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Collagen-induced Arthritis: A Model for Murine Autoimmune Arthritis
胶原蛋白诱导的关节炎:构建鼠自身免疫性关节炎的模型   

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Abstract

Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund’s adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund’s adjuvant (IFA) 21 days after the first injection. These mice typically develop disease 26 to 35 days after the initial injection. C57BL/6J mice are resistant to arthritis induced by type II bovine collagen, but can develop arthritis when immunized with type II chicken collagen in CFA, and receive a boost of type II chicken collagen in IFA 21 days after the first injection. The concentration of heat-killed Mycobacterium tuberculosis H37RA (MT) in CFA also differs for each strain. DBA/1J mice develop arthritis with 1 mg/ml MT, while C57BL/6J mice require and 3-4 mg/ml MT in order to develop arthritis. CIA develops slowly in C57BL/6J mice and cases of arthritis are mild when compared to DBA/1J mice. This protocol describes immunization of DBA/1J mice with type II bovine collagen and the immunization of C57BL/6J mice with type II chicken collagen.

Materials and Reagents

  1. Interchangeable Syringes (Micro-Mate, catalog number: 148251A )
    Note: Currently, it is “Thermo Fisher Scientific, Micro-Mate™, catalog number: 148251A ”.
  2. Nylon 3-Way Stopcock (Kimble Chase, catalog number: 4201634503 )
    Note: Currently, it is “Thermo Fisher Scientific, Kimble Chase™, catalog number: 4201634503 ”.
  3. Needle (BD, catalog number: 305109 )
  4. Disposable syringe (BD, catalog number: 309623 )
  5. DBA/1J mice (male, 8 weeks old)
  6. C57BL/6 (male, 8 weeks old)
  7. Bovine Collagen Type II (Chondrex, catalog number: 20021 )
  8. Chicken Collagen Type II (Chondrex, catalog number: 20011 )
  9. Complete Freund's Adjuvant (CFA) (Chondrex, catalog number: 7008 -1 mg/ml and 7001 -4 mg/ml)
  10. Incomplete Freund's Adjuvant (IFA) (Sigma-Aldrich, catalog number: F5506 )
  11. Ketamine (KetaVed) (Vedco, catalog number: 078908598 )
  12. Xylazine (AnaSed) (LLOYD, catalog number: 078081939 )
  13. Glacial acetic acid (Thermo Fisher Scientific, catalog number: A38-212 )

Equipment

  1. Filtration System (Corning, catalog number: 431096 )
  2. Tailveiner Restrainer (Braintree Scientific, catalog number: TV-150 )
  3. Caliper (Kafer, catalog number: 217901 )

Procedure

  1. Collagen-induced arthritis: Immunization of DBA/1J mice with type II bovine collagen

    Preparation of bovine collagen stock
    1. Prepare 0.01 N acetic acid in ddH2O and filter through a 0.2 µM membrane to sterilize diluent. Store at 4 °C.
    2. Dilute 10 mg of bovine collagen with 2.5 ml of 0.01 N acetic acid for a final concentration of 4 mg/ml collagen.
    3. Secure lid and wrap the collagen bottle in aluminum foil to avoid light exposure.
    4. Rotate the bottle overnight at 4 °C or until collagen is completely dissolved.
    5. Aliquot collagen solution (500 µl aliquots) and freeze at -20 °C.
      1. Aliquots of collagen can be stored at -20 °C for 6 months.
      2. Aliquots should not be thawed more than 2 times prior to injection.

    Preparation of anesthetic
    1. Prepare a working solution of 25 mg/ml ketamine and 2.5 mg/ml xylazine in PBS. Store working solution for 2 weeks at room temperature, or 1-3 months at 4 °C.
      1. For a 10 ml solution, combine 2.5 ml of ketamine stock, and 0.25 ml of xylazine stock, with 7.25 ml PBS.
      2. Each mouse will be anesthetized through an intraperitoneal (i.p.) injection with 0.1 ml of stock solution for a final concentration of 2.5 mg ketamine and 0.25 mg xylazine per mouse.

    Preparation of bovine collagen emulsion with CFA for Day 0 immunization
    *Important: Perform in sterile tissue culture hood
    1. Connect two autoclaved 2 ml glass luer lock syringes to the 3-way stopcock.
    2. Place glass plunger into one of the syringes and stand the opposite syringe against a 50 ml tube rack. The syringe with the plunger should lay on the surface of the hood, while the second syringe stands at a 90° angle to the syringe with the plunger (Figure 1A).
    3. Create a working solution of collagen by adding 4 mg/ml collagen stock to 0.01 N acetic acid at a 1:1 ratio in the standing syringe (Figure 1B).
    4. Add CFA to the syringe at a 1:1 ratio of collagen working solution in the standing syringe.
      1. Example: 500 µl 4 mg/ml collagen, 500 µl 0.01 N acetic acid, and 1 ml CFA.
      2. Actual volumes of the working solution and CFA will be determined by the number of mice to be immunized, where each DBA/1J mouse receives 0.1 ml of the final emulsion.
      3. Mixing the emulsion will cause 0.4 ml of the emulsion to be lost in the stopcock, be sure to account for this when determining volumes of reagents.
    5. Turn the stopcock lever to close the valve opening not connected to a syringe.
    6. Place the plunger in the standing syringe.
    7. Mix the solution between the two syringes slowly, by plunging the entire solution into the opposite syringe 20 times. This will create a white emulsion (Figure 1C).
    8. Plunge all of the collagen into a single syringe. While holding the syringe containing the collagen stopcock end up (Figure 1D), remove the stopcock and connect a 27 gauge ½ inch luer lock needle to the end of the syringe.
    9. Gently and slowly lower the plunger to remove air from syringe until the collagen emulsion fills the needle (Figure 1E).
    10. Place collagen emulsion on ice and away from light until immunization.


      Figure 1. Syringe assembly. Attach 2 of 2 ml leur-lock syringes with a stopcock at a 90° angle. Insert the plunger to the syringe perpendicular to the standing syringe. Do no insert plunger into standing syringe (A). Fill the standing syringe with the collagen working solution and CFA (B). Insert the plunger into the standing syringe and exchange the solution to the opposite syringe 20 times, until a white emulsion forms (C). Turn the syringe so leur-lock faces upward and the entire emulsion falls against a single plunger (D). Remove the stopcock and syringe without the emulsion and assemble the syringe with the 27 ½’ gauge needle (E).

    Immunization of DBA/1J mice with Bovine Collagen Emulsion in CFA: Day 0
    1. Anesthetize each mouse with a 0.1 ml injection of the ketamine/xylazine working solution described in step A6a.
    2. Place the fully anesthetized mouse in the restraint with tail exposed.
    3. Turn the bevel of the needle upwards and inject 0.1 ml of the collagen emulsion subcutaneously in the tail, approximately 2 cm from the base of the tail. Be careful not to inject into tail veins, as this will result in death.

    Preparation and injection of DBA/1J mice with Type II Bovine Collagen Emulsion in IFA for Day 21 Boost
    1. Prepare bovine collagen emulsion for 21-day boost in the same concentrations as the day 0 immunization, substituting CFA for IFA.
    2. Mix the collagen slowly, and assemble the needle with the same methods described for day 0 immunization.
    3. Anesthetize each mouse with an 0.1 ml injection of the ketamine/xylazine working solution described in step A6a.
    4. Place the fully anesthetized mouse in the restraint with tail exposed.
    5. Inject 0.1 ml of collagen emulsion into the tail of the mouse closer to the base of the tail than the original injection site.
      If the boost injection is administered further from the base of the tail than the original injection spot, arthritis may develop slowly, or to a lesser extent than boost injections given closer to the base of the tail.

  2. Collagen-induced arthritis: Immunization of C57BL/6J mice with type II chicken collagen
    The procedure to elicit CIA in C57BL/6J mice is modeled after the DBA/1J procedure, but contains two major changes. Immunization of C57BL/6J mice uses type II chicken collagen in place of type II bovine collagen. Additionally, the concentration of MT in CFA is increased to 3-4 mg/ml for C57BL/6J mice from than 1 mg/ml used for DBA/1J mice. All other details are identical to the DBA/1J procedure.

  3. Paw measurement and scoring arthritis
    1. Anesthetize mice with the drop method of isoflurane anesthesia (add approximately 0.3 ml to a 500 µl container per IACUC guidelines).
    2.  Weigh each mouse, score paws, and measure paw thickness on day 0 and once a week until day 21. After day 21, weigh, measure and score paws twice a week.
    3. Apply an arthritis score to each paw with the following criteria:
      1. 0: Normal, no inflammation or redness
      2. 1: Redness and swelling in one digit
      3. 2: Redness and swelling in more than one digit or redness and swelling in one digit and ankle and wrist joint.
      4. 3: Redness and swelling present in all digits and joints
    4. Record the severity of paw swelling by measuring paw thickness at wrist joint of the front paws and the ankle joints of the hind paws. Each measurement should be noted in mm.
      1. To measure the front paws, the caliper should touch the back of the paw and the “palm” of the paw (Figure 2A).
      2. To measure the hind paws, the caliper should be placed on either side of the ankle joint, measuring the width of the ankle joint from one side to the other (Figure 2B).


        Figure 2. Paw measurement. Measure front paws by placing the front paw in the caliper so the palm and back of paw touch either side of the caliper (A). Read measurement in mm. Measure the hind paws by placing the ankle joint in the caliper so either side of the ankle touches the caliper (B). Read measurement in mm.

  4. Data analysis
    1. Plot the arthritis score data by adding the score of each paw together, for a maximum score of 12.
    2. Display the data in an XY plot, where Y is arthritis score and X is days after immunization (Figure 3A).
    3. Plot the measurement data by subtracting the thickness of the paw in mm from the original day 0 thickness.
    4. Display the data in an XY plot, where Y is swelling in mm and X is days after immunization (Figure 3B).

Representative data


Figure 3. DBA/1J mice were immunized with type II bovine collagen in CFA on day 0, and boosted with type II bovine collagen in IFA on day 21. Over the course of disease, an arthritis score was attributed to each paw of each mouse, based on the features described in section C. The score of each paw was summed for a score of 0-12 for each mouse (A). The thickness of each paw was also measured and the increase in paw thickness in mm was plotted (B). The displayed results represent three individual mice.

Acknowledgments

This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIAMS-NIH) under Award Number 1R01AR063132.

References

  1. Brand, D. D., Latham, K. A. and Rosloniec, E. F. (2007). Collagen-induced arthritis. Nat Protoc 2(5): 1269-1275.
  2. Crook, K. R., Jin, M., Weeks, M. F., Rampersad, R. R., Baldi, R. M., Glekas, A. S., Shen, Y., Esserman, D. A., Little, P., Schwartz, T. A. and Liu, P. (2015). Myeloid-derived suppressor cells regulate T cell and B cell responses during autoimmune disease. J Leukoc Biol 97(3): 573-582.
  3. Inglis, J. J., Simelyte, E., McCann, F. E., Criado, G. and Williams, R. O. (2008). Protocol for the induction of arthritis in C57BL/6 mice. Nat Protoc 3(4): 612-618.

简介

胶原诱导的关节炎(CIA)是用于研究类风湿性关节炎(RA)的常见的自身免疫动物模型。 CIA的发展涉及巨噬细胞和嗜中性粒细胞渗入关节,以及T和B细胞对II型胶原的反应。在鼠CIA中,用完全弗氏佐剂(CFA)中的II型牛胶原乳剂免疫遗传易感小鼠(DBA/1J),并在第一次给药21天后接受不完全弗氏佐剂(IFA)中II型牛胶原的加强免疫注射。这些小鼠通常在初次注射后26至35天发展疾病。 C57BL/6J小鼠对由II型牛胶原蛋白诱导的关节炎具有抗性,但当在CFA中用II型鸡胶原免疫时可产生关节炎,并且在首次注射后21天在IFA中接受II型鸡胶原蛋白的加强。热死亡的结核分枝杆菌H37RA(MT)在CFA中的浓度对于每个菌株也不同。 DBA/1J小鼠发展具有1mg/ml MT的关节炎,而C57BL/6J小鼠需要和3-4mg/ml MT以形成关节炎。 CIA在C57BL/6J小鼠中缓慢发展,并且与DBA/1J小鼠相比,关节炎病例轻度。该方案描述了用II型牛胶原免疫DBA/1J小鼠和用II型鸡胶原免疫C57BL/6J小鼠。

材料和试剂

  1. 可互换注射器(Micro-Mate,目录号:148251A)
    注意:目前,它是"Thermo Fisher Scientific,Micro-Mate?,目录号:148251A"。
  2. 尼龙3通旋塞阀(Kimble Chase,目录号:4201634503)
    注意:目前,它是"Thermo Fisher Scientific,Kimble Chase?,目录号:4201634503"。
  3. 针(BD,目录号:305109)
  4. 一次性注射器(BD,目录号:309623)
  5. DBA/1J小鼠(雄性,8周龄)
  6. C57BL/6(雄性,8周龄)
  7. 牛胶原II型(Chondrex,目录号:20021)
  8. 鸡胶原II型(Chondrex,目录号:20011)
  9. 完全弗氏佐剂(CFA)(Chondrex,目录号:7008-1mg/ml和7001-4mg/ml)
  10. 不完全弗氏佐剂(IFA)(Sigma-Aldrich,目录号:F5506)
  11. 氯胺酮(KetaVed)(Vedco,目录号:078908598)
  12. 赛拉嗪(Anaed)(LLOYD,目录号:078081939)
  13. 冰乙酸(Thermo Fisher Scientific,目录号:A38-212)

设备

  1. 过滤系统(Corning,目录号:431096)
  2. Tailveiner Restrainer(Braintree Scientific,目录号:TV-150)
  3. 卡尺(Kafer,目录号:217901)

程序

  1. 胶原诱导的关节炎:用II型牛胶原免疫DBA/1J小鼠
    牛胶原蛋白原液的制备
    1. 在ddH 2 O中制备0.01N乙酸,并通过0.2μM膜过滤以对稀释剂灭菌。储存于4°C。
    2. 用2.5ml 0.01N乙酸稀释10mg牛胶原,最终浓度为4mg/ml胶原。
    3. 固定盖子并将胶原蛋白瓶包装在铝箔中,以避免暴露于光。
    4. 在4℃下将瓶子旋转过夜,或直到胶原完全溶解
    5. 等分胶原溶液(500μl等分试样),并在-20℃下冷冻
      1. 胶原蛋白的等分试样可以在-20℃保存6个月
      2. 等分试样在注射前不应解冻超过2次。

    麻醉剂的准备
    1. 制备25mg/ml氯胺酮和2.5mg/ml甲苯噻嗪的工作溶液 在PBS中。将工作溶液在室温下储存2周,或1-3 个月。
      1. 对于10ml溶液,将2.5ml氯胺酮储液和0.25ml甲苯胺储液与7.25ml PBS混合。
      2. 每只小鼠将通过腹膜内(i.p.) 注射0.1ml原液,最终浓度为2.5 ?mg氯胺酮和0.25mg xyalzine。

    使用CFA进行第0天免疫的牛胶原乳液的制备
    * 重要提示:在无菌组织培养罩中预成型
    1. 将两个高压灭菌的2 ml玻璃路厄锁定注射器连接到三通活栓
    2. 将玻璃柱塞放入其中一个注射器,并站在相反的位置 注射器针对50毫升管架。带柱塞的注射器 放置在罩的表面上,而第二注射器以90°放置 ?角度与注射器与柱塞(图1a)。
    3. 创建一个 通过加入4mg/ml胶原原料至0.01N的胶原工作溶液 乙酸以1:1的比例在静置注射器中(图1b)。
    4. 在注射器中以1:1的胶原工作溶液比例将CFA加入到注射器中。
      1. 实施例:500μl4mg/ml胶原,500μl0.01N乙酸和1ml CFA
      2. 工作解决方案和CFA的实际量将由 ?待免疫小鼠的数目,其中每个DBA/1J小鼠接受0.1ml ?的最终乳液
      3. 混合乳液将产生0.4ml的 乳液在活塞中损失,一定要考虑到这一点 当确定试剂的体积时。
    5. 转动停止旋塞杆,关闭未连接到注射器的阀门开口。
    6. 将柱塞放入站立式注射器中。
    7. 混合溶液在两个注射器之间缓慢,通过突然 整个溶液进入相对的注射器20次。这将创建一个 白色乳剂(图1c)
    8. 把所有的胶原蛋白倒入 单注射器。当拿着包含胶原的注射器时 (图1d),取下活塞并连接一个27号? ?英寸luer锁针到注射器的末端
    9. 轻轻慢慢地降低柱塞,以从注射器中除去空气,直到胶原乳液填充针(图1e)。
    10. 将胶原蛋白乳液放在冰上,远离光,直到免疫

      图1.注射器组件。将2个2 ml leur锁注射器与a 停止旋塞成90°角。将柱塞插入注射器 垂直于站立式注射器。不要插入柱塞 (A)。用胶原填充立式注射器 工作溶液和CFA(B)。将柱塞插入支架 注射器并将溶液交换到相对的注射器20次, 直到白色乳液形成(C)。转动注射器,使leur-lock面 并且整个乳液落在单个柱塞(D)上。 取出没有乳液的止动阀和注射器,并组装 注射器用27?'号针(E)

    在CFA中用牛胶原乳液免疫DBA/1J小鼠:第0天
    1. 用0.1ml注射的步骤A6a中所述的氯胺酮/赛拉嗪工作溶液麻醉每只小鼠。
    2. 将完全麻醉的鼠标在尾部暴露的约束。
    3. 向上转动针的斜面,并注射0.1毫升 胶原乳液在尾部皮下,距离大约2厘米 尾部的基部。小心不要注入尾静脉,因为这 将导致死亡。

    在IFA中制备和注射II型牛胶原蛋白乳胶的DBA/1J小鼠,用于第21天增强
    1. 准备牛胶原乳液21天增强在相同的 浓度作为第0天免疫,用CFA代替IFA。
    2. 慢慢混合胶原蛋白,用与第0天免疫相同的方法组装针头。
    3. 用0.1ml注射的步骤A6a中所述的氯胺酮/赛拉嗪工作溶液麻醉每只小鼠。
    4. 将完全麻醉的鼠标在尾部暴露的约束。
    5. 注入0.1毫升胶原乳液到小鼠的尾巴 比原始注射部位更靠近尾部的基部 如果 从尾部的基部进一步施用增强注射 比原来的注射点,关节炎可能发展缓慢,或者 较少的增强注射给予更接近的基地 尾巴。

  2. 胶原诱导的关节炎:用II型鸡胶原免疫C57BL/6J小鼠 在C57BL/6J小鼠中引起CIA的程序在DBA/1J程序后建模,但包含两个主要变化。 C57BL/6J小鼠的免疫使用II型鸡胶原蛋白代替II型牛胶原蛋白。此外,对于C57BL/6J小鼠,CFA中MT的浓度从用于DBA/1J小鼠的1mg/ml增加至3-4mg/ml。所有其他详细信息与DBA/1J过程相同。

  3. 爪子测量和得分关节炎
    1. 用异氟醚麻醉的滴剂麻醉麻醉小鼠(添加 根据IACUC指南,约0.3ml至500μl容器)
    2.  称重每只小鼠,对爪子进行评分,并在第0天测量爪厚度 每周一次,直到第21天。第21天后,称重,测量和记分爪 每周两次。
    3. 按照以下标准对每只脚施加关节炎评分:
      1. 0:正常,无炎症或发红
      2. 1:一位数发红和肿胀
      3. 2:在一个以上的指中发红和肿胀,在一个指和踝和腕关节发红和肿胀。
      4. 3:所有手指和关节都有发红和肿胀
    4. 通过测量爪厚度来记录爪肿胀的严重程度 前爪的腕关节和后爪的踝关节。 每个测量值应以mm为单位。
      1. 为了测量前爪,卡钳应该接触爪的后部和爪的"掌"(图2a)。
      2. 要测量后爪,应该将卡尺放在其上 侧的踝关节,从一个测量踝关节的宽度 ?侧到另一侧(图2b)

        图2.爪子测量。 通过将前爪放置在卡尺中以测量前爪,以便掌心 并且爪的背部接触制动钳(A)的任一侧。读取测量 mm。通过将踝关节放置在卡尺中来测量后爪 因此踝的任一侧接触卡钳(B)。读取测量 ?mm。

  4. 数据分析
    1. 通过将每个爪子的得分加在一起来绘制关节炎得分数据,最大得分为12.
    2. 在XY图中显示数据,其中Y是关节炎评分,X是免疫后的天数(图3a)
    3. 通过从原始第0天的厚度减去爪的厚度(mm)来绘制测量数据
    4. 在XY图中显示数据,其中Y以mm为单位膨胀,X是免疫后的天数(图3b)

代表数据


图3.在第0天用CFA中的II型牛胶原免疫DBA/1J小鼠,并在第21天用IFA中的II型牛胶原增强。在疾病过程中,关节炎评分归于每只小鼠的每只爪子,基于C节中描述的特征。每只小鼠的得分总计为每只小鼠的0-12分(A)。还测量每个爪的厚度,并绘制以mm为单位的爪厚度的增加(B)。显示的结果代表三只个体小鼠。

致谢

这项工作得到国家卫生研究院关心和肌肉骨骼和皮肤疾病国家研究所(NIAMS-NIH)的支持,奖号为1R01AR063132。

参考文献

  1. Brand,D.D.,Latham,K.A。和Rosloniec,E.F。(2007)。 胶原诱导的关节炎 Nat Protoc 2(5 ):1269-1275。
  2. Crook,KR,Jin,M.,Weeks,MF,Rampersad,RR,Baldi,RM,Glekas,AS,Shen,Y.,Esserman,DA,Little,P.,Schwartz,TA和Liu, 。 骨髓衍生抑制细胞在自身免疫性疾病期间调节T细胞和B细胞反应。 em J J Leukoc Biol 97(3):573-582。
  3. Inglis,J.J.,Simelyte,E.,McCann,F.E.,Criado,G.and Williams,R.O。(2008)。 C57BL/6小鼠中关节炎诱导的方案。Nat Protoc 3(4):612-618。

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pietrosimone, K. M., Jin, M., Poston, B. and Liu, P. (2015). Collagen-induced Arthritis: A Model for Murine Autoimmune Arthritis. Bio-protocol 5(20): e1626. DOI: 10.21769/BioProtoc.1626.
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