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

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CTX 75 mg/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(6×105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40 µL(每侧20 µL，2×104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 morphogenetic protein 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，C 3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的表达明显增多，Bax的表达明显减少(均P<0.01)，大鼠卵巢颗粒细胞的凋亡率明显降低(P<0.01)；B组与C组之间比较，大鼠体重增长值，血清雌二醇及卵泡刺激素水平，各级卵泡计数，BMP-15，Bcl-2及Bax的表达水平，卵巢颗粒细胞凋亡率差异均无统计学意义(均P>0.05)。结论：脂肪间充质干细胞移植能够有效地修复CTX化疗所致的卵巢功能损伤，该作用可能是通过抑制颗粒细胞的线粒体凋亡途径实现的。

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

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 (6×105 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, 2×104 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 follicles, and pre-sinusoidal follicles were dramatically increased in the Group B. The follicle at all levels count was increased in the Group C than that in the Group A (all P<0.01). Comparing with the Group A, the expressions of BMP-15 and Bcl-2 were increased (all P<0.01), the expressions of Bax was decreased (both P<0.01), and the apoptosis rates of follicular cells were decreased in the Group B and C (both P<0.01). However, there was no difference between the Group B and the Group C in the above indexes (all P>0.05). Conclusion: ADMSCs transplantation can effectively repair ovarian damage induced by CTX in rats, which may be achieved by inhibiting mitochondrial apoptosis of granulosa cells.