CN114525244A - Mechanism research method of pyroptosis product of mesenchymal stem cells in periodontal dental pulp combined diseased tissue in occurrence and development of diseases - Google Patents
Mechanism research method of pyroptosis product of mesenchymal stem cells in periodontal dental pulp combined diseased tissue in occurrence and development of diseasesDownload PDFInfo
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明涉及生物技术领域，具体为一种牙周牙髓联合病变组织中间充质干细胞的焦亡产物在疾病发生发展中的机制研究方法。The invention relates to the field of biotechnology, in particular to a method for studying the mechanism of the pyroptosis products of mesenchymal stem cells in periodontal pulp combined with diseased tissue in the occurrence and development of diseases.
背景技术Background technique
牙周炎和牙髓炎是口腔科常见病,牙周组织和牙髓组织在解剖上相通,可相互感染。原发的牙髓病变可通过这些解剖因素波及牙周组织，而牙周组织的病变也可能影响牙髓。这也就是牙周牙髓联合病变的解剖学基础。因此一旦牙周或牙髓发生联合病变，容易相互扩散和影响，往往病变破坏范围较大、病程较长且迁延不愈，治疗难度大、周期长、费用高且预后不确切，容易导致牙齿缺失的结局，是临床上一个较为棘手的问题。Periodontitis and pulpitis are common diseases in stomatology, periodontal tissue and pulp tissue are anatomically connected and can infect each other. The primary pulp lesions can spread to the periodontal tissue through these anatomical factors, and the lesions of the periodontal tissue may also affect the pulp. This is also the anatomical basis of periodontal and endodontic lesions. Therefore, once a periodontal or pulpal joint lesion occurs, it is easy to spread and influence each other, and the lesions often have a large range of damage, a long course of disease, and prolonged unhealing. The outcome is a more difficult clinical problem.
细胞的程序性死亡分为细胞凋亡、细胞焦亡、程序性细胞坏死、铁死亡等等，细胞焦亡(Pyroptosis)通常是病原体感染时可能引发的细胞死亡的主要模式，其特征在于死亡细胞的破裂和由此产生的有效炎症诱导剂的释放，在细胞受感染的情况下还包括病原体及其不同成分，已有研究表明牙周炎的发生发展过程中伴随着焦亡的产生，此种效应导致了牙周膜干细胞的丧失、促进破骨细胞形成，加重了炎症程度；不仅如此，细胞焦亡还导致了根尖周炎的发生，并且焦亡的严重程度与根尖周炎的病情严重程度呈正相关。牙周牙髓联合病变作为口腔中较为严重的一种炎性疾病，其发生发展机制与焦亡及焦亡产物的相关性尚无报道。Programmed cell death is divided into apoptosis, pyroptosis, programmed cell necrosis, ferroptosis, etc. Pyroptosis is usually the main mode of cell death that may be caused by pathogen infection, which is characterized by dead cells The rupture and the resulting release of potent inflammatory inducers, including pathogens and their different components in the case of cell infection, have been shown to be accompanied by pyroptosis in the development and progression of periodontitis. The effect leads to the loss of periodontal ligament stem cells, promotes the formation of osteoclasts, and aggravates the degree of inflammation; not only that, cell pyroptosis also leads to the occurrence of apical periodontitis, and the severity of pyroptosis is related to the condition of apical periodontitis. Severity was positively correlated. The periodontal-pulp joint disease is a serious inflammatory disease in the oral cavity, and there is no report on the relationship between its occurrence and development mechanism and pyroptosis and pyroptosis products.
细胞外囊泡是一组异质的细胞衍生膜结构，包括外泌体、微囊泡、凋亡小体等，存在于生物体液中并参与多种生理和病理过程。细胞外囊泡现在被认为是细胞间通讯的额外机制，允许细胞交换蛋白质、脂质和遗传物质。它介导的是一种远程的、长效的、低免疫原性的细胞间通讯新方式5，在细胞间通讯、疾病进展或作为生物标志物方面的作用引起了人们的极大兴趣。本课题组先前的研究发现细胞在焦亡的过程中会释放一些具有免疫调节功能的EVs，我们暂且称之为焦亡囊泡(Pyrop-EVs)，并且已经成功在体外诱导牙周牙髓联合病变组织中的中间充质干细胞(MSCs)发生焦亡。Extracellular vesicles are a heterogeneous group of cell-derived membrane structures, including exosomes, microvesicles, apoptotic bodies, etc., that exist in biological fluids and participate in a variety of physiological and pathological processes. Extracellular vesicles are now recognized as additional mechanisms for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. It mediates a new mode of long-range, long-lasting, and low immunogenicity of cell-to-cell communication5, and its role in cell-to-cell communication, disease progression, or as a biomarker has aroused great interest. Previous research by our group found that cells release some EVs with immunomodulatory functions during the process of pyroptosis, which we call Pyrop-EVs for the time being, and have successfully induced periodontal endodontic association in vitro Mesenchymal stem cells (MSCs) in diseased tissues undergo pyroptosis.
MSCs的免疫调节特性与炎症的状态有关，在轻微炎症中，MSCs发挥抗炎作用，有助于炎症的清除，而在严重的疾病环境中，MSCs也许发挥促炎作用，组织位置和炎性状态是MSCs免疫调节特性的关键决定因素。目前许多类型的人牙组织来源MSCs已被分离和表征，包括牙髓干细胞、脱落乳牙干细胞、牙周膜干细胞、牙囊祖细胞、牙槽骨来源的间充质干细胞、来自牙乳头的干细胞、牙胚祖细胞和牙龈间充质干细胞12。发现在牙周牙髓联合病变的肉芽组织中也存在MSCs，其形态与增殖分化能力都与正常的牙周膜干细胞无明显差异。The immunomodulatory properties of MSCs are related to the state of inflammation. In mild inflammation, MSCs play an anti-inflammatory role and contribute to the clearance of inflammation, while in severe disease settings, MSCs may play a pro-inflammatory role, tissue location and inflammatory state. is a key determinant of the immunomodulatory properties of MSCs. Many types of human dental tissue-derived MSCs have been isolated and characterized, including dental pulp stem cells, exfoliated deciduous tooth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, alveolar bone-derived mesenchymal stem cells, stem cells from dental papilla, Tooth germ progenitor cells and gingival mesenchymal stem cells 12. It was found that MSCs also existed in the granulation tissue of periodontal pulp combined lesions, and their morphology and proliferation and differentiation ability were not significantly different from those of normal periodontal ligament stem cells.
根据目前的研究，猜想在牙周牙髓联合病变这样一个炎性环境中，往往伴随着MSCs免疫调节特性的改变，目前从牙周牙髓联合病变的组织中已经成功分离出MSCs，分析此种炎性环境下MSCs的焦亡产物特性与探究牙周牙髓联合病变的致病机制密切相关。According to the current research, it is speculated that in an inflammatory environment such as periodontal and pulpal combined lesions, it is often accompanied by changes in the immunoregulatory properties of MSCs. At present, MSCs have been successfully isolated from the tissue of periodontal and pulpal combined lesions. The characteristics of pyroptosis products of MSCs in inflammatory environment are closely related to the exploration of the pathogenic mechanism of periodontal and pulpal combined lesions.
因此应当认为，Pyrop-EVs在牙周牙髓联合病变的炎性微环境下对于颌骨骨髓间充质干细胞的免疫调节功能及成骨分化能力具有影响，研究牙周牙髓联合病变组织的中间充质干细胞焦亡囊泡的是否在牙周牙髓联合病变疾病进展中发挥重要作用及其具体机制是急需解决的科学问题。Therefore, it should be considered that Pyrop-EVs have an impact on the immune regulation function and osteogenic differentiation ability of jaw bone marrow-derived mesenchymal stem cells in the inflammatory microenvironment of periodontal pulp combined lesions. Whether mesenchymal stem cell pyroptotic vesicles play an important role in the progression of periodontal and pulpal combined diseases and its specific mechanism are urgent scientific questions to be solved.
发明内容SUMMARY OF THE INVENTION
为解决背景技术提出的上述问题，本发明提出一种牙周牙髓联合病变组织中间充质干细胞的焦亡产物在疾病发生发展中的机制研究方法。In order to solve the above problems raised by the background art, the present invention proposes a mechanism research method of the pyroptotic products of mesenchymal stem cells in periodontal pulp combined with diseased tissue in the occurrence and development of diseases.
为实现上述目的，本发明提供如下技术方案：一种牙周牙髓联合病变组织中间充质干细胞的焦亡产物在疾病发生发展中的机制研究方法，所述方法包括以下步骤：(1)分离、培养人牙周牙髓联合病变肉芽组织干细胞，利用焦亡诱导液诱导其焦亡，并提取其焦亡囊泡(Pyrop-EVs)悬液；(2)分离、培养人颌骨骨髓干细胞，将焦亡囊泡(Pyrop-EVs)以不同浓度梯度注射至人颌骨骨髓干细胞中共同培养，并检测其细胞的变化；(3)诱导小鼠溃疡性结肠炎后，将小鼠分为实验组和对照组，给实验组的小鼠注射不同浓度梯度的焦亡囊泡(Pyrop-EVs)悬液，给对照组的小鼠注射PBS缓冲液，并检测实验组和对照组小鼠的各项生理指标。In order to achieve the above object, the present invention provides the following technical solutions: a method for studying the mechanism of the pyroptosis product of mesenchymal stem cells in periodontal pulp combined with diseased tissue in the occurrence and development of disease, the method comprises the following steps: (1) separating , culture human periodontal pulp combined with diseased granulation tissue stem cells, use pyroptosis-inducing solution to induce their pyroptosis, and extract the pyrop-EVs suspension; (2) isolate and culture human jaw bone marrow stem cells, The pyroptotic vesicles (Pyrop-EVs) were injected into human jaw bone marrow stem cells with different concentration gradients for co-culture, and the cell changes were detected; (3) After inducing ulcerative colitis in mice, the mice were divided into experimental groups. group and control group, the mice in the experimental group were injected with different concentration gradients of pyrop-EVs suspension, and the mice in the control group were injected with PBS buffer, and the experimental group and the control group were tested for different levels of Pyrop-EVs. Physiological indicators.
优选的，所述步骤1中分离、培养人牙周牙髓联合病变肉芽组织干细胞的方法为：选取根据临床体格检查及影像学资料，诊断为牙周牙髓联合病变，且无系统性疾病，身体状况良好的患者，拔除其患牙上附带的肉芽组织，剁碎、消化、培养、传代、冻存后使用。Preferably, in the step 1, the method for separating and culturing human periodontal pulp combined diseased granulation tissue stem cells is as follows: according to clinical physical examination and imaging data, a diagnosis of periodontal pulp combined lesion, and no systemic disease, is selected. For patients in good physical condition, the granulation tissue attached to the teeth was extracted, minced, digested, cultured, passaged, and frozen for use.
优选的，所述步骤1中取P3代长势良好的人牙周牙髓联合病变肉芽组织干细胞，加入焦亡诱导液诱导其焦亡后，并鉴定细胞确实发生焦亡，利用差速离心的方法，分离焦亡囊泡。Preferably, in the step 1, the P3 generation of human periodontal pulp combined with diseased granulation tissue stem cells with good growth is taken, and pyroptosis-inducing solution is added to induce their pyroptosis, and it is identified that the cells have indeed undergone pyroptosis, and the method of differential centrifugation is used. , to isolate pyroptotic vesicles.
优选的，所述步骤2中分离、培养人颌骨骨髓干细胞的方法为：选取临床因正颌手术或牙槽突修整手术中废弃的健康颌骨，将其清洗、剪碎、离心、培养、传代、冻存后使用。Preferably, the method for separating and culturing human jaw bone marrow stem cells in the step 2 is as follows: selecting healthy jaws discarded in clinical orthognathic surgery or alveolar process surgery, washing, shredding, centrifuging, culturing, Use after passage and cryopreservation.
优选的，所述步骤2中取长势良好的人颌骨骨髓干细胞分为两组，根据浓度梯度加入焦亡囊泡(Pyrop-EVs)悬液诱导24小时后；一组人颌骨骨髓干细胞，收集其上清液，提取上清液中的细胞蛋白后，进行ELISA(酶联免疫吸附实验)和WB(免疫印迹)检测；另一组人颌骨骨髓干细胞，用成骨诱导液及成脂诱导培养后，进行茜素红染色、油红O染色。Preferably, in the step 2, the well-growing human jaw bone marrow stem cells are divided into two groups, and the pyroptotic vesicle (Pyrop-EVs) suspension is added according to the concentration gradient to induce 24 hours; one group of human jaw bone marrow stem cells, The supernatant was collected, and the cell proteins in the supernatant were extracted, and then ELISA (enzyme-linked immunosorbent assay) and WB (immunoblotting) were performed; another group of human jaw bone marrow stem cells were treated with osteogenic induction solution and adipogenic After the induction culture, Alizarin Red staining and Oil Red O staining were performed.
优选的，所述步骤2中ELISA(酶联免疫吸附实验)和WB(免疫印迹)检测时，检测细胞因子IL-6或RUNX2或ALP中的一种或多种。Preferably, one or more of cytokines IL-6, RUNX2 or ALP are detected during ELISA (enzyme-linked immunosorbent assay) and WB (immunoblotting) detection in the step 2.
优选的，所述步骤3中通过含有2.5％dss的无菌水喂养小鼠，诱导其患溃疡性结肠炎。Preferably, in the step 3, the mice are induced to suffer from ulcerative colitis by feeding the mice with sterile water containing 2.5% dss.
优选的，所述步骤3中检测的实验组和对照组小鼠的生理指标包括：疾病活动指数、组织学损伤指数、免疫指标的变化。Preferably, the physiological indicators of the mice in the experimental group and the control group detected in the step 3 include: disease activity index, histological damage index, and changes in immune indicators.
优选的，所述步骤3中检测实验组和对照组小鼠的生理指标时，操作方法为：在疾病进展最严重的小鼠体重下降约30％左右时，同时处死所有小鼠，收集小鼠血清、结肠、脾脏、肠系膜淋巴结；检测血清里炎症相关细胞因子；结肠长度测量、结肠HE染色检测；脾脏研磨成单细胞悬液后，用流式细胞术检测淋巴细胞；肠系膜淋巴结用流式细胞术观察淋巴细胞的变化。Preferably, when the physiological indicators of the mice in the experimental group and the control group are detected in the step 3, the operation method is as follows: when the weight of the mice with the most severe disease progression drops by about 30%, all the mice are killed at the same time, and the mice are collected. Serum, colon, spleen, and mesenteric lymph nodes; detection of inflammation-related cytokines in serum; colon length measurement, colon HE staining detection; spleen grinded into single cell suspension, and lymphocytes were detected by flow cytometry; mesenteric lymph nodes were detected by flow cytometry The changes of lymphocytes were observed by surgery.
优选的，所述步骤1中诱导人牙周牙髓联合病变肉芽组织干细胞焦亡的焦亡诱导液由α-MEM、LPS、nigericin配制而成。Preferably, in the step 1, the pyroptosis-inducing solution for inducing pyroptosis of human periodontal pulp combined with diseased granulation tissue stem cells is prepared from α-MEM, LPS, and nigericin.
与现有技术相比，本发明的有益效果如下：Compared with the prior art, the beneficial effects of the present invention are as follows:
本发明提供的牙周牙髓联合病变组织中间充质干细胞的焦亡产物在疾病发生发展中的机制研究方法，探究牙周牙髓联合病变组织中间充质干细胞焦亡囊泡的免疫调节功能影响及介导骨吸收机制，为寻找牙周牙髓联合病变的具体发生发展机制奠定基础，将来为发现口腔炎性病变致病因素提供新的见解，同时该方法整体步骤简单，易于操作。The present invention provides a method for studying the mechanism of the pyroptotic products of periodontal pulp combined with diseased tissue mesenchymal stem cells in the occurrence and development of diseases, and explores the impact of the immune regulation function of periodontal pulp combined with diseased tissue mesenchymal stem cell pyroptosis vesicles And the mechanism of mediating bone resorption, laying a foundation for finding the specific occurrence and development mechanism of periodontal-endodontic lesions, and providing new insights for the discovery of the pathogenic factors of oral inflammatory lesions in the future. At the same time, the overall steps of this method are simple and easy to operate.
附图说明Description of drawings
图1为本发明方法步骤1和步骤2的流程图；Fig. 1 is the flow chart of step 1 and step 2 of the method of the present invention;
图2为本发明步骤3的流程图。FIG. 2 is a flowchart of step 3 of the present invention.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
如图1-2所示，本发明提出了一种牙周牙髓联合病变组织中间充质干细胞的焦亡产物在疾病发生发展中的机制研究方法，所述方法包括以下步骤：(1)分离、培养人牙周牙髓联合病变肉芽组织干细胞，利用焦亡诱导液诱导其焦亡，并提取其焦亡囊泡(Pyrop-EVs)悬液；(2)分离、培养人颌骨骨髓干细胞，将焦亡囊泡(Pyrop-EVs)以不同浓度梯度注射至人颌骨骨髓干细胞中共同培养，并检测其细胞的变化；(3)诱导小鼠溃疡性结肠炎后，将小鼠分为实验组和对照组，给实验组的小鼠注射不同浓度梯度的焦亡囊泡(Pyrop-EVs)悬液，给对照组的小鼠注射PBS缓冲液，并检测实验组和对照组小鼠的各项生理指标。As shown in Figures 1-2, the present invention proposes a method for studying the mechanism of the pyroptosis product of mesenchymal stem cells in periodontal pulp combined with diseased tissue in the occurrence and development of the disease. The method includes the following steps: (1) Separation , culture human periodontal pulp combined with diseased granulation tissue stem cells, use pyroptosis-inducing solution to induce their pyroptosis, and extract the pyrop-EVs suspension; (2) isolate and culture human jaw bone marrow stem cells, The pyroptotic vesicles (Pyrop-EVs) were injected into human jaw bone marrow stem cells with different concentration gradients for co-culture, and the cell changes were detected; (3) After inducing ulcerative colitis in mice, the mice were divided into experimental groups. group and control group, the mice in the experimental group were injected with different concentration gradients of pyrop-EVs suspension, and the mice in the control group were injected with PBS buffer, and the experimental group and the control group were tested for different levels of Pyrop-EVs. Physiological indicators.
所述步骤1中分离、培养人牙周牙髓联合病变肉芽组织干细胞的方法为：选取根据临床体格检查及影像学资料，诊断为牙周牙髓联合病变，且无系统性疾病，身体状况良好的患者，拔除其患牙上附带的肉芽组织，剁碎、消化、培养、传代、冻存后使用。The method for separating and culturing human periodontal pulp combined diseased granulation tissue stem cells in the step 1 is as follows: according to clinical physical examination and imaging data, the diagnosis is periodontal pulp combined lesion, and there is no systemic disease, and the physical condition is good. The granulation tissue attached to the teeth was extracted, minced, digested, cultured, passaged, and cryopreserved before use.
所述步骤1中取P3代长势良好的人牙周牙髓联合病变肉芽组织干细胞，加入焦亡诱导液诱导其焦亡后，利用差速离心的方法，分离焦亡囊泡。In the step 1, the well-grown human periodontal pulp combined with diseased granulation tissue stem cells of the P3 generation was taken, and pyroptotic vesicles were separated by differential centrifugation after adding pyroptosis-inducing solution to induce their pyroptosis.
需要说明的是，利用差速离心的方法，分离焦亡囊泡的方法为：首先第一次差速离心19小时后，弃第一次差速离心的上清液，对第一次离心后的沉淀继续加入焦亡诱导液，反应24小时后，进行第二次差速离心10分钟，将第二次差速离心的上清液继续进行第三次差速离心10分钟，将第三次差速离心的上清液继续进行第四次差速离心30分钟，弃第四次差速离心的上清液，将第四次差速离心的沉淀，进行第五次差速离心30分钟，弃第五次差速离心的上清液，第五次差速离心的沉淀即为分离出的焦亡囊泡。It should be noted that the method of using differential centrifugation to separate pyroptotic vesicles is as follows: first, after the first differential centrifugation for 19 hours, discard the supernatant of the first differential centrifugation, and then discard the supernatant of the first differential centrifugation. Continue to add the pyroptosis induction solution to the precipitation of the precipitation, and after 24 hours of reaction, carry out the second differential centrifugation for 10 minutes, and continue the third differential centrifugation for the supernatant of the second differential centrifugation for 10 minutes. The supernatant of the differential centrifugation was further subjected to the fourth differential centrifugation for 30 minutes, the supernatant of the fourth differential centrifugation was discarded, and the precipitate of the fourth differential centrifugation was subjected to the fifth differential centrifugation for 30 minutes. The supernatant of the fifth differential centrifugation was discarded, and the precipitate of the fifth differential centrifugation was the separated pyroptotic vesicles.
需要说明的是，分离、培养人牙周牙髓联合病变肉芽组织干细胞，利用焦亡诱导液诱导其焦亡后，需要做焦亡鉴定，具体包括如下步骤：It should be noted that, after isolating and culturing human periodontal pulp combined with diseased granulation tissue stem cells, and inducing their pyroptosis with pyroptosis-inducing solution, pyroptosis identification is required, which specifically includes the following steps:
1.取第一次差速离心后的上清液做LDH检测；2.取第一次差速离心后的沉淀做western blot:GSDMD,ASC,caspase 1；3.ELISA:IL-1β,IL-18。根据焦亡鉴定的结果可调整差速离心的实验参数。1. Take the supernatant after the first differential centrifugation for LDH detection; 2. Take the precipitate after the first differential centrifugation for western blot: GSDMD, ASC, caspase 1; 3. ELISA: IL-1β, IL -18. The experimental parameters of differential centrifugation can be adjusted according to the results of pyroptosis identification.
需要进一步说明的是，所述步骤1中诱导人牙周牙髓联合病变肉芽组织干细胞焦亡的药物由α-MEM、LPS、nigericin配制而成。It should be further noted that the drug for inducing pyroptosis of human periodontal pulp combined with diseased granulation tissue stem cells in the step 1 is prepared from α-MEM, LPS and nigericin.
所述步骤2中分离、培养人颌骨骨髓干细胞的方法为：选取临床因正颌手术或牙槽突修整手术中废弃的健康颌骨，将其清洗、剪碎、离心、培养、传代、冻存后使用。The method of separating and culturing human jaw bone marrow stem cells in the step 2 is as follows: selecting healthy jaws discarded in clinical orthognathic surgery or alveolar process trimming surgery, washing, shredding, centrifuging, culturing, passaged, freezing. Use after saving.
所述步骤2中取长势良好的人颌骨骨髓干细胞分为两组，根据浓度梯度加入焦亡囊泡(Pyrop-EVs)悬液诱导24小时后；一组人颌骨骨髓干细胞，收集其上清液，提取上清液中的细胞蛋白后，进行ELISA(酶联免疫吸附实验)和WB(免疫印迹)检测；另一组人颌骨骨髓干细胞，用成骨诱导液及成脂诱导培养后，进行茜素红染色、油红O染色。需要说明的是，所述步骤2中ELISA(酶联免疫吸附实验)和WB(免疫印迹)检测时，检测细胞因子IL-6或RUNX2或ALP中的一种或多种。In the step 2, the well-growing human jaw bone marrow stem cells were divided into two groups, and after induction for 24 hours by adding pyroptotic vesicles (Pyrop-EVs) suspension according to the concentration gradient; one group of human jaw bone marrow stem cells was collected. The supernatant, after extracting the cell proteins in the supernatant, was subjected to ELISA (enzyme-linked immunosorbent assay) and WB (immunoblotting) detection; another group of human jaw bone marrow stem cells were cultured with osteogenic induction solution and adipogenic induction , Alizarin red staining and Oil red O staining were performed. It should be noted that, in the ELISA (enzyme-linked immunosorbent assay) and WB (immunoblotting) detection in the step 2, one or more of the cytokines IL-6, RUNX2 or ALP are detected.
所述步骤3中通过含有2.5％dss的无菌水喂养小鼠，诱导其患溃疡性结肠炎。In the step 3, the mice were fed with sterile water containing 2.5% dss to induce ulcerative colitis.
具体地，溃疡性结肠炎模型表现：结肠充血、水肿、变短、变脆、重量长度比增加，出现不同程度的结肠溃疡，黏膜水肿、杯状细胞缺失、隐窝肿胀破坏，黏膜和黏膜下层出现不同程度的炎症细胞浸润，上皮细胞损伤。Specifically, the ulcerative colitis model showed: colon hyperemia, edema, shortening, brittleness, increased weight-to-length ratio, different degrees of colon ulceration, mucosal edema, goblet cell loss, crypt swelling and destruction, mucosal and submucosa Different degrees of inflammatory cell infiltration and epithelial cell damage occurred.
所述步骤3中检测的实验组和对照组小鼠的各项生理指标包括疾病活动指数、组织学损伤指数、免疫指标的变化。The physiological indicators of the mice in the experimental group and the control group detected in the step 3 include changes in disease activity index, histological damage index, and immune index.
需要说明的是，所述疾病活动指数(DAI)的评估包括体重、大便性状和血便；所述组织学损伤指数(HI)的评估：包括观察结肠内有无出血，溃疡等，同时取一小块组织常规石蜡包埋、切片、HE染色，观察组织学改变并进行评分；所述免疫指标的变化：包括血清及结肠部位炎症细胞因子，如IFN-γ的变化情况。It should be noted that the evaluation of the disease activity index (DAI) includes body weight, stool characteristics and bloody stool; the evaluation of the histological injury index (HI) includes observing whether there is bleeding, ulcers, etc. in the colon, and taking a small The block tissue was routinely embedded in paraffin, sectioned, and stained with HE. Histological changes were observed and scored; the changes of the immune indexes included changes in serum and inflammatory cytokines in the colon, such as IFN-γ.
具体地，所述步骤3中检测实验组和对照组小鼠的生理指标时，操作方法为：在疾病进展最严重的小鼠体重下降约30％左右时，同时处死所有小鼠，收集小鼠血清、结肠、脾脏、肠系膜淋巴结；检测血清里炎症相关细胞因子；结肠长度测量、结肠HE染色检测；脾脏研磨成单细胞悬液后，用流式细胞术检测淋巴细胞；肠系膜淋巴结用流式细胞术观察淋巴细胞的变化。Specifically, when detecting the physiological indicators of the mice in the experimental group and the control group in the step 3, the operation method is as follows: when the weight of the mice with the most severe disease progression drops by about 30%, all the mice are killed at the same time, and the mice are collected. Serum, colon, spleen, and mesenteric lymph nodes; detection of inflammation-related cytokines in serum; colon length measurement, colon HE staining detection; spleen grinded into single cell suspension, and lymphocytes were detected by flow cytometry; mesenteric lymph nodes were detected by flow cytometry The changes of lymphocytes were observed by surgery.
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any relationship between these entities or operations. any such actual relationship or sequence exists. Moreover, the terms "comprising", "comprising" or any other variation thereof are intended to encompass a non-exclusive inclusion such that a process, method, article or device that includes a list of elements includes not only those elements, but also includes not explicitly listed or other elements inherent to such a process, method, article or apparatus.
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made in these embodiments without departing from the principle and spirit of the invention and modifications, the scope of the present invention is defined by the appended claims and their equivalents.
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