还原型谷胱甘肽和乌司他丁对冻融人类卵巢组织异种移植的保护作用

Protection of reduced glutathione and ulinastatin on frozen-thawed ovarian tissues after xenotransplantation

背景:还原型谷胱甘肽是有效的抗氧化剂,可直接消除氧自由基,具有抑制炎症细胞因子的作用。乌司他丁能抑制氧自由基的产生,抑制多种炎症递质的产生,维持细胞膜和溶酶体膜的稳定性,还可抑制大量亢进的酶的活性,发挥了对移植器官的保护作用。目的:探讨还原型谷胱甘肽和乌司他丁在改善冻融人类卵巢组织异种移植方面的效果。方法:收集6名来源于中山大学附属第一医院年龄(31.8±5.7)岁妇女的卵巢组织,慢速程序化冻融后每一份卵巢组织随机分成4组:还原型谷胱甘肽组(谷胱甘肽组)、乌司他丁组、谷胱甘肽+乌司他丁组、空白对照组,卵巢组织培养箱中平衡15 min后移植在雄性NOD-SCID小鼠背部皮下,移植后4组小鼠分别连续5 d给药还原型谷胱甘肽、乌司他丁、还原型谷胱甘肽和乌司他丁、生理盐水。移植后第1,3,7,14,28,56和85天回收移植物,用苏木精-伊红染色法及酶消化分离荧光染色法观察卵泡组织学形态、各阶段卵泡比例及卵泡的存活率。结果与结论:(1)解冻后的卵巢组织中以始基卵泡为主(79.5±14.3)%。新鲜卵巢组织切片的卵泡存活率为97.6%,解冻后卵巢组织的卵泡存活率为(84.4±5.7)%;(2)还原型谷胱甘肽组、乌司他丁组及谷胱甘肽+乌司他丁组卵泡存活率均高于空白对照组(P=0.022),联合用药与单独用药比较无显著差异(P>0.05);移植后第3天卵泡存活率达到低值(P <0.05),移植1周后有所上升;(3)用药各组在各时间点分离卵泡后的平均卵泡存活率稍高于对照组但差异无显著性意义(P=0.114),与组织学切片部分比较,分离卵泡的存活率明显更高;(4)结果说明,使用还原型谷胱甘肽和乌司他丁可以改善人类卵巢组织异种移植后早期的卵泡存活率,联合用药暂未见优势。

BACKGROUND: Reduced glutathione, as an effective antioxidant can inhibit inflammatory cytokines. Ulinastatin can directly remove oxygen free radicals and inhibit production of oxygen free radicals and various inflammatory mediators, sustain the stability of cell membrane and lysosomal membrane, and inhibit the activity of active enzymes, thus protecting the transplanted organ. OBJECTIVE: To evaluate the effect of reduced glutathione and ulinastatin on improving the xenotransplantation efficiency of frozen-thawed human ovarian tissue. METHODS: Ovarian tissue samples from 6 women aged 31.8±5.7 years old in the First Affiliated Hospital, Sun Yat-sen University, were collected and each tissue sample was randomly divided into four groups after slow freezing: reduced glutathione, ulinastatin, glutathione + ulinastatin and blank control (normal saline) groups. Ovarian tissues were xenotransplantated under the back skin of male NOD-SCID mice after 15-minute cultivation. The mice were administered with corresponding drugs for 5 consecutive days. The grafts were collected at 1, 3, 7, 14, 28, 56, and 85 days after transplantation. Histomorphology, growth situation and viability of the follicle were observed through hematoxylin-eosin staining and enzymatic isolation method. RESULTS AND CONCLUSION: The majority of the frozen-thawed ovarian tissues were primordial follicles (79.5±14.3%). The viability of the follicle in fresh ovarian tissue was 97.6%, and (84.4±5.7)% in thawed ovarian tissue. The reduced glutathione, ulinastatin, and glutathione + ulinastatin groups showed significantl improvement in the viability of the follicle compared with the control group (P=0.022). There was no significant difference between single and combined drug groups (P > 0.05). The follicle viability reached a lowest value at 3 days after transplantation (P < 0.05), and increased after 1 week later. The mean survival rate of the follicle in the isolated follicle at different time points showed no significant difference among groups (P=0.114). Compared with the histological sections, the isolated follicle showed highest survival rate. Our results suggest that reduced glutathione and ulinastatin can increase the early viability of the follicles after xenotransplantation, and combined administration shows no extra benefit.