Smad2/3与Smad7在大鼠化疗性卵巢早衰卵泡中的表达和意义
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摘要
目的探讨顺铂对大鼠卵巢结构和功能损伤的影响及其可能的分子机制。方法 3月龄SD雌性大鼠随机分为对照组(C组,5只)和化疗组(H组,5只)。H组给予每只大鼠腹腔注射顺铂2 mg/(kg.d),C组给予腹腔注射等体积生理盐水,连续注射7 d后检测大鼠卵巢指数(卵巢湿重/体重);酶联免疫吸附法检测血清雌二醇(E2)和促卵泡刺激素(FSH)水平;苏木素-伊红染色观察卵泡发育并计数各级卵泡;免疫组织化学和Real-time PCR法检测各组Smad2、磷酸化Smad2(p-Smad2)、Smad3、磷酸化Smad3(p-Smad3)、Smad4和Smad7的蛋白和mRNA表达。结果与C组相比,H组大鼠卵巢体积减小,生长卵泡数明显减少(P<0.05),大鼠卵巢指数下降(P<0.05),血清中E2水平降低和FSH水平上升(P<0.05)。免疫组织化学和Real-time PCR结果显示,Smad2(p-Smad2)、Smad3(p-Smad3)、Smad4和Smad7蛋白在卵巢各级卵泡均有表达,H组Smad2蛋白和mRNA表达增加(P<0.05),Smad2磷酸化水平(p-Smad2)表达增高(P<0.05);Smad3、Smad4和Smad7蛋白和mRNA表达减少(P<0.05),p-Smad3表达降低(P<0.05)。结论顺铂诱导大鼠卵泡损伤并促进卵巢早衰,引起卵泡细胞内Smad2表达增加,Smad3、Smad7表达降低,Smad细胞内信号通路参与了化疗性卵巢损伤的过程。
Objective To study the effect of cisplatin on ovarian structure and function in rats and its possible molecular mechanism. Methods Three-month-old SD female rats were divided into control group( C group,n = 5) and chemotherapy group( H group,n = 5). Rats in H group were given intraperitoneal injection of cisplatin 2 mg/( kg. d),rats in C group were given intraperitoneal injection of equal volume of saline. After 7 days continuous injection,rat ovarian index( ovarian wet weight/body weight) was measured. The levels of serum estradiol( E2) and follicle stimulating hormone( FSH) were tested by enzyme-linked immunosorbent assay( ELISA). The follicle development was observed by hematoxylin-eosin staining. The expression of Smad2,p-Smad2,Smad3,p-Smad3,Smad4 and Smad7 proteins/mRNAs in ovary tissue were detected by immunohistochemistry and Real-time PCR. Results Compared with C group,the ovarian volume in H group was decreased and the number of growing follicles was significantly decreased( P < 0. 05). The ovarian index decreased( P < 0. 05),and the levels of E2 and FSH in serum increased( P < 0. 05). Immunohistochemistry and Real-time PCR showed that Smad2( p-Smad2),Smad3( p-Smad3),Smad4 and Smad7 were expressed in ovarian follicles at all levels,and the expression of Smad2 protein and mRNA in H group increased( P < 0. 05). The expressions of Smad3,Smad4 and Smad7 protein and mRNA were decreased( P < 0. 05),and the expression of P-Smad3 was decreased( P < 0. 05). Conclusion Cisplatin-induced follicle injury in rats promotes premature ovarian failure,which result in the increase of Smad2 expression,and decrease of Smad3 and Smad7 expression in the follicle cells. The Smad intracellularsignal pathway is involved in the process of chemotherapy ovarian injury.
Objective To study the effect of cisplatin on ovarian structure and function in rats and its possible molecular mechanism. Methods Three-month-old SD female rats were divided into control group( C group,n = 5) and chemotherapy group( H group,n = 5). Rats in H group were given intraperitoneal injection of cisplatin 2 mg/( kg. d),rats in C group were given intraperitoneal injection of equal volume of saline. After 7 days continuous injection,rat ovarian index( ovarian wet weight/body weight) was measured. The levels of serum estradiol( E2) and follicle stimulating hormone( FSH) were tested by enzyme-linked immunosorbent assay( ELISA). The follicle development was observed by hematoxylin-eosin staining. The expression of Smad2,p-Smad2,Smad3,p-Smad3,Smad4 and Smad7 proteins/mRNAs in ovary tissue were detected by immunohistochemistry and Real-time PCR. Results Compared with C group,the ovarian volume in H group was decreased and the number of growing follicles was significantly decreased( P < 0. 05). The ovarian index decreased( P < 0. 05),and the levels of E2 and FSH in serum increased( P < 0. 05). Immunohistochemistry and Real-time PCR showed that Smad2( p-Smad2),Smad3( p-Smad3),Smad4 and Smad7 were expressed in ovarian follicles at all levels,and the expression of Smad2 protein and mRNA in H group increased( P < 0. 05). The expressions of Smad3,Smad4 and Smad7 protein and mRNA were decreased( P < 0. 05),and the expression of P-Smad3 was decreased( P < 0. 05). Conclusion Cisplatin-induced follicle injury in rats promotes premature ovarian failure,which result in the increase of Smad2 expression,and decrease of Smad3 and Smad7 expression in the follicle cells. The Smad intracellularsignal pathway is involved in the process of chemotherapy ovarian injury.
引文
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[19]Li J,Tang X,Chen X.Comparative effects of TGF-β2/Smad2 and TGF-β2/Smad3 signaling pathways on proliferation,migration,and extracellular matrix production in a human lens cell line[J].Exp Eye Res,2011,92(3):173-179.
[2]Howard-Anderson J,Ganz PA,Bower JE,et al.Quality of life,fertility concerns,and behavioral health outcomes in younger breast cancer survivors:a systematic review[J].J Natl Cancer Inst,2012,104(5):386-405.
[3]Yeh J,Kim B,Liang YJ,et al.Müllerian inhibiting substance as a novel biomarker of cisplatin-induced ovarian damage[J].Biochem Biophys Res Commun,2006,348(2):337-344.
[4]Liang D,Cao BL,Li JK,et al.Experimental study of cisplatin inducement in establishment of the rat model of premature ovarian failure[J].Modern Journal of Integrated Traditional Chinese and Western Medicine,2012,21(26):2873-2875.(in Chinese)梁丹,曹保利,李继坤,等.顺铂诱导大鼠卵巢早衰的实验研究[J].现代中西医结合杂志,2012,21(26):2873-2875..
[5]Li Dong,Chen Yan,Qi,Lei,et al.Differentially expressed genes in cisplatin-induced premature ovarian failure in rats[J].Anim Reprod Sci,2013,137(3-4):205-213.
[6]Jurisicova A,Lee HJ,D’Estaing SG,et al.Molecular requirements for doxorubicin-mediated death in murine oocytes[J].Cell Death Differ,2006,13(9):1466-1474.
[7]Ben-AharonⅠ,Shalgi R.What lies behind chemotherapy-induced ovarian toxicity[J]?Reproduction,2012,144(2):153-163.
[8]Hasky N,Uri-Belapolsky S,Goldberg K,et al.Gonadotrophinreleasing hormone agonists for fertility preservation:unraveling the enigma[J]?Hum Reprod,2015,30(5):1089-1101.
[9]Ben-AharonⅠ,Bar-Joseph H,Tzarfaty G,et al.Doxorubicininduced ovarian toxicity[J].Reprod Biol Endocrinol,2010,8(20):2-7.
[10]Morgan S,Anderson RA,Gourley C,et al.How do chemotherapeutic agents damage the ovary[J]?Hum Reprod Update,2012,18(5):525-535.
[11]Oktem O,Oktay K.Quantitative assessment of the impact of chemotherapy on ovarian follicle reserve and stromal function[J].Cancer,2007,110(10):2222-2229.
[12]Xu PL,Liu JM,Derynck R.Post-translational regulation of TGFbeta receptor and Smad signaling[J].FEBS Lett,2012,586(14):1871-1884.
[13]Pardali E,Goumans MJ,ten Dijke P.Signaling by members of the TGF-beta family in vascular morphogenesis and disease[J].Trends Cell Biol,2010,20(9):556-567.
[14]Massague J.TGFbeta signalling in context[J].Nat Rev Mol Cell Biol,2012,13(10):616-630.
[15]Massague J,Gomis RR.The logic of TGFbeta signaling[J].FEBS Lett,2006,580(12):2811-2820.
[16]Tomic D,Miller KP,Kenny HA,et al.Ovarian follicle development requires Smad3[J].Mol Endocrinol,2004,18(9):2224-2240.
[17]Li Q,Pangas SA,Jorgez CJ,et al.Redundant roles of SMAD2 and SMAD3 in ovarian granulosa cells in vivo[J].Mol Cell Biol,2008,28(23):7001-7011.
[18]Wei XH,Qi LH,Chi XCh,et al.The effects of FSH on the phosphorylation of Smad2/Smad3 protein in rat ovarian granulose cells[J].Acta Anatomica Sinica,2007,38(2):205-208.(in Chinese)卫晓红,祁丽花,迟晓春,等.Smad2/Smad3蛋白在大鼠卵巢颗粒细胞中的表达及FSH对其活化的影响[J].解剖学报,2007,38(2):205-208.
[19]Li J,Tang X,Chen X.Comparative effects of TGF-β2/Smad2 and TGF-β2/Smad3 signaling pathways on proliferation,migration,and extracellular matrix production in a human lens cell line[J].Exp Eye Res,2011,92(3):173-179.
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