WO2023004847A1 - 一种小鼠早衰模型及其构建方法与应用 - Google Patents

一种小鼠早衰模型及其构建方法与应用 Download PDF

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WO2023004847A1
WO2023004847A1 PCT/CN2021/110471 CN2021110471W WO2023004847A1 WO 2023004847 A1 WO2023004847 A1 WO 2023004847A1 CN 2021110471 W CN2021110471 W CN 2021110471W WO 2023004847 A1 WO2023004847 A1 WO 2023004847A1
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mouse
mice
hbxip
aging
model
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史丽云
张薇
沙舟
冀鹏
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浙江树人学院（浙江树人大学）
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  • the invention relates to the field of biotechnology, in particular to a mouse premature aging model and its construction method and application.
  • Premature aging is premature aging, which refers to the phenomenon of premature physical, physical and psychological weakness of the body due to various internal or external reasons, and its pathological mechanism is not yet clear. Therefore, aging-related diseases have become hotspots and difficulties that urgently need to be solved and breakthroughs in the global scientific research field. Delaying aging is a necessary condition for ensuring social stability and sustainable economic development.
  • mouse aging models mainly include D-galactose mouse aging model, ⁇ -ray irradiation-induced mouse aging model, and thymectomy-induced mouse aging model.
  • the current common mouse aging models mainly include D-galactose injection mouse aging model, thymectomy-induced aging mouse model, gamma-ray irradiation-induced aging mouse model, etc., these three common
  • the construction method and existing defects of the mouse aging model are as follows: 1) D-galactose injection mouse aging model: mice are injected intraperitoneally with 500 mg/kg/day of D-galactose normal saline solution for 2 consecutive months Molded.
  • Thymectomy mice cannot be qualitatively or quantitatively measured for some immune indicators of aging, and it is difficult to judge whether the ideal aging state has been achieved, which affects the credibility of the model; 3) ⁇ -ray irradiation-induced mouse aging model: mice given 3Gy whole body irradiation (irradiation area is 25cm ⁇ 25cm, height is 80cm, time is 5min), once every 10 days, a total of 8 times, a total of 24Gy, and the model is formed after 80 days.
  • the radiation dose is too large, the experimental animals will easily die and the experiment cannot be carried out, so the degree and time of radiation must be well controlled.
  • Hepatitis B X-interacting protein is a constitutive protein mainly found in mammalian cells, with a molecular weight of about 19kDa.
  • HBXIP can inhibit the replication of hepatitis B virus, promote the proliferation and migration of tumor cells, and inhibit the acute lung injury and endotoxemia induced by LPS.
  • HBXIP accelerates the growth of breast cancer by promoting the degradation of tumor suppressor p53 through MDM2.
  • HBXIP is a late endosome/lysosomal adapter and a member of the MAPK and mammalian target of rapamycin LAMTOR complex, which is involved in regulating the activation of mTORC1 and affecting the anabolism, translation, transcription, and lipid synthesis of cells , autophagy and other processes. Therefore, HBXIP is a regulatory factor involved in important life activities of the body.
  • the invention provides a mouse progeria model construction based on HBXIP conditional knockout and related applications.
  • the technical problem to be solved by the present invention is to provide a mouse premature aging model for the deficiencies of the prior art.
  • the technical problem to be solved by the present invention is to provide a method for constructing the above mouse progeria model.
  • the final technical problem to be solved by the present invention is to provide the application of the above mouse progeria model.
  • the present invention discloses a mouse premature aging model, which is characterized in that the hepatitis B virus X protein binding protein (HBXIP) in mouse bone marrow-derived cells is knocked out.
  • HBXIP hepatitis B virus X protein binding protein
  • the knockout is to knock out the second exon in the gene encoding the hepatitis B virus X protein binding protein.
  • the mRNA sequence of the hepatitis B virus X protein binding protein coding gene is Accession: NM_026774.2 in Gene Bank, and its genome sequence is Accession: NC_000069.7 in Gene Bank.
  • the knockout is through the Cre-loxp system knockout; preferably, the knockout is through the bone marrow cell-specific expression of Cre recombinase mouse LysM cre/+ knockout.
  • the present invention discloses a method for constructing the above-mentioned mouse premature aging model, comprising the following steps:
  • the HBXIP fl/+ mouse is the endogenous endogenous protein on both sides of the second exon (1013-1074) of the hepatitis B virus X protein binding protein genome (NC_000069.7) in the C57BL/6 mouse
  • the loxp1 site and the loxp2 site are added to the containing subregion respectively, that is, the nucleotide sequence (loxp1 site) as shown in SEQ ID No.1 is inserted after the 762nd nucleotide of the HBXIP genome sequence, and the 1250th core Insert the nucleotide sequence (loxp2 site) as shown in SEQ ID No.2 after the nucleotide.
  • the insertion method of the loxp sequence can be obtained by various known and recognized methods.
  • the present invention discloses the application of the above-mentioned mouse progeria model.
  • the application is the application of the mouse premature aging model in the development or screening of products for the prevention and/or treatment of premature aging; wherein, the products include but not limited to drugs, food, health products, and cosmetics.
  • the application is the application of the mouse progeria model in developing or screening technical methods for preventing and/or treating progeria.
  • the application is the application of the mouse progeria model in progeria research.
  • the present invention has the following advantages:
  • the progeria mice exhibit typical progeria phenotypes such as alopecia, significantly up-regulated expression of senescence-related genes, up-regulation of senescence-related secretory phenotype factors in serum, and aging of tissues and organs at the age of 4-8 months .
  • the myeloid cell conditional knockout HBXIP mice provided by the present invention have a short feeding cycle (6-8 months old), small individual differences, and few interfering factors , Low cost and other advantages.
  • the myeloid cell conditional knockout HBXIP mouse premature aging model provided by the present invention has 1) no complicated modeling process, stable reproduction, and spontaneous aging phenotype; 2) The experimental technology is not demanding and easy to control, which can avoid the death of mice caused by operation and other reasons during the modeling process; 3) The aging state is easy to detect and characterize, and the aging state of mice can be accurately judged, and the model is reliable 4) Small individual differences in gene knockout background mice; 5) Conditional HBXIP knockout in myeloid cells means that the knockout of HBXIP is cell-specific and only occurs in myeloid cell lines, including single nuclear cells, mature macrophages, and granulocytes.
  • the present invention induces premature aging in mice by conditionally knocking out HBXIP by manipulating bone marrow-derived immune cells, which has significant innovation and good application prospects, and can be used to establish personalized drugs/foodstuffs/technical methods for anti-aging screening
  • the platform at the same time, can be used to study the pathogenesis of aging-related diseases, such as neurodegenerative diseases, metabolic diseases, autoimmune and inflammatory diseases, as well as technology and product development for the prevention, diagnosis and treatment of related diseases.
  • Figure 1 shows the preparation method and genotype identification of HBXIP ⁇ LysM mice.
  • Figure 2 is the transcript level verification of HBXIP knockout effect in BMDM of HBXIP ⁇ LysM mice.
  • Figure 3 is the protein level verification of HBXIP knockout effect in BMDM of HBXIP ⁇ LysM mice.
  • Figure 4 shows the expression of aging-related genes in BMDM of wild-type mice (WT) and HBXIP ⁇ LysM mice.
  • Figure 5 shows the levels of secreted phenotypic factors related to aging in the serum of wild-type mice and HBXI P ⁇ LysM mice.
  • Figure 6 is the detection of liver function in wild-type mice and HBXIP ⁇ LysM mice.
  • Figure 7 is the detection of kidney function in wild-type mice and HBXIP ⁇ LysM .
  • Figure 8 is H&E staining of wild-type mice and HBXIP ⁇ LysM tissue sections.
  • Example 1 Preparation method and genotype identification of myeloid cell conditional knockout HBXIP mice.
  • Bone marrow cell-specific Cre recombinase expression mouse (LysM cre/+ ): English name B6.129P2-Lyz2 tm1(cre)Ifo/J, Stock No: 004781, purchased from The Jackson Laboratory.
  • the strain mouse genome is inserted with a Cre enzyme gene, and the gene can express Cre recombinase in bone marrow cells through a bone marrow cell-specific promoter.
  • the Cre-mediated recombination can delete the target gene sequence. These sequence deletions are cell-specific and only occur in myeloid cell lines, including monocytes, mature macrophages and granulocytes, etc. .
  • mice Preparation of loxp transgene heterozygous (HBXIP fl/+ ) mice: the mouse strain is C57BL/6, in the second exon (1013- 1074) loxp1 site and loxp2 site were added to the intron regions on both sides, that is, the nucleotide sequence of SEQ ID No.1 (loxp1 site) was inserted after the 762nd nucleotide of the HBXIP genome sequence, the 1250th The nucleotide sequence of SEQ ID No.2 (loxp2 site) is inserted after the first nucleotide, and the nucleotide sequences of the two loxp sites are shown in SEQ ID No.1 and SEQ ID No.2 respectively.
  • the insertion method of the above loxp sequence is as follows: using Optimized Cas9/CRISPR System (OCAS) technology, two gRNA sequences are designed, which are gRNA target 1: tagagcatgcttctataccaAGG (for the forward sequence of the genome, gRNA-1, nucleoside The acid sequence is shown in SEQ ID No.3), gRNA target 2: GGAACAGTTGTAGAACTGGGAGG (for the reverse sequence of the genome, gRNA-2, the nucleotide sequence is shown in SEQ ID No.4).
  • OCAS Optimized Cas9/CRISPR System
  • the Case9 mRNA, two gRNA sequences, and the SSODN homologous recombination template (the nucleotide sequence is shown in SEQ ID No. 5) were introduced into the fertilized mouse eggs, and the DNA repair mechanism was used to insert the two The complete sequence of loxp is inserted into the genome.
  • the embryos are cultured in vitro for 1-2 hours, they are transplanted into the fallopian tubes of pseudopregnant mother mice, and the surrogate mice are ready to give birth after the wound is sutured. After calving, F0 generation verification, mouse cross verification, etc. are carried out.
  • HBXIP fl/+ is mated with HBXIP fl/+ individuals to obtain HBXIP fl/fl homozygous mice
  • HBXIP fl/fl is mated with LysM cre/+ individuals
  • Individuals that contain loxp sites and express Cre enzymes can be obtained, that is, (HBXIP fl/+ -LysM cre/+ ) individuals
  • (HBXIP fl/+ -LysM cre/+ ) individuals are combined with (HBXIP fl/ + -LysM cre/+ ) individuals were mated to obtain (HBXIP fl/fl -LysM cre/+ ) individuals.
  • the primers for Cre mouse tail identification are as follows:
  • Heterozygote 300bp and 750bp
  • mice 2.3 Extraction, isolation and culture of mouse BMDMs.
  • PBS fetal bovine serum
  • M-CSF mouse M-CSF (macrophage colony stimulating factor) 25ng/ml
  • Example 2 Phenotype verification of progeria model in myeloid cell conditional knockout HBXIP mice.
  • the reagents required for real-time quantitative PCR are the same as those described in 1.1 in Example 1.
  • the ELISA detection kit was purchased from Lianke Biotech, the alanine aminotransferase activity detection kit (BC1555), the aspartate aminotransferase activity detection kit (BC1565), and the Bradford protein concentration quantification kit (PC0010) were purchased from Beijing Solaibao Technology Co., Ltd.
  • Real-time quantitative PCR was used to detect the expression of aging-related genes in BMDM of wild-type mice and HBXIP ⁇ LysM mice.
  • the detection method of real-time quantitative PCR is the same as 2.2 in Example 1.
  • ELISA was used to detect the levels of secreted phenotypic factors related to aging in the serum of wild-type mice and HBXIP ⁇ LysM mice.
  • the levels of secreted phenotype factors related to aging in mouse serum were measured by ELISA experimental method. Soak the ELISA plate, add standard, add sample, incubate, wash, add enzyme to incubate, wash, add substrate for color development, add stop solution, and detect reading in sequence. The detection reading should be within 30 minutes.
  • Use a microplate reader to perform dual-wavelength detection, and measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm. The OD value after calibration is the measured value at 450 nm minus the measured value at 570 nm or 630 nm.
  • ALT activity detection first dilute the alanine standard to 2 ⁇ mol/mL with distilled water, and dilute the standard to 1.5, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0 mM and other concentration gradients. A certain volume of alanine (200mM), ⁇ -ketoglutaric acid solution (2mM) and 2,4-dinitrophenylhydrazine solution was added to the standard tube, measurement tube and control tube respectively.
  • ALT activity unit the amount of catalyzed production of 1 ⁇ mol pyruvate per mL of serum sample per hour is an activity unit.
  • Aspartate transaminase (AST) activity detection first dilute the sodium pyruvate standard to 2 ⁇ mol/mL with distilled water, and dilute the standard to a concentration gradient of 1.5, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, and 0 mM. Add a certain volume of ⁇ -ketoglutaric acid (2mM), aspartic acid (200mM) and 2,4-dinitrophenylhydrazine solutions into the standard tube, measurement tube and control tube respectively.
  • AST activity unit the amount of catalyzed production of 1 ⁇ mol pyruvate per mL of serum sample per hour is an activity unit.
  • the concentration of urinary protein in mice was detected by Bradford method. Completely dissolve the protein standard to a final concentration of 0.2 mg/ml. Add 0, 2, 4, 6, 8, 12, 16, and 20 microliters of the standard to the 96-well plate, and add PBS diluent to make up to 20 microliters. The mouse urine sample was properly diluted (2 times, 4 times, 8 times dilution), and 20 microliters were added to the sample wells of the 96-well plate. Add 200 microliters of diluted 1 ⁇ G250 Coomassie Brilliant Blue staining solution to each well, and place at room temperature for 3-5 minutes. A595 was measured with a microplate reader. Calculate the protein concentration in the sample according to the standard curve.
  • the present invention provides a mouse premature aging model and its construction method and ideas and methods for its application.

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Abstract

提供了一种小鼠早衰模型及其构建方法与应用，所述小鼠早衰模型是通过敲除小鼠骨髓来源细胞中的乙肝病毒X蛋白结合蛋白而获得的。该早衰小鼠在4-8个月龄时出现脱毛、衰老相关基因表达显著上调、血清中衰老相关的分泌表型因子上调、组织器官衰老等典型的早衰表型。通过操控骨髓来源的免疫细胞条件性敲除HBXIP，诱导小鼠早衰具有显著的创新性和良好的应用前景，可用于建立延缓衰老的个性化药物/食材/技术方法等的筛选平台，同时，可用于研究衰老相关疾病，如神经退行性疾病、代谢性疾病、自身免疫及炎症性疾病等发病机制研究，以及相关疾病的预防、诊断和治疗等技术和产品研发。

Description

一种小鼠早衰模型及其构建方法与应用 技术领域
本发明涉及生物技术领域中，具体涉及一种小鼠早衰模型及其构建方法与应用。
背景技术
早衰即过早衰老，是指由于各种内在的或外在的原因使机体过早地出现生理上、体质上和心理上衰弱的现象，且其病理机制尚未明确。因此，衰老相关疾病已成为全球科研领域亟需解决和突破的热点及难点，延缓衰老是保证社会稳定、经济持续性发展的必要条件。
利用小鼠等模式生物衰老模型能更好地认识人类衰老的进程，为临床转化研究提供有效的工具和理论基础。常见的小鼠衰老模型主要包括D-半乳糖小鼠衰老模型、γ射线照射诱导小鼠衰老模型、胸腺摘除诱导小鼠衰老模型。除自然衰老小鼠模型以外，目前常见的小鼠衰老模型主要包括D-半乳糖注射小鼠衰老模型、胸腺摘除诱导小鼠衰老模型、γ射线照射诱导小鼠衰老模型等，这三种常见的小鼠衰老模型的构建方法及存在的缺陷如下所述：1)D-半乳糖注射法小鼠衰老模型：小鼠腹腔注射D-半乳糖生理盐水溶液500毫克/千克/天，连续2个月成模。每天注射D-半乳糖比较繁琐，注射造模时间较长，需要2个月左右，且由于小鼠的个性差异较大，不易控制；2)胸腺摘除小鼠衰老模型：麻醉小鼠，剥离并取出胸腺，缝合切口并滴加青霉素，继续常规饲养6个月成模。该造模方法对试验技术要求较高，在摘除胸腺时容易导致小鼠死亡，有一定的差异性。其耗费时间更长，需要6个月。胸腺摘除小鼠，对于衰老的某些免疫指标无法进行定性或定量的测定，难以判断是否达到理想的衰老状态，影响模型的可信度；3)γ射线照射诱导小鼠衰老模型：小鼠给予3Gy全身照射(照射面积为25cm×25cm，高度为80cm，时间为5min)，每10天一次，共8次，总计24Gy，80天后成模型。但是如果辐射剂量过大，则会使试验动物容易死亡，无法进行试验，因此要掌握好辐射的度和时间。
乙肝病毒X蛋白结合蛋白(hepatitis B X-interacting protein,HBXIP)，是一种主要存在于哺乳动物的细胞组成型蛋白，分子量约为19kDa。HBXIP能够抑制乙型肝炎病毒的复制，促进肿瘤细胞的增殖和迁移，抑制LPS诱导的急性肺损伤和内毒素血症等。研究表明HBXIP促进细胞周期蛋白上调，抑制细胞周期负调控因子p21、p27表达，从而 促进细胞增殖。HBXIP通过MDM2促进肿瘤抑制因子p53的降解，加速乳腺癌的生长。此外，HBXIP是晚期内体/溶酶体适配器以及MAPK和哺乳动物雷帕霉素靶蛋白LAMTOR复合物的成员之一，参与调控mTORC1的活化，影响细胞的合成代谢、翻译、转录、脂质合成、自噬等过程。因此，HBXIP是参与机体重要生命活动的调节因子。本发明提供了一种基于HBXIP条件敲除的小鼠早衰模型构建及相关应用。
发明内容
发明目的：本发明所要解决的技术问题是针对现有技术的不足，提供一种小鼠早衰模型。
本发明还要解决的技术问题是提供上述小鼠早衰模型的构建方法。
本发明最后要解决的技术问题是提供上述小鼠早衰模型的应用。
为了解决上述第一个技术问题，本发明公开了一种小鼠早衰模型，其特征在于，敲除小鼠骨髓来源细胞中的乙肝病毒X蛋白结合蛋白(HBXIP)。
其中，所述敲除为敲除乙肝病毒X蛋白结合蛋白编码基因中的第二个外显子。
其中，所述乙肝病毒X蛋白结合蛋白编码基因的mRNA序列为Gene Bank中的Accession:NM_026774.2，其基因组序列为Gene Bank中的Accession:NC_000069.7。
其中，所述敲除为通过Cre-loxp系统敲除；优选地，所述敲除为通过骨髓细胞特异性表达Cre重组酶小鼠LysM cre/+敲除。
为了解决上述第二个技术问题，本发明公开了上述小鼠早衰模型的构建方法，包括如下步骤：
(1)将HBXIP fl/+小鼠与HBXIP fl/+小鼠交配，获得HBXIP fl/fl纯合子小鼠；
(2)将HBXIP fl/fl小鼠与LysM cre/+小鼠交配，获得BXIP fl/+-LysM cre/+小鼠；
(3)将HBXIP fl/+-LysM cre/+小鼠与HBXIP fl/+-LysM cre/+小鼠交配，获得HBXIP fl/fl-LysM cre/+小鼠(HBXIP △LysM)。
步骤(1)中，所述HBXIP fl/+小鼠为将C57BL/6小鼠中乙肝病毒X蛋白结合蛋白基因组(NC_000069.7)的第二个外显子(1013-1074)两侧的内含子区域分别添加loxp1位点和loxp2位点，即在HBXIP基因组序列的第762位核苷酸后插入如SEQ ID No.1所示的核苷酸序列(loxp1位点)，第1250位核苷酸后插入如SEQ ID No.2所示的核苷酸序列(loxp2位点)。
其中，所述loxp序列的插入方法可以用多种已知的、被大家公认的方法获得。
为了解决上述第三个技术问题，本发明公开了上述小鼠早衰模型的应用。
其中，所述应用为所述小鼠早衰模型在开发或筛选预防和/或治疗早衰产品中的应用；其中，所述产品包括但不限于药物、食品、保健品、化妆品。
其中，所述应用为所述小鼠早衰模型在开发或筛选预防和/或治疗早衰技术方法中的应用。
其中，所述应用为所述小鼠早衰模型在早衰研究中的应用。
有益效果：与现有技术相比，本发明具有如下优势：
(1)本发明中，所述早衰小鼠在4-8个月龄时出现脱毛、衰老相关基因表达显著上调、血清中衰老相关的分泌表型因子上调、组织器官衰老等典型的早衰表型。
(2)对比自然衰老小鼠(18-24月龄)，本发明所提供的髓系细胞条件性敲除HBXIP小鼠具有饲养周期短(6-8月龄)、个体差异小、干扰因素少、成本低等优势。
(3)对比目前常见的三种小鼠衰老模型，本发明所提供的髓系细胞条件性敲除HBXIP小鼠早衰模型具有1)无需繁杂的造模过程，可稳定繁殖，自发衰老表型；2)对实验技术要求不高且易于控制，可避免造模过程中因操作等原因所导致的小鼠死亡；3)衰老状态易于检测和表征，可准确判断小鼠的衰老状态，模型可信度高；4)基因敲除背景小鼠个体差异小等优势；5)且髓系细胞条件性HBXIP敲除，是指HBXIP的敲除具有细胞特异性，仅在骨髓细胞系中发生，包括单核细胞、成熟的巨噬细胞和粒细胞等。
(4)本发明通过操控骨髓来源的免疫细胞条件性敲除HBXIP，诱导小鼠早衰具有显著的创新性和良好的应用前景，可用于建立延缓衰老的个性化药物/食材/技术方法等的筛选平台，同时，可用于研究衰老相关疾病，如神经退行性疾病、代谢性疾病、自身免疫及炎症性疾病等发病机制研究，以及相关疾病的预防、诊断和治疗等技术和产品研发。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明，本发明的上述和/或其他方面的优点将会变得更加清楚。
图1是HBXIP △LysM鼠的制备方法及基因型鉴定。
图2是HBXIP △LysM鼠BMDM中HBXIP敲除效果的转录水平验证。
图3是HBXIP △LysM鼠BMDM中HBXIP敲除效果的蛋白水平验证。
图4是野生型小鼠(WT)与HBXIP △LysM鼠BMDM中衰老相关基因的表达。
图5是野生型小鼠与HBXI P△LysM鼠血清中衰老相关的分泌表型因子水平。
图6是野生型小鼠与HBXIP △LysM鼠肝功能检测。
图7是野生型小鼠与HBXIP △LysM肾功能检测。
图8是野生型小鼠与HBXIP △LysM组织切片H&E染色。
具体实施方式
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
实施例1：髓系细胞条件性敲除HBXIP小鼠的制备方法及基因型鉴定。
1、实验材料
1.1实验用试剂：SYBR GREEN PCR Master Mix(QPK-201)购于日本TOYOBO公司；主要引物合成于上海捷瑞生物工程有限公司。抗体HBXIP(#14633)，β-Actin(#3700)，购于CST公司。蛋白酶K(10401ES60)购于翌圣生物科技股份有限公司。鼠尾裂解液(50mL的配方为：1M Tris-Hcl 0.5mL，0.5M EDTA 1mL，10％SDS 2.5mL，5M Nacl 1mL，H 2O 41ml，调PH至8.0)。Case9 mRNA购自于Sigma-Aldrich，货号为CAS9MRNA-1EA。
1.2实验用动物及饲养：野生型小鼠、髓系细胞特异性敲除Cre工具鼠(LysM cre/+)、HBXIP fl/+、HBXIP fl/fl、HBXIP △LysM鼠。全价营养颗粒饲料：由江苏省协同医药生物技术有限公司提供；饲养条件：室温20±2℃，湿度55-65％，明暗交替，光度适度，通风洁净良好。
2、实验方法
2.1髓系细胞条件性敲除HBXIP小鼠的制备方法。
(1)骨髓细胞特异性表达Cre重组酶小鼠(LysM cre/+)：英文名B6.129P2-Lyz2 tm1(cre)Ifo/J，Stock No:004781，购自The Jackson Laboratory。该品系小鼠基因组插入 Cre酶基因，并且该基因可以通过骨髓细胞特异性的启动子在骨髓细胞表达Cre重组酶。与loxp转基因小鼠交配，则Cre介导的重组能够将目的基因序列删除，这些序列删除具有细胞特异性，仅在骨髓细胞系中发生，包括单核细胞、成熟的巨噬细胞和粒细胞等。
(2)loxp转基因杂合子(HBXIP fl/+)小鼠的制备：小鼠品系为C57BL/6，在其乙肝病毒X蛋白结合蛋白基因组(NC_000069.7)的第二个外显子(1013-1074)两侧的内含子区域分别添加loxp1位点和loxp2位点，即在HBXIP基因组序列的第762位核苷酸后插入SEQ ID No.1核苷酸序列(loxp1位点)，第1250位核苷酸后插入SEQ ID No.2核苷酸序列(loxp2位点)，两个loxp位点的其核苷酸序列分别如SEQ ID No.1和SEQ ID No.2所示。
SEQ ID No.1 Loxp1插入位置及序列：
Figure PCTCN2021110471-appb-000001
SEQ ID No.2 Loxp2插入位置及序列：
Figure PCTCN2021110471-appb-000002
其中，上述loxp序列的插入方法如下：运用Optimized Cas9/CRISPR System(OCAS)技术，设计了两个gRNA序列，分别是gRNA靶点1:tagagcatgcttctataccaAGG(为基因组的正向序列，gRNA-1，核苷酸序列如SEQ ID No.3所示)，gRNA靶点2：GGAACAGTTGTAGAACTGGGAGG(为基因组的反向序列，gRNA-2，核苷酸序列如SEQ ID No.4所示)。通过显微注射，将Case9 mRNA、两个gRNA序列、SSODN同源重组模板(核苷酸序列别如SEQ ID No.5所示)导入小鼠受精卵中，利用DNA修复机制把带有2个loxp的完整序列插入到基因组中去。胚胎体外培养1-2小时后，移植入假孕母鼠的输卵管中，代孕鼠伤口缝合后待产。产仔后，进行F0代验证，小鼠杂交验证等。
(3)交配方法：第一步，将HBXIP fl/+与HBXIP fl/+个体交配，可以获得HBXIP fl/fl纯合子小鼠；第二步，HBXIP fl/fl与LysM cre/+个体交配，可以获得既含有loxp位点又表达Cre酶的个体，即(HBXIP fl/+-LysM cre/+)个体；第三步，(HBXIP fl/+-LysM cre/+)个体再与(HBXIP fl/+-LysM cre/+)个体交配，即可获得(HBXIP fl/fl-LysM cre/+)个体。研究已经证实，loxp位点与Cre酶结合，就会发生基因删除(Clausen et al.，1999)。因此，(HBXIP fl/+-LysM cre/+)个体为骨髓细胞特异性敲除HBXIP基因的杂合子， (HBXIP fl/fl-LysM cre/+)个体则为骨髓细胞特异性敲除HBXIP基因的纯合子(简写为HBXIP △LysM)。
2.2小鼠基因型鉴定。从HBXIP △LysM小鼠剪取0.5cm左右的尾巴，加入鼠尾裂解液和蛋白酶K(500μl，体积比为100:1)，55℃摇动消化过夜。消化完全后，5000r，离心3min，取200μl上清置于干净EP管中。在上清中加入400μl无水乙醇，上下颠倒混匀，此时可见絮状DNA沉淀。12000r离心5min，弃去上清。在沉淀中加入400μl 70％乙醇，混匀，12000r离心5min，弃去上清。放在超净台晾干，加入200μl ddH 2O溶解备用。PCR法检测相关基因的表达。
loxp小鼠鼠尾鉴定的引物如下：
F:CACTTTCACGCACATCTTCCCGC
R:CAGGCACCTCTGCTTCACAAAGACAC
HBXIP fl/+＝296bp和348bp
HBXIP fl/fl＝348bp
WT＝296bp
Cre小鼠鼠尾鉴定的引物如下：
F:CTTGGGCTGCCAGAATTTCTC
R1:CCAGAAATGCCAGATTACG
R2:TTACAGTCGGCCAGGCTGAC
Mutant＝750bp
Heterozygote＝300bp和750bp
WT＝300bp
2.3小鼠BMDMs的提取、分离与培养。取野生型小鼠C57BL/6和HBXIP △LysM小鼠的腿骨，PBS冲出骨髓，裂解红细胞，DMEM培养基(10vt％FBS(胎牛血清)，小鼠M-CSF(巨噬细胞集落刺激因子)25ng/ml)培养3天，待多数细胞贴壁，更换新鲜培养基，再培养3天，约6天左右BMDMs分化为M0型。
2.4实时定量PCR法验证HBXIP △LysM小鼠BMDM中HBXIP敲除效果。提取小鼠BMDM的总RNA并进行反转录，制备成cDNA。使用SYBR Green PCR Master Mix进行实时定量PCR检测，β-actin为内参，用2 -△△Ct法计算mRNA的相对表达水平。
2.5蛋白免疫印迹法验证HBXIP △LysM小鼠BMDM中HBXIP敲除效果。裂解小鼠 BMDM，BCA定量试剂盒测定蛋白浓度；按40μg的总蛋白上样量，依次进行电泳、转膜、封闭、一抗孵育、二抗孵育、显色、曝光。
3、实验结果
3.1 HBXIP △LysM小鼠鼠尾基因型鉴定结果。通过2.1所述的繁育方式(图1左)，得几种不同基因型的小鼠。根据loxp与Cre重组酶的敲除原理，经鼠尾鉴定，只要在基因组DNA中检测loxp位点和Cre重组酶基因的有无就可判断幼鼠的基因型。如实施例1的2.1和图1右所示，若用loxp引物进行扩增：HBXIP fl/+＝296bp和348bp；HBXIP fl/fl＝348bp；WT＝296bp。若用Cre引物进行扩增：Mutant＝750bp；Heterozygote＝300bp和750bp；WT＝300bp。若用loxp小鼠鼠尾鉴定的引物扩增出348bp条带，同时用Cre小鼠鼠尾鉴定的引物扩增出750bp条带的小鼠则为HBXIP △LysM小鼠。
3.2 HBXIP △LysM小鼠BMDM中HBXIP敲除效果验证。收取BMDMs细胞，分别进行RNA提取和蛋白提取。结果显示，与野生型小鼠相比，HBXIP △LysM小鼠BMDM中不表达HBXIP的蛋白(图2)，其转录水平也显著下调(图3)，证明BMDM中HBXIP的敲除效果显著。
实施例2：髓系细胞条件性敲除HBXIP小鼠早衰模型的表型验证。
1、实验材料
1.1实验用试剂：实时定量PCR所需试剂同实施例1中的1.1所述。ELISA检测试剂盒购于联科生物，谷丙转氨酶活性检测试剂盒(BC1555)、谷草转氨酶活性检测试剂盒(BC1565)、Bradford蛋白浓度定量试剂盒购(PC0010)自北京索莱宝科技有限公司。
1.2实验用动物及饲养：野生型小鼠与HBXIP △LysM鼠，7-8月龄。全价营养颗粒饲料：由江苏省协同医药生物技术有限公司提供；饲养条件：室温20±2℃，湿度55-65％，明暗交替，光度适度，通风洁净良好。
2、实验方法
2.1实时定量PCR法检测野生型小鼠与HBXIP △LysM鼠BMDM中衰老相关基因的表达。实时定量PCR的检测方法同实施例1中2.2。
2.2 ELISA法检测野生型小鼠与HBXIP △LysM鼠血清中衰老相关的分泌表型因子水平。根据细胞因子检测试剂盒的说明书，用ELISA的实验方法，测定小鼠血清中衰老 相关的分泌表型因子水平。依次进行浸泡酶标板、加标准品、加样本、孵育、洗涤、加酶孵育、洗涤、加底物显色、加终止液、检测读数。检测读数应在30分钟之内，使用酶标仪进行双波长检测，测定450nm最大吸收波长和570nm或630nm参考波长下的OD值。校准后的OD值为450nm的测定值减去570nm或630nm的测定值。
2.3小鼠血清中谷丙、谷草转氨酶的活性检测。谷丙转氨酶(ALT)活性检测：首先将丙氨酸标准品用蒸馏水稀释至2μmol/mL，将标准品稀释为1.5,1,0.8,0.6,0.4,0.2,0.1,0.05,0mM等浓度梯度。在标准管、测定管、对照管中分别加入一定体积的丙氨酸(200mM)，α-酮戊二酸溶液(2mM)和2,4-二硝基苯肼溶液。按血清体积计算，ALT活力单位：每小时每mL血清样本催化产生1μmol丙酮酸的量为一个活力单位。谷草转氨酶(AST)活性检测：首先将丙酮酸钠标准品用蒸馏水稀释至2μmol/mL，将标准品稀释为1.5,1,0.8,0.6,0.4,0.2,0.1,0.05,0mM等浓度梯度。在标准管、测定管、对照管中分别加入一定体积的化α-酮戊二酸(2mM)、天门冬氨酸(200mM)和2,4-二硝基苯肼溶液。按血清体积计算，AST活力单位：每小时每mL血清样本催化产生1μmol丙酮酸的量为一个活力单位。
2.4 Bradford法检测小鼠尿蛋白浓度。完全溶解蛋白标准品，使终浓度为0.2mg/ml。将标准品按0,2,4,6,8,12,16,20微升分别加到96孔板中，加PBS稀释液补足到20微升。将小鼠尿液样品作适当稀释(2倍、4倍、8倍稀释)，加20微升到96孔板的样品孔中。各孔加入200微升稀释后的1×G250考马斯亮蓝染色液，室温放置3-5分钟。用酶标仪测定A595。根据标准曲线计算出样品中的蛋白浓度。
3、实验结果
3.1野生型小鼠与HBXIP △LysM鼠BMDM中衰老相关基因的表达。提取小鼠BMDMs(8个月龄)中的RNA，并进行发转录。实时定量方法检测野生型小鼠与HBXIP △LysM鼠BMDM中衰老相关基因的表达。结果显示，HBXIP △LysM鼠BMDM中衰老相关基因p16，p21，MCP-1，IL-6，TNF-α，IL-1β的表达水平显著升高(图4)。
3.2野生型小鼠与HBXI P△LysM鼠血清中衰老相关的分泌表型因子水平。收集野生型小鼠与HBXI P△LysM鼠(8个月龄)的血清，ELISA法检测血清中衰老相关的分泌表型因子水平。结果显示，HBXIP △LysM鼠血清中衰老相关的分泌表型因子水平IL-6，TNF-α，IL-1β显著升高(图5)。
3.3野生型小鼠与HBXIP △LysM鼠肝、肾功能检测。收集野生型小鼠与HBXIP △LysM鼠 (8个月龄)的血清，分别用谷丙转氨酶(ALT)活性和谷草转氨酶(AST)活性检测试剂盒检测小鼠肝脏功能。结果显示，HBXIP △LysM鼠血清中谷草转氨酶活性显著高于野生型小鼠，其谷丙转氨酶活性与野生型小鼠相比，无显著性差异(图6)。收集野生型小鼠与HBXIP △LysM鼠的尿液，Bradford法检测尿液中的蛋白浓度。结果显示，HBXIP △LysM鼠尿液中的蛋白浓度显著高于野生型小鼠(图7)。另外，对野生型小鼠与HBXIP △LysM鼠的皮肤、肺、肝脏、肾脏的组织切片进行H&E染色，结果显示HBXIP △LysM鼠毛囊萎缩，肺部有大量炎性细胞浸润，肝肾组织结构改变等(图8)。以上实验结果表明，HBXIP △LysM鼠出现了衰老小鼠的症状，即肝肾功能损伤。
本发明提供了一种小鼠早衰模型及其构建方法与应用的思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和拓展其应用，这些改进和应用也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。

Claims (10)

  1. 一种小鼠早衰模型，其特征在于，敲除小鼠骨髓来源细胞中的乙肝病毒X蛋白结合蛋白。
  2. 根据权利要求1所述小鼠早衰模型，其特征在于，所述敲除为敲除乙肝病毒X蛋白结合蛋白编码基因中的第二个外显子。
  3. 根据权利要求2所述小鼠早衰模型，其特征在于，所述乙肝病毒X蛋白结合蛋白编码基因的mRNA序列为Gene Bank中的Accession:NM_026774.2，其基因组序列为Gene Bank中的Accession:NC_000069.7。
  4. 根据权利要求1所述小鼠早衰模型，其特征在于，所述敲除为通过Cre-loxp系统敲除。
  5. 根据权利要求1所述小鼠早衰模型，其特征在于，所述敲除为通过骨髓细胞特异性表达Cre重组酶小鼠LysM cre/+敲除。
  6. 权利要求1-5中任意一项所述小鼠早衰模型的构建方法，其特征在于，包括如下步骤：
    (1)将HBXIP fl/+小鼠与HBXIP fl/+小鼠交配，获得HBXIP fl/fl纯合子小鼠；
    (2)将HBXIP fl/fl小鼠与LysM cre/+小鼠交配，获得BXIP fl/+-LysM cre/+小鼠；
    (3)将HBXIP fl/+-LysM cre/+小鼠与HBXIP fl/+-LysM cre/+小鼠交配，获得HBXIP fl/fl-LysM cre/+小鼠。
  7. 根据权利要求6所述方法，其特征在于，步骤(1)中，所述HBXIP fl/+小鼠为将C57BL/6小鼠中乙肝病毒X蛋白结合蛋白基因组序列的第762位核苷酸后插入如SEQ ID No.1所示的核苷酸序列，第1250位核苷酸后插入如SEQ ID No.2所示的核苷酸序列。
  8. 权利要求1-5中任意一项所述小鼠早衰模型在开发或筛选预防和/或治疗早衰产品中的应用。
  9. 权利要求1-5中任意一项所述小鼠早衰模型在开发或筛选预防和/或治疗早衰技术方法中的应用。
  10. 权利要求1-5中任意一项所述小鼠早衰模型在早衰研究中的应用。
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