Tal Reshef, Liu Ying, Pluchino Nicola, Shaikh Shafiq, Mamillapalli Ramanaiah, Taylor Hugh S
Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine, New Haven, Connecticut 06510.
Endocrinology. 2016 Oct;157(10):3749-3759. doi: 10.1210/en.2016-1418. Epub 2016 Jul 18.
Bone marrow (BM)-derived cells (BMDCs) contribute to endometrial regeneration. Our objective was to develop a nongonadotoxic mouse BM transplant (BMT) model using 5-fluorouracil (5-FU) for investigating BMDCs trafficking in reproduction. Female C57BL/6J mice received either single (CTX-1) or paired (CTX-2) 5-FU (150 mg/kg) dose, or single (CTX-1+SCF) or paired-dose (CTX-3+SCF) 5-FU with stem cell factor (SCF). Control mice received BMT only or saline. BM cells (20 × 10) from transgenic green-fluorescent protein (GFP) mice were injected iv. For fertility experiment, mice were mated on day 28 after BMT. Alternatively, mice were killed 1 month after BMT and BMDCs recruitment to the uterus was determined. Mice receiving 5-FU ± SCF showed intact ovarian function and fertility. CTX-3+SCF resulted in greatest BM donor chimerism at 1 month (∼45%). Flow cytometry analysis demonstrated that 6.6% of total uterine cells in CTX-3+SCF mice were GFP+ BMDCs. Remarkably, this was about 40- and 80-fold greater than BMDCs in uterus of CTX-1 or BMT only mice (6.6% vs 0.16% vs 0.08%, respectively, P < .001). Immunohistochemical analysis showed that BMDCs in the uterus were mostly localized to the endometrial stroma (71.8%). The majority of endometrial BMDCs colocalized with the pan-leuokocyte CD45 marker (58.5%), but 41.5% were CD45-negative. Cytokeratin and CD31 staining showed that the GFP+CD45- cells were not epithelial or endothelial, confirming their stromal identity. We demonstrate that paired-dose 5-FU regimen results in efficient BM donor chimerism while maintaining ovarian function and fertility. This model could be used for studying BMDCs trafficking to the uterus in various reproductive physiological and pathological conditions.
骨髓（BM）来源的细胞（BMDCs）有助于子宫内膜再生。我们的目标是开发一种使用5-氟尿嘧啶（5-FU）的无性腺毒性小鼠骨髓移植（BMT）模型，用于研究BMDCs在生殖过程中的迁移。雌性C57BL/6J小鼠接受单次（CTX-1）或配对（CTX-2）剂量的5-FU（150mg/kg），或单次（CTX-1+SCF）或配对剂量（CTX-3+SCF）的5-FU与干细胞因子（SCF）。对照小鼠仅接受BMT或生理盐水。将来自转基因绿色荧光蛋白（GFP）小鼠的骨髓细胞（20×10）静脉注射。对于生育实验，小鼠在BMT后第28天进行交配。或者，在BMT后1个月处死小鼠，确定BMDCs向子宫的募集情况。接受5-FU±SCF的小鼠显示卵巢功能和生育能力完好。CTX-3+SCF在1个月时导致最大的骨髓供体嵌合率（约45%）。流式细胞术分析表明，CTX-3+SCF小鼠子宫中6. 6%的总细胞为GFP+BMDCs。值得注意的是，这比CTX-1或仅接受BMT的小鼠子宫中的BMDCs分别高出约40倍和80倍（分别为6.6%对0.16%对0.08%，P<.001）。免疫组织化学分析表明，子宫中的BMDCs大多定位于子宫内膜基质（71.8%）。大多数子宫内膜BMDCs与全白细胞CD45标记物共定位（58.5%），但41.5%为CD45阴性。细胞角蛋白和CD31染色表明，GFP+CD45-细胞不是上皮细胞或内皮细胞，证实了它们的基质身份。我们证明，配对剂量的5-FU方案可导致有效的骨髓供体嵌合，同时维持卵巢功能和生育能力。该模型可用于研究在各种生殖生理和病理条件下BMDCs向子宫的迁移。
