Does adipose tissue-derived stem cell therapy improve graft quality in freshly grafted ovaries?
Does adipose tissue-derived stem cell therapy improve graft quality in freshly grafted ovaries?
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DOI：
10.1186/s12958-015-0104-2
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发表时间：
2015-09-23
期刊：
影响因子：
--
通讯作者：
Baracat EC
中科院分区：
文献类型：
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作者：
Damous LL;Nakamuta JS;Carvalho AE;Carvalho KC;Soares JM Jr;Simões Mde J;Krieger JE;Baracat EC
卵巢移植的一个主要问题是在低氧初期大量的卵泡丢失。脂肪组织来源的干细胞(ASCs)已经被用于促进血管生成，当注射到缺血组织中。本研究评价了脂肪组织来源的干细胞(ASCs)在注射后30天对新鲜移植卵巢的安全性和有效性。将绿色荧光蛋白转基因大鼠(5 × 10~4个/卵巢)获得的大鼠卵巢干细胞(RASCs)注射于30只12周龄成年雌性Wistar大鼠的异体卵巢或新鲜移植卵巢内。卵巢切除后立即将整个卵巢移植于腹膜后，不吻合血管。每天进行阴道涂片检查，以评估发情周期的恢复。分析雌二醇水平、移植物形态、卵泡存活率和密度。免疫组织化学方法检测RASC-GFP+、血管内皮生长因子组织表达、细胞凋亡(裂解caspase-3和TUNEL)和细胞增殖(Ki-67)。用逆转录聚合酶链式反应检测血管内皮细胞生长因子A、bcl2、表皮生长因子和转化生长因子β1的定量基因表达，用免疫荧光双标记法检测血管内皮细胞生长因子和血管内皮细胞生长因子的表达。经rasc-gfp+处理的移植物卵巢表现出更早的恢复发情期(p <； 0.05)，血管内皮生长因子-A表达增加(移植物卵巢是对照组的11倍，异位卵巢是对照组的5倍)，卵巢组织中血管数量(p <； 0.05)增加，而不会导致细胞凋亡或细胞增殖(p >； 0.05)。各组间雌二醇水平相似(p >； 0.05)。RASC-GFP+在移植卵巢和移植卵巢中的表达数量相似(p >； 0.05)，并且在两个注射组中均观察到绿色荧光蛋白和vWF的双重标记。RASC在自体新鲜卵巢移植物中的治疗是可行和安全的，可以诱导大鼠动情期提前恢复，并增强血管。这项先导性研究可能会对未来冷冻解冻卵巢组织的新研究有所帮助。本文的在线版本(doi：10.1186/s12958-0150104-2)包含补充材料，授权用户可以使用。
A major concern in ovarian transplants is substantial follicle loss during the initial period of hypoxia. Adipose tissue-derived stem cells (ASCs) have been employed to improve angiogenesis when injected into ischemic tissue. This study evaluated the safety and efficacy of adipose tissue-derived stem cells (ASCs) therapy in the freshly grafted ovaries 30 days after injection. Rat ASCs (rASCs) obtained from transgenic rats expressing green fluorescent protein (GFP)-(5 × 104 cells/ovary) were injected in topic (intact) or freshly grafted ovaries of 30 twelve-week-old adult female Wistar rats. The whole ovary was grafted in the retroperitoneum without vascular anastomosis, immediately after oophorectomy. Vaginal smears were performed daily to assess the resumption of the estrous cycle. Estradiol levels, grafts morphology and follicular viability and density were analyzed. Immunohistochemistry assays were conducted to identify and quantify rASC-GFP+, VEGF tissue expression, apoptosis (cleaved caspase-3 and TUNEL), and cell proliferation (Ki-67). Quantitative gene expression (qPCR) for VEGF-A, Bcl2, EGF and TGF-β1 was evaluated using RT-PCR and a double labeling immunofluorescence assay for GFP and Von Willebrand Factor (VWF) was performed. Grafted ovaries treated with rASC-GFP+ exhibited earlier resumption of the estrous phase (p < 0.05), increased VEGF-A expression (11-fold in grafted ovaries and 5-fold in topic ovaries vs. control) and an increased number of blood vessels (p < 0.05) in ovarian tissue without leading to apoptosis or cellular proliferation (p > 0.05). Estradiol levels were similar among groups (p > 0.05). rASC-GFP+ were observed in similar quantities in the topic and grafted ovaries (p > 0.05), and double-labeling for GFP and vWF was observed in both injected groups. rASC therapy in autologous freshly ovarian grafts could be feasible and safe, induces earlier resumption of the estrous phase and enhances blood vessels in rats. This pilot study may be useful in the future for new researches on frozen-thawed ovarian tissue. The online version of this article (doi:10.1186/s12958-015-0104-2) contains supplementary material, which is available to authorized users.