comparatively new technology which applies high concentration cryoprotectant and rapid refrigeration. By the method, the cells were quickly frozen and to avoid damage by ice crystals inside and outside.
OBJECTIVE: To compare the effect of four cryoprotectants on morphology and function of ovarian tissue in rats after vitrification.
METHODS: The rats were randomly assigned into six groups with 6 rats for each: DMSO + EG, DMSO + EG + sucrose, DMSO + EG + sucrose + acetamide, EG + sucrose + acetamide, ovariectomized, and normal control groups. The ovarian tissues of four freezing groups were treated with the corresponding cryoprotectants, the vitrified ovarian tissues were then resected but not frozen and transplanted; otherwise, tissues were not treated with any treatment in the normal control group. Two weeks after freezing, the tissues were thawed and heterotopic-transplanted into femoribus internus of hind limb. At 30 days after implantation, vaginal epithelial cells and estrus cycle were observed, while after three months, blood were collected to detect the level of estradiol (E2) and the ovarian tissues were reclaimed to analyze their morphological changes.
RESULTS AND CONCLUSION: All ovarian tissues were damaged after cryoprerservation in four freezing groups. The rates of healthy primordial follicles were 67.9%, 71.6%, 80.5%, and 59.4%, respectively, while healthy primary follicles were 41.6%, 52.3%, 55.9%, and 36.7%, respectively. In all freezing groups, the rate of the healthy follicles in DMSO + EG + sucrose + acetamide group was higher than DMSO + EG group and EG + sucrose + acetamide group (P < 0.05). No significant difference was found in the proportion of follicles at different development stages among four groups. The typical secondary follicle was not found in four groups. Damaged ovotid showed oocyte pyknosis and vacuolation in cytoplasmic area. There was not typical cell type of all freezing groups. Ovarian autografting gained visible vascularity from surrounding tissue that connected ovarian tissue to form net. There was a lot of blood capillary in transplanted ovarian tissues and clumped primordial follicles in cortical substance. The rates of primary follicles and secondary follicles were lower than primordial follicles. The level of serum estradiol was obviously decreased compared with normal control group (P < 0.01). There was significant difference between DMSO + EG + sucrose + acetamide group and other three freezing groups (P < 0.05). Four kinds of freezing methods have poor effects on different stages of follicles and the structure of ovarian tissue. DMSO + EG + sucrose + acetamide group is an optimal protocol for cryoprerserving ovarian tissue. Freezing methods still need to explore further because the rats had not appeared disciplinary estrus cycle after ovarian autografting.

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