CN101932331B - 卵泡体外成熟方法 - Google Patents
卵泡体外成熟方法 Download PDFInfo
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- CN101932331B CN101932331B CN2009801024299A CN200980102429A CN101932331B CN 101932331 B CN101932331 B CN 101932331B CN 2009801024299 A CN2009801024299 A CN 2009801024299A CN 200980102429 A CN200980102429 A CN 200980102429A CN 101932331 B CN101932331 B CN 101932331B
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Abstract
本发明涉及用于使卵泡和卵母细胞成熟的方法。更具体地,本发明涉及磷酸酶PTEN抑制剂(如氧钒酸和过氧钒酸配合物)在使卵泡和卵母细胞体外和体内成熟之方法中的用途。
Description
技术领域
本发明涉及用于使卵泡和卵母细胞体外成熟的方法。更具体地,本发明涉及磷酸酶PTEN抑制剂在用于使卵泡和卵母细胞体外成熟之方法中的用途。
背景技术
不论是从研究角度看还是在众多应用领域中,诱导卵泡活化以实现成熟都是非常期望的。原始卵泡(primordial follicle)潜在地可用作用于体外受精之卵母细胞的来源,但是能够利用活化和成熟卵泡用于其它应用(例如癌症的化疗后治疗或放射性治疗)也是非常重要的。然而,目前还没有用于原始卵泡活化的方法,这意味着卵母细胞材料的一个潜在重要来源仍尚未被开发。鉴于有关体外受精的争论,上述考虑愈显重要。
在临床上,如果女性的原始卵泡不能开始生长,即不能从休眠状态活化,那么她的卵泡将不能对激素(如促卵泡激素(FSH))产生应答。因此,该女性不能生育,并且她不能使用自己的卵母细胞用于体外受精。目前,如上所述,尚没有技术将原始卵泡用作体外受精之卵母细胞的来源。因此,现有技术需要开发用于诱导卵泡在体内和/或体外活化和成熟的方法以用于各种应用。
现有技术描述了脂质激酶磷脂酰肌醇3-激酶(PI3K),其使肌醇磷脂的肌醇环上的3’-OH基团磷酸化。PTEN(10号染色体上缺失的磷酸酶和张力蛋白同源物)是一种脂质磷酸酶,其逆转上述过程并因此用作PI3K作用的主要负调节剂(Cantley,Science 2002,296:1655-1657)。
发明内容
本发明人已经鉴定PTEN为抑制原始卵泡活化的因子。在卵母细胞特异性缺失Pten基因的小鼠模型中,所有原始卵泡均被过早活化。
因此,本发明提供了用于使卵泡和卵母细胞体外活化和成熟的方法,其包括使用一种或多种PTEN抑制剂。所述方法可用于活化卵泡体外成熟过程中的卵泡以及活化用于体外受精的卵母细胞。所述卵泡可以是非生长中的卵泡(non-growing follicle),如原始卵泡、间期卵泡(intermediate follicle)和初级卵泡(primary follicle)。
所述方法可包括在生理可接受的介质中孵育卵泡和/或卵母细胞,所述介质包含一种或者多种PTEN抑制剂。所述介质可进一步包含促性腺激素,比如FSH和/或CG。
孵育时间可以是足以使卵泡和/或卵母细胞实现活化和/或成熟的任何时间。
所述PTEN抑制剂可以提供卵泡和/或卵母细胞活化和/或成熟的任意浓度存在,例如1nM至1.0mM的浓度,尤其是1.0至100μM的浓度。
在本发明的一个优选实施方案中,所述卵泡和/或卵母细胞的活化和/或成熟包括如下步骤:(i)从合适的对象中获得用于卵泡和/或卵母细胞之活化和/或成熟的合适细胞和/或器官,(ii)在加湿的孵育器中(例如在温度基本保持37℃以及约5%CO2下)培养所述细胞和/或器官,(iii)以1nM至1.0mM之浓度、尤其是1.0至100μM之浓度的PTEN抑制剂瞬时或持续孵育所述细胞和/或器官,从而诱导所述卵泡和/或卵母细胞的活化和/或成熟,以及(iv)利用所获取的本发明范围内的材料。
本发明方法可用于人卵泡或来自动物(如家畜或濒危动物)的卵泡。例如,所述家畜可以是马、牛、猪、猫、狗。
本发明还提供了一种或者多种PTEN抑制剂在制备用于促进卵泡和卵母细胞之体外成熟的药物中的用途。
本发明还提供了这样的组合物,其包含用于促进卵泡和卵母细胞之体外成熟(特别是非生长中的卵泡(如原始卵泡、间期卵泡和初级卵泡)之体外活化)的一种或多种PTEN抑制剂。该组合物可进一步包含可药用载体、赋形剂或稀释剂。
PTEN抑制剂描述在WO 2005/097119及相应的美国申请10/599,748,以及WO2004/075897及相应的US 10/546,632中,其全部通过引用并入本文。
PDZ结构域相互作用(尤其是PTEN相关MAGI(膜相关反向鸟苷酸激酶蛋白)中PDZ结构域之间的相互作用)抑制剂可用作本发明的PTEN活性抑制剂。这些抑制剂描述于WO2004/092346、US7,141,600、WO2006/07542中,其全部通过引用并入本文。
过氧钒配合物(peroxovanadium complex)已显示为PTEN的强效抑制剂(Schmid等,FEBS Lett 2004,566:35-38;Rosivatz等人,ACSChem Biol 2006,1:780-790)。
过氧钒配合物的合成可如Shaver等(Inorg Chem 1993,32:3109-3113)、Rosivatz等(ACS Chem Biol 2006,1:780-790)以及Posner等(J Biol Chem 1994,269:4596-604)所述进行。
可根据本发明使用的化合物的实例是:
bpV(bipy),双过氧(联吡啶)氧钒酸钾(V),K[VO(O2)2C10H8N2];
bpV(phen),双过氧(1,10-菲咯啉)氧钒酸钾(V),K[VO(O2)2C12H8N2];
bpV(pic),双过氧(吡啶甲酸)氧钒酸二钾(V),K2[VO(O2)2C6H4NO2];
bpV-HOpic,双过氧(5-羟基吡啶-2-羧基)氧钒酸二钾(V),K2[VO(O2)2C6H4NO3];
VO-pic,二-(吡啶甲酸)氧钒酸盐(IV),VOC10H10NO4;
VO-OHpic,二-(3-羟基吡啶甲酸)氧钒酸盐(IV),VOC10H10NO6;
bpV-双胍,双过氧(苯基双胍)氧钒酸钾(V),K[VO(O2)2C8H11N5];
VO-双胍,二-(苯基双胍)氧钒酸盐(IV),VOC16H20N10;
bpV-异喹啉,双过氧(异喹啉羧酸)氧钒酸二钾(V),K2[VO(O2)2C10H7NO2];
下式的化合物
其中:
R是环烷基,
A是低级亚烷基,
以及在喹诺酮核的3-和4-位之间的键代表单键或双键;
选自以下的抗坏血酸衍生物或脱氢抗坏血酸衍生物:
其中,
R1表示H、C1-C3烷基、芳基、烷基芳基、(CH2)nCOXR3、(CH2)nXCOR3、(CH2)nCOR3、(CH2)nSO2R3、(CH2)nXR3、(CH2)nSO2XR3、(CH2)nXSO2R3、(CH2)nNR3R4或(CH2)nCO(CH2)mXR3;
R2表示H、C1-C3烷基、芳基、烷基芳基、(CH2)nCOXR3、(CH2)nXCOR3、(CH2)nCOR3、(CH2)nSO2R3、(CH2)nXR3、(CH2)nSO2XR3、(CH2)nXSO2R3、(CH2)nNR3R4或(CH2)nCO(CH2)mXR3;
R3、R5、R6独立地为H、C1-C4烷基、芳基或烷基芳基;
R4表示H、C1-C4烷基、芳基、烷基芳基、NHSO2R5、NHCO2R5或NR5R6;
m=0至3;n=0至3;以及X表示O或NR4;
根据下式的化合物
其中,
R1表示H、C1-C4烷基、芳基、烷基芳基、COXR2、COR2、SO2XR2、SO2R2;
R2表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR4、(CH2)nXCOR4、(CH2)nXR4、(CH2)nSO2XR4、(CH2)nXSO2R4、NHSO2R4、NHCOR4、NHCO2R4、NHCOCO2R4或NR4R5;
R3表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR4、(CH2)nXCOR4、(CH2)nXR4、(CH2)nSO2XR4、(CH2)nXSO2R4、NHSO2R4、NHCOR4、NHCO2R4、NHCOCO2R4或NR4R5;
R4表示H、C1-C4烷基、芳基或烷基芳基;
R5表示H、C1-C4烷基、芳基、烷基芳基、NHSO2R6、NHCOR6、NHCO2R6、NR6R7或N=C(R6R7);
R6表示H、C1-C4烷基、芳基或烷基芳基;
R7表示H、C1-C4烷基、芳基或烷基芳基;
n=0至3;以及X表示O或NR5;
根据下式的化合物
其中,
A是五元或六元环;R1表示H、C1-C3烷基、芳基、烷基芳基、(CH2)nCOXR3、(CH2)nXCOR3、(CH2)nCOR3、(CH2)nSO2R3、(CH2)nXR3、(CH2)nSO2XR3、(CH2)nXSO2R3、NHSO2R3、NHCO2R3、NHCOR3、NHCOCO2R3、NR3R4或(CH2)nCO(CH2)mX3;
R2表示H、C1-C3烷基、芳基、烷基芳基、(CH2)nCOXR3、(CH2)nXCOR3、(CH2)nCOR3、(CH2)nSO2R3、(CH2)nXR3、(CH2)nSO2XR3、(CH2)nXSO2R3、NHSO2R3、NHCO2R3、CHCOR3、NHCOCO2R3、NR3R4或(CH2)nCO(CH2)mX3;
R3表示H、C1-C4烷基、芳基或烷基芳基;
R4表示H、C1-C4烷基、芳基、烷基芳基、NHSO2R5、NHCO2R5或NR5R6;
R5表示H、C1-C4烷基、芳基或烷基芳基;
R6表示H、C1-C4烷基、芳基或烷基芳基;
n=0至3;m=0至3;以及X表示O或者NR4;
A环可以是饱和的、不饱和的或芳香性的,并且可任选地包含N和O;
根据下式的化合物
其中,
R1表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR3、(CH2)mXCOR3、(CH2)mXR3、(CH2)nCOR3、(CH2)nSO2XR3或(CH2)mXSO2R3;
R2表示H、C1-C4烷基、芳基或烷基芳基;
R3表示H、C1-C烷基、芳基或烷基芳基;
R4表示H、C1-C4烷基、芳基、烷基芳基、NHSO2R5、NHCO2R5、N=C(R5R6)或NR5R6;
R5表示H、C1-C4烷基、芳基或烷基芳基;
R6表示H、C1-C4烷基、芳基或烷基芳基;
m=1-3;n=0-3;以及X表示O或NR4;
下式的取代的1,10-菲咯啉-5,6-二酮:
其中,
R1表示O、C 1-C4烷基、(CH2)nCOXR2、(CH2)nXCOR2、(CH2)nXR2、(CH2)nCOR2、(CH2)nSO2XR2、(CH2)nXSO2R2或(CH2)nSO2R2;
R2表示H、C1-C4烷基、芳基、烷基芳基、NHSO2R4、NHCOR4、NHCO2R4、NHCOCO2R4或NR4R5;
R3表示H、C1-C4烷基、芳基、烷基芳基、NHSO2R4、NHCOR4、NHCO2R4、NHCOCO2R4或NR4R5;
R4表示H、C1-C4烷基、芳基或烷基芳基;
R5表示H、C1-C4烷基、芳基或烷基芳基;
R6每次出现时独立地选自氢、卤素、NO2、R4R10、C1-C4烷基、NH(CH2)pCO(CH2)qXR2、(CH2)pCOXR2、(CH2)pXCOR2、(CH2)pXR2、(CH2)pCOR2、(CH2)pSO2XR2或(CH2)pXSO2R2;
R7表示H、C1-C4烷基、芳基、烷基芳基、SO2R4、NHSO2R4、NHCO2R4或NR8R9;
R8独立地表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2或(CH2)nXR2;
R9独立地表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nXR2、(CH2)pCOXR2、(CH2)pXCOR2、(CH2)pXR2、(CH2)pCOR2、(CH2)pSO2XR2、(CH2)pXSO2R2或(CH2)pSO2R2;
R10表示H、C1-C4烷基、芳基、烷基芳基、SO2R4、NHSO2R4、NHCO2R4或NR8R9;
m独立地表示0或1;n=1-5;p=0-5;q=0-5;X表示O或NR3;以及Z表示O或NR7;
下式的取代的菲-9,10-二酮:
其中,
R1表示H、NO2、NR5R6、卤素、氰基、烷基、烷基芳基、羰基、羧基、COR2或CONR5R6;
R2和R3独立地表示H、C1-C4烷基、芳基或烷基芳基;
R4表示H、C1-C4烷基、芳基、烷基芳基、SO2R2、NHSO2R2、NHCOR2、NHCO2R2、N=CR2R3或NR5R6;
R5表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nXR2、(CH2)nCO(CH2)mXR2、SO2R2、(CH2)nCO(CH2)nCOXR2或(CH2)nCOR2;
R6表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nXR2、(CH2)nCO(CH2)mXR2、SO2R2、(CH2)nCO(CH2)nCOXR2或(CH2)nCOR2;
m=0-3;n=0-3;以及X表示CR2R3、O或NR4;
如下式的化合物:
其中,
R1表示H、NO2、NR5R6、卤素、氰基、烷基、烷基芳基、羰基、羧基、COR2、CONR5R6、SO3R2或SO2NR2R3;
R2和R3独立地表示H、C1-C4烷基、芳基或烷基芳基;
R4表示H、C1-C4烷基、芳基、烷基芳基、SO2R2、NHSO2R2、NHCOR2、NHCO2R2、N=CR2R3或NR5R6;
R5表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nXR2、(CH2)nCO(CH2)mXR2、SO2R2、(CH2)nCO(CH2)nCOXR2或(CH2)nCOR2;
R6表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nXR2、(CH2)nCO(CH2)mXR2、SO2R2、(CH2)nCO(CH2)nCOXR2或(CH2)nCOR2;
m=0-3;n=0-3;以及X表示CR2R3、O或NR4;
下式的取代的菲-9-醇:
其中,
R1表示H、NO2、NR5R6、卤素、氰基、烷基、烷基芳基、羰基、羧基、COR2或CONR5R6;
R2和R3独立地表示H、C1-C4烷基、芳基或烷基芳基;
R4表示H、C1-C4烷基、芳基、烷基芳基、SO2R2、NHSO2R2、NHCOR2、NHCO2R2、N=CR2R3或NR5R6;
R5表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nXR2、(CH2)nCO(CH2)mXR2、SO2R2、(CH2)nCO(CH2)nCOXR2或(CH2)nCOR2;
R6表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nXR2、(CH2)nCO(CH2)mXR2、SO2R2、(CH2)nCO(CH2)nCOXR2或(CH2)nCOR2;
m=0-3;n=0-3;以及X表示CR2R3、O或NR4;
下式的取代的萘-1,2-二酮:
其中,
R1表示H、NO2、NR3R4、卤素、氰基、烷基、烷基芳基、羰基、羧基、(CH2)nCOXR3、COR2、SO3R2、SO2NR3R4、NHSO2R3、NHCO2R3、NHCOR3、NHCOCO2R3、NR3R4或CONR3R4;
R2表示H、C1-C4烷基、芳基或烷基芳基;
R3和R4独立地表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nOR2或(CH2)nCO(CH2)mXR2;
m=0-3;n=0-3;以及X表示O或NR2;
下式的取代的萘-1,4-二酮:
其中,
R1表示H、NO2、NR3R4、卤素、氰基、烷基、烷基芳基、羰基、羧基、(CH2)nCOXR3、COR2、SO3R2、SO2NR3R4、NHSO2R3、NHCO2R3、NHCOR3、NHCOCO2R3、NR3R4或CONR3R4;
R2表示H、C1-C4烷基、芳基或烷基芳基;
R3和R4独立地表示H、C1-C4烷基、芳基、烷基芳基、(CH2)nCOXR2、(CH2)nOR2或(CH2)nCO(CH2)mXR2;
m=0-3;n=0-3;以及X表示O或者NR2;以及
具有下式的化合物:
其中,
n是0、1或2;
X1是NH、N(CH3)、CH2、CH(CH3)、C(CH3)2、O、S、S(O)或SO2;
R0选自C1-C3烷基、环丙基、卤素、OR5以及S(O)mR5,其中m是0、1或2;
R1和R2独立地选自C2-C8烯基、苯基环丙基、苯基丙烯基、R6-X2-C(R8)(R8)-R7-、R6-X2-N(R8)-R7-和R10X3R7-;
R3和R4独立地为氢、甲基或乙基;
R5是甲基或乙基;
R6选自氢、C1-C10烷基、芳基、W、Y、NH2、NHCONR3R4、NHCOOR3和NHSO2R9;
R7选自直接键合、具有1至10个碳原子的烷基、芳基、-(NH)p(CH2CH2O)q(NH)p-和W,其中p是0或1,q是从1至4的整数;
R8选自H、Y、OH、-NHCONR3R4、-NHCOOR3、-NHSO2R9、-(CH2)rCO2R3和(CH2)rCONR3R4,其中r是1至3的整数;
R9是芳基或C1-C6烷基;
R10选自C1-C10烷基、芳基和W;
X2选自直接键合、-NH-、-N(CH3)-、-NCONR3R4、-NCOOR3和-NSO2R9;
X3选自O、S、SO和SO2;
X4选自-CH-、C-卤素、-C(CH3)或-C(C2H5);
W是饱和的碳环或杂环基;
Y选自COOH、COOR3、CONR3R4、CONHSO2R5、羟甲基、-CH2COOH、CH2CONR3R4和5-四唑基;
Z是-CH2-、-CH(CH3)-、-C(CH3)2-或-CO-
可将靶向编码PTEN之核酸的反义寡核苷酸和双链RNA用作本发明的PTEN抑制剂。这些化合物描述于WO 01/07457和WO 01/90341、WO 2004/27030和WO 2004/63329及其相应的美国专利US 6,020,199和US6,284,538中,其全部通过引用并入本文。用作PTEN特异性抑制剂的抗体和/或抗体片段也在本发明的范围中。
本发明还提供了用于鉴定潜在可用于体外活化原始卵泡之化合物的方法。所述方法包括测量所述化合物用作PTEN活性之抑制剂的能力。
如Schmid等(FEBS Lett 2004,566:35-38)所述,PTEN活性可通过磷酸释放来测量。基本上,重组PTEN的酶活性于30℃在含有50ng/μlBSA、150μM合成的二棕榈酰基-PtdIns(3,4,5)P3(Cell Signals)和0.25%(w/v)辛基糖苷(Sigma)的200mM Tris(pH 7.4)中30分钟来测量。为了终止酶反应,向测定中加入0.7体积的显色剂(2.3mg/ml孔雀绿,在3.6M HCl以及17mM钼酸铵中)。使此混合物显色20分钟并在625nm下测量吸光度。
如果化合物的IC50小于100μM,优选IC50小于10μM,或甚至更优选IC50小于1μM,则该化合物定义为PTEN抑制剂。
PTEN抑制剂的功能可通过测量在PTEN存在下增加的PI3激酶活性来验证,PI3激酶活性也可通过丝氨酸473上Akt的磷酸化水平来测量。
本发明还提供了用于使卵母细胞体外成熟的方法,其包括根据本发明的方法活化原始卵泡。
本发明还提供了体外受精的方法,其包括植入有此需要的胚,其中所述胚通过包括用精子处理成熟卵母细胞的方法产生,其中所述卵母细胞通过包括根据本发明方法活化原始卵泡的方法而产生。
本发明还提供了体外受精或胚移入雌性哺乳动物中后促进胚发育的方法,包括将源自卵母细胞的胚植入所述雌性哺乳动物中,其中所述卵母细胞通过包括根据本发明方法活化原始卵泡的方法而产生。
本发明还提供了用于卵泡和卵母细胞之体内活化和成熟的方法,包括施用含有一种或多种PTEN抑制剂的组合物。优选地,所述施用通过局部注射进行,更优选通过卵泡内或囊内注射进行。所述卵泡可以是非生长中的卵泡,比如原始卵泡、间期卵泡和初级卵泡。所述方法可用于人或者动物,比如家畜或者濒危动物。所述家畜动物可以是例如马、牛、猪、猫、狗。
所述局部注射可借助于腹腔镜或超声来进行。
本发明还提供了一种或多种PTEN抑制剂在制备用于卵泡和卵母细胞之体内活化和成熟(特别是用于非生长中的卵泡(如原始卵泡、间期卵泡和初级卵泡)之体内活化)的药物组合物中的用途。所述组合物可旨在通过局部注射或其它类型的局部递送来施用,优选通过卵泡内或囊内注射来施用。所述组合物可旨在用于人用或兽医用。
本发明还提供了含有一种或多种PTEN抑制剂的用于卵泡和卵母细胞之体内活化和成熟(特别是用于非生长中的卵泡(如原始卵泡、间期卵泡和初级卵泡)之体内活化)的组合物。所述组合物还可包含可药用载体、赋形剂或稀释剂。所述组合物可被配制成用于局部注射,优选用于卵泡内或囊内注射。所述组合物可旨在用于人用或兽医用。
附图说明
图1.在卵母细胞缺少PTEN的小鼠中原始卵泡的过度活化。
将来自35日龄小鼠的卵巢包埋到石蜡中,制备8μm厚的切片并用苏木精染色。A.对照小鼠:在对照正常小鼠中,存在原始卵泡(插图中的箭头)。B-C.卵母细胞中缺少PTEN的小鼠。在缺少PTEN的卵母细胞中,所有卵泡均过早长大(C,箭头),以及整个卵巢变得更大(B)。
图2.在PtenloxP/loxP;GCre+卵母细胞中提高的Akt信号传导
从PD5和PD12-14的PtenloxP/loxP;GCre+和PtenloxP/loxP小鼠的卵巢中分离出卵母细胞并进行western印迹。(A)在PD12-14PtenloxP/loxP;GCre+和PtenloxP/loxP卵母细胞中p-Akt(丝氨酸473)和总Akt的水平。(B)在PD12-14ptenloxP/loxP;GCre+和PtenloxP/loxP卵母细胞中经Kit配体(KL)处理(100ng/ml,2分钟)后的Akt(p-Akt,丝氨酸473)活化。Akt水平用作内部对照。(C)对PD5和PD12-14PtenloxP/loxP;GCre+和PtenloxP/loxP卵母细胞的信号传导研究,显示了p-Akt(丝氨酸473)、rpS6、p-rpS6(丝氨酸235/6)、p-mTOR(丝氨酸2448)、p-TSC2(苏氨酸1462)以及p-S6K(苏氨酸389)的水平。总的Akt、mTOR、TSC2、S6K和β-肌动蛋白的水平用作内部对照。所有实验均重复至少3次。对于用于western印迹的PD5卵母细胞的分离而言,每个泳道使用10-15只PtenloxP/loxP;GCre+和PtenloxP/loxP小鼠。对于PD12-14卵母细胞的分离而言,每个泳道使用3-5只ptenloxP/loxP;GCre+小鼠或6-10只ptenloxP/loxP小鼠。在每个泳道中,加样30-40μg蛋白质样品。
图3.PTEN抑制剂促进培养的小鼠卵巢中原始卵泡的存活和发育。
将出生后四天的含有原始卵泡的小鼠卵巢与载体(A)或PTEN抑制剂bpv(Hopic)(B)一起培养8天。所述PTEN抑制剂提高了卵泡在培养的卵巢中的存活率(B的箭头与A的箭头指明在经PTEN抑制剂处理的卵巢中拯救的坏死部分);并且所述PTEN抑制剂刺激扁平的前颗粒细胞(pregranulosa cell)(箭头,A)增殖和分化成为立方颗粒细胞(箭头,B),这是原始卵泡活化进入生长阶段的至关重要的步骤。
发明详述
目前,如果将动物或人原始卵泡在体外培养,则其很难开始它们的生长,并经历成熟。根据本发明,在PTEN抑制剂存在下,可将来自人或家畜/濒危动物的原始卵泡进行体外活化,即启动原始卵泡的生长。然后可将这些活化的卵泡进行进一步培养,直到其成熟,其可用于体外受精。
所述方法是将包含来自人或家畜/濒危动物之原始卵泡的卵巢切片用一种或多种PTEN抑制剂进行临时处理,以启动其生长。卵泡生长一经诱发,即撤除PTEN抑制剂,利用现有技术常规地维持进一步的卵泡培养。
所述方法可用于原始卵泡不能天然活化的女性,或者用于将要进行癌症化疗或放疗的女性。所述方法还可用于体外活化家畜或濒危动物的原始卵泡,以增加卵泡来源和提高体外受精的成功率。
可利用在同一时间培养的小鼠卵巢切片对培养的卵巢切片中PTEN活性的有效抑制进行监测。
实施例
实施例1.卵母细胞PI3K途径在哺乳动物卵泡活化中的功能性作用
为了研究卵母细胞PI3K途径在哺乳动物卵泡活化中的功能性作用,通过将PtenloxP/loxP小鼠(Groszer等,Science 2001,294:2186)与表达互长分化因子9(Gdf-9)启动子介导之Cre重组酶(其特异性地在卵母细胞中有活性(Lan等,Biol.Reprod.2004,71:1469))的转基因小鼠(称为GCre小鼠)杂交而将Pten基因从小鼠卵母细胞中剔除,。已经发现,在6至34周龄测试阶段中,PtenloxP/loxP;GCre+雌性(即其卵母细胞中缺少PTEN的小鼠)产出最多的一窝幼仔,但是在成年期早期(即在12-13周龄后)变为不育。为了研究卵母细胞的Pten的缺失如何阻碍小鼠生育,我们比较了PtenloxP/loxP;GCre+小鼠与对照(PtenloxP/loxP)小鼠出生后的第一波卵泡发育。在出生后(postnatal day,PD)5天的PtenloxP/loxP;GCre+小鼠与对照小鼠卵巢中没有发现明显的形态差异。两种基因型的卵巢大部分都具有数目相当的含有被扁平前体粒细胞所围绕之小卵母细胞的原始卵泡,以及一些含有增大的卵母细胞的活化卵泡。直到PD35,所述PtenloxP/loxP;GCre+卵巢(图1,B)比对照卵巢大,并且包含了显著更多的活化卵泡(图1,C)。实际上,在突变的卵巢中不能鉴定出原始卵泡,而在对照卵巢中大多数卵泡则仍处于原始阶段。因此,在PtenloxP/loxP;GCre+卵巢中的整个原始卵泡库都已被活化。
因此,原始卵泡库的活化终止于卵泡的耗竭。这引起PtenloxP/loxP;GCre+小鼠的POF(premature ovarian failure,卵巢早衰)。在这些小鼠中观察到的表型与人POF类似(Beck-Peccoz和Persani,Orphanet.J.Rare.Dis.2006,1:9)。为了阐明促进PtenloxP/loxP;GCre+卵巢中卵母细胞增大的分子机制,研究了从PD12-14PtenloxP/loxP;GCre+小鼠和对照小鼠的卵巢中分离的卵母细胞的Akt信号传导。发现在体外培养的以及血清饥饿的PtenloxP/loxP;GCre+卵母细胞中,磷酸-Akt(p-Akt,丝氨酸473)的水平提高(图2A)。此外,与对照卵母细胞中相比,在PtenloxP/loxP;GCre+卵母细胞中的Kit配体(KL)(其可通过其卵母细胞表面受体Kit来活化生长中卵母细胞的PI3K途径(Reddy等,Dev.Biol.2005,281:160)),更大程度地将Akt活化。因此,卵母细胞中Pten的缺失导致卵母细胞PI3K/Akt信号传导增强。为了研究在PtenloxP/loxP;GCre+卵巢中促进卵母细胞生长的原因,研究了增强的PI3K/Akt信号发生是否导致增加的核糖体蛋白S6(rpS6)活化。在PD5(PtenloxP/loxP;GCre+卵巢与对照卵巢之间没有明显形态差别的发育阶段(图2,A-C))中,PtenloxP/loxP;GCre+卵母细胞中Akt的活化已被提高(图2C,PD5,p-Akt)。这与rpS6的表达增强(图2C,PD5,rpS6)和磷酸化增强(表明活化)(图2C,PD5,p-rpS6,丝氨酸235/6)有关。此结果提示,当卵母细胞中Gdf-9-Cre-介导的Pten缺失刚刚发生时,蛋白质翻译的提高就已经开始了。相似地,在PD12-14从PtenloxP/loxP;GCre+卵巢中分离出的卵母细胞中,提高的PI3K/Akt信号导致rpS6的表达和磷酸化均提高(图2C,PD12-14)。然而,雷帕霉素的哺乳动物靶标(mTOR)-p70 S6激酶(S6K)级联的活化并未因Pten缺失而增加,因为在PtenloxP/loxP;GCre+卵母细胞和对照卵母细胞中,PD5和PD12-14的磷-mTOR(p-mTOR,丝氨酸2448)、磷酸-马铃薯球蛋白/TSC2(p-TSC2,苏氨酸1462)和磷酸-S6K(p-S6K,苏氨酸389)的水平相似(图2C)。因此,rpS6活化的提高由rpS6表达本身的提高引起(图2C)。然而,在突变的卵母细胞中rpS6和S6K的磷酸化对PI3K特异性抑制剂LY294002和mTOR特异性抑制剂雷帕霉素敏感,这表明rpS6在PtenloxP/loxP;GCre+卵母细胞中的活化依赖PI3K和mTOR的活性。先前已经假设,未知的卵巢内因素刺激一些原始卵泡开始生长,而其余的卵泡保持静息。
实施例2.PTEN抑制剂促进培养的小鼠卵巢中原始卵泡的存活和发育
将出生4天后小鼠的卵巢在无菌条件下除去,将所述整个卵巢培养在1ml α-MEM培养基(Gibco-BRL)中的细胞过滤网(cell strainer)(40μm孔径)(BD Biosciences,Stockholm,Sweden)中,所述培养基补充有28mM抗坏血酸和0.3%(w/v)BSA,并且添加或不添加10μM的PTEN抑制剂bpV-HOpic(双过氧(5-羟基吡啶基-2-羧基)氧钒酸二钾(V),K2[VO(O2)2C6H4NO3])。将所述培养的卵巢在加湿的孵箱(5%CO2,37℃)中孵育,在培养期间每天将三分之一的培养基更换成新鲜培养基。为了固定,将所述卵巢用PBS清洗一次并用4%多聚甲醛固定过夜,并进行包埋用于形态分析。
所述PTEN抑制剂提高了卵泡在经培养卵巢中的存活率(图3B的箭头与图3A的箭头),这表明在经PTEN抑制剂处理的卵巢中被拯救的坏死部分。所述PTEN抑制剂还刺激扁平的前颗粒细胞(箭头,3A)增殖和分化成立方颗粒细胞(箭头,图3B),这是原始卵泡活化进入生长阶段的至关重要的步骤。
结论
本发明数据表明卵母细胞PTEN起到卵泡活化抑制剂的作用。卵母细胞内的PTEN/PI3K信号级联似乎在启动卵母细胞生长方面发挥作用。我们认为,每个单独的卵母细胞中PI3K途径的活化对于决定原始卵泡的命运(其是否保持休眠、其是否在特定的时间被活化或者其是否从始基阶段直接经历闭锁)而言可能是至关重要的。此外,表明了在卵母细胞特异性剔除Pten的小鼠中POF的独特卵巢表型,其由原始卵泡的过度活化和耗竭而引起。因此,本研究的发现具有广泛的生理学和临床上的意义,有助于深入理解正常卵巢生理和卵巢疾病的发生。在人中,POF被定义为原发卵巢缺陷,其特征在于无月经初潮(原发闭经)或在40岁之前卵泡早衰/卵泡生成受阻(继发闭经),发病率估计为1%(Beck-Peccoz和Persani,Orphanet.J.Rare.Dis.2006,1:9)。在POF的众多可能原因中,导致卵泡过度活化和耗竭的基因变异可能是人当中的病因之一。另一方面,卵泡活化的迟滞和/或过度的原始卵泡闭锁这两者均可通过卵母细胞中PI3K途径的活化不足而引起,它们也可导致POF,虽然是从相反的方向。对于卵母细胞中PTEN/PI3K信号传导网络之重要性的认识开拓了理解哺乳动物卵巢的生理学和病理学过程的新前景。
Claims (8)
1.一种体外鉴定潜在地可用于卵泡体外成熟之化合物的方法,所述方法包括测量所述化合物抑制PTEN酶活性的能力。
2.一种或多种PTEN酶活性抑制剂在制备用于提高卵泡体外成熟之药物中的用途,其中所述PTEN酶活性抑制剂选自:
双过氧(联吡啶)氧钒酸盐,
双过氧(1,10-菲咯啉)氧钒酸盐,
双过氧(吡啶甲酸)氧钒酸盐,
双过氧(5-羟基吡啶-2-羧基)氧钒酸盐,
二-(吡啶甲酸)氧钒酸盐,
二-(3-羟基吡啶甲酸)氧钒酸盐,
双过氧(苯基双胍)氧钒酸盐,
二-(苯基双胍)氧钒酸盐,和
双过氧(异喹啉羧酸)氧钒酸盐。
3.一种或多种PTEN酶活性抑制剂在制备使非生长中的卵泡体外活化和/或体外成熟之药物中的用途,其中所述PTEN酶活性抑制剂选自:
双过氧(联吡啶)氧钒酸盐,
双过氧(1,10-菲咯啉)氧钒酸盐,
双过氧(吡啶甲酸)氧钒酸盐,
双过氧(5-羟基吡啶-2-羧基)氧钒酸盐,
二-(吡啶甲酸)氧钒酸盐,
二-(3-羟基吡啶甲酸)氧钒酸盐,
双过氧(苯基双胍)氧钒酸盐,
二-(苯基双胍)氧钒酸盐,和
双过氧(异喹啉羧酸)氧钒酸盐。
4.根据权利要求3的用途,其中所述非生长中的卵泡选自原始卵泡、间期卵泡和初级卵泡。
5.含有一种或多种PTEN酶活性抑制剂的组合物在制造用于使非生长中的卵泡体内活化和/或成熟的药物中的用途,其中所述PTEN酶活性抑制剂选自:
双过氧(联吡啶)氧钒酸盐,
双过氧(1,10-菲咯啉)氧钒酸盐,
双过氧(吡啶甲酸)氧钒酸盐,
双过氧(5-羟基吡啶-2-羧基)氧钒酸盐,
二-(吡啶甲酸)氧钒酸盐,
二-(3-羟基吡啶甲酸)氧钒酸盐,
双过氧(苯基双胍)氧钒酸盐,
二-(苯基双胍)氧钒酸盐,和
双过氧(异喹啉羧酸)氧钒酸盐。
6.根据权利要求5的用途,其中所述非生长中的卵泡选自原始卵泡、间期卵泡和初级卵泡。
7.根据权利要求5的用途,其中所述药物用于局部注射。
8.根据权利要求7的用途,其中所述药物用于卵泡内或囊内注射。
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US61/024,210 | 2008-01-29 | ||
PCT/SE2009/050049 WO2009091332A1 (en) | 2008-01-17 | 2009-01-19 | Methods for in vitro maturation of ovarian follicles |
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CN102533642B (zh) * | 2011-11-28 | 2014-07-09 | 李晶 | 原始卵泡体外激活试剂盒 |
EA201590549A1 (ru) * | 2012-09-13 | 2015-08-31 | Зе Боард Оф Трастиз Оф Зе Лилэнд Стенфорд Джуниор Юниверсити | Стимуляция развития овариального фолликула и созревания ооцита |
CN104744505B (zh) * | 2015-02-13 | 2017-07-11 | 武汉弘跃医药科技有限公司 | 一种氧钒类配合物及其应用 |
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US20160130560A1 (en) | 2016-05-12 |
CN101932331A (zh) | 2010-12-29 |
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US20110015169A1 (en) | 2011-01-20 |
RU2010134371A (ru) | 2012-02-27 |
BRPI0906613A8 (pt) | 2015-10-06 |
KR101699676B1 (ko) | 2017-01-24 |
KR20160039696A (ko) | 2016-04-11 |
EP2242496B1 (en) | 2017-04-05 |
KR20100103593A (ko) | 2010-09-27 |
US9259438B2 (en) | 2016-02-16 |
AU2009205771B2 (en) | 2014-12-11 |
BRPI0906613A2 (pt) | 2015-07-14 |
AU2009205771A1 (en) | 2009-07-23 |
JP5560203B2 (ja) | 2014-07-23 |
CA2711646A1 (en) | 2009-07-23 |
JP2011509670A (ja) | 2011-03-31 |
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KR101639820B1 (ko) | 2016-07-14 |
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