WO2020073780A1 - 用于子宫内膜异位症小鼠模型建立的缓释混液、制备方法以及小鼠模型建立方法 - Google Patents

用于子宫内膜异位症小鼠模型建立的缓释混液、制备方法以及小鼠模型建立方法 Download PDF

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WO2020073780A1
WO2020073780A1 PCT/CN2019/106729 CN2019106729W WO2020073780A1 WO 2020073780 A1 WO2020073780 A1 WO 2020073780A1 CN 2019106729 W CN2019106729 W CN 2019106729W WO 2020073780 A1 WO2020073780 A1 WO 2020073780A1
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temperature
estrogen
sensitive hydrogel
sustained
powder
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PCT/CN2019/106729
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French (fr)
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戴永东
张松英
黄倩梦
林翔
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浙江大学
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Priority to CH01157/20A priority Critical patent/CH716073B1/de
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    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
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    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/566—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol having an oxo group in position 17, e.g. estrone
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    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
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    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue

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  • the invention relates to the field of biology, in particular to provide a stable and convenient estrogen temperature-sensitive hydrogel mixture for the establishment of a mouse model of endometriosis.
  • Endometriosis is an important cause of pelvic pain and infertility in women of childbearing age. At present, the pathogenesis of endometriosis is still unclear, and there is a lack of effective treatment. Due to ethical constraints, it is difficult to conduct research on the causes, metabolic changes, drug efficacy, and disease outcomes of patients. In vitro animal models have solved this limitation to a certain extent. The mouse model of endometriosis is widely used in the research related to endometriosis.
  • Endometriosis is an estrogen-dependent disease.
  • the construction of a mouse model of endometriosis has a fundamental role in studying the pathogenesis of endometriosis and developing new treatment methods.
  • the experimentally constructed mouse model of endometriosis needs the support of exogenous estrogen to maintain the continuous growth of ectopic lesions.
  • exogenous estrogen There are three main ways to support exogenous estrogen: 1. Intraperitoneal injection of aqueous estrogen solution; 2. Subcutaneous injection of estrogen oil solution; 3. Subcutaneous implantation of estrogen sustained-release tablets.
  • the estrogen aqueous solution needs to be injected intraperitoneally daily or every other day. Subcutaneous injection of estrogen oil solution can be administered once a week, but subcutaneous injection of oil is more difficult and local irritation.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the above-mentioned background technology, to provide an estrogen temperature-sensitive hydrogel mixture using a temperature-sensitive hydrogel as a carrier and a preparation method thereof to provide stability for the establishment of an endometriosis mouse model Convenient and low-cost exogenous estrogen support.
  • the sustained-release mixture used for the establishment of a mouse model of endometriosis includes the following components:
  • Estrogen powder 0.5-1.0g / ml, the rest is temperature sensitive hydrogel.
  • the sustained-release mixture used for the establishment of a mouse model of endometriosis includes the following components:
  • Estrogen powder 0.8g / ml, the rest is temperature sensitive hydrogel.
  • the preparation method of the sustained-release mixed liquid used for establishing the mouse model of endometriosis includes the following steps:
  • the temperature of the refrigerator is below -30 ° C.
  • the invention overcomes the defect that the existing endometriosis mouse model lacks a simple and economical support method of exogenous estrogen during the establishment of the endometriosis mouse model. , It has a long-lasting sustained-release effect, and the temperature-sensitive hydrogel mixture of estrogen concentration of 0.5-1.0mg / ml weekly subcutaneous injection method can effectively support the construction of a mouse model of endometriosis, with economic (5 yuan / Time), simple operation, time saving, good effect and other advantages, it is worth promoting.
  • Fig. 1 is a comparison state diagram of the temperature-sensitive hydrogel at different temperatures (A is the state of the temperature-sensitive hydrogel at normal temperature, and B is the state of the temperature-sensitive hydrogel at 37 ° C).
  • Figure 2 is a schematic diagram of the release rule of different concentrations of estrogen temperature-sensitive hydrogel mixture.
  • Fig. 3 is a schematic diagram of the release time concentration curve of the estrogen temperature-sensitive hydrogel mixed solution.
  • Figure 4 is a picture of the formation of gel after subcutaneous injection of mouse estrogen temperature-sensitive hydrogel.
  • FIG. 5 is a general picture of an ectopic cyst of a model mouse in which a mixture of a model mouse injected daily with an estrogen solution and a temperature-sensitive hydrogel of estrogen is injected weekly.
  • Figure 6 is a comparison of HE staining pictures of the ectopic cyst tissue of the model mice with daily intraperitoneal injection of estrogen solution and the model mice with subcutaneous injection of estrogen temperature-sensitive hydrogel weekly and normal mice. schematic diagram.
  • temperature-sensitive hydrogel As a new material, temperature-sensitive hydrogel is gradually used for drug delivery.
  • the temperature-sensitive hydrogel can change state with the temperature change.
  • the temperature-sensitive hydrogel At room temperature, the temperature-sensitive hydrogel is in a transparent solution state, and the drug can be easily mixed into the solution. With the increase of temperature, it can change to a milky white gel state at about 37 ° C. The drug can be slowly released from it.
  • the degradation products of the temperature-sensitive hydrogel material are harmless to the human body, and it is not easy to cause irritation or other adverse immune reactions in the body tissues, which provides the possibility of being used as a subcutaneous drug delivery carrier.
  • Polyethylene glycol-polyester two-block temperature-sensitive hydrogel (mPEG-PLGA, Shanghai Funing Technology Co., Ltd.);
  • the temperature-sensitive hydrogel is a transparent liquid at normal temperature (25C in Fig. A), and is a milky white gel at 37 ° C (Fig. B).
  • ICR mice were injected subcutaneously with 0mg / ml, 0.15mg / ml, 0.3mg / ml, 0.6mg / ml, 0.9mg / ml, 1.5mg / ml, 3mg / ml estrogen temperature sensitive hydrogel mixture 0.2ml, measured after 3 days
  • Blood estrogen concentrations were A: 11pg / ml, B: 19pg / ml, C: 267pg / ml, D: 323pg / ml, E: 1076pg / ml, F: 2784pg / ml, G: 3377pg / ml, mice There is a linear relationship between the concentration of blood estrogen and the concentration of estrogen temperature-sensitive hydrogel (see Figure 2).
  • Literature has shown that after 21 days of subcutaneous implantation of low-dose subcutaneous estrogen sustained-release tablets in mice, the blood estrogen concentration can reach 792.5 ⁇ 743.3pg / ml (14). Therefore, the inventors used 0.8mg / ml estrogen temperature-sensitive hydrogel mixture and injected subcutaneously once a week to obtain stable blood estrogen concentration and support the growth of ectopic lesions according to the release regularity curve of estrogen temperature-sensitive hydrogel mixture. .
  • mice in the experimental group were survived during the modeling process.
  • the abdominal incisions healed well without obvious signs of infection.
  • mice were injected subcutaneously with estrogen temperature-sensitive hydrogel, see Figure 4 for gel formation.
  • Figure 5 shows the ectopic cysts of the model mice in which the estrogen solution is injected daily intraperitoneally and the model mice in which the estrogen temperature-sensitive hydrogel is subcutaneously injected weekly. Among them:
  • a and B are ectopic cysts of mice in the estrogen solution group injected intraperitoneally;
  • C and D are ectopic cysts of mice in the subcutaneous injection group of estrogen temperature-sensitive hydrogel.
  • mice in the estrogen injection group and the estrogen temperature-sensitive hydrogel mixed injection group all showed ectopic cyst formation, with a single spherical lesion with solid white and gray cysts, and visible neovascularization.
  • the average diameter of the lesions in the estrogen injection group was 5 mm, and the average diameter of the lesions in the estrogen temperature-sensitive hydrogel mixed injection group was 5 mm. There was no significant difference between the two.
  • Ectopic cysts of mice in the estrogen injection group and the estrogen temperature-sensitive hydrogel mixed injection group were paraffin sectioned and observed under the microscope after HE staining. Typical glandular structure and interstitial tissue structure similar to endometrial tissue were seen (see figure 6).
  • FIG. 6 is a HE staining diagram of the ectopic cyst tissue of the model mice and the normal endometrium of the normal mice in which the estrogen solution is injected intraperitoneally daily and the estrogen temperature-sensitive hydrogel mixture is injected subcutaneously weekly. among them:
  • A, B HE staining of normal mouse endometrial tissue
  • E, F HE staining of heterogeneous lesions in mice of subcutaneous injection group of estrogen temperature-sensitive hydrogel.
  • the estrogen temperature-sensitive hydrogel mixture prepared by this method can support the growth of endometriotic lesions in the subcutaneous injection group of mice, and the formed lesions are not significantly different from those formed by the methods provided by the predecessors.
  • the estrogen temperature-sensitive hydrogel mixture can be used to establish a mouse model of endometriosis.
  • the present invention explores the changes of blood estrogen levels with the concentration and time of injection after subcutaneous injection of estrogen temperature-sensitive hydrogel mixed mice, and through comparative experiments, established 0.5-1.0g / ml estrogen temperature-sensitive hydrogel mixed liquid (0.2ml injection every 7 days) Injecting mice subcutaneously can successfully construct a mouse model of endometriosis with typical endometriosis pathological features.

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Abstract

提供一种用于子宫内膜异位症小鼠模型建立中提供外源性雌激素支持的缓释混液、制备方法以及小鼠模型的建立方法。缓释混液包括：雌激素粉末0.5-1.0g/ml，其余是温敏水凝胶。制备方法包括以下步骤：1）从冷藏冰箱中取出温敏水凝胶，常温下化开至透明液态；2）将雌激素粉末加入水凝胶中；3）置于震荡器上振荡使雌激素粉末与水凝胶充分混匀；4）分装后置于冷藏冰箱保存。小鼠模型的建立方法包括供体小鼠内膜片获取、受体小鼠手术移植和将缓释混液注入受体小鼠背部皮下的步骤。

Description

用于子宫内膜异位症小鼠模型建立的缓释混液、制备方法以及小鼠模型建立方法 技术领域
本发明涉及生物领域，具体是为子宫内膜异位症小鼠模型的建立提供稳定方便外源性雌激素支持的雌激素温敏水凝胶混液。
背景技术
子宫内膜异位症是生育年龄妇女盆腔疼痛以及不孕的一个重要原因。目前，子宫内膜异位症的发病机理尚不明确，缺乏有效的治疗手段。由于伦理约束，难以在患者身上进行发病原因、代谢变化、药物疗效、疾病转归等方面的研究。体外动物模型在一定程度上解决了这一局限。子宫内膜异位症小鼠模型在子宫内膜异位症相关研究中被广泛应用。
子宫内膜异位症是一种雌激素依赖性疾病。子宫内膜异位症小鼠模型的构建对研究子宫内膜异位症的发病机制及开发新的治疗手段具有基础性作用。实验构建的子宫内膜异位症小鼠模型需要外源性雌激素的支持，才能维持异位病灶的持续生长。已有的外源性雌激素支持方式主要有3种：1.雌激素水溶液腹腔注射；2.雌激素油溶液皮下注射；3.雌激素缓释药片皮下植入。雌激素水溶液需每日或隔日腹腔注射给药。雌激素油溶液皮下注射可每周给药一次，但油剂皮下注射较困难，局部刺激性大。雌激素缓释药片皮下植入后缓慢释放能够维持稳定血雌激素浓度，但需手术植入，且价格昂贵(单盒100片共4157美元，平均单片单次价格近300元人民币)，限制了其在实验研究中的应用。一种操作简便、经济且雌激素支持效果明确的外源性雌激素补充方式将更有利于学者对子宫内膜异位症的研究。
技术解决方案
本发明所要解决的技术问题是克服上述背景技术的不足，提供一种以温敏水凝胶为载体的雌激素温敏水凝胶混液及其制备方法，为子宫内膜异位症小鼠模型的建立提供稳定方便以及成本低廉的外源性雌激素支持。
本发明提供的技术方案是：
用于子宫内膜异位症小鼠模型建立的缓释混液，包括的成分是：
雌激素粉末0.5-1.0g/ml，其余是温敏水凝胶。
用于子宫内膜异位症小鼠模型建立的缓释混液，包括的成分是：
雌激素粉末0.8g/ml，其余是温敏水凝胶。
用于子宫内膜异位症小鼠模型建立的缓释混液的制备方法，包括以下步骤：
1)从冷藏冰箱中取出温敏水凝胶，常温下化开至透明液态；
2)按雌激素粉末与温敏水凝胶的比例，将雌激素粉末加入水凝胶中；
3)置于震荡器上振荡使雌激素粉末与水凝胶充分混匀，配置成雌激素温敏水凝胶混液；
4)分装后置于冷藏冰箱保存。
所述冷藏冰箱的温度低于-30℃。
所述子宫内膜异位症小鼠模型的建立步骤是：
1)供体小鼠内膜片获取：取供体小鼠ICR 5只，移植前一周每天腹腔注射0.1ml(30μg/ml)雌激素溶液；1周后将小鼠处死后进行手术，分离出小鼠子宫，在PBS中漂洗3遍，纵型剪开子宫使内膜面翻卷朝外，每只小鼠获得2段等长的子宫片，置于无菌PBS中等待移植；
2)受体小鼠手术移植：腹腔注射麻醉受体ICR小鼠9只后行开腹手术，将处理好的1段子宫片内膜面朝外用无菌线固定于肠系膜上，逐层连续缝合腹部切口，PVP-I消毒后放回鼠笼；禁食24小时，可自由饮水；24小时后常规喂养。
有益效果
本发明的有益效果是：
本发明克服了现有子宫内膜异位症小鼠模型建立过程中缺乏简单经济的外源性雌激素支持方式的缺陷，提供的温敏水凝胶承载雌激素缓释混液在ICR小鼠皮下注射后，具有持久的缓释效果，且雌激素浓度为0.5-1.0mg/ml的温敏水凝胶混液每周皮下注射法，能够有效支持子宫内膜异位症小鼠模型的构建，具有经济(5元/次)、操作简单、节约时间、效果良好等优点，值得推广。
附图说明
图1是温敏水凝胶在不同温度下的对比状态图(A为温敏水凝胶在常温状态，B为温敏水凝胶在37℃时的状态)。
图2是不同浓度雌激素温敏水凝胶混液释放规律示意图。
图3是雌激素温敏水凝胶混液释放时间浓度曲线示意图。
图4是小鼠皮下注射雌激素温敏水凝胶后，凝胶形成情况图片。
图5是雌激素溶液每日腹腔注射的建模小鼠与雌激素温敏水凝胶混液每周皮下注射的建模小鼠的异位囊肿的大体图片。
图6是雌激素溶液每日腹腔注射的建模小鼠与雌激素温敏水凝胶混液每周皮下注射的建模小鼠的异位囊肿组织以及正常小鼠在位子宫内膜的HE染色图片对比示意图。
本发明的实施方式
温敏水凝胶作为一种新型材料逐渐被人们用于药物的递送。温敏水凝胶能够随着温度改变发生状态转变，在室温下，温敏水凝胶呈透明溶液状态，药物可以方便地混入溶液，随着温度的升高，在37℃左右能转变为乳白色凝胶状态，药物能缓慢地从中释放出来。温敏水凝胶材料自身降解的产物对人体是无害，在体内组织中不易引起刺激或者引起其他不良免疫反应，为其作为皮下给药载体提供了可能。
以下结合附图所示的实施例进一步说明。
一、本发明提供的以温敏水凝胶为载体的雌激素温敏水凝胶缓释混液，制作及使用过程包括以下步骤：
1、关键试剂及仪器
聚乙二醇-聚酯类两嵌段温敏水凝胶(mPEG-PLGA，上海复凝科技有限公司)；
β-雌二醇(Sigma-Aldrich,Milano,Italy)。
2、雌激素温敏水凝胶混液的制备
1)-30℃冰箱中取出瓶装温敏水凝胶，常温下化开至透明溶液状态，分装，避免反复冻融，操作过程中避免手直接接触凝胶管壁，防止凝胶凝固成胶。
2)称取8mg雌二醇粉末，加入10ml温敏水凝胶，置于振荡器上震荡，使雌激素粉末与温敏水凝胶充分混匀，分装至1.5ml ep管中，置于-30℃冰箱中保存。
3、雌激素温敏水凝胶混液的使用
1)从-30℃冰箱中取出配制好的0.8mg/ml的雌激素温敏水凝胶混液，室温下化开，操作过程中避免手直接接触凝胶管壁，防止凝胶凝固成胶。
2)小鼠建模后，将雌激素温敏水凝胶混液注入建模小鼠背部皮下，将小鼠制动30s，使凝胶更好地原位成胶，每只小鼠注射0.2ml的0.8mg/ml雌激素温敏水凝胶混液，每周注射一次。
二、分析与研究
1、温敏水凝胶在不同温度下的状态变化
从图1可知：温敏水凝胶在常温下(图A为25℃)为透明液态，而37℃时(图B)为乳白色凝胶。
2、雌激素温敏水凝胶混液在小鼠体内的释放规律
为探索雌激素温敏水凝胶混液在ICR小鼠皮下注射后的释放规律，可通过注射不同雌激素浓度的温敏水凝胶混液以及注射后不同时间，观察小鼠血中雌激素的浓度。
2.1浓度效应：
ICR小鼠皮下注射0mg/ml、0.15mg/ml、0.3mg/ml、0.6mg/ml、0.9mg/ml、1.5mg/ml、3mg/ml雌激素温敏水凝胶混液0.2ml，3天后测得血雌激素浓度分别为A:11pg/ml,B:19pg/ml,C:267pg/ml,D:323pg/ml,E:1076pg/ml,F:2784pg/ml,G:3377pg/ml，小鼠血雌激素浓度与雌激素温敏水凝胶混液浓度呈线性关系(见图2)。有文献表明，低剂量皮下雌激素缓释药片小鼠皮下植入21天后，血雌激素浓度能够达到792.5±743.3pg/ml(14)。故发明人根据不同浓度雌激素温敏水凝胶混液释放规律曲线，使用0.8mg/ml的雌激素温敏水凝胶混液，每周皮下注射一次的方式来获得稳定血雌激素浓度，支持异位病灶的生长。
2.2时间效应：
在初步确定ICR小鼠皮下注射温敏水凝胶混液雌激素为0.8mg/ml后，分别于第1、3、5、7、14天采用摘眼球取血法采血，测得血雌激素浓度分别为a:4714pg/ml,b:768pg/ml,c:453pg/ml,d:562pg/ml,e:32pg/ml。0.2ml(0.8mg/ml)雌激素水凝胶溶液皮下注射成胶后，第一天呈突释状态，后缓慢释放，血液雌激素浓度维持在接近文献报道的792.5±743.3pg/ml水平，并能持续释放时间为7天(见图3)；
3、雌激素温敏水凝胶混液对子宫内膜异位症小鼠模型的建模效果
3.1构建子宫内膜异位症小鼠模型
1)供体小鼠内膜片获取：取供体小鼠ICR 5只,移植前一周每天腹腔注射0.1ml(30μg/ml)雌激素溶液。1周后用颈椎脱臼法将小鼠处死后进行手术，无菌眼科剪逐层打开下腹部约1cm，分离出小鼠子宫，剪断子宫系膜及血管，于子宫卵巢连接处及宫颈上方剪断子宫，在PBS中漂洗3遍，纵型剪开子宫使内膜面翻卷朝外，每只小鼠获得2段等长的子宫片(小鼠子宫为Y字形)，置于无菌PBS中等待移植。
2)受体小鼠手术移植：腹腔注射麻醉受体ICR小鼠9只后行开腹手术，无菌眼科剪逐层打开上腹部约1cm，暴露小鼠肠系膜，将处理好的1段子宫片内膜面朝外用无菌线固定于肠系膜上，逐层连续缝合腹部切口，PVP-I消毒后放回鼠 笼。禁食24小时，可自由饮水。24小时后常规喂养。
3.2不同雌激素补充处理
将受体9只小鼠分为PBS注射组(每天腹腔注射0.1ml PBS)，雌激素注射组(每天腹腔注射0.1ml雌激素，30μg/ml)和雌激素温敏水凝胶混液注射组(每周皮下注射一次0.2ml雌激素温敏水凝胶混液，0.8mg/ml)。
3.3结果观察
以上3组实验处理持续21天后，处死小鼠进行结果观察。
1)建模小鼠一般情况
实验组小鼠共9只，建模过程中3组小鼠均成活，腹部切口愈合良好，无明显感染征象。雌激素温敏水凝胶混液注射组小鼠皮下注射部位无明显感染征象。
小鼠皮下注射雌激素温敏水凝胶后，凝胶形成情况参见图4。
2)异位囊肿大体观察
图5显示了雌激素溶液每日腹腔注射的建模小鼠与雌激素温敏水凝胶混液每周皮下注射的建模小鼠的异位囊肿情况，其中：
A、B为雌激素溶液腹腔注射组小鼠异位囊肿；
C、D为雌激素温敏水凝胶混液皮下注射组小鼠异位囊肿。
雌激素注射组和雌激素温敏水凝胶混液注射组小鼠均可见异位囊肿形成，病灶呈单个球形，灰白色囊实性凸起，可见新生血管包绕。雌激素注射组病灶平均直径为5mm，雌激素温敏水凝胶混液注射组病灶平均直径为5mm，两者无显著差异。
PBS注射组小鼠未见明显异位囊肿形成(图片省略)。
3)异位囊肿病理观察
雌激素注射组和雌激素温敏水凝胶混液注射组小鼠异位囊肿分别进行石蜡切片，HE染色后镜下观察，可见类似子宫内膜组织的典型的腺体结构和间质组织结构(参见图6)。
图6为雌激素溶液每日腹腔注射的建模小鼠与雌激素温敏水凝胶混液每周皮下注射的建模小鼠的异位囊肿组织以及正常小鼠在位子宫内膜的HE染色图。其中：
A、B：正常小鼠在位子宫内膜组织HE染色；
C、D：雌激素溶液腹腔注射组小鼠内异病灶HE染色；
E、F：雌激素温敏水凝胶混液皮下注射组小鼠内异病灶HE染色。
由图可知：本法制备的雌激素温敏水凝胶混液在小鼠皮下注射组可支持内异症病灶的生长，形成的病灶与前人提供的方法形成的病灶无明显差异，参照本法制备的雌激素温敏水凝胶混液可用于子宫内膜异位症小鼠模型的建立。
结论：本发明探索了雌激素温敏水凝胶混液小鼠皮下注射后血液雌激素水平随注射的浓度和时间的变化规律，并通过比较实验，确立了0.5-1.0g/ml雌激素温敏水凝胶混液(每7天注射0.2ml)注射小鼠皮下可成功构建具有典型子宫内膜异位症病理特征的子宫内膜异位症小鼠模型。

Claims (8)

  1. 一种用于子宫内膜异位症小鼠模型建立的缓释混液，其特征在于，所述缓释混液包括的成分是：
    雌激素粉末0.5-1.0g/ml，其余是温敏水凝胶。
  2. 如权利要求1所述的缓释混液，其特征在于，包括的成分是：
    雌激素粉末0.8g/ml，其余是温敏水凝胶。
  3. 一种用于子宫内膜异位症小鼠模型建立的缓释混液的制备方法，其特征在于，包括以下步骤：
    1)从冷藏冰箱中取出温敏水凝胶，常温下化开至透明液态；
    2)按雌激素粉末与温敏水凝胶的比例，将雌激素粉末加入水凝胶中；其中，所述雌激素粉末0.5-1.0g/ml，其余是温敏水凝胶；
    3)置于震荡器上振荡使雌激素粉末与水凝胶混匀，配置成雌激素温敏水凝胶混液；
    4)将所述雌激素温敏水凝胶混液分装后置于冷藏冰箱保存。
  4. 根据权利要求3所述的用于子宫内膜异位症小鼠模型建立的缓释混液的制备方法，其特征在于，所述冷藏冰箱的温度低于-30℃。
  5. 根据权力要求3所述的用于子宫内膜异位症小鼠模型建立的缓释混液的制备方法，其特征在于，所述将雌激素粉末加入水凝胶的步骤中包括：
    称取8mg雌激素粉末，加入10ml温敏水凝胶；其中，所述雌激素粉末为β-雌二醇粉末；所述温敏水凝胶为聚乙二醇-聚酯类两嵌段温敏水凝胶。
  6. 一种子宫内膜异位症小鼠模型的建立方法，其特征在于，所述建立方法包括以下步骤：
    1)供体小鼠内膜片获取：取供体ICR小鼠5只，移植前一周每天腹腔注射0.1ml(30μg/ml)雌激素溶液；1周后将小鼠处死后进行手术，分离出小鼠子宫，在PBS中漂洗3遍，纵型剪开子宫使内膜面翻卷朝外，每只小鼠获得2段等长的子宫片，置于无菌PBS中等待移植；
    2)受体小鼠手术移植：腹腔注射麻醉受体ICR小鼠9只后行开腹手术，将处理好的1段子宫片内膜面朝外用无菌线固定于肠系膜上，逐层连续缝合腹部切口，PVP-I消毒后放回鼠笼；禁食24小时，可自由饮水；24小时后常规喂养；
    3)受体小鼠完成手术移植后，将缓释混液注入所述受体小鼠背部皮下，将所述受体小鼠制动30s，使凝胶更好地原位成胶；按预定周期给每只所述受体小鼠注射预定量的所述缓释混液；其中，所述缓释混液包括的成分是：雌激素粉末0.5-1.0g/ml，其余是温敏水凝胶。
  7. 如权利要求6所述的子宫内膜异位症小鼠模型的建立方法，其特征在于，所述按预定周期给每只所述受体小鼠注射预定量的所述缓释混液的步骤包括：
    按每周一次的预定周期给每只所述受体小鼠注射0.2ml所述缓释混液，其中所述缓释混液包含0.8g/ml的雌激素粉末，其余是温敏水凝胶。
  8. 如权利要求7所述的子宫内膜异位症小鼠模型的建立方法，其特征在于，所述缓释混液的通过以下步骤获得：
    称取8mg雌激素粉末加入10ml温敏水凝胶来；其中，所述雌激素粉末为β-雌二醇粉末，所述温敏水凝胶为聚乙二醇-聚酯类两嵌段温敏水凝胶；
    将所述雌激素粉末放入所述温敏水凝胶后，置于振荡器上震荡，使所述雌激素粉末与所述温敏水凝胶充分混匀。
PCT/CN2019/106729 2018-10-10 2019-09-19 用于子宫内膜异位症小鼠模型建立的缓释混液、制备方法以及小鼠模型建立方法 WO2020073780A1 (zh)

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