GDF-9转染的脂肪间充质干细胞治疗化疗性卵巢早衰大鼠

GDF-9 transfected adipose mesenchymal stem cells therapy for chemotherapy-induced premature ovarian failure in rats

目的:研究上调生长分化因子-9(GDF-9)表达的大鼠脂肪间充质干细胞(ASC)输注治疗化疗性卵巢早衰(POF)大鼠的效果。方法:酶解贴壁法从大鼠脂肪中培养ASC,脂质体法转染p EGFP-N3-GDF-9质粒入ASC,并验证GDF-9表达。腹腔注射顺铂建立大鼠POF模型,1周后通过尾静脉分别回输ASC和ASC-GDF-9细胞,观察大鼠体重、卵巢重量、血清中雌二醇(E_2)、卵泡刺激素(FSH)和促黄体激素(LH)值,并分析卵泡形成情况。结果:ASC细胞生长旺盛能向成骨和脂肪细胞分化,转染后可顺利表达GDF-9蛋白,转染效率为(43±9.68)%。顺铂腹腔注射可降低大鼠体重伴随卵巢体积缩小,各级卵泡减少,E_2降低,FSH和LH明显升高。输注ASC-GDF-9细胞4次后,可显著增加各级卵泡数目和基质细胞密度,降低LH和FSH激素水平。结论:转染GDF-9的ASC经尾静脉输注后,可更好地改善卵巢功能,恢复各级卵泡的发育,改善激素紊乱状态。

Objective：Isolate rat adipose derived mesenchymal stem cells,and enhance the growth differentiation factor-9 （GDF-9） expression to treat the chemotherapy-induced pre- mature ovarian failure （POF） rats. Method ： Using eollagenase digestion and adherent culture method,adipose derived mesenehymal stem cells （ASC） were obtained from rat adipose tissue. Rat GDF-9 gene was cloned into the pEGFP-N3 eukaryotic expression plasmid and sequenced. pEGFP-N3-GDF-9 plasmid was transfected into ASC using liposome method. Intraperitoneal in- jection of eisplatin daily was used to establish a rat model of POF. ASC and ASC-GDF9 cells were transfused by tail vein one week after the modeling process is over. Body weight, ovaries weight and serum levels of rats estradiol （E2） , follicle stimulating hormone （FSH） and luteini- zing hormone （LH） value, and the follicles in ovary sections were analyzed. Results：Obtained ASC had strong growth ability and could differentiate into osteoblasts and adipocytes. The GDF- 9 protein expressed successfully after transfection with pEGFP-N3-GDF-9 plasmid. The transfec- tion rate was （43_＋9.68）%. Intraperitoneal injection of cisplatin could make the POF rat, ac- companied by weight loss and follicle reduction. Estradiol （E2 ） concentration decreased signifi- cantly ,follicle stimulating hormone （FSH） and luteinizing generation （LH） increased signifi- cantly in POF rat serum. After continuous infusion of ASC-GDF-9 for four times once a week, the number of follicles in the ovary increased significantly. The density of granulosa cells also increased in ASC-GDF-9 group,while the level of LH and FSH hormone decreased. Conclu- sions：Intraperitoneal injection of GDF-9 transfeted ASC could improve the ovarian function,re- store the development of all levels of follicle, and improve the disorder of hormone in POF rat.