抑癌基因pten在原始卵泡启动生长中的作用及机制
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摘要
目的:
本课题拟通过原始卵泡体外培养模型，探究调控原始卵泡启动生长的重要基因pten在原始卵泡的表达；利用携带pten-shRNA慢病毒载体转染离体卵巢，观察pten下调对原始卵泡启动生长的影响，进而探讨pten在原始卵泡启动生长中的作用及机制。
方法:
1.无菌条件下获取2日龄SD雌性大鼠卵巢，分别在Waymouth培养体系中培养0、4、8天。采用HE染色法观察0,4,8天卵巢中原始卵泡的表达和数量变化；采用PCR法观察pten mRNA在原始卵泡的表达；采用免疫组化法观察0,4,8天卵巢中PTEN蛋白在原始卵泡的定位和表达。
2．有效干扰片段的筛选，pten-shRNA慢病毒的包装,选择最佳感染时间和最佳感染复数(MOI：感染时病毒和细胞数量的比值),应用RT-PCR和western blot法验证pten-shRNA慢病毒基因沉默效率。应用荧光显微镜观察GFP表达变化，并放射免疫检测0,4,8天培养液中激素含量的变化。
3．应用PI3K抑制剂LY294002，慢病毒转染最佳干扰片段pten-shRNA。应用HE染色法观察各处理组原始卵泡启动生长发育，用RT-PCR法观察ptenmRNA的表达含量的变化，用western blot观察PTEN蛋白的表达变化及信号通路PI3K蛋白的表达变化。
结果：
1.原始卵泡数在卵泡总数中的比例随着培养天数的增加而减少；pten mRNA在原始卵泡中有表达,随着卵泡的发育表达强度逐渐减少；PTEN蛋白在0,4,8天组原始卵泡中均有表达，表达部位由卵母细胞胞浆转向颗粒细胞胞浆，随着卵泡的发育表达量逐渐减少。
2. Pten-shRNA慢病毒感染后，最佳感染时间为卵巢取出后的36h,最佳感染复数MOI=1.0×10~9×20×10~(-3)/1×10~6=20.用HE染色，RT-PCR和western blot法检测，与对照组相比，原始卵泡数占卵泡总数的比例降低，pten mRNA和PTEN蛋白表达量明显下降。Pten-shRNA慢病毒感染后的培养液中雌激素含量降低，孕激素显著降低。
3.加入PI3K抑制剂LY294002后，与空白组相比，pten mRNA表达变化不明显，原始卵泡数占所有卵泡总数的比例升高，原始卵泡发育受抑制。Pten-shRNA慢病毒感染后，用western blot观察PTEN蛋白表达在8天正常组比0天正常组下降，8天最佳干扰组比8天正常组下降。PI3K蛋白表达在8天最佳干扰组比8天正常组下降。
结论:
1. Pten mRNA在原始卵泡中有表达，PTEN蛋白的表达量随着原始卵泡启动生长而降低。
2. Pten-shRNA慢病毒转染后，原始卵泡数量减少，促进了原始卵泡的启动生长。
3.抑癌基因pten通过PI3K信号通路对原始卵泡的发育起作用，其上下游关系可能是pten→PI3K；还可同时通过卵泡细胞分泌的孕激素对原始卵泡的发育起调控作用。
Objective:
This topic to be of primordial follicles in vitro culture model to explore theregulation of primordial follicles initiation and growth of an important gene ptenexpression in primordial follicles, and used the carrying of pten-shRNA lentiviralvector to transfect vitro ovary, observed pten down impact the initiation and growthof primordial follicles, and then explore the pten in the role and mechanism ofprimordial follicles initiation and growth.
Methods:
1. Took2days old female SD rat ovaries under sterile conditions, appliedWaymouth culturing system to culture0,4,8days. Applied HE staining to observenormal0,4,8days expression and change of the primordial follicles in the ovary.Applied PCR method to observe pten mRNA expression in the primordial follicle.Applied immunohistochemistry to observe0,4,8days localization and expression ofPTEN protein in the primordial follicle.
2. Selected effective interference fragment, pten-shRNA lentiviral packaged,choosed the best transfer time and best multiplicity of infection (MOI: infected virusand the ratio of the number of cells)，applied RT-PCR and western blot method toverify pten-shRNA lentiviral gene silencing efficiency. Applied fluorescencemicroscope to observe GFP expression and change, meanwhile,the levels of hormonein the0,4,8days of culture solution were detected by radioimmunoassay.
3. Applied PI3K inhibitor LY294002, the best interference fragment oflentiviral transduction pten-shRNA. Applied HE staining to observe growth anddevelopment of the primordial follicle, applied RT-PCR method to observe changesin mRNA expression of contents, applied western blot to observe PTEN protein andPI3K protein expression.
The result:
1. The number of primordial follicles in the proportion of the total number offollicles reduced with the culture days increasing; ptenmRNA expression in theprimordial follicle, with the development of follicles expression intensity wasgradually reduced;0,4,8days group of PTEN protein expressed in the primordialfollicle, expression site by oocyte cytoplasm turned granular cytoplasm, expressionamount reduced.
2. Pten-shRNA lentiviral packaged, best time to transfer was the ovary removed36h, the best multiplicity of infection was MOI=1.0×10~9×20×10~(-3)/1×10~6=20. AppliedHE staining, RT-PCR and western blot, compared with the control group, primordialfollicle proportional of the total number of follicles reduced, the expression ofpten-shRNA group pten mRNA and PTEN protein decreased markedly. Estrogencontent in the media reduced, progesterone reduced significantly.
3. Added the PI3K inhibitor LY294002, compared with the control group,ptenmRNA expression did not change significantly, but primordial follicleproportional of the total number of follicles increased, primordial follicle growthwas suppressed. After pten-shRNA lentiviral transduction, observed by western blotPTEN protein expression in normal8days group than normal0day group fell, thebest interference8days group than normal8days group fell. PI3K proteinsexpression in the best interference8days group than normal8days group fell.
Conclusions:
1. Pten mRNA express in the primordial follicles, and the amount of PTENprotein expression reduce with the primordial follicles initiation and growth.
2. After pten-shRNA lentiviral transfection, the number of primordial folliclesreduce, promote the initiation and growth of primordial follicles.
3. Tumor suppressor gene pten by PI3K signaling pathway on primordial follicledevelopment, its upstream and downstream relationship may be the pten→PI3K.Meanwhile, through progesterone secretion in the follicle cells in the regulationof primordial follicle development.
本课题拟通过原始卵泡体外培养模型，探究调控原始卵泡启动生长的重要基因pten在原始卵泡的表达；利用携带pten-shRNA慢病毒载体转染离体卵巢，观察pten下调对原始卵泡启动生长的影响，进而探讨pten在原始卵泡启动生长中的作用及机制。
方法:
1.无菌条件下获取2日龄SD雌性大鼠卵巢，分别在Waymouth培养体系中培养0、4、8天。采用HE染色法观察0,4,8天卵巢中原始卵泡的表达和数量变化；采用PCR法观察pten mRNA在原始卵泡的表达；采用免疫组化法观察0,4,8天卵巢中PTEN蛋白在原始卵泡的定位和表达。
2．有效干扰片段的筛选，pten-shRNA慢病毒的包装,选择最佳感染时间和最佳感染复数(MOI：感染时病毒和细胞数量的比值),应用RT-PCR和western blot法验证pten-shRNA慢病毒基因沉默效率。应用荧光显微镜观察GFP表达变化，并放射免疫检测0,4,8天培养液中激素含量的变化。
3．应用PI3K抑制剂LY294002，慢病毒转染最佳干扰片段pten-shRNA。应用HE染色法观察各处理组原始卵泡启动生长发育，用RT-PCR法观察ptenmRNA的表达含量的变化，用western blot观察PTEN蛋白的表达变化及信号通路PI3K蛋白的表达变化。
结果：
1.原始卵泡数在卵泡总数中的比例随着培养天数的增加而减少；pten mRNA在原始卵泡中有表达,随着卵泡的发育表达强度逐渐减少；PTEN蛋白在0,4,8天组原始卵泡中均有表达，表达部位由卵母细胞胞浆转向颗粒细胞胞浆，随着卵泡的发育表达量逐渐减少。
2. Pten-shRNA慢病毒感染后，最佳感染时间为卵巢取出后的36h,最佳感染复数MOI=1.0×10~9×20×10~(-3)/1×10~6=20.用HE染色，RT-PCR和western blot法检测，与对照组相比，原始卵泡数占卵泡总数的比例降低，pten mRNA和PTEN蛋白表达量明显下降。Pten-shRNA慢病毒感染后的培养液中雌激素含量降低，孕激素显著降低。
3.加入PI3K抑制剂LY294002后，与空白组相比，pten mRNA表达变化不明显，原始卵泡数占所有卵泡总数的比例升高，原始卵泡发育受抑制。Pten-shRNA慢病毒感染后，用western blot观察PTEN蛋白表达在8天正常组比0天正常组下降，8天最佳干扰组比8天正常组下降。PI3K蛋白表达在8天最佳干扰组比8天正常组下降。
结论:
1. Pten mRNA在原始卵泡中有表达，PTEN蛋白的表达量随着原始卵泡启动生长而降低。
2. Pten-shRNA慢病毒转染后，原始卵泡数量减少，促进了原始卵泡的启动生长。
3.抑癌基因pten通过PI3K信号通路对原始卵泡的发育起作用，其上下游关系可能是pten→PI3K；还可同时通过卵泡细胞分泌的孕激素对原始卵泡的发育起调控作用。
Objective:
This topic to be of primordial follicles in vitro culture model to explore theregulation of primordial follicles initiation and growth of an important gene ptenexpression in primordial follicles, and used the carrying of pten-shRNA lentiviralvector to transfect vitro ovary, observed pten down impact the initiation and growthof primordial follicles, and then explore the pten in the role and mechanism ofprimordial follicles initiation and growth.
Methods:
1. Took2days old female SD rat ovaries under sterile conditions, appliedWaymouth culturing system to culture0,4,8days. Applied HE staining to observenormal0,4,8days expression and change of the primordial follicles in the ovary.Applied PCR method to observe pten mRNA expression in the primordial follicle.Applied immunohistochemistry to observe0,4,8days localization and expression ofPTEN protein in the primordial follicle.
2. Selected effective interference fragment, pten-shRNA lentiviral packaged,choosed the best transfer time and best multiplicity of infection (MOI: infected virusand the ratio of the number of cells)，applied RT-PCR and western blot method toverify pten-shRNA lentiviral gene silencing efficiency. Applied fluorescencemicroscope to observe GFP expression and change, meanwhile,the levels of hormonein the0,4,8days of culture solution were detected by radioimmunoassay.
3. Applied PI3K inhibitor LY294002, the best interference fragment oflentiviral transduction pten-shRNA. Applied HE staining to observe growth anddevelopment of the primordial follicle, applied RT-PCR method to observe changesin mRNA expression of contents, applied western blot to observe PTEN protein andPI3K protein expression.
The result:
1. The number of primordial follicles in the proportion of the total number offollicles reduced with the culture days increasing; ptenmRNA expression in theprimordial follicle, with the development of follicles expression intensity wasgradually reduced;0,4,8days group of PTEN protein expressed in the primordialfollicle, expression site by oocyte cytoplasm turned granular cytoplasm, expressionamount reduced.
2. Pten-shRNA lentiviral packaged, best time to transfer was the ovary removed36h, the best multiplicity of infection was MOI=1.0×10~9×20×10~(-3)/1×10~6=20. AppliedHE staining, RT-PCR and western blot, compared with the control group, primordialfollicle proportional of the total number of follicles reduced, the expression ofpten-shRNA group pten mRNA and PTEN protein decreased markedly. Estrogencontent in the media reduced, progesterone reduced significantly.
3. Added the PI3K inhibitor LY294002, compared with the control group,ptenmRNA expression did not change significantly, but primordial follicleproportional of the total number of follicles increased, primordial follicle growthwas suppressed. After pten-shRNA lentiviral transduction, observed by western blotPTEN protein expression in normal8days group than normal0day group fell, thebest interference8days group than normal8days group fell. PI3K proteinsexpression in the best interference8days group than normal8days group fell.
Conclusions:
1. Pten mRNA express in the primordial follicles, and the amount of PTENprotein expression reduce with the primordial follicles initiation and growth.
2. After pten-shRNA lentiviral transfection, the number of primordial folliclesreduce, promote the initiation and growth of primordial follicles.
3. Tumor suppressor gene pten by PI3K signaling pathway on primordial follicledevelopment, its upstream and downstream relationship may be the pten→PI3K.Meanwhile, through progesterone secretion in the follicle cells in the regulationof primordial follicle development.
引文
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[1]朱敬,李利,赵昕车.抑癌基因PTEN与人类肿瘤关系的研究现状[J].广东医学，2008，29（10）：1756～1758.
[2]王晓川，曹萍，万屏. PTEN在肿瘤的研究进展[J].中国现代医生，2008,46（17）:40～44.
[3]苏连明，朱雯，王程程.抑癌基因PTEN研究进展[J].牡丹江医学院学报，2010，31（2）：77～78.
[4]田新霞，吴浩强.新的肿瘤抑制基因PTEN[J].中华病理学杂志，2000,29（10）455～457.
[5]李曼，徐宁，黄宪章.抑癌基因PTEN的研究进展[J].现代检验医学杂志，2009,5（24）：12～14.
[6] Vitt UA,McGee EA,Hayashi M,et al. In vivo treatment with GDF-9stimulates primordialand primary follicle progression and the cacell maker CYP17inovariesofimmaturerats[J].Endocrinology,2000,141(10):3814～3820.
[7] Schmidt D,Ocitt C E,Anlag K,et al.The murine winged-helix transcription factor Foxl2isrequired for granulose cell differentiation and ovary maintenance [J].Development,2004,131(9):933～942.
[8] LiS,Maruo T,Ladies-Llave CA,et al.Stage expression of myc on coprotein in the humanovary during follicular growth,regression and atresia[J]. Endocr J,1994,41(1):83～92.
[9] Roby K F,Son D S,Taylor CC,et al.Alteration in reproductive functionin SRC tyrosine kinaseknockout mice[J].Endocrine,2005,26(2):169～76.
[10]郑莉萍，吴磊，郑月慧.原癌基因c-erbB2，c-myb对小鼠卵母细胞体外成熟的影响[J].生殖与避孕，2005,12（25）：712～715.
[11] Salmon NA,Handyside AH,Joyce IM.Oocyte regulation of anti-Mulerian hormoneexpression in granulose cells during ovarian follicle development in mice[J].Dev Biol,2004,266(1):201～208.
[12]罗丽莉，黄菊，隋旭霞,等. KL与其受体Kit在不同发育阶段大鼠卵巢卵泡中的表达研究[J].生殖与避孕，2007,8（27）：502～507.
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[1]朱敬,李利,赵昕车.抑癌基因PTEN与人类肿瘤关系的研究现状[J].广东医学，2008，29（10）：1756～1758.
[2]王晓川，曹萍，万屏. PTEN在肿瘤的研究进展[J].中国现代医生，2008,46（17）:40～44.
[3]苏连明，朱雯，王程程.抑癌基因PTEN研究进展[J].牡丹江医学院学报，2010，31（2）：77～78.
[4]田新霞，吴浩强.新的肿瘤抑制基因PTEN[J].中华病理学杂志，2000,29（10）455～457.
[5]李曼，徐宁，黄宪章.抑癌基因PTEN的研究进展[J].现代检验医学杂志，2009,5（24）：12～14.
[6] Vitt UA,McGee EA,Hayashi M,et al. In vivo treatment with GDF-9stimulates primordialand primary follicle progression and the cacell maker CYP17inovariesofimmaturerats[J].Endocrinology,2000,141(10):3814～3820.
[7] Schmidt D,Ocitt C E,Anlag K,et al.The murine winged-helix transcription factor Foxl2isrequired for granulose cell differentiation and ovary maintenance [J].Development,2004,131(9):933～942.
[8] LiS,Maruo T,Ladies-Llave CA,et al.Stage expression of myc on coprotein in the humanovary during follicular growth,regression and atresia[J]. Endocr J,1994,41(1):83～92.
[9] Roby K F,Son D S,Taylor CC,et al.Alteration in reproductive functionin SRC tyrosine kinaseknockout mice[J].Endocrine,2005,26(2):169～76.
[10]郑莉萍，吴磊，郑月慧.原癌基因c-erbB2，c-myb对小鼠卵母细胞体外成熟的影响[J].生殖与避孕，2005,12（25）：712～715.
[11] Salmon NA,Handyside AH,Joyce IM.Oocyte regulation of anti-Mulerian hormoneexpression in granulose cells during ovarian follicle development in mice[J].Dev Biol,2004,266(1):201～208.
[12]罗丽莉，黄菊，隋旭霞,等. KL与其受体Kit在不同发育阶段大鼠卵巢卵泡中的表达研究[J].生殖与避孕，2007,8（27）：502～507.
[13]高辉，郁琦，徐苓.原始卵泡的形成与发育[J].生殖医学杂志，2007,16（6）：453～456.
[14]娄超，黄高，王哲，等.人卵子发生过程中卵母细胞PTEN基因的表达及其意义[J].第四军医大学学报，2005,26（10）：884～887.
[15] Wei Ding,Wei Wang,Bo Zhou, et al. Formation of primordial follicles and immunolocal-ization of PTEN,PKB and FOXO3A protein in the ovaries of fetal and neonatal pigs[J].Journal of Reproduction and Development,2010,56(1):162～168.
[16] Reddy,P.et al.Oocyte-specific deletion of PTEN causes premature activation of theprimordial follicle pool[J].Science,2008,319(3):611～613.
[17] Pradeep Reddy,Wenjing Zheng and Kui Liu.Mechanisms maintaining the dormancy andsurvival of mammalian primordial follicles[J].Cell press2009,10(5):1～8.
[18] VIVANCO I,SAWYERSCL.The phosphatidy linosite3-kinase AKT pathway in humancancer[J]Nat Rev Cancer,2002,2(5):489～501.
[19] LESLIENR,BENNETTD,LNDSAYYE,et al. Redox regulation of PI3-kinase signaling viainactivation of PTEN[J]. EMBO2003,22(20):5501～5510.
[20] Li DM,Sum H.TEP1,encode by a candidate tumor suppressor locus,is a novel proteintyrosine phosphatase regulated by transforming growth actor beta[J]. Cancer Res,1997,57(11):2124～2129.
[21] Eppig JJ,Brien MJ.Development in vitro of mouse oocytes from primordial follicles[J]. BiolReprod,1996,54(1):197～207.
[22] Maki Goto,Akira Iwase,Hisao Ando, et al. PTEN and Akt expression during growth ofhuman ovarian follicles[J]. Assist Reprod Genet,2007,24(3):541～546.
[23]陈国庭，尹浩然，朱正刚. PTEN基因的研究进展[J].国外医学遗传分册，2001,24（9）：71～73.
[24]李祥勇，林观平，周克元.抑癌基因PTEN与细胞凋亡关系的研究进展[J].广东医学院学报，2005,23（6）：676～678.
[25] Adhikari,D.etal. Tsc/mTORC1signaling in oocytes governs the quiescence and activationof primordial follicles[J]. Hum. Mol. Genet.2009，10（5）：213～218.