CN115413623A - ***样小鼠伴糖脂代谢异常模型构建方法 - Google Patents
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明属于动物模型构建技术领域,公开了一种***样小鼠伴糖脂代谢异常模型构建方法,***样小鼠伴糖脂代谢异常模型构建方法包括:将双氢睾酮粉剂用DMSO溶解为50μg/μL,再用玉米油稀释为1μg/μL,震荡直至充分溶解,得双氢睾酮混合溶液;选取小鼠,并饲喂选取的小鼠高脂饲料;通过皮下注射预先制备的双氢睾酮混合溶液,得到***样小鼠伴糖脂代谢异常模型。本发明联合DHT皮下注射和高脂饮食成功诱导出了小鼠卵巢多囊样改变及动情周期改变,伴随糖耐量及胰岛素敏感性下降,血脂谱及体脂肪质量亦明显升高,造模时间较短,为PCOS发病机制的实验研究提供了一种较好的小鼠模型。
Description
技术领域
本发明属于动物模型构建技术领域,尤其涉及一种***样小鼠伴糖脂代谢异常模型构建方法。
背景技术
***(polycystic ovary syndrome,PCOS)是育龄期女性主要的生殖障碍性疾病,以高雄激素症状、***及多囊卵巢为主要特点,其临床表型和生化特点异质性较大,发病机制现仍未完全阐明,可能与遗传、母体激素和代谢紊乱环境、表观遗传和环境影响等因素相关。动物模型有助于帮助学者研究其病因和病理生理学机制,了解其发生的起源,以及对其发病进行预测和预防,但现目前因缺乏容易获得的实验诱导的涵盖PCOS多种复杂表型的动物模型,很大程度上阻碍了PCOS的基础研究及临床治疗进展。此外,PCOS患者存在β细胞功能受损及胰岛素敏感性下降,发生2型糖尿病(type2 diabetesmellitus,T2DM)、高脂血症、代谢综合征及心血管疾病风险也会增高,故亦是一种代谢障碍性疾病,这大大增加了PCOS临床管理的复杂性。随着BMI的增加,PCOS的患病率可从正常BMI的5%增加到肥胖BMI的15%,同时会增加PCOS表型的严重程度,还能独立于高雄激素影响PCOS女性的生殖功能。考虑到30%~50%的PCOS患者体型是正常的,故体重正常和肥胖PCOS相关的发病机制可能有不同的分子基础。肥胖和糖代谢异常、脂代谢异常有密切关系,因此,建立反映一系列代谢表型的PCOS样模型是至关重要的。
然而,现目前尚无成熟的PCOS样伴糖脂代谢异常的模型。新发表的国际 PCOS评估和管理指南推荐以下3项满足2项,同时排除其他内分泌相关疾病即可诊断PCOS:稀发***或无***;高雄激素的临床表现和(或)高雄激素血症;超声检查卵巢多囊样改变。因没有特定某些物种PCOS的诊断标准,PCOS的动物模型并没有金标准,学者们往往是力求复制出2个或更多类似于PCOS的临床诊断标准的特征组合,称为PCOS样模型。啮齿类动物是一种经济、易于操作、生殖周期短的通用工具,已有多种PCOS的大鼠及小鼠模型研究,包括雄激素、***、芳香化酶抑制剂、光暴露改变和遗传操作等干预方法。高雄激素血症是PCOS最一致的特征,因此最直接的造模方式就是使用雄激素来建立模型,包括睾酮、丙酸睾酮、脱氢表雄酮(dehydroepiandrosterone,DHEA)、双氢睾酮(dihydrotestosterone,DHT)和来曲唑。因为雄激素、丙酸睾酮均可在体内转化为***,故难以界定PCOS病理生理中雄激素介导的机制到底是通过雄激素受体或者***受体而发挥作用,此法已很少使用。PCOS女性血清的DHEA水平明显升高,虽然有报道DHEA的***大鼠的短期(20天)干预可诱导PCOS无周期和无***的生殖特征,但卵巢主要是大囊状卵泡为主,与 PCOS女性的窦前卵泡和小窦卵泡增多不符,且DHEA***小鼠长期干预(90 天)根本没有任何卵巢、生殖内分泌及糖脂代谢紊乱的改变,说明DHEA对生殖影响可能是短期的。来曲唑是一种芳香化酶抑制剂,可以阻止雄激素向***的转化,从而提高循环和卵巢雄激素水平。用来曲唑干预***小鼠或者大鼠,虽可见多囊样卵巢改变,但不能诱导腹型肥胖、胰岛素抵抗及血脂等改变,在代谢方面的表型不明显,说明来曲唑诱导的PCOS样模型在研究代谢机制方面存在局限性。DHT为雄激素的非芳香化产物,不能转换为***,对雄激素受体亲和力高,在体内的生物活性明显强于睾酮。现有的啮齿类PCOS模型认可度较高的主要是给予皮下植入可持续90天释放DHT控释剂(含2.5mg双氢睾酮,27.5μg/d)83μg)(购置于Innovative Research of America,Sarasota,FL)来实现。但Innovative Research of America公司已不再出口本国以外的国家雄激素缓释剂型,导致建立模型受阻。另外,同以上使用其他雄激素方法一样,单独使用DHT 并不能完全体现代谢方面的特征。
通过上述分析,现有技术存在的问题及缺陷为:现有的PCOS模型构建方法构建时间长,且构建过程受到限制,构建的模型不能准确、全面的反应PCOS 生殖和代谢方面的特征。
解决以上问题及缺陷的难度为:
1、皮下注射DHT的剂量的稳定性。
2、DHT联合高脂饮食是否可诱导出生殖和代谢两方面的特征。
解决以上问题及缺陷的意义为:
1、建立一种新的可以全面反映代谢表型和生殖表型的PCOS模型。
2、不需进口缓释剂,节约科研资金及实验时间。
3、该模型可为PCOS的发病机制及药物筛选等研究提供一种可操作性的实验基础。
发明内容
针对现有技术存在的问题,本发明提供了一种***样小鼠伴糖脂代谢异常模型构建方法。
本发明是这样实现的,一种***样小鼠伴糖脂代谢异常模型构建方法,所述***样小鼠伴糖脂代谢异常模型构建方法包括:
步骤一,制备双氢睾酮混合溶液;选取小鼠,并饲喂选取的小鼠高脂饲料;
步骤二,通过皮下注射预先制备的双氢睾酮混合溶液,得到***样小鼠伴糖脂代谢异常模型。
进一步,步骤一中,所述双氢睾酮混合溶液制备方法包括:
将双氢睾酮粉剂用DMSO溶解为50μg/μL,再用玉米油稀释为1μg/μL,震荡直至充分溶解,即可得双氢睾酮混合溶液。
进一步,步骤一中,所述选取小鼠包括:选取3周龄的雌性小鼠。
进一步,步骤一中,所述饲喂环境包括:饲养温度为20~24℃,环境湿度为 40%~60%,每日光照及黑暗时间各12h。
进一步,所述高脂饲料为60%脂肪热能饲料。
进一步,所述高脂饲料饲喂时间为:持续6周。
进一步,所述双氢睾酮混合溶液注射剂量为50μg,注射次数为每日一次,持续6周。
本发明的另一目的在于提供一种利用所述***样小鼠伴糖脂代谢异常模型构建方法构建的***样小鼠伴糖脂代谢异常模型。
本发明的另一目的在于提供一种所述***样小鼠伴糖脂代谢异常模型在筛选用于治疗或改善***的药物以及验证用于治疗***的药物药效中的应用。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:本发明构建了一种具备以上诊断诊断标准的生殖表型,同时伴有糖脂代谢异常的PCOS 样小鼠模型,为PCOS的发病机制及药物筛选等研究提供了一种可操作性的实验基础。本发明联合DHT皮下注射和高脂饮食成功诱导出了小鼠卵巢多囊样改变及动情周期改变,伴随糖耐量及胰岛素敏感性下降,血脂谱及体脂肪质量亦明显升高,造模时间较短,为PCOS发病机制的实验研究提供了一种较好的小鼠模型。
附图说明
图1是本发明实施例提供的***样小鼠伴糖脂代谢异常模型构建方流程图。
图2是本发明实施例提供的***(A)及性激素水平(B、C、D)。
图3是本发明实施例提供的14天中动情周期情况;完成1个周期(A)、2 个周期(B)和3个周期(C)的小鼠比例,P值为第14天时两组比较值;D:平均周期数;E:四个阶段所占天数;F:对照组小鼠的典型动情周期;G:实验组小鼠的典型动情周期;E:动情期;D:动情间期。
图4是本发明实施例提供的卵巢质量和卵巢组织形态学;A:卵巢质量;B:黄体计数;C:卵泡计数。D:不健康卵泡比例;E:膜细胞和颗粒细胞面积占比;F:HE染色。bar=50μm。
图5是本发明实施例提供的体重(A)、脂肪质量(B)及血脂(C、D); BW:体重;Retroperitoneal:腹膜后;Parametrial:子宫旁;Inguinal:腹股沟。
图6是本发明实施例提供的糖代谢;A:空腹血糖;B和C:葡萄糖耐量和曲线下面积;D和E:胰岛素耐量和曲线下面积。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种***样小鼠伴糖脂代谢异常模型构建方法,下面结合附图对本发明作详细的描述。
本发明实施例提供的***样小鼠伴糖脂代谢异常模型构建方法包括:
利用皮下注射双氢睾酮联合饲喂高脂饲料构建***样小鼠伴糖脂代谢异常模型。
如图1所示,本发明实施例提供的***样小鼠伴糖脂代谢异常模型构建方法包括以下步骤:
S101,将双氢睾酮粉剂用DMSO溶解为50μg/μL,再用玉米油稀释为1μg/μL,震荡直至充分溶解,得双氢睾酮混合溶液;
S102,选取3周龄的雌性小鼠,并饲喂选取的小鼠高脂饲料;
S103,通过皮下注射预先制备的双氢睾酮混合溶液,得到***样小鼠伴糖脂代谢异常模型。
本发明实施例提供的饲喂环境包括:饲养温度为20~24℃,环境湿度为 40%~60%,每日光照及黑暗时间各12h。
本发明实施例提供的高脂饲料为60%脂肪热能饲料。
本发明实施例提供的高脂饲料饲喂时间为:持续6周。
本发明实施例提供的双氢睾酮混合溶液注射剂量为50μg,注射次数为每日一次,持续6周。
下面结合具体实施例对本发明的技术方案做进一步说明。
实施例1:
1材料与方法
1.1实验材料
1.1.1实验动物
16只3周龄清洁级雌性C57BL/6小鼠购于重庆医科大学动物实验中心,体重为8.4~10.5g,饲养温度为20~24℃,环境湿度为40%~60%,每日光照及黑暗时间各12h,自由进食水。所有实验过程均经重庆医科大学动物实验伦理委员会批准。
1.1.2实验主要试剂及器材
5α-DHT粉剂购于中国索莱宝公司(ID0310);玉米油购于中国索莱宝公司(C7030);二甲基亚砜(dimethyl sulfoxide,DMSO)购于中国赛维尔公司 (WD2650);高脂饲料(60%由脂肪供能,D12492)和标准饲料(10%由脂肪供能,D12450J)购于美国ResearchDiets公司;甲苯胺蓝染色液购于中国索莱宝公司(G3665);诺和灵R购于丹麦诺和诺德公司;血糖仪购于美国强生公司 (OneTouch Ultra);改良苏木精-伊红(hematoxylin-eosin,HE)染色试剂盒购于中国索莱宝公司(G1121);多聚甲醛中性固定液购于中国赛维尔公司(G1101);甘油三酯(triglyceride,TG)测试盒(E-BC-K261-M)和总胆固醇 (totalcholesterol,TC)测试盒(E-BC-K109-M)均购于中国伊莱瑞特生物科技股份有限公司;血清总睾酮(total testosterone,T)试剂盒购于美国Cayman公司(No.582701);DHT试剂盒购于美国Biovision公司(E4604-100);***(luteinizing hormone,LH)试剂盒购于英国abbexa公司(abx254243)。
1.2实验方法
1.2.1动物分组及干预
将DHT粉剂用DMSO溶解为50μg/μL,再用玉米油稀释为1μg/μL,充分震荡直至充分溶解以当日使用。小鼠适应性喂养3天后被随机分为两组并分别干预6周:实验组(n=8)每日予以腹部皮下注射DHT50μg+高脂饲料干预,对照组(n=8)予以每日皮下注射同等剂量有机溶剂+标准饲料干预。
1.2.2体质量测定
每周测量小鼠体质量并记录数值,监测小鼠体质量变化。测量体质量前晚至当日清晨至少禁食12h。
1.2.3动情周期监测
喂养第5~6周对每只小鼠进行***涂片检查,观察动情周期变化。用沾有生理盐水的棉球擦小鼠外阴去除粪便等杂物,然后用移液器吸取生理盐水约 15μL,移液器枪头置于小鼠***内反复抽吸3~5次,见白色分泌浑浊物吸出后将其平涂至载玻片上风干,甲苯胺蓝染色后显微镜下观察细胞形态。每日10am 涂片,连续14天。判读标准:动情前期:涂片全是有核上皮细胞,偶有少量角化细胞;动情期:全是无核角化细胞或间有少量上皮细胞;动情后期:角化细胞、有核上皮细胞、白细胞同时存在;动情间期:白细胞为主。统计各小鼠完成动情周期次数,并计算动情周期各个时期所占时间比例。
1.2.4糖代谢测定
每周用葡萄糖氧化酶法测定实验小鼠空腹血糖水平(禁食8h)。喂养第6 周对每只小鼠分别进行腹腔葡萄糖耐量实验(intraperitoneal glucose tolerance test,IPGTT)和腹腔胰岛素耐量实验(intraperitoneal insulintolerance test,IPITT)。实验小鼠饥饿6h后,尾静脉针刺取血测量基础血糖水平(0min),再通过腹腔注射2g/kg体重的葡糖糖(配成20%葡萄糖溶液)或0.5iu/kg体重的胰岛素后,测取30、60及120min的血糖水平。IPGTT及IPITT各时间点的血糖值均取0min 时的百分数计算。
1.2.5卵巢组织学检查
小鼠喂养第6周末用***麻醉处死后解剖、分离并称重双侧卵巢,然后用生理盐水冲洗后置于4%多聚甲醛中充分固定,常规脱水、石蜡包埋。将卵巢在最大纵切面附近以5μm厚度连续切10个切面放在一个玻片上进行HE染色。在光镜下观察卵泡的病理学改变,计数黄体、原始卵泡、小窦前卵泡、大窦前卵泡、小窦卵泡、大窦卵泡的数量,并计算窦前卵泡和窦状卵泡各自的不健康卵泡比例,最后用ImageJ软件计算两组小鼠大窦卵泡的颗粒细胞和膜细胞在***核层面的面积占比。各阶段发育卵泡的分类依据:①原始卵泡(primordialfollicle):单层扁平颗粒细胞包围***;②初级卵泡(primaryfollicle):单层立方颗粒细胞包围***;③小窦前卵泡(small preantral): 1.5~2层立方颗粒细胞包围***;④大窦前卵泡(large preantral):由2~5 层立方颗粒细胞包围***;⑤小窦卵泡(small antral):5层以上立方颗粒细胞包围***,和/或1~2个小面积的卵泡腔;⑥大窦卵泡(large antral):单个大的窦腔形成;⑦***前卵泡(preovulatory):单个大的卵泡腔,壁颗粒细胞形成的茎状结构相连有卵丘细胞包围的***(***逐渐脱离颗粒细胞层)。健康卵泡定义为:卵泡含有完整的***,颗粒细胞有组织分布且固缩比例极少(<10%)。不健康卵泡(闭锁卵泡)定义为:不同发育阶段的卵泡含有退化的***,颗粒细胞层分布散乱和/或固缩比例较大(≥10%)。闭锁囊样卵泡(Cyst)为卵巢内充满囊液的大囊泡,表现为颗粒细胞层变薄,膜细胞层分散,***与颗粒细胞缺乏连接。为方便统计,本发明中将②和③统称为小窦前卵泡(smallpreantral),⑥和⑦统称为大窦卵泡(large antral),闭锁囊样卵泡归类于不健康的大窦卵泡中。
1.2.6性激素、血脂及脂肪重量测定
小鼠处死前摘眼球取血离心后留血清EP管分装,于-80℃保存备用。血清T、 DHT和LH均采用ELISA法测定。血清TG采用单试剂GPO-PAP比色法测定,血清TC采用单试剂COD-PAP比色法测定。处死后立即分离双侧腹股沟脂肪库、子宫旁脂肪库、腹膜后脂肪库并称重。腹股沟脂肪贮库位于后肢上段的前方,代表小鼠的皮下脂肪组织。子宫旁脂肪库位于子宫水平;腹膜后脂肪库位于背部背壁,包裹在一层薄膜中。后二者可用来表示小鼠的内脏脂肪组织。
1.3统计学处理
所有数据经SPSS22.0进行统计分析。计量资料采用表示,计数资料采用例(%)表示。计量资料的正态性检验采用shapiro-wilk检验法。正态分布使用独立样本t检验进行比较;非正态分布采用Wilcoxon秩和检验进行比较。计数资料采用Fisher’s确切概率法比较差异。采用两因素重复测量方差分析空腹血糖、IPGTT及IPITT的血糖变化情况。取双侧α值为0.05,P<0.05为差异有统计学意义。
2结果
2.1两组小鼠一般情况及性激素水平变化
两组小鼠均发育良好,体毛润泽,反应灵活,行为无明显差异。体征方面,实验组小鼠的***明显增大(图2A)。实验组血清DHT水平较对照组升高,约为其3倍(46.549±16.398pg/ml vs.16.089±6.896pg/ml,t=4.843,P=0.000) (图2B);而两组间血清T(51.520±14.314pg/ml vs.58.458±11.376pg/ml, t=-1.073,P=0.301)(图2C)和LH(101.295±14.700pg/ml vs.108.155±15.270 pg/ml,t=-0.915,P=0.375)(图2D)水平差异无统计学意义。
2.2两组动情周期变化
***涂片14天判断动情周期。对照组所有小鼠均有规律动情周期,而光镜下实验组主要以白细胞为主,提示该组小鼠主要处于动情间期,均无动情周期 (图3F、3G)。其中,对照组完成1个周期(100%vs.0%,P=0.000)和2个周期(75%vs.0%,P=0.007)的小鼠比例均高于实验组(图3A、3B),而两组完成3个周期(12.5%vs.0%)的小鼠比例无统计学意义(P=0.467)(图3C)。 14天中对照组的平均周期数(1.875±0.690天)明显高于实验组(0.000±0.000 天)(Z=-3.556,P=0.000)(图3D)。进一步分析两组小鼠动情周期中四个阶段的所占时间发现,实验组动情前期(1.125±0.991天vs.3.563±1.590天, Z=-2.732,P=0.006)和动情期(0.000±0.000天vs.3.938±1.761天,Z=-3.596, P=0.000)的持续时间均短于对照组,而动情间期明显长于对照组,是其4.2倍 (9.750±1.282天vs.2.313±0.799天,Z=-3.388,P=0.001)(图3E)。
2.3两组卵巢质量和卵巢组织形态学变化
对照组小鼠卵巢表面颜色红润,而实验组卵巢表面苍白,隐约可见大小不等囊状卵泡形成。以矫正体质量后的卵巢质量[(卵巢重量)mg/(体重)g]更能客观反应卵巢质量大小。本研究中,实验组(0.1992±0.045mg/g)的卵巢质量小于对照组(0.268±0.048mg/g),差异有统计学意义(t=-2.954,P=0.010)(图 4A)。
光镜下,实验组小鼠卵巢呈囊性改变,颗粒细胞层数减少,颗粒细胞固缩、退化,排列疏松,膜细胞增生、分散、厚度增加,与周围***分界不明(图 4F)。与对照组相比,实验组小鼠未见黄体(0.000±0.000vs.2.500±0.926, Z=-3.614,P=0.000)(图4B),而小窦卵泡(8.625±1.685vs.6.875±1.458,t=2.222, P=0.043)和大窦卵泡(4.625±1.188vs.2.125±0.835,t=4.871,P=0.000)数量明显升高(图4C);同样地,小窦卵泡中不健康卵泡比例(25.889±8.890vs. 12.669±5.830%,Z=-2.537,P=0.011)和大窦卵泡中不健康卵泡比例 (62.083±5.893vs.12.500±17.252%,Z=-3.459,P=0.001)也明显升高(图4D)。实验组小鼠大窦卵泡的颗粒细胞面积占比较对照组减少(49.479±11.973vs.70.063±9.309%,t=-5.758,P=0.000),而膜细胞面积占比增多(22.253±5.166vs.17.429±3.017%,t=3.386,P=0.002)(图4E、4F)。
2.4两组体重及糖脂代谢变化
干预结束后实验组小鼠增加的体重明显大于对照组(10.063±2.225g vs. 6.713±1.796g,t=3.314,P=0.005)(图5A)。实验组矫正体重后的腹膜后 (6.775±1.501mg/gvs.4.732±1.029mg/g,t=3.175,P=0.007)、子宫旁 (17.691±2.945mg/g vs.13.336±2.339mg/g,t=3.275,P=0.006)及腹股沟 (20.519±2.648mg/g vs.15.748±4.291mg/g,t=2.676,P=0.018)脂肪库的质量均大于对照组(图5B)。同样的,血清TG(0.879±0.233mmol/L vs. 0.588±0.052mmol/L,t=3.436,P=0.009)和TC(3.013±0.422mmol/Lvs. 1.655±0.232mmol/L,t=7.980,P=0.000)也明显升高(图5C、5D)。
实验组(2.06±0.14mmol/L)和对照组(2.04±0.18mmol/L)的基线空腹血糖差异无统计学意义(F=0.104,P=0.756)(图6A)。从6周龄到9周龄,实验组的空腹血糖均高于对照组(分别为第6周:4.350±1.338mmol/L vs. 3.150±0.355mmol/L,F=7.565,P=0.028;第7周:5.275±0.947mmol/L vs. 3.450±0.504mmol/L,F=24.952,P=0.002;第8周:5.225±1.095mmol/L vs. 3.413±0.318mmol/L,F=17.985,P=0.004;第9周:5.338±0.515mmol/L vs. 3.725±0.450mmol/L,F=28.727,P=0.001)(图6A);同样的,干预结束后IPGTT 提示,实验组60min(210.852±58.887%vs.126.199±21.439%,F=15.858, P=0.005)和120min(132.531±25.997%vs.101.222±21.171%,F=8.472,P=0.023) 的血糖值明显高于对照组(图6B),其曲线下面积明显增大(24044.471±5216.302vs.17921.021±1579.386,t=3.178,P=0.012),表明实验组糖耐量下降(图6C)。而在整个IPITT过程中,实验组的血糖值较对照组高4.970%(95%置信区间: 1.543~8.397%,F=11.759,P=0.011)(图6D),其曲线下面积亦增大 (6316.115±365.952vs.5557.661±574.619,t=3.149,P=0.007),表明实验组胰岛素敏感性下降(图6E)。
近期有研究表明,DHT的持续暴露是PCOS的一种可复制的啮齿类模型干预方式,能有效地模拟PCOS患者的许多生殖特征,如不规律的动情周期、黄体数量减少、闭锁卵泡数量增加、小窦卵泡数量增加、卵巢多囊样改变等,以及体重的增加。但是,该方法能否引起明显的糖脂代谢异常的改变,因评估方法及DHT剂量不同,不同实验结果有所差异。现有技术1将DHT的持续缓释剂(7.5mg DHT)植入***(19天)雌性Wistar大鼠皮下90天,用正糖高胰岛素钳夹实验评估胰岛素抵抗程度,发现DHT处理大鼠胰岛素敏感性下降。现有技术2将DHT的持续缓释剂(2.5mg DHT)植入***(19天)雌性C57BL/6 小鼠皮下90天,DHT的血清浓度较安慰剂组升高了6倍,同时闭锁小窦和大窦卵泡的数量增加了2-4倍,颗粒细胞层分散,膜细胞层增厚,可见增生性***。虽然IPGTT后各点血糖均明显升高,提示糖耐量下降,但两组小鼠的空腹血糖及胰岛素无明显变化,并未对胰岛素敏感性影响进一步阐明。将含有DHT的硅胶植入物(10mg DHT)植入***(21-23天)雌性小鼠皮下90天,DHT 的血清浓度较安慰剂组升高了8倍左右,同时表现为动情周期消失、典型的多囊卵巢、闭锁囊样卵泡、厚度变薄的退化颗粒细胞层和增厚的膜细胞层,但是两组空腹血糖无明显差别,且IPITT提示胰岛素敏感性无明显变化。另外,DHT 干预啮齿动物的性腺、腹膜后和腹股沟脂肪库的脂肪细胞直径及重量均明显增大,但貌似对血清TG及TC并无明显影响。
故本发明在前人基础上,使用皮下注射DHT联合高脂饮食的方式诱导 PCOS样小鼠,旨在模型复制的生殖表型和代谢表型更加明显。有研究报道,单独用高脂高糖饮食喂养23周龄SD大鼠90天,发现实验组大鼠空腹血糖、空腹胰岛素、HOMA-IR均明显升高,而血清雄激素水平无明显改变,卵巢的病理改变以大囊泡增多为主要表现,并不是典型的多囊样改变。但是,高脂饮食本身可以加重啮齿类PCOS模型的糖脂代谢异常。同样的,非人灵长类动物在***时予以雄激素干预不会导致代谢功能障碍和体重增加,而联用高脂饮食后可造成代谢相关改变。本发明表明,实验组小鼠明显表现出***明显变大、动情周期消失、无***、小窦卵泡和大窦卵泡增加、颗粒细胞面积减少、膜细胞面积增大的生殖特征;同时表现出空腹血糖升高、糖耐量减低、胰岛素敏感下降、体重增加、内脏脂肪和皮下脂肪重量增加、TG和TC升高的糖脂代谢异常特征。实验组黄体消失、小窦卵泡和大窦卵泡不健康比例增多,说明这些卵泡在***前均已出现成熟障碍,不能***形成黄体;但与PCOS患者相反,这些实验小鼠的卵巢质量减少、表面苍白,可能与无黄体、囊状卵泡增多和卵巢血管关注减少有关。
PCOS女性DHT水平增高,约为正常女性的2-3倍左右。因DHT缓释剂型暂不能进口,故本发明以皮下注射方式代替。虽然在理论上每日给予50μg/d的 DHT皮下注射剂量较之前的25ug/d的DHT皮下缓释剂量大1倍,但最终实验组小鼠DHT水平仅约为对照组的3倍,反而更接近PCOS患者的真实情况,这可能与不同的给药方式导致药物利用率不同有关,这也很好地解决了本发明的一个难度,就是皮下注射DHT一样可以达到比较理想的血清DHT药物浓度。另外,较3个月的DHT缓释剂干预相比,本发明联合高脂饮食,共诱导了6周,不但从时间上明显缩短了干预周期,而且糖脂代谢异常也较DHT单药诱导的 PCOS样模型更加明显。
PCOS患者中,大于75%的女性LH明显升高,可能是由于GnRH神经元活动和分泌增加,导致LH脉冲频率增加。本发明中,实验组T和LH水平并无明显升高,和之前的研究一致。T水平无变化可能与DHT代谢物的负反馈有关。虽然小鼠***暴露于DHT会导致LH、GnRH受体基因在垂体的表达增加,但循环中的LH和LH/FSH比值没有改变,这表明在这个模型中GnRH信号并没有增加,可能与本发明中高水平的DHT暴露可激活下丘脑雄激素受体,从而抑制GnRH神经元兴奋性有关。事实上,PCOS女性的性腺类固醇激素负反馈敏感性受损也可能提高GnRH神经元的兴奋性。故如想探索神经内分泌在PCOS中的作用,可在本PCOS样模型基础上联合基因操作等方式来实现。
综上,本发明联合DHT皮下注射和高脂饮食成功诱导出了小鼠卵巢多囊样改变及动情周期改变,伴随糖耐量及胰岛素敏感性下降,血脂谱及体脂肪质量亦明显升高,造模时间较短,为PCOS发病机制的实验研究提供了一种较好的小鼠模型。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
Claims (9)
1.一种***样小鼠伴糖脂代谢异常模型构建方法,其特征在于,所述***样小鼠伴糖脂代谢异常模型构建方法包括:
步骤一,制备双氢睾酮混合溶液;选取小鼠,并饲喂选取的小鼠高脂饲料;
步骤二,通过皮下注射预先制备的双氢睾酮混合溶液,得到***样小鼠伴糖脂代谢异常模型。
2.如权利要求1所述***样小鼠伴糖脂代谢异常模型构建方法,其特征在于,步骤一中,所述双氢睾酮混合溶液制备方法包括:将双氢睾酮粉剂用DMSO溶解为50μg/μL,再用玉米油稀释为1μg/μL,震荡直至充分溶解,得双氢睾酮混合溶液。
3.如权利要求1所述***样小鼠伴糖脂代谢异常模型构建方法,其特征在于,步骤一中,所述选取小鼠包括:选取3周龄的雌性小鼠。
4.如权利要求1所述***样小鼠伴糖脂代谢异常模型构建方法,其特征在于,步骤一中,所述饲喂环境包括:饲养温度为20~24℃,环境湿度为40%~60%,每日光照及黑暗时间各12h。
5.如权利要求1所述***样小鼠伴糖脂代谢异常模型构建方法,其特征在于,所述高脂饲料为60%脂肪热能饲料。
6.如权利要求1所述***样小鼠伴糖脂代谢异常模型构建方法,其特征在于,所述高脂饲料饲喂时间为:持续6周。
7.如权利要求1所述***样小鼠伴糖脂代谢异常模型构建方法,其特征在于,所述双氢睾酮混合溶液注射剂量为50μg,注射次数为每日一次,持续6周。
8.一种利用如权利要求1-7任意一项所述***样小鼠伴糖脂代谢异常模型构建方法构建的***样小鼠伴糖脂代谢异常模型。
9.一种如权利要求8所述***样小鼠伴糖脂代谢异常模型在筛选用于治疗或改善***的药物以及验证用于治疗***的药物药效中的应用。
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