人冻融卵巢组织异种异位移植血管生成的研究

Study on revascularization of frozen-thawed human ovarian tissues after heterogenous heterotopic transplantation in NOD-SICD mice

目的对人冻融卵巢组织在非肥胖糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠异位移植后血管重建模式的观察分析。方法收集2005至2007年在中山大学附属第一医院行卵巢切除手术的6例肿瘤患者部分外观正常的卵巢组织,人卵巢组织经慢速程序化冻融后,将其移植在雄性NOD-SCID小鼠背部皮下,在移植后第1、3、7、14、28、56和85天回收移植物。用免疫组化检测小鼠血管组织中CD34和CD31的表达,并在图像中计算微血管密度(MVD);观察小鼠背部卵巢组织移植物中人血管及小鼠新生血管随时间变化的情况,免疫组化检测其中血管内皮生长因子(VEGF)的表达,并采用Histochemical Score(HS)进行半定量评价;观察人卵巢组织中小鼠血管的重建过程,免疫组化检测其中VEGF的表达。结果抗人CD34标记的MVD在移植后逐渐下降(F=2.871,P=0.024)。抗小鼠CD31标记的MVD在移植后显著增加(F=155.710,P<0.001),移植后第1天观察到极少量新生血管形成,第3天增加明显(F=75.543,P<0.001),第14天可观察到有肌层大血管形成。移植后人卵巢组织中VEGF的表达呈先增加后下降的过程,在移植后第3天VEGF的表达增加明显(F=11.072,P=0.001),第14天后开始有所下降(F=18.564,P=0.001),各时间点之间VEGF的表达差异有统计学意义(F=45.981,P<0.05)。结论人冻融卵巢组织在雄性NOD-SCID小鼠皮下移植后可快速建立血供,第14天可观察到有肌层大血管形成,VEGF的表达在移植早期明显增加,为卵巢移植早期预防及治疗缺血再灌注损伤提供了理论基础。

Objective To analyze revascularization of frozen-thawed human ovarian tissue after heterogenous heterotopic transplantation in non-obese diabetes/severe combined immunodeficient(NOD-SICD)mice.Methods 6 cases of seemingly normal ovarian tissues were collected from tumor patients who underwent oophorectomy in the First Affiliated Hospital of Sun Yat-sen University in aperiod from 2005 to 2007.Human ovarian tissues were frozen and thawed in a slow freezing procedure,then they were transplanted into subcutaneous tissues on the back of male NOD-SCID mice.The grafts were retrieved on the 1 st,3 rd,7 th,14 th,28 th,56 th and 85 th days after transplantation.(1)Immunohistochemistry was used to detect expression of CD34 and CD31 in the vascular tissues of mice,and microvessel density(MVD)was calculated in the image;(2)The changes in human blood vessels and newborn mouse blood vessels in the back graft of mice with time were observed.The expression of vascular endothelial growth factor(VEGF)was detected by using immunohistochemistry and semi-quantitative evaluation was performed by Histochemical Score(HS).(3)The remodeling process of mouse blood vessels in human ovarian tissues was observed,and the expression of VEGF in the tissues was detected by immunohistochemistry.Results The anti-human antibody CD34-labelled MVD in the grafts gradually decreased after transplantation(F=2.871,P=0.024).While the anti-mouse antibody CD31-labelled MVD in the grafts remarkably increased after transplantation(F=155.710,P<0.001):very few murine vessels were observed on day1 immediately,profuse microvessels were observed on day 3(F=75.543,P<0.001)and large vessels with muscular layer were observed on day 14.The expression of VEGF shown an increase first and then decrease tendency:it increased on day 3(F=11.072,P=0.001),then decreased on day 14(F=18.564,P=0.001),in VEGF expression at different time points there were statistically significant differences(F=45.981,P<0.05).Conclusion Revascularization in human cryopreserved ovarian grafts occurred rapidly after subcutaneous transplantation into male NOD-SCID mice,large vessels with muscular layer were observed on day 14,and the expression of VEGF markedly increased at the early phase of transplantation,which provides a theoretical basis for prevention and treatment of ischemic-reperfusion injury occurred at early phase of ovarian transplantation.