WO2023241565A1 - Use of reagent for detecting content of apoc3 protein in preparation of reagent for predicting development of oocytes - Google Patents

Use of reagent for detecting content of apoc3 protein in preparation of reagent for predicting development of oocytes Download PDF

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WO2023241565A1
WO2023241565A1 PCT/CN2023/099895 CN2023099895W WO2023241565A1 WO 2023241565 A1 WO2023241565 A1 WO 2023241565A1 CN 2023099895 W CN2023099895 W CN 2023099895W WO 2023241565 A1 WO2023241565 A1 WO 2023241565A1
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apoc3
reagent
oocytes
development
pcos
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PCT/CN2023/099895
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French (fr)
Chinese (zh)
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李荔
莫蕙
缪华章
欧燕兰
李小芳
刘姜伶
李远成
王友烽
范明皓
邱琳
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广东省妇幼保健院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides

Definitions

  • ApoC3 has achieved many clinical and basic studies in reducing blood lipids and has been identified as a new lipid-lowering target. ApoC3 is generally considered a direct risk factor for cardiovascular disease. Currently, relevant studies have linked ApoC3 levels in the peripheral circulation to atherosclerosis and adverse cardiovascular outcomes. ApoC3 is important for regulating fatty acid metabolism, and local lipid metabolism in the ovary provides a powerful energy source for the resumption of oocyte meiosis. Fatty acids produced by lipid lipolysis in oocytes are further metabolized through ⁇ -oxidation in mitochondria to produce ATP, which is used for oocyte maturation and ensures its quality. PCOS patients have abnormal lipid metabolism. Excessive lipid deposition in oocytes will increase the content of reactive oxygen species and damage the functions of mitochondria and endoplasmic reticulum, ultimately hindering the development of oocytes.
  • ApoC3 is also closely related to glucose metabolism. Elevated concentrations of ApoC3 in plasma can aggravate the development of insulin resistance and diabetes. In a small study of subjects with type 2 diabetes, it was found that inhibiting ApoC3 improves systemic insulin resistance (IR). ApoC3 may be involved in the pathophysiological process of IR by promoting islet cell apoptosis and glucose homeostasis disorder. Literature has reported the damage of ApoC3 to blood sugar in pathological conditions such as kidney disease, systemic lupus erythematosus, diabetes and liver disease. Glucose metabolism disorders in PCOS patients can cause changes in glucose metabolite levels in the local ovarian environment. The development process of eggs requires glucose to provide energy. However, due to the local insulin resistance and defects in the glucose transport pathway in the ovaries of PCOS patients, the utilization rate of glucose is reduced, which is detrimental to the growth and development of dry oocytes.
  • a reagent for detecting ApoC3 protein content in ovarian tissue in preparing a reagent for predicting oocyte development.
  • the second object of the present invention is to provide the application of ApoC3 protein as a biological marker for predicting oocyte development.
  • the third object of the present invention is to provide a reagent for predicting oocyte development, which includes a reagent for detecting ApoC3 protein content.
  • the present invention found that ApoC3 expression gradually increased during mouse development, reached a peak on the 21st day of age, and then slowly decreased. Therefore, ApoC3 can be used as a biological marker to predict oocyte development.
  • Figure 1 shows immunohistochemical staining of ovarian tissue from PCOS patients and controls. Brown-yellow staining indicates the ApoC3-positive area.
  • A Immunohistochemical staining of the control group and PCOS group;
  • B Immunohistochemical ApoC3 positive area analysis of the two groups.
  • Figure 3 shows the expression and localization of ApoC3 in ovarian tissue of PCOS mice and control mice at different stages of modeling.
  • A Immunohistochemical staining of ApoC3 in ovarian tissue. The brown-yellow staining indicates the ApoC3-positive area, and the red arrow indicates the oocyte. Scale bar: 200 ⁇ m; 50 ⁇ m.
  • B Immunofluorescence staining of ApoC3 in ovarian tissue. ApoC3 stains in red; cell nuclei stain in blue. White arrows indicate oocytes. Scale bar: 100 ⁇ m; 75 ⁇ m.
  • C ApoC3-positive area analyzed by immunohistochemical staining.
  • D Immunofluorescence staining analysis of ApoC3-positive areas.
  • Figure 4 shows the dynamic expression of ApoC3 in ovarian tissue.
  • B Immunohistochemical staining analysis of ApoC3-positive areas.
  • Figure 5 is an experimental design diagram.
  • PCOS patients aged (28.66 ⁇ 1.23) years old, who were admitted to the Department of Gynecology of Guangdong Maternal and Child Health Hospital from 2014 to 2016 were collected as the PCOS group. They underwent laparoscopic ovarian wedge resection, and a small part of the ovarian tissue from the wedge resection was collected for research. . During the same period, 30 non-PCOS patients whose age and weight matched those of the PCOS group were collected. Patients who underwent laparoscopic surgery for unilateral ovarian teratoma were the control group, aged (28.56 ⁇ 1.15) years old.
  • the control group underwent laparoscopic ovarian teratoma removal on the affected side and ovarian tissue dissection on the contralateral side, and the ovarian tissue from the contralateral side was taken for study.
  • the cases in the PCOS group and the control group were from Guangdong Maternal and Child Health Hospital.
  • the size should reach one-tenth of the entire ovarian tissue. Take a piece of tissue with a pyramid-like structure on the surface of the ovarian tissue. This tissue should include granulosa cells, theca cells, and oocytes. , interstitial cells and other various structures.
  • the surgery was performed by a chief gynecological endocrinologist from the Guangdong Maternal and Child Health Hospital with Class IV laparoscopy qualifications to ensure the accuracy and representativeness of clinical sample collection.
  • Patients signed a surgical consent form before collecting samples.
  • Sample collection was carried out after obtaining informed consent from the patients and approval from the Ethics Committee of Guangdong Maternal and Child Health Hospital (ethical approval number: 201401011).
  • Exclusion criteria (1) Other hyperandrogen diseases such as congenital adrenal hyperplasia, Cushing's syndrome, androgen-secreting tumors, etc.; (2) Other diseases causing ovulation disorders such as hyperprolactinemia, premature ovarian failure, or hypothalamic Amenorrhea and abnormal thyroid function; (3) vascular abnormalities such as essential hypertension and type 1 diabetes; (4) history of radiotherapy or chemotherapy for ovarian tumors.
  • DHEA dehydroepiandrosterone
  • mice were raised in the animal facility of the SPF Animal Laboratory (License Number: SYXK (GZ) 2019-0144) of the International Institute of Translational Traditional Chinese Medicine (Guangzhou), Guangzhou University of Chinese Medicine (Guangzhou). Animal experiments were approved by the Animal Care and Use Professional Committee of Guangzhou University of Traditional Chinese Medicine (No. 20220504) and were conducted in accordance with ethical standards and national guidelines. All mice were anesthetized with 3% isoflurane (ISO) and euthanized by exsanguination before dissection.
  • ISO isoflurane
  • Enzyme-linked immunoassay kit serum apolipoprotein C3 (SEB890Mu; CloudClone, Texas, USA), luteinizing hormone (H206-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), anti -Müllerian hormone (CEA228Mu; Cloud-Clone, Texas, USA), spectrophotometer (VL0000D0; ThermoFisher, Singapore) was used according to the manufacturer's instructions.
  • the primer sequence was obtained from PrimerBank (https://pga, mgh, harvard, edu/primerbank/) and synthesized by Invitrogen. The primer sequences are as follows:
  • Paraffin embedding, sectioning, dewaxing, antigen retrieval and blocking of mouse ovarian tissue were performed according to the above immunohistochemical staining methods.
  • Primary antibodies (ApoC3, Servicebio, GB112005, 1:300; DDX4, abcam, ab180462, 1:50) were incubated for 12 hours at 4°C. Wash three times with PBS, and incubate with Alexa Fluor 594-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature in the dark.
  • the nuclei were stained with DAPI-containing anti-fluorescence quenching mounting solution (P0131, Beyotime, Shanghai) at room temperature and protected from light, and the slides were mounted.
  • Mouse ovary tissue was frozen and homogenized by adding RIPA lysis buffer (9806, CST, Beverly, Massachusetts, USA), lysed, and centrifuged at 14000g for 10 minutes. The total protein concentration of the supernatant was detected by BCA (P0012, Beyotime, Shanghai), and the loading amount was 30 ⁇ g. SDS-PAGE electrophoresis conditions were 55V for 30min, 120V for 60min, transfer membrane, conditions were 300mA for 45min, blocked at room temperature for 2h, and washed 3 times with TBST.
  • the PVDF membrane was placed in an antibody incubation box containing internal reference GAPDH (sc-47724; Santa, USA, 1:2000) and primary anti-ApoC3 antibody (GB112005, Servicebio, Wuhan, 1:1000), incubated at 4°C overnight, and rewarmed. , washed 3 times with TBST, incubated with secondary antibodies anti-rabbit IgG-HRP and anti-mouse IgG-HRP (CST, 1:10000) for 1 hour at room temperature, washed 3 times with TBST, and collected images by ECL chemiluminescence.
  • GAPDH internal reference GAPDH
  • primary anti-ApoC3 antibody GB112005, Servicebio, Wuhan, 1:1000
  • mice 7-42 days old mice were purchased from Guangdong Sijiajingda Biotechnology Co., Ltd. (License Number: SCXK (Guangdong) 2020-0052). Animal experiments were approved by the Animal Care and Use Professional Committee of Guangzhou University of Traditional Chinese Medicine (No. 20220719) and were conducted in accordance with ethical standards and national guidelines. Changes in ApoC3 in mouse ovarian tissue were observed every 7 days. All mice were anesthetized with 3% isoflurane (ISO) and euthanized by exsanguination before dissection. The experimental design is shown in Figure 5 (day after birth Age: day of post partum, dpp).
  • ISO isoflurane
  • ApoC3 is mainly located in the cytoplasm of oocytes (red arrows in Figure 3A, white arrows in Figure 3B mark oocytes), and also after the second week of modeling, ApoC3 in the ovaries of the PCOS group was significantly higher than that of the control group. ApoC3 increased more significantly in the PCOS group after the third week of modeling ( Figure 3C, D).

Abstract

The use of a reagent for detecting the content of an ApoC3 protein in the preparation of a reagent for predicting the development of oocytes. A study found that the expression of ApoC3 in ovarian tissue gradually increases during mouse development, reaches a peak when mice are 21 days old, and then slowly decreases. Therefore, ApoC3 can be used as a biological marker for predicting the development situation of oocytes.

Description

检测ApoC3蛋白含量的试剂在制备预测***发育试剂中的应用Application of reagents for detecting ApoC3 protein content in preparing reagents for predicting oocyte development 技术领域：Technical areas:
本发明属于医药领域，具体涉及检测ApoC3蛋白含量的试剂在制备预测***发育试剂中的应用。The invention belongs to the field of medicine, and specifically relates to the application of a reagent for detecting ApoC3 protein content in preparing a reagent for predicting oocyte development.
背景技术：Background technique:
血清载脂蛋白C(apolipoprotein C，ApoC)是载脂蛋白家族中的一员。ApoC中，以ApoC3浓度最高，其对稳定脂蛋白结构、结合和转运脂质、调节脂蛋白代谢都有重要作用。ApoC3最初在肝脏和小肠中合成，其基因位于第11号染色体长臂11q23.3，长约3.1kb，有4个外显子和3个内含子，是含有79个氨基酸的肽链，大小为8.8kDa的小分子糖蛋白。ApoC3在血液中含量最高，主要调节富含甘油三酯脂蛋白(triglyceride-rich lipoproteins，TRLs)的分解与代谢。Serum apolipoprotein C (ApoC) is a member of the apolipoprotein family. Among ApoC, ApoC3 has the highest concentration, which plays an important role in stabilizing lipoprotein structure, binding and transporting lipids, and regulating lipoprotein metabolism. ApoC3 is initially synthesized in the liver and small intestine. Its gene is located on the long arm of chromosome 11, 11q23.3. It is about 3.1kb long, has 4 exons and 3 introns, and is a peptide chain containing 79 amino acids. It is a small molecule glycoprotein of 8.8kDa. ApoC3 has the highest content in the blood and mainly regulates the breakdown and metabolism of triglyceride-rich lipoproteins (TRLs).
①ApoC3与脂代谢：ApoC3在降低血脂方面取得了许多临床与基础研究，已被确定为新的降脂靶点。ApoC3被普遍认为是心血管疾病的直接危险因素。目前，已有相关研究将外周循环中ApoC3水平与动脉粥样硬化以及不良心血管结局联系起来。ApoC3对于调节脂肪酸代谢十分重要，卵巢局部脂质代谢为***减数***的恢复提供了一种强有力的能量来源。***中的脂质脂解产生的脂肪酸通过线粒体中的β-氧化进一步代谢以产生ATP，供***成熟并保证其质量。PCOS患者脂代谢异常，***中脂质沉积过多会增加活性氧的含量并且破坏线粒体和内质网的功能，最终使***的发育受阻。①ApoC3 and lipid metabolism: ApoC3 has achieved many clinical and basic studies in reducing blood lipids and has been identified as a new lipid-lowering target. ApoC3 is generally considered a direct risk factor for cardiovascular disease. Currently, relevant studies have linked ApoC3 levels in the peripheral circulation to atherosclerosis and adverse cardiovascular outcomes. ApoC3 is important for regulating fatty acid metabolism, and local lipid metabolism in the ovary provides a powerful energy source for the resumption of oocyte meiosis. Fatty acids produced by lipid lipolysis in oocytes are further metabolized through β-oxidation in mitochondria to produce ATP, which is used for oocyte maturation and ensures its quality. PCOS patients have abnormal lipid metabolism. Excessive lipid deposition in oocytes will increase the content of reactive oxygen species and damage the functions of mitochondria and endoplasmic reticulum, ultimately hindering the development of oocytes.
②ApoC3与糖代谢：ApoC3与葡萄糖代谢也有密切关系。血浆中ApoC3浓度升高，可加重胰岛素抵抗以及糖尿病的发展。在一项针对2型糖尿病受试者的小型研究中发现，抑制 ApoC3可改善全身胰岛素抵抗(IR)。ApoC3可能通过促进胰岛细胞凋亡和葡萄糖稳态紊乱参与IR的病理生理过程。已有文献在肾病、***性红斑狼疮、糖尿病及肝病等病理状态下报道ApoC3对血糖的损害。PCOS患者的糖代谢紊乱会引起卵巢局部内环境中的葡萄糖代谢物水平的变化。卵子的发育过程需要葡萄糖提供能量，而PCOS患者由于卵巢存在局部胰岛素抵抗状态、葡萄糖转运途径缺陷，导致葡萄糖的利用率降低，不利干***的生长发育。②ApoC3 and glucose metabolism: ApoC3 is also closely related to glucose metabolism. Elevated concentrations of ApoC3 in plasma can aggravate the development of insulin resistance and diabetes. In a small study of subjects with type 2 diabetes, it was found that inhibiting ApoC3 improves systemic insulin resistance (IR). ApoC3 may be involved in the pathophysiological process of IR by promoting islet cell apoptosis and glucose homeostasis disorder. Literature has reported the damage of ApoC3 to blood sugar in pathological conditions such as kidney disease, systemic lupus erythematosus, diabetes and liver disease. Glucose metabolism disorders in PCOS patients can cause changes in glucose metabolite levels in the local ovarian environment. The development process of eggs requires glucose to provide energy. However, due to the local insulin resistance and defects in the glucose transport pathway in the ovaries of PCOS patients, the utilization rate of glucose is reduced, which is detrimental to the growth and development of dry oocytes.
③ApoC3与炎症反应：ApoC3促进促炎核因子kappa B(NF-κB)信号转导，进而激活单核细胞，促进局部巨噬细胞募集和炎症发生。ApoC3还可诱导活性氧介导的血管平滑肌增殖，加期动脉粥样硬化的进程。Kelly等学者在2001年首次提出PCOS患者处于慢性低度炎症状态，并且与IR和腹部肥胖有关。肥胖可能加重PCOS患者的炎症状态，并增加远期心血管疾病的发病风险。PCOS脂代谢紊乱患者血清中有更高水平的ApoC3，ApoC3的升高可能通过激活内皮细胞蛋白激酶(PKC-β)和骨骼肌细胞外信号调节激酶(ERK1/2)，诱导炎症反应，促进炎症因子：肿瘤坏死因子(TNF-α)、核因子(NF-κB)进一步加重PCOS胰岛素抵抗、脂质异常的发生。③ApoC3 and inflammatory response: ApoC3 promotes pro-inflammatory nuclear factor kappa B (NF-κB) signal transduction, thereby activating monocytes and promoting local macrophage recruitment and inflammation. ApoC3 can also induce reactive oxygen species-mediated vascular smooth muscle proliferation and accelerate the progression of atherosclerosis. Kelly and other scholars first proposed in 2001 that PCOS patients are in a state of chronic low-grade inflammation and are related to IR and abdominal obesity. Obesity may aggravate the inflammatory state in PCOS patients and increase the risk of long-term cardiovascular disease. Patients with PCOS lipid metabolism disorder have higher levels of ApoC3 in the serum. The increase in ApoC3 may induce inflammatory response and promote inflammation by activating endothelial cell protein kinase (PKC-β) and skeletal muscle extracellular signal-regulated kinase (ERK1/2). Factors: Tumor necrosis factor (TNF-α) and nuclear factor (NF-κB) further aggravate the occurrence of insulin resistance and lipid abnormalities in PCOS.
④PCOS患者存在卵巢局部IR，与高表达的ApoC3密切相关，导致***功能障碍。多项研究表明在患有PCOS的女性中，胰岛素活性在经典靶器官(脂肪组织和骨骼肌等)中缺失引起胰岛素抵抗。PCOS同时具有卵巢局部胰岛素抵抗，影响卵巢局部葡萄糖代谢、胆固醇摄取、甾体激素生成以及细胞***增殖。有研究发现PCOS患者卵巢颗粒细胞存在受体后胰岛素信号传导分子的改变，导致下游激酶活性的改变，减弱胰岛素信号转导，促进卵巢局部胰岛素抵抗发生。胰岛素在细胞内的信号传导途径包括调节葡萄糖代谢的促代谢途径和引起细胞***增殖作用的促***途径等。卵巢局部IR时表现为细胞内胰岛素的促代谢作用途径受 损，而促***作用途径发生放大现象，使卵巢体积增加、卵泡发育停滞、***障碍、及卵巢局部高雄激素状态。④ PCOS patients have local ovarian IR, which is closely related to the high expression of ApoC3, leading to ovulatory dysfunction. Multiple studies have shown that in women with PCOS, loss of insulin activity in classic target organs (adipose tissue, skeletal muscle, etc.) causes insulin resistance. PCOS also has local insulin resistance in the ovary, which affects local glucose metabolism, cholesterol uptake, steroid hormone production, and cell division and proliferation in the ovary. Some studies have found that the presence of post-receptor insulin signaling molecules in ovarian granulosa cells in PCOS patients leads to changes in downstream kinase activity, weakens insulin signal transduction, and promotes the occurrence of local insulin resistance in the ovary. Insulin's intracellular signaling pathways include metabolic pathways that regulate glucose metabolism and mitogenic pathways that cause cell division and proliferation. Local IR in the ovary shows that the metabolic pathway of intracellular insulin is affected. Damage occurs, and the mitogenic pathway is amplified, resulting in increased ovarian volume, stagnant follicular development, ovulation disorders, and local hyperandrogen status in the ovary.
发明内容：Contents of the invention:
本发明的目的是提供检测ApoC3蛋白含量的试剂在制备预测***发育试剂中的应用。The purpose of the present invention is to provide the application of a reagent for detecting ApoC3 protein content in preparing a reagent for predicting oocyte development.
本发明前期对PCOS患者卵巢组织进行免疫组化研究：发现PCOS患者卵巢组织中ApoC3蛋白高表达。进一步通过对PCOS小鼠和正常对照小鼠卵巢组织行免疫组化和免疫荧光定位检查，发现：ApoC3定位于小鼠***胞浆内，我们推测ApoC3表达与***发育相关。我们随后采用出生后7至42日龄的小鼠进行验证，小鼠出生第7至42日，是卵巢的发育阶段。我们研究发现：ApoC3在小鼠不同发育时期表达水平不同，ApoC3表达在小鼠卵泡发育过程中逐渐升高，在卵巢组织中第21日龄达到高峰，随后缓慢下降。第21日即小鼠***启动期，此时期***接受***刺激(***LH)，从初期***第一次减数***前期发展为次级***的关键时间窗可作为预测***发育的生物学标志物。In the early stage of the present invention, an immunohistochemical study was conducted on the ovarian tissue of PCOS patients: it was found that ApoC3 protein was highly expressed in the ovarian tissue of PCOS patients. Further immunohistochemistry and immunofluorescence localization examination of ovarian tissues of PCOS mice and normal control mice revealed that ApoC3 is located in the cytoplasm of mouse oocytes. We speculate that ApoC3 expression is related to oocyte development. We then used mice aged 7 to 42 days after birth for verification. The 7th to 42nd day after birth is the development stage of the ovary. Our study found that the expression levels of ApoC3 are different in different developmental stages of mice. The expression of ApoC3 gradually increases during the development of mouse follicles, reaches a peak at the 21st day of age in ovarian tissue, and then slowly decreases. Day 21 is the initiation period of mouse puberty. During this period, oocytes receive gonadotropin stimulation (luteinizing hormone LH) and develop from the early stage of the first meiotic prophase of the initial oocyte to the critical time window of secondary oocytes. It can be used as a biological marker to predict oocyte development.
因此，本发明的第一个目的是提供检测ApoC3蛋白含量的试剂在制备预测***发育试剂中的应用。Therefore, the first object of the present invention is to provide the application of a reagent for detecting ApoC3 protein content in preparing a reagent for predicting oocyte development.
优选，是检测卵巢组织中的ApoC3蛋白含量的试剂在制备预测***发育试剂中的应用。Preferably, it is the application of a reagent for detecting ApoC3 protein content in ovarian tissue in preparing a reagent for predicting oocyte development.
本发明的第二个目的是提供ApoC3蛋白作为预测***发育的生物学标志物的应用。The second object of the present invention is to provide the application of ApoC3 protein as a biological marker for predicting oocyte development.
本发明的第三目的是提供一种预测***发育试剂，其包括检测ApoC3蛋白含量的试剂。 The third object of the present invention is to provide a reagent for predicting oocyte development, which includes a reagent for detecting ApoC3 protein content.
本发明发现ApoC3表达在小鼠发育过程中逐渐升高，在第21日龄达到高峰，随后缓慢下降。因此，ApoC3可作为预测***发育的生物学标志物，用于预测***发育情况。The present invention found that ApoC3 expression gradually increased during mouse development, reached a peak on the 21st day of age, and then slowly decreased. Therefore, ApoC3 can be used as a biological marker to predict oocyte development.
附图说明Description of the drawings
图1是PCOS患者和对照组卵巢组织免疫组化染色。棕黄色染色为ApoC3阳性面积。(A)对照组和PCOS组免疫组化染色；(B)两组免疫组化ApoC3阳性面积分析。Figure 1 shows immunohistochemical staining of ovarian tissue from PCOS patients and controls. Brown-yellow staining indicates the ApoC3-positive area. (A) Immunohistochemical staining of the control group and PCOS group; (B) Immunohistochemical ApoC3 positive area analysis of the two groups.
图2是PCOS小鼠和对照小鼠在造模不同阶段卵巢组织的ApoC3蛋白表达水平。(A)两组小鼠在三个造模阶段：造模第一周(1W)，造模第二周(2W)，造模第三周(3W)的ApoC3蛋白表达水平。(B)不同是造模阶段ApoC3相对蛋白表达量。Figure 2 shows the ApoC3 protein expression levels in ovarian tissue of PCOS mice and control mice at different stages of modeling. (A) ApoC3 protein expression levels of two groups of mice during three modeling stages: the first week of modeling (1W), the second week of modeling (2W), and the third week of modeling (3W). (B) The difference is the relative protein expression of ApoC3 during the modeling stage.
图3是PCOS小鼠和对照小鼠在造模不同阶段卵巢组织的ApoC3表达及定位。(A)卵巢组织中ApoC3的免疫组化染色。棕黄色染色为ApoC3阳性区域，红色箭头所示为***。比例尺:200μm；50μm。(B)卵巢组织中ApoC3免疫荧光染色。ApoC3呈红色染色；细胞核呈蓝色染色。白色箭头所示为***。比例尺:100μm；75μm。(C)免疫组化染色分析的ApoC3阳性区域。(D)免疫荧光染色分析ApoC3染色阳性区域。Figure 3 shows the expression and localization of ApoC3 in ovarian tissue of PCOS mice and control mice at different stages of modeling. (A) Immunohistochemical staining of ApoC3 in ovarian tissue. The brown-yellow staining indicates the ApoC3-positive area, and the red arrow indicates the oocyte. Scale bar: 200 μm; 50 μm. (B) Immunofluorescence staining of ApoC3 in ovarian tissue. ApoC3 stains in red; cell nuclei stain in blue. White arrows indicate oocytes. Scale bar: 100 μm; 75 μm. (C) ApoC3-positive area analyzed by immunohistochemical staining. (D) Immunofluorescence staining analysis of ApoC3-positive areas.
图4是ApoC3在卵巢组织中的动态表达情况。(A)第7至42日龄小鼠卵巢组织免疫组化染色，棕色染色的为ApoC3阳性区域，红色箭头指示为***；比例尺＝200μm；50μm。(B)免疫组化染色分析ApoC3阳性区域。Figure 4 shows the dynamic expression of ApoC3 in ovarian tissue. (A) Immunohistochemical staining of ovarian tissue from mice aged 7 to 42 days. The brown stained areas are ApoC3-positive areas, and the red arrows indicate oocytes; scale bar = 200 μm; 50 μm. (B) Immunohistochemical staining analysis of ApoC3-positive areas.
图5是实验设计图。Figure 5 is an experimental design diagram.
具体实施方式：Detailed ways:
以下实施例是对本发明的进一步说明，而不是对本发明的限制。The following examples further illustrate the present invention, rather than limiting the present invention.
实施例1 Example 1
一、研究方法1. Research methods
1.临床样本1.Clinical samples
收集2014年至2016年就诊于广东省妇幼保健院妇科的30名PCOS患者作为PCOS组，年龄(28.66±1.23)岁，行腹腔镜下卵巢楔形切除术，取楔形切除的小部分卵巢组织进行研究。同期收集30名年龄和体重与PCOS组匹配的非PCOS患者，因单侧卵巢畸胎瘤行腹腔镜手术患者为对照组，年龄(28.56±1.15)岁。对照组行腹腔镜下患侧卵巢畸胎瘤剔除术及健侧卵巢组织剖探术，取剖视活检的健侧卵巢组织进行研究。PCOS组及对照组病例来源于广东省妇幼保健院。腹腔镜手术收集卵巢组织样本时，其大小应达到整个卵巢组织的十分之一，在卵巢组织表面取一块类似金字塔样结构的组织，该组织应包括有颗粒细胞、卵泡膜细胞、***、***等各种结构。手术由广东省妇幼保健院具有IV类腹腔镜执业资格的妇科内分泌主任医师进行，以确保临床样本收集的准确性及代表性。收集样本前，患者已签署手术同意书。样本采集征得患者知情同意和广东省妇幼保健院伦理委员会批准后实施(伦理审批号：201401011)。Thirty PCOS patients, aged (28.66±1.23) years old, who were admitted to the Department of Gynecology of Guangdong Maternal and Child Health Hospital from 2014 to 2016 were collected as the PCOS group. They underwent laparoscopic ovarian wedge resection, and a small part of the ovarian tissue from the wedge resection was collected for research. . During the same period, 30 non-PCOS patients whose age and weight matched those of the PCOS group were collected. Patients who underwent laparoscopic surgery for unilateral ovarian teratoma were the control group, aged (28.56±1.15) years old. The control group underwent laparoscopic ovarian teratoma removal on the affected side and ovarian tissue dissection on the contralateral side, and the ovarian tissue from the contralateral side was taken for study. The cases in the PCOS group and the control group were from Guangdong Maternal and Child Health Hospital. When collecting ovarian tissue samples during laparoscopic surgery, the size should reach one-tenth of the entire ovarian tissue. Take a piece of tissue with a pyramid-like structure on the surface of the ovarian tissue. This tissue should include granulosa cells, theca cells, and oocytes. , interstitial cells and other various structures. The surgery was performed by a chief gynecological endocrinologist from the Guangdong Maternal and Child Health Hospital with Class IV laparoscopy qualifications to ensure the accuracy and representativeness of clinical sample collection. Patients signed a surgical consent form before collecting samples. Sample collection was carried out after obtaining informed consent from the patients and approval from the Ethics Committee of Guangdong Maternal and Child Health Hospital (ethical approval number: 201401011).
纳入标准：(1)PCOS组为接受腹腔镜治疗，并且符合2003鹿特丹诊断标准诊断为PCOS的患者；(2)对照组为同期就诊的年龄相匹配，月经规律，临床及生化检查排除高雄激素血症。其中，因输卵管不通畅就诊患者30例。Inclusion criteria: (1) The PCOS group consists of patients who received laparoscopic treatment and were diagnosed with PCOS according to the 2003 Rotterdam diagnostic criteria; (2) The control group consists of patients who were diagnosed with PCOS during the same period, have regular menstruation, and have clinical and biochemical examinations to rule out hyperandrogenism. disease. Among them, 30 patients were treated due to fallopian tube obstruction.
排除标准:(1)其他高雄激素疾病如先天性肾上腺皮质增生、库欣综合征、分泌雄激素的肿瘤等；(2)其他引起***障碍疾病如高催乳素血症、卵巢早衰或下丘脑性闭经及甲状腺功能异常；(3)原发性高血压、1型糖尿病等血管异常性疾病；(4)卵巢肿瘤放疗或化疗史。 Exclusion criteria: (1) Other hyperandrogen diseases such as congenital adrenal hyperplasia, Cushing's syndrome, androgen-secreting tumors, etc.; (2) Other diseases causing ovulation disorders such as hyperprolactinemia, premature ovarian failure, or hypothalamic Amenorrhea and abnormal thyroid function; (3) vascular abnormalities such as essential hypertension and type 1 diabetes; (4) history of radiotherapy or chemotherapy for ovarian tumors.
2.实验动物2. Experimental animals
2.1第一部分2.1 Part 1
所有C57BL/6小鼠(21日龄小鼠)购自广东斯嘉景达生物技术有限公司(许可证号:SCXK(粤)2020-0052)。将21日龄小鼠(9～13g)随机分为2组(每组n＝18):对照组(C)，PCOS组(P)。PCOS小鼠每日皮下注射脱氢表雄酮(DHEA，6mg/100g体重)溶解于芝麻油21天，对照组皮下注射芝麻油21天。动物置于温度湿度控制的房间(22±1℃，湿度45～60％)，一套12h的明暗循环。饲养于广州中医药大学(广州)国际中医药转化研究所SPF动物实验室(许可证号:SYXK(GZ)2019-0144)动物设施中。动物实验经广州中医药大学动物护理与使用专业委员会(No.20220504)批准，按照伦理标准和国家指南进行。所有小鼠均用3％异氟醚(ISO)麻醉，并在解剖前放血安乐死。All C57BL/6 mice (21-day-old mice) were purchased from Guangdong Sijiajingda Biotechnology Co., Ltd. (license number: SCXK (Guangdong) 2020-0052). 21-day-old mice (9-13g) were randomly divided into 2 groups (n=18 in each group): control group (C), PCOS group (P). PCOS mice were daily subcutaneously injected with dehydroepiandrosterone (DHEA, 6 mg/100g body weight) dissolved in sesame oil for 21 days, and the control group was injected subcutaneously with sesame oil for 21 days. The animals were placed in a temperature and humidity controlled room (22±1°C, humidity 45-60%) with a 12-h light-dark cycle. They were raised in the animal facility of the SPF Animal Laboratory (License Number: SYXK (GZ) 2019-0144) of the International Institute of Translational Traditional Chinese Medicine (Guangzhou), Guangzhou University of Chinese Medicine (Guangzhou). Animal experiments were approved by the Animal Care and Use Professional Committee of Guangzhou University of Traditional Chinese Medicine (No. 20220504) and were conducted in accordance with ethical standards and national guidelines. All mice were anesthetized with 3% isoflurane (ISO) and euthanized by exsanguination before dissection.
2.1.1 ELISA检测2.1.1 ELISA detection
EDTA采血管眼眶穿刺采集血样，3000转离心15分钟，-80℃保存上清，待生化和激素分析。酶联免疫检测试剂盒：血清载脂蛋白C3(SEB890Mu；云克隆公司，德克萨斯州，美国)，***(H206-1-2；南京建成生物工程研究所，中国南京)，anti-Müllerian激素(CEA228Mu；Cloud-Clone公司，德克萨斯州，美国)，分光光度计(VL0000D0；ThermoFisher，新加坡)根据制造商的说明使用。Blood samples were collected by orbital puncture with EDTA blood collection tubes, centrifuged at 3000 rpm for 15 minutes, and the supernatant was stored at -80°C for biochemical and hormone analysis. Enzyme-linked immunoassay kit: serum apolipoprotein C3 (SEB890Mu; CloudClone, Texas, USA), luteinizing hormone (H206-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), anti -Müllerian hormone (CEA228Mu; Cloud-Clone, Texas, USA), spectrophotometer (VL0000D0; ThermoFisher, Singapore) was used according to the manufacturer's instructions.
2.1.2 HE染色2.1.2 HE staining
用PBS冰上清洗卵巢两次，然后将一侧卵巢用4％多聚甲醛固定24小时。石蜡包埋组织采用梯度乙醇脱水、二甲苯透化、石蜡包埋的顺序进行。切片厚度3μm(CM1850；Leica，Nussloch，德国)，在二甲苯中脱蜡，用梯度乙醇水化，然后用苏木精和伊红染料染色，用 中性树脂封片。在显微镜下观察卵泡形态和数量(DM750，Leica，Wetzlar，Germany)。The ovaries were washed twice with PBS on ice, and then one ovary was fixed with 4% paraformaldehyde for 24 hours. Paraffin-embedded tissues were dehydrated with gradient ethanol, permeabilized with xylene, and embedded in paraffin in this order. Sections with a thickness of 3 μm (CM1850; Leica, Nussloch, Germany) were deparaffinized in xylene, hydrated with graded ethanol, and then stained with hematoxylin and eosin dyes and stained with Seal slides in neutral resin. Follicle morphology and number were observed under a microscope (DM750, Leica, Wetzlar, Germany).
2.1.3 RNA提取和qRT-PCR2.1.3 RNA extraction and qRT-PCR
总RNA从卵巢中分离，用TRIzol试剂提取RNA(15596026；Invitrogen，卡尔斯巴德，加州)。使用RevertAidcDNA合成试剂盒反转录(K1622；美国ThermoFisher)。通过实时PCR***(Applied Biosystems，CA，USA)监测SYBR绿色荧光的增加，进行定量实时PCR。引物序列从PrimerBank(https://pga，mgh，harvard，edu/primerbank/)获得，由Invitrogen公司合成。引物序列如下:
Total RNA was isolated from ovaries, and RNA was extracted using TRIzol reagent (15596026; Invitrogen, Carlsbad, CA). Reverse transcription was performed using RevertAid cDNA synthesis kit (K1622; ThermoFisher, USA). Quantitative real-time PCR was performed by monitoring the increase in SYBR green fluorescence using a real-time PCR system (Applied Biosystems, CA, USA). The primer sequence was obtained from PrimerBank (https://pga, mgh, harvard, edu/primerbank/) and synthesized by Invitrogen. The primer sequences are as follows:
每个样本扩增三次。数据采用ΔΔCt方法进行相对定量分析。Each sample was amplified three times. The data were analyzed relatively quantitatively using the ΔΔCt method.
2.1.4免疫组化染色2.1.4 Immunohistochemical staining
按上述方法脱蜡。采用pH为6.0的柠檬酸钠缓冲液在微波中进行抗原修复，100℃，20min，用3％的H2O2淬灭内源性过氧化物酶。正常山羊血清在室温下封闭10分钟，用一抗ApoC3(Novusbio；NB600-610，1:1000；GB112005；Servicebio，武汉，1:300)在4℃孵育2h。生物素标记山羊抗兔IgG抗体(CW2069S；切片中加入CWBio，江苏，中国)，室温孵育2h。PBS冲洗后，加入链霉亲和素-HRP，最后用DAB混合液进行显色反应。Dewax as above. Antigen retrieval was performed in the microwave using sodium citrate buffer with pH 6.0, 100°C, 20 min, and endogenous peroxidase was quenched with 3% H 2 O 2 . Normal goat serum was blocked for 10 minutes at room temperature and incubated with primary antibody ApoC3 (Novusbio; NB600-610, 1:1000; GB112005; Servicebio, Wuhan, 1:300) for 2 hours at 4°C. Biotin-labeled goat anti-rabbit IgG antibody (CW2069S; CWBio, Jiangsu, China) was added to the sections and incubated at room temperature for 2 h. After washing with PBS, streptavidin-HRP was added, and finally DAB mixture was used for color development reaction.
2.1.5免疫荧光染色2.1.5 Immunofluorescence staining
小鼠卵巢组织石蜡包埋、切片、脱蜡、抗原修复及封闭按上述免疫组化染色方法进行。一抗(ApoC3,Servicebio，GB112005,1:300；DDX4,abcam，ab180462,1:50)在4℃孵育12h， PBS洗3次，Alexa Fluor 594偶联山羊抗兔和Alexa Fluor 488偶联山羊抗小鼠IgG二抗(Jackson ImmunoResearch)室温避光孵育1h。用含DAPI抗荧光淬灭封片液(P0131，碧云天，上海)室温避光染核并封片。Paraffin embedding, sectioning, dewaxing, antigen retrieval and blocking of mouse ovarian tissue were performed according to the above immunohistochemical staining methods. Primary antibodies (ApoC3, Servicebio, GB112005, 1:300; DDX4, abcam, ab180462, 1:50) were incubated for 12 hours at 4°C. Wash three times with PBS, and incubate with Alexa Fluor 594-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature in the dark. The nuclei were stained with DAPI-containing anti-fluorescence quenching mounting solution (P0131, Beyotime, Shanghai) at room temperature and protected from light, and the slides were mounted.
2.1.6蛋白印迹2.1.6 Western blotting
小鼠卵巢组织加入RIPA裂解液(9806，CST，Beverly,Massachusetts，USA)冷冻匀浆、裂解，冷冻离心14000g，10分钟。上清液用BCA(P0012，碧云天，上海)检测蛋白总浓度，上样量为30μg。SDS-PAGE电泳条件55V 30min，120V 60min，转膜，条件300mA45min，室温封闭2h，TBST洗3次。PVDF膜放入装有内参GAPDH(sc-47724；Santa,USA，1∶2000)和一抗ApoC3抗体(GB112005，Servicebio，武汉，1：1000)的抗体孵育盒中，4℃孵育过夜，复温、TBST洗3次，二抗为抗兔IgG-HRP和抗鼠IgG-HRP(CST，1∶10000)室温孵育1h，TBST洗3次，ECL化学发光采集图像。Mouse ovary tissue was frozen and homogenized by adding RIPA lysis buffer (9806, CST, Beverly, Massachusetts, USA), lysed, and centrifuged at 14000g for 10 minutes. The total protein concentration of the supernatant was detected by BCA (P0012, Beyotime, Shanghai), and the loading amount was 30 μg. SDS-PAGE electrophoresis conditions were 55V for 30min, 120V for 60min, transfer membrane, conditions were 300mA for 45min, blocked at room temperature for 2h, and washed 3 times with TBST. The PVDF membrane was placed in an antibody incubation box containing internal reference GAPDH (sc-47724; Santa, USA, 1:2000) and primary anti-ApoC3 antibody (GB112005, Servicebio, Wuhan, 1:1000), incubated at 4°C overnight, and rewarmed. , washed 3 times with TBST, incubated with secondary antibodies anti-rabbit IgG-HRP and anti-mouse IgG-HRP (CST, 1:10000) for 1 hour at room temperature, washed 3 times with TBST, and collected images by ECL chemiluminescence.
2.1.7统计学方法2.1.7 Statistical methods
两组比较采用独立样本t检验。用pearson相关系数(R)进行相关性分析，用ImageJ软件进行灰度值分析。采用SPSS 25和GraphPad Prism 7进行统计计算。P<0.05被认为有统计学意义。The independent samples t test was used to compare the two groups. Pearson correlation coefficient (R) was used for correlation analysis, and ImageJ software was used for gray value analysis. Statistical calculations were performed using SPSS 25 and GraphPad Prism 7. P<0.05 was considered statistically significant.
2.2实验部分22.2 Experimental part 2
2.2.1所有C57BL/6小鼠(出生7-42日龄小鼠)购自广东斯嘉景达生物技术有限公司(许可证号:SCXK(粤)2020-0052)。动物实验经广州中医药大学动物护理与使用专业委员会(No.20220719)批准，按照伦理标准和国家指南进行。每7日观察小鼠卵巢组织ApoC3的变化。所有小鼠均用3％异氟醚(ISO)麻醉，并在解剖前放血安乐死。实验设计如图5(出生后日 龄：day of post partum，dpp)。2.2.1 All C57BL/6 mice (7-42 days old mice) were purchased from Guangdong Sijiajingda Biotechnology Co., Ltd. (License Number: SCXK (Guangdong) 2020-0052). Animal experiments were approved by the Animal Care and Use Professional Committee of Guangzhou University of Traditional Chinese Medicine (No. 20220719) and were conducted in accordance with ethical standards and national guidelines. Changes in ApoC3 in mouse ovarian tissue were observed every 7 days. All mice were anesthetized with 3% isoflurane (ISO) and euthanized by exsanguination before dissection. The experimental design is shown in Figure 5 (day after birth Age: day of post partum, dpp).
2.2.2 ELISA检测2.2.2 ELISA detection
同上Same as above
2.2.3免疫组化染色2.2.3 Immunohistochemical staining
同上Same as above
2.2.4统计学方法2.2.4 Statistical methods
同上Same as above
二、研究结果2. Research results
1、PCOS组患者卵巢组织和ApoC3的平均光密度明显高于对照组人群(图1)。1. The average optical density of ovarian tissue and ApoC3 in the PCOS group was significantly higher than that in the control group (Figure 1).
2、PCOS小鼠卵巢组织的ApoC3蛋白表达在造模第二周后，较对照组明显升高(图2)。2. The ApoC3 protein expression in the ovarian tissue of PCOS mice was significantly higher than that in the control group after the second week of modeling (Figure 2).
3、ApoC3主要定位在***的细胞浆内(图3A红色箭头，图3B白色箭头标注为***)，且同样在造模第二周后PCOS组ApoC3卵巢较对照组明显升高，ApoC3在PCOS组造模第三周后升高更为显著(图3C、D)。3. ApoC3 is mainly located in the cytoplasm of oocytes (red arrows in Figure 3A, white arrows in Figure 3B mark oocytes), and also after the second week of modeling, ApoC3 in the ovaries of the PCOS group was significantly higher than that of the control group. ApoC3 increased more significantly in the PCOS group after the third week of modeling (Figure 3C, D).
4、在卵巢组织中，ApoC3表达在小鼠发育过程中逐渐升高，在卵巢组织中ApoC3表达第21日龄达到高峰，随后缓慢下降(图4)。 4. In ovarian tissue, ApoC3 expression gradually increased during mouse development. ApoC3 expression in ovarian tissue reached a peak at the 21st day of age, and then decreased slowly (Figure 4).

Claims (4)

  1. 检测ApoC3蛋白含量的试剂在制备预测***发育试剂中的应用。Application of reagents for detecting ApoC3 protein content in preparing reagents for predicting oocyte development.
  2. 根据权利要求1所述的应用，其特征在于，是检测卵巢组织中的ApoC3蛋白含量的试剂在制备预测***发育试剂中的应用。The application according to claim 1, characterized in that it is an application of a reagent for detecting ApoC3 protein content in ovarian tissue in preparing a reagent for predicting oocyte development.
  3. ApoC3蛋白作为预测***发育的生物学标志物的应用。Application of ApoC3 protein as a biological marker for predicting oocyte development.
  4. 一种预测***发育试剂，其特征在于，包括检测ApoC3蛋白含量的试剂。 A reagent for predicting oocyte development, which is characterized by including a reagent for detecting ApoC3 protein content.
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