CN107156041A - 一种胰岛素抵抗状态下子宫内膜增殖培育方法 - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
本发明涉及一种胰岛素抵抗状态下子宫内膜增殖培育方法,所述方法包括:步骤1,胰岛素抵抗动物模型的建立;步骤2,将所述大鼠去势及给予雌激素;步骤3,阴道脱落细胞巴氏染色;步骤4,分离肝脏、内脏脂肪和子宫;步骤5,组织学染色。因此,本发明的子宫内膜增殖培养方法实现了有效和快速的实现子宫内膜增殖培养。
Description
技术领域
本发明涉及一种培养方法,尤其涉及一种胰岛素抵抗状态下子宫内膜增殖培育方法。
背景技术
子宫内膜癌是最常见的妇科恶性肿瘤之一,发病率呈逐年上升且年轻化的趋势。大量流行病学研究表明肥胖、糖尿病和胰岛素抵抗是子宫内膜癌的危险因素。近年来,随着生活水平的提高及生活方式的改变、膳食结构的变化,超重和肥胖人群逐年增多。大量脂肪摄入是胰岛素抵抗形成的主要因素,胰岛素作为生长因子家族的成员调控细胞生长和能量代谢,在肿瘤形成过程中起重要作用,子宫内膜增殖是子宫内膜癌的癌前病变,饮食引起的代谢异常与子宫内膜增殖和子宫内膜癌的关系仍不明确。因此在验证利用长期高脂饮食诱导的胰岛素抵抗对子宫内膜增殖影响时需要建立一种动物模型,并为进一步深入的胰岛素抵抗和高胰岛素血症与子宫内膜增殖和子宫内膜癌发生之间的相互作用机制研究奠定实验室基础。
发明内容
本发明的目的是针对现有技术的缺陷,提供一种建立胰岛素抵抗和子宫内膜增殖动物模型方法,可以有效和快速的实现子宫内膜增殖。
为实现上述目的,本发明提供了一种胰岛素抵抗状态下子宫内膜增殖培育方法,所述方法包括:
步骤1,胰岛素抵抗动物模型的建立;
1.1,将大鼠适应性喂养1周后分为2组,对照组(N组)、雌激素组(NE组)和胰岛素抵抗+雌激素组组(FE组);
1.2,给予对照组和雌激素组正常维持饲料饮食:碳水化合物66%、脂肪10%、蛋白质24%,胰岛素抵抗+雌激素组高脂饮食:碳水化合物20%、脂肪60%、蛋白质20%;
1.3,大鼠喂养40周,禁食10~12h,内眦静脉取血检测血脂、空腹血糖及胰岛素水平,计算HOMA-IR指数,腹腔注射葡萄糖后于30min、60min、90min和120min尾静脉取血测定血糖值并计算血糖曲线下面积;
1.4,测定糖耐量1周,两组大鼠禁食5~7h,尾静脉取血测空腹血糖后,腹腔注射胰岛素,测定30min、60min、90min和120min血糖值并计算曲线下面积;
步骤2,将所述大鼠去势及给予雌激素;
2.1,大鼠取仰卧位,消毒后5%戊巴比妥钠(50mg/kg)腹腔注射麻醉,于尿道口上方1cm处向上行正中纵形切口皮肤3cm,分离皮下组织及肌肉进入腹腔,在子宫角与输卵管交界靠子宫角侧结扎,摘除大鼠双侧卵巢,关闭腹腔。对照组仅切开皮肤暴露腹腔取出与卵巢等量的脂肪组织后关闭腹腔;
2.2,术后1周,灌胃给予雌激素和胰岛素抵抗+雌激素组大鼠17β-雌二醇【800μg/kg(体重)】4周,对照组给予等量溶剂橄榄油;
步骤3,阴道脱落细胞巴氏染色;
3.1,给予溶剂或17β-雌二醇3天后,每天行阴道涂片的巴氏染色:95%酒精5min,碳酸锂溶液5min,95%酒精5min,橘黄染液3min,95%酒精Ⅰ、Ⅱ各5min,EA-50 5min,95%酒精Ⅰ、Ⅱ各5min,无水乙醇Ⅰ、Ⅱ各5min,二甲苯Ⅰ、Ⅱ各5min,中性树胶封片;
步骤4,分离肝脏、内脏脂肪和子宫;
4.1,给予雌激素4周后,暴露腹腔处理,分离腹腔脂肪并称重,分离子宫并称量子宫的湿重和干重,取子宫角中段8-10mm和5mm×5mm×5mm肝脏固定于10%中性福尔马林中用于HE染色,观察肝脏脂肪样变和子宫上皮鳞状化生,测量子宫腔上皮细胞和腺上皮细胞高度、固有层和肌层的厚度。剩余组织经液氮迅速冷冻后置于-80℃冰箱保存,断颈处死大鼠;
步骤5,组织学染色;
5.1,固定于10%中性福尔马林中的肝脏和子宫组织经石蜡包埋、切片,烤片2h后,脱蜡入水,苏木精染色后盐酸酒精分化至切片呈粉红色,氨水返蓝后,伊红染色,梯度酒精脱水,二甲苯透明处理,中性树胶封片;
进一步的,所述方法还包括:步骤6,统计学处理;
6.1,定量资料以均值±标准差表示,两组比较采用独立样本的t检验,三组比较采用单因素方差分析。
因此,本发明的一种胰岛素抵抗状态下子宫内膜增殖培育方法方法实现了有效和快速的实现子宫内膜增殖培养。
附图说明
图1为本发明一种胰岛素抵抗状态下子宫内膜增殖培育方法的流程图。
具体实施方式
下面通过附图和实施例,对本发明的技术方案做进一步的详细描述。
长期高脂饮食诱导的胰岛素抵抗是否影响子宫内膜增殖,代谢异常与子宫内膜增殖及其癌变之间的关系目前尚不明确。本发明通过对正常Sprague-Dawley大鼠进行长期的高脂饮食,诱导大鼠胰岛素抵抗,观察在胰岛素抵抗状态下,大鼠子宫内膜增殖的变化,为进一步发明高胰岛素环境及糖代谢异常对子宫内膜的作用机制提供实验依据。
图1为本发明子宫内膜增殖培养方法的流程图,如图所示,本发明具体包括如下步骤:
材料的准备。材料是SPF级性成熟雌性性成熟Sprague-Dawley大鼠(8周龄)27只,购自北京维通利华公司。大鼠体重无明显差异,自由进食水、光照12h/天,饲养环境为室温20~25℃、湿度40-50%。高脂饲料(60%脂肪含量)购自北京华阜康公司,葡萄糖、17β-雌二醇购自美国Sigma公司,血糖试纸和血糖仪购自美国罗氏公司。
步骤1,胰岛素抵抗动物模型的建立;
1.1,适应性喂养1周后,大鼠分为3组,即对照组(N组,n=9)、雌激素组(NE组,n=9)和胰岛素抵抗+雌激素组(FE组,n=9);
1.2,给予对照组和雌激素组正常维持饲料饮食(碳水化合物66%,脂肪10%,蛋白质24%)、给予胰岛素抵抗+雌激素组高脂饮食(碳水化合物20%,脂肪60%,蛋白质20%);
1.3,大鼠喂养40周时,禁食10~12h后,内眦静脉取血检测血脂(酶法)、空腹血糖(氧化酶法)及胰岛素水平(碘125放射免疫法),计算HOMA-IR指数【空腹血糖(mmol/L)×空腹胰岛素(mU/ml)/22.5)】,腹腔注射葡萄糖(2g/kg)后于30min、60min、90min和120min测定血糖值并计算血糖曲线下面积;
1.4,测定糖耐量1周后,各组大鼠禁食7h后,尾静脉取血测空腹血糖水平后,腹腔注射胰岛素(0.75U/kg),测定30min、60min、90min和120min血糖值并计算曲线下面积;
步骤2,将所述大鼠去势及给予雌激素;
2.1,大鼠取仰卧位,消毒后5%戊巴比妥钠(50mg/kg)腹腔注射麻醉,于尿道口上方约1cm处向上行正中纵形切口皮肤约3cm,分离皮下组织及肌肉进入腹腔,在子宫角与输卵管交界靠子宫角侧结扎,摘除大鼠双侧卵巢,关闭腹腔。对照组仅切开皮肤暴露腹腔,取出卵巢旁等量脂肪组织后关闭腹腔。
2.2,术后1周,灌胃给予雌激素组和胰岛素+雌激素组大鼠17β-雌二醇【800μg/kg(体重)】4周,对照组给予等量溶剂橄榄油。
步骤3,阴道脱落细胞巴氏染色;
3.1,给予溶剂或17β-雌二醇3天后,每天行阴道涂片后行脱落细胞的巴氏染色,95%酒精固定5min,碳酸锂溶液5min,95%酒精5min,橘黄染液3min,95%酒精Ⅰ、Ⅱ各5min,EA-50 5min,95%酒精Ⅰ、Ⅱ各5min,无水乙醇Ⅰ、Ⅱ各5min,二甲苯Ⅰ、Ⅱ各5min,中性树胶封片。
步骤4,分离肝脏、内脏脂肪和子宫;
4.1,给予溶剂或17β-雌二醇(800μg/kg【体重】)4周后,暴露腹腔操作如前,分离腹腔脂肪并称重,分离子宫并称量子宫的湿重和干重。取子宫角中段8~10mm和5mm×5mm×5mm的肝脏固定于10%中性福尔马林中用于HE染色,观察肝脏脂肪样变和子宫上皮鳞状化生,测量子宫腔上皮细胞和腺上皮细胞高度、固有层和肌层的厚度。剩余组织经液氮迅速冷冻后置于-80℃冰箱保存。最终,断颈处死大鼠。
步骤5,组织学(HE)染色;
5.1,固定于10%中性福尔马林中的肝脏和子宫组织经石蜡包埋、切片(5μm),烤片2h后,脱蜡(二甲苯Ⅰ、Ⅱ,各15min)入水(无水乙醇Ⅰ、Ⅱ、95%、90%、80%、75%乙醇各1次,各5min),漂洗(自来水5min),苏木精(5min),自来水漂洗3min,盐酸酒精分化(浓盐酸1ml,75%酒精99ml,2s)至切片呈粉红色,自来水漂洗(3min)后氨水返蓝(氨水0.2ml,自来水99.8ml,10s),自来水漂洗(3min)后,伊红染色5min,自来水漂洗(3min)后,梯度酒精脱水(90%、95%酒精各1次,无水乙醇Ⅰ、Ⅱ,各3min),透明(二甲苯Ⅰ、Ⅱ,各10min),中性树胶封片。
步骤6,统计学处理。
具体的,采用SPSS 16.0软件,定量资料以均值±标准差表示,两组比较采用独立样本的t检验,三组比较采用单因素方差分析。P<0.05为差异有统计学意义。
处理结果如下:
结果一,胰岛素抵抗大鼠模型的建立。
饲养第2周,胰岛素抵抗+雌激素组大鼠体重大于对照组和雌激素组(271.41±21.16g vs 251.71±13.94g,P<0.05),饲养至第40周末,与对照组和雌激素组相比,胰岛素抵抗组大鼠体重显著增加(462.50±86.99g vs 379.46±53.19g,P<0.01)。饲养至第胰岛素抵抗组糖负荷在60min、90min和120min血糖比对照组和雌激素组明显升高(P<0.01),且血糖恢复至正常水平时间延迟,葡萄糖曲线下面积比对照组和雌激素组也明显增大(P<0.01),见表1。胰岛素抵抗组腹腔注射胰岛素后90min和120min血糖比对照组和雌激素组明显升高(P<0.05),血糖恢复至正常水平时间延迟,葡萄糖曲线下面积比对照组和雌激素组也明显增多(P<0.05),见表2。饲养至第40周末,胰岛素抵抗组大鼠胰岛素抵抗指数10.01±3.04较对照组和雌激素组的7.16±2.97显著升高(P<0.05)。胰岛素抵抗组大鼠甘油三酯、胆固醇、高密度脂蛋白和低密度脂蛋白与对照组和雌激素组无显著差异。胰岛素抵抗组内脏脂肪含量大于对照组和雌激素组(P<0.001),见表3。
表1高脂饮食对大鼠葡萄糖耐量的影响
**P<0.01vs N+NE。N,对照组;NE,雌激素组;FE,胰岛素抵抗+雌激素组。
表2高脂饮食对大鼠胰岛素耐量的影响
*P<0.05vs N+NE。N,对照组;NE,雌激素组;FE,胰岛素抵抗+雌激素组。
表3大鼠内脏脂肪、子宫重量和子宫各指标的变化
*P<0.01vs N组、NE组;#P<0.05vs N组;**P<0.05vs NE组。N,对照组;NE,雌激素组;FE,胰岛素抵抗+雌激素组。
结果二,子宫内膜增殖情况。
去势后的大鼠给予雌激素灌胃3天后,阴道脱落细胞经巴氏染色后,镜下可见满视野的细胞质粉染的完全角化的上皮细胞,说明给予雌激素有效。给予雌激素4周后,雌激素组和胰岛素抵抗+雌激素组大鼠子宫角与对照组相比明显增粗,内含或清亮无色液体,子宫的重量显著增加(P<0.05)。见表3。HE染色可见雌激素组和胰岛素抵抗+雌激素组大鼠宫腔扩张,黏膜层向腔内凸出,腔上皮和腺上皮细胞高柱状,可呈假复层或复层排列,细胞内可有空泡出现,腺腔内可有大量分泌物,细胞浆核比增高。和对照组相比,雌激素组和胰岛素抵抗+雌激素组大鼠子宫腔上皮细胞和腺上皮细胞高度均变大(P<0.01)。雌激素组和胰岛素抵抗+雌激素组大鼠的子宫内膜腔上皮和腺上皮出现了鳞状细胞化生。此外,雌激素组和胰岛素抵抗+雌激素组大鼠子宫内膜间质内嗜酸性粒细胞增多。对照组、雌激素组和胰岛素抵抗+雌激素组大鼠子宫固有层、环形肌层和纵形肌层厚度无显著性差异。胰岛素抵抗+雌激素组腔上皮细胞和腺上皮细胞厚度显著大于雌激素组(P<0.05),见表3。此外,雌激素组和胰岛素抵抗+雌激素组大鼠的子宫内膜出现鳞状细胞化生的比例为66.7%(7/9),大于雌激素组33.3%(3/9)的鳞状上皮化生比例。对照组大鼠子宫内膜腔上皮和腺上皮细胞呈矮柱状,未见鳞状上皮化生。
长期高热量和低纤维饮食可使与代谢紊乱有关的慢性疾病发病率增加。机体胰岛素抵抗的存在使胰岛分泌更多的胰岛素代偿,从而发生高胰岛素血症。高脂饮食的胰岛素抵抗大鼠与人类肥胖时出现胰岛素抵抗病因学类似。发明表明,肥胖和运动减少等因素导致的Ⅱ型糖尿病、胰岛素抵抗和高胰岛素血症等可增加子宫内膜癌的风险。本发明通过单纯高脂饮食诱导大鼠胰岛素抵抗,观察长期胰岛素抵抗状态下,大鼠发生子宫内膜增值时子宫内膜形态学等方面的改变,以证实胰岛素抵抗可加速子宫内膜增殖的病变。
与正常饮食相比,高脂饮食脂肪含量高、热量大,使骨骼肌葡萄糖摄取减少、胰岛素抑制肝糖原输出的能力减弱,影响葡萄糖刺激胰岛细胞的胰岛素分泌。本发明中大鼠经过长期的高脂喂养后,大鼠体内出现了显著的胰岛素抵抗。大鼠的体重增加,内脏脂肪含量也明显增加。大鼠出现的糖耐量异常,一方面表现口服糖耐量发明和腹腔注射胰岛素发明后,胰岛素抵抗大鼠的血糖曲线下面积增加,另一方面表现在HOMA-IR指数的增大。胰岛素抵抗常伴有脂代谢异常,胰岛素状态下肝脂酶的活性增加使TG合成增加也反应了大鼠胰岛素抵抗的状态。子宫的结构和功能受到雌激素和孕激素的双重调控,子宫内膜增殖症则是由于雌激素水平增高而无孕激素拮抗引起的子宫内膜腺体或间质增生,是子宫内膜癌的癌前病变,如无对抗的雌激素继续持续作用,子宫内膜上皮和腺体可发生恶性转化导致子宫内膜癌发生。本发明中给予雌激素使大鼠持续处于动情期,即雌激素分泌水平高、孕激素分泌水平低的状态,刺激子宫内膜出现增殖,子宫增重以增加子宫内膜腺细胞分泌活动为主,对子宫固有层和肌层的影响不大。
胰岛素抵抗以及继发的高胰岛素血症作为肥胖、糖尿病和PCOS等的共同病理生理学基础,与代谢紊乱有关,也与多种肿瘤的发生有关。胰岛素作为生长因子家族成员之一,与胰岛素受体结合后诱发细胞下游信号,激活PI3K/Akt、Ras/MAPK等通路使信息传递到细胞核,激活转录因子,启动相关基因转录翻译,以适应细胞增殖和细胞周期等反应。本发明中大鼠行去势手术以排除自身不同水平雌激素的影响,通过外源性给予17β-雌二醇,除了子宫内膜增殖的改变外,同时出现了鳞状上皮细胞化生,有胰岛素抵抗的大鼠出现鳞状上皮化生的比例高于未发生胰岛素抵抗的大鼠。绝经后妇女体内雌激素水平明显降低,但在肥胖等因素作用下,脂肪中的芳香化酶使雄激素转化为雌激素的量明显增高,绝经后的部分子宫内膜仍处于较活跃的增殖状态,即使无外源性雌激素作用,仍可能引起子宫内膜的增生甚至癌变。本发明中在胰岛素抵抗状态下大鼠在发生子宫内膜增殖时,子宫腔上皮和腺上皮细胞的高度显著大于无胰岛素抵抗的大鼠,且子宫内膜鳞状上皮化生的比例升高,说明胰岛素抵抗或者血循环中高胰岛素的水平可使子宫内膜在已有增殖的基础上进一步加重病变程度。
综上所述,本发明通过长期高脂饮食诱导大鼠胰岛素抵抗的情况下观察大鼠在发生子宫内膜增殖时子宫内膜的形态学改变,提示在胰岛素抵抗存在时,子宫内膜增殖的表现更显著,主要表现在子宫腔上皮和腺上皮细胞的高度增大以及子宫内膜的鳞状上皮化生的比例升高,为高胰岛素环境对子宫内膜增殖病变提供实验依据。此外,通过合理的饮食和运动改善改善胰岛素抵抗的状态,积极预防子宫内膜癌前病变的发生。
最后所应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围。
Claims (3)
1.一种胰岛素抵抗状态下子宫内膜增殖培育方法,其特征在于,所述方法包括:
步骤1,胰岛素抵抗动物模型的建立;
1.1,将大鼠适应性喂养1周后,编号并随机、平均分为3组:对照组、雌激素组和胰岛素抵抗+雌激素组;
1.2,给予对照组和雌激素组正常维持饲料饮食:碳水化合物66%、脂肪10%、蛋白质24%;给予胰岛素抵抗+雌激素组高脂饮食:碳水化合物20%、脂肪60%、蛋白质20%;
1.3,喂养大鼠40周,禁食10~12h,内眦静脉取血检测血脂、空腹血糖及胰岛素水平,计算HOMA-IR指数,腹腔注射葡萄糖后于30min、60min、90min和120min尾静脉取血测定血糖值并计算血糖曲线下面积;
1.4,测定葡萄糖耐量1周后,大鼠禁食7h,尾静脉取血测空腹血糖,腹腔注射胰岛素,测定30min、60min、90min和120min血糖值并计算曲线下面积;
步骤2,将所述大鼠去势及给予雌激素;
2.1,大鼠取仰卧位,消毒腹部后用50mg/kg(体重)戊巴比妥钠腹腔注射麻醉,于尿道口上方1cm处向上行正中纵形切口皮肤3cm,分离皮下组织及肌肉进入腹腔,在子宫角与输卵管交界靠子宫角侧结扎,摘除大鼠双侧卵巢后关闭腹腔,对照组仅切开皮肤暴露腹腔取出与卵巢等量的脂肪组织后关闭腹腔;
2.2,术后1周,灌胃给予雌激素和胰岛素抵抗+雌激素组大鼠17β-雌二醇4周,对照组给予等量溶剂橄榄油;
步骤3,阴道脱落细胞巴氏染色;
3.1,给予溶剂或17β-雌二醇3天后,每天行阴道涂片后行如下步骤:95%酒精5min,碳酸锂溶液5min,95%酒精5min,橘黄染液3min,95%酒精Ⅰ、Ⅱ各5min,EA-50 5min,95%酒精Ⅰ、Ⅱ各5min,无水乙醇Ⅰ、Ⅱ各5min,二甲苯Ⅰ、Ⅱ各5min,中性树胶封片;
步骤4,分离肝脏、内脏脂肪和子宫;
4.1,给予溶剂或17β-雌二醇4周,用50mg/kg(体重)戊巴比妥钠麻醉大鼠后再次暴露其腹腔,分离腹腔脂肪并称重,分离子宫并称量子宫的湿重和干重后,取子宫角中段8~10mm和5mm×5mm×5mm肝脏固定于10%中性福尔马林中用于HE染色,观察肝脏脂肪样变和子宫上皮鳞状化生,测量子宫腔上皮细胞和腺上皮细胞高度、固有层和肌层的厚度;
4.5,剩余组织经液氮迅速冷冻后置于-80℃冰箱保存,断颈处死大鼠。
步骤5,组织学染色;
5.1,固定于10%中性福尔马林中的肝脏和子宫组织经石蜡包埋、切片、烤片后脱蜡入水,苏木精染色后盐酸酒精分化至切片呈粉红色,氨水返蓝后,伊红染色,梯度酒精脱水,二甲苯透明处理,中性树胶封片。
2.根据权利要求1所述的方法,其特征在于,所述方法还包括:
步骤6,统计学处理;
6.1,定量资料以均值±标准差表示,两组比较采用独立样本的t检验,三组比较采用单因素方差分析。
3.根据权利要求1所述的方法,其特征在于,所述方法还包括:
摘除大鼠双侧卵巢后3天,去势的大鼠因无雌激素作用,阴道脱落细胞为非角化上皮细胞;术后1周开始给予雌激素3天后,去势大鼠在雌激素的作用下,阴道脱落细胞为角化上皮细胞。
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