不同冷冻方法对人卵巢组织血管内皮生长因子表达及血管生成的影响

Vascular endothelial growth factor expression and angiogenesis in human ovarian tissue after different cryopreservations

背景:人卵巢组织冷冻已成为一种保存生育能力的手段,卵巢组织移植后必须经过血管重建过程才能恢复血供,冷冻保存和复苏技术是影响移植后卵巢组织血管重建的关键。目的:比较新型玻璃化冷冻和慢速冷冻后人卵巢组织血管内皮生长因子表达情况及微血管密度,探讨不同冷冻方法对人卵巢组织血管重建的影响。方法:8份卵巢组织标本取自子宫内膜癌患者手术切除的正常卵巢组织,将每份卵巢皮质切成1.5mm×1.5mm×1.0mm大小的组织块共12块后,随机数字表法分为3组:对照组(新鲜组)、新型玻璃化冷冻组和慢速冷冻组。将新型玻璃化冷冻组卵巢组织块依次在含体积分数7.5%乙二醇+体积分数7.5%二甲基亚砜+体积分数20%胎牛血清的TCM-199培养液和含体积分数15%乙二醇+体积分数15%二甲基亚砜+0.5mol/L蔗糖的TCM-199培养液中平衡脱水,然后直接浸入液氮并装入冷冻管保存,解冻时按浓度梯度1.0,0.5,0.25mol/L蔗糖和含体积分数20%牛血清的TCM-199培养液依次洗脱冷冻保护剂。将慢速冷冻组人卵巢组织块放入盛有1mL冷冻液(含1.5mol/L二甲基亚砜+体积分数20%牛血清+0.1mol/L蔗糖的TCM-199培养液)的1.8mL冷冻管内平衡,然后放入程序冷冻仪中按设定程序进行冷冻。解冻时按浓度梯度1.0mol/L二甲基亚砜+0.1mol/L蔗糖、0.5mol/L二甲基亚砜+0.1mol/L蔗糖、0.25mol/L二甲基亚砜+0.1mol/L蔗糖和0.1mol/L蔗糖依次洗脱冷冻保护剂。冻融组及对照组均进行体外培养。免疫组织化学观察培养0,2,4,6d各组人卵巢组织血管内皮生长因子和CD34表达情况,并进行微血管计数。结果与结论:3组人卵巢组织培养前后间质细胞中均见血管内皮生长因子成斑片状弱表达,培养2d血管内皮生长因子表达均增加并达到峰值,培养4d均开始减弱,6d时进一步减弱;与慢速冷冻组相比,新型玻璃化冷冻组血管内皮生长因子表达更接近对照组。3组人卵巢组织培养2d微血管密度均增加并达到峰值,对照组和新型玻璃化冷冻组明显高于慢速冷冻组(P<0.05);慢速冷冻组培养4d时微血管密度较培养2d时显著下降(P<0.05);3组培养6d时微血管密度较培养2d时显著下降(P<0.05)。与慢速冷冻法相比,新型玻璃化冷冻法能更好地保存人卵巢组织间质细胞和细胞外基质,对卵巢组织血管内皮生长因子表达及微血管生成的影响更少。

BACKGROUND：The cryopreservation of human ovarian tissue has become an attractive method to preserve female fertility. Human ovarian tissue experiences neovascularization after transplantation to recover blood supply,cryopreservation and resuscitation technique is a key for the neovascularization of human ovarian tissue following transplantation. OBJECTIVE：To investigate vascular endothelial growth factor （VEGF） expression and microvessel density in human ovarian tissue following novel needle immersed vitrification （NIV） and slow-freezing,to explore the influence of two cryopreservation methods play in the neovascularization of human ovarian tissue after transplantation. METHODS：Eight normal human ovarian tissues from patients with carcinoma of endometrium were cut into 12 fragments in the size of 1.5 mm × 1.5 mm × 1.0 mm,then randomly assigned to 3 groups：fresh control group,NIV group and slow-freezing group. In the NIV group,pieces of ovarian tissue strips were dehydrated in an equilibration solution consisting of 7.5% ethylene glycol and 7.5% dimethyl sulfoxide in TCM-199 supplement with 20% fetal bovine serum and a vitrification solution consisting of 15% ethylene glycol,15% dimethyl sulfoxide and 0.5 mol/Lsucrose,then were plunged in liquid nitrogen directly and sealed in liquid nitrogen-filled cryovials. For thawing,the needles holding ovarian tissues were serially transferred into 1.0,0.5,0.25 mol/L sucrose solution and TCM-199 supplemented with 20% fetal bovine serum. In the slow-freezing group,pieces of human ovarian cortex fragments were placed in a 1.8-mL cryovial containing 1 mL of TCM-199 medium supplemented with 0.1 mol/L sucrose,20% fetal bovine serum and 1.5 mol/L dimethyl sulfoxide,the cryovials were placed in the programmable freezer and cryopreserved by pre-set slow-cooling protocol. For thawing,the ovarian tissue strips were washed in a stepwise manner：1.0 mol/L dimethyl sulfoxide ＋ 0.1 mol/L sucrose,0.5 mol/L dimethyl sulfoxide ＋ 0.1 mol/L sucrose,0.25 mol/L dimethyl sulfoxide ＋ 0.1 mol/L sucrose and 0.1 mol/L sucrose. The frozen-thawed and fresh controlled human ovarian tissues were cultured in vitro. The expression of VEGF and CD34,as well as microvessel density,was analyzed by immunohistochemistry. RESULTS AND CONCLUSION：There was patchy and mild expression of VEGF in the stromal cells of all the three groups before and after culture. The expression of VEGF increased and reached peak value after culture for 2 days,began to decrease after culture for 4 days and further attenuated after culture for 6 days in all the three groups. Compared with slow-freezing group,the expression of VEGF in NIV group was closer to that in fresh control group. Microvessel density of all the three groups increased and reached peak value after culture for 2 days,and the microvessel density of fresh control group and NIV group was significantly higher than that of slow-freezing group （P 0.05）. The microvessel density of slow-freezing group after culture for 4 days and that of all the three groups after culture for 6 days significantly decreased compared with after culture for 2 days （P 0.05）. NIV is superior to slow-freezing to preserve stromal cells and extracellular matrix of human ovarian tissue,and plays less influence in VEGF expression and angiogenesis in human ovarian tissue.