脂肪间充质干细胞移植对环磷酰胺诱导的大鼠卵巢功能损伤的影响  

Effect of adipose-derived mesenchymal stem cell transplantation on cyclophosphamide-induced ovarian damage in rats

作　　者：章婷婷[1] 章蒙蒙 方小玲[1] 朱付凡[1] 夏晓梦[1] 王西 王思雪 ZHANG Tingting;ZHANG Mengmeng;FANG Xiaoling;ZHU Fufan;XIA Xiaomeng;WANG Xi;WANG Sixue(Department of Obstetrics and Gynecology,Second Xiangya Hospital,Central South University,Changsha 410011,China)

机构地区：[1]中南大学湘雅二医院妇产科

出　　处：《中南大学学报（医学版）》2019年第7期731-740,共10页Journal of Central South University ：Medical Science

基　　金：湖南省自然科学基金(2018JJ3739)~~

摘　　要：目的:探讨脂肪间充质干细胞对化学药物治疗(以下简称化疗)药物环磷酰胺(cyclophosphamide,CTX)诱导的卵巢功能损伤的影响及其作用机制。方法:从雌性SD大鼠脂肪组织中分离脂肪间充质干细胞并进行体外培养,将其分为空白对照组和CTX组,每组15只;再选取45只正常动情周期的SD大鼠,用CTX75mg/kg腹腔注射给药1次的方式建立模型,再随机分为对照组(不再予其他处理,A组)、尾静脉注射组[尾静脉注射脂肪间充质干细胞0.6 mL(6×10^5个细胞),B组]和卵巢原位注射组[经卵巢原位注射脂肪间充质干细胞40μL(每侧20μL,2×10^4个细胞),C组],每组15只。比较A,B,C 3组大鼠用药前后生存质量、体重变化;ELISA法测定血清卵泡刺激素、雌二醇水平;HE染色观察卵巢组织及卵泡计数;免疫组织化学法、real-time PCR法及蛋白质印迹法测定骨形态发生蛋白-15(bone morphogeneticprotein 15,BMP-15),Bcl-2和Bax的表达;TdT介导的脱氧尿嘧啶缺口末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)检测A,B,C3组大鼠卵泡细胞凋亡率。结果:尾静脉注射或卵巢原位注射脂肪间充质干细胞后,与A组比较,B组和C组大鼠生存质量提高,移植前后的体重增长值明显增加,差异均有统计学意义(均P<0.01);与A组比较,B组大鼠血清雌二醇水平明显升高,卵泡刺激素水平明显下降(均P<0.01),C组大鼠血清雌二醇水平升高,卵泡刺激素水平明显下降(分别P<0.05,P<0.01);B组和C组大鼠卵巢组织切片HE染色见颗粒细胞层明显增多,黄体数量增多,各级卵泡计数均增加,可见多个新生卵泡及成熟的卵母细胞;与A组比较,B组大鼠的原始卵泡数、窦状和排卵前卵泡数以及总卵泡数均明显增加,窦前卵泡数也增加(分别P<0.01,P<0.05),C组大鼠的原始卵泡数、窦前卵泡数、窦状和排卵前卵泡数以及总卵泡数均明显增加(均P<0.01);与A组比较,B组和C组大鼠卵巢组织BMP-15和Bcl-2的表达明显增�Objective:To explore the effect of adipose-derived mesenchymal stem cells(ADMSCs)on ovarian damage induced by cyclophosphamide(CTX)and its mechanism.Methods:ADMSCs isolated from adipose tissue of female SD rats were cultured and divided into a blank group and a CTX group(n=15 in each group).CTX(75 mg/kg)was injected intraperitoneally to establish a model of ovarian damage in rats.A total of 45 female SD rats were also divided into 3 groups:Group A(15 rats,only injected intraperitoneally with 75 mg/kg CTX diluted with 1 mL 0.9%saline),Group B[15 rats,injected intraperitoneally with 75 mg/kg CTX diluted with 1 mL 0.9%saline,after 4 estrus cycles,injected 0.6 mL ADMSCs(6×10^5 cells)by the tail vein],and Group C[15 rats,injected intraperitoneally with 75 mg/kg CTX diluted with 1 mL 0.9%saline,after 4 estrus cycles,injected 40 mL ADMSCs(20 mL per side,2×10^4 cells)in situ ovarian].After 4 estrus cycles,the changes of quality of life,ponderal growth were recorded,the sex hormone levels[estradiol(E2),follicle-stimulating hormone(FSH)]were tested by ELISA,and the morphology of ovarian tissue and follicle count were observed by HE staining.The expression of BMP-15,Bcl-2 and Bax in ovarian tissues were tested by immunohistochemistry,real-time PCR or Western blotting.The apoptosis rate of follicular cells was detected by TdT-mediated dUTP nick end labeling(TUNEL)assay.Results:After transplantation of ADMSCs,compared with the Group A,their quality of life of rats in the Group B and C was improved,and the ponderal growth was increased(both P<0.01).Compared with the Group A,the serum E2 levels in the Group B and the Group C were increased(P<0.01,P<0.05),and the FSH levels in the Group B and C were decreased(both P<0.01).The granular cell layer,the number of corpus lutein and the count of various grade follicles were significantly increased,and many new follicles and mature oocytes were observed in the Group B and C.Compared with Group A,the count of primitive follicles,sinusoidal follicles,pre-ovulation follicles and total f

关 键 词：环磷酰胺 卵巢功能损伤 脂肪间充质干细胞移植 颗粒细胞凋亡 

分 类 号：R73[医药卫生—肿瘤]