非清髓预处理及人脐带血造血干细胞移植对小鼠生精功能的影响

张萌　姚观平　何方

[摘要] 目的 探讨化疗药非清髓预处理与人脐带血造血干细胞移植（HSCT）对小鼠生精功能的影响。 方法 将30只Balb/c小鼠按照随机数字表法分為三组，每组10只，分别为对照组、白消安（BU）联合环磷酰胺（CY）非清髓预处理（BU+CY）组、HSCT组。除对照组外，BU+CY组和HSCT组使用BU 10 mg/kg和CY 120 mg/kg预处理造成睾丸损伤，HSCT组进行人脐带血HSCT。HSCT移植3周后，称重法计算睾丸指数，进行小鼠精液质量常规分析及睾丸、附睾的病理组织学检查。 结果 与对照组比较，BU+CY组小鼠睾丸指数、精子密度和精子活力均显著降低，而精子畸形率升高（P < 0.01）。HSCT组小鼠移植物抗宿主病（GVHD）评分较高，睾丸指数、精子密度和活力均高于BU+CY组（P < 0.05），精子畸形率低于BU+CY组（P < 0.01）。病理学检查发现，BU+CY组小鼠睾丸体积较小，生精小管破坏明显，生精细胞肿胀、排列紊乱，生精上皮变薄，生精小管与附睾管内精子数目大大减少；与BU+CY组比较，HSCT组睾丸体积增大，生精小管完整性较好，生精小管内精子数量较高，但生精上皮层数和精母细胞数量仍较少。 结论 BU+CY非清髓预处理可造成明显睾丸损伤，轻度急性GVHD情况下，脐带血HSCT可一定程度促进睾丸生精功能的恢复。

[关键词] 白消安；环磷酰胺；脐带血造血干细胞；睾丸损伤

[中图分类号] R737.21 [文献标识码] A [文章编号] 1673-7210（2018）05（a）-0009-05

The effects of non-myeloablative conditioning and cord blood hematopoietic stem cells transplantation on spermatogenic function in mice

ZHANG Meng1* YAO Guanping2* HE Fang1 LI Yuxiang3 FAN Zhenhai1 YU Limei1

1.Key Laboratory of Cell Engineering in Guizhou Province， Affiliated Hospital of Zunyi Medical University， Guizhou Province， Zunyi 563000， China； 2.Human Reproductive Medicine Center， Affiliated Hospital of Zunyi Medical University， Guizhou Province， Zunyi 563000， China； 3.Department of Phylaxiology and Pediatric Dentistry， Hospital of Stomatology， Zunyi Medical University， Guizhou Province， Zunyi 563000， China

[Abstract] Objective To explore the effects of chemotherapeutic drugs non-myeloablative pretreatment and human cord blood hematopoietic stem cells transplantation （HSCT） on the spermatogenic function in mice. Methods Thirty Balb/c mice were divided into 3 groups by random digital table method， with 10 mice in each group， which contained control group， Busulfan （BU） combined with Cyclophosphamide （CY） non-myeloablative conditioning （BU+CY） group and HSCT group. In addition to the control group， BU+CY group and HSCT group were pretreated with BU 10 mg/kg and CY 120 mg/kg to induce testicular lesion， and HSCT group was taken human umbilical cord blood HSCT. After HSCT for three weeks， the testicular index was calculated by weighting method. The routine quality analysis of semen and histopathological examination of the testis and epididymis were carried out. Results Compared with the control group， the testicular index， sperm density and sperm viability in BU+CY group were significantly decreased， but the sperm malformation rate was significantly increased （P < 0.01）. The graft versus host disease （GVHD） score was higher in HSCT group. The testicular index， sperm density and sperm viability in HSCT group were higher than those in BU+CY group （P < 0.05）， while the sperm malformation rate was lower than that of BU+CY group （P < 0.01）. The pathological examination found that the testicular volume in BU+CY group was smaller， the seminiferous tubules were obviously impaired， the spermatogenic cells were swelled and arranged in disorder， the seminiferous epithelia was thinned， the number of sperm was significantly reduced in seminiferous tubule and epididymal tubule. Compared with the BU+CY group， the testicular volume in HSCT group was increased， the seminiferous tubule was more integrity， and the number of sperm was increased， but the number of layers seminiferous epithelia cells and spermatocyte were still less. Conclusion BU+CY non-myeloablative conditioning can cause obvious testis injury. Cord blood HSCT can promote a certain extent recovery of testicular spermatogenesis in mild acute GVDH mice.

[Key words] Busulfan； Cyclophosphamide； Cord blood hematopoietic stem cell； Testis injury

不孕不育的夫妇中，男性因素约占50%，且有60%～65%的男性不育为特发性，其中大部分被认为是由遗传缺陷和表观遗传学异常所致[1]，也有相当一部分患者存在由药物和环境等因素所致病。白血病、再生障碍性贫血等疾病治疗中常用较大剂量白消安（Busulfan，BU）和环磷酰胺（Cyclophosphamide，CY）进行清髓或非清髓预处理，杀死大量肿瘤细胞，清除受者大量免疫细胞的同时，也会造成包括生殖器官在内的多种组织器官损伤[2]。单次腹腔注射较大剂量的CY或BU及二者联用也是建立非梗阻性无精症动物模型的常用方法[3]，一次性腹腔注射BU 10 mg/kg和CY 120 mg/kg，第4周小鼠出现睾丸生精小管内精子及精子细胞消失、睾丸指数下降，直至第12周产生间质纤维化和20周仍处于无精子状态[4]。非清髓处理后进行异基因脐带血造血干细胞移植（hematopoietic stem cells transplantation，HSCT），不但可明……