胶原蛋白诱导小鼠关节炎实验方案（三）
C. Immunization Schedule
免疫程序
根据小鼠品系和实验目的的不同，诱导高发病率和严重关节炎的方法也各有不同。
1.通过单次免疫（无需加强免疫）诱发关节炎（易感品系）
注射胶原和完全弗式佐剂混合而成的含2mg/ml结合杆菌乳剂。对于易感品系小鼠DBA/1（H-2q）和B10.RIII(H-2r)注射乳剂后28-35天会引发关节炎。42-56天发病率达90-100%。关节炎严重程度高，临床评分达10-12分 （最高16分）。
注：由于高浓度的结核杆菌会引起免疫部位严重的炎症反应，因此根据各研究机构对动物实验的要求，可选用方案2或方案4。
2.通过加强免疫诱发关节炎（易感品系）
注射胶原和完全佐剂混合而成的含0.5mg/ml结合杆菌乳.在初次注射后第21天，加强注射胶原和不完全佐剂（不含结核杆菌）混合而成的乳剂。尾部皮下注射0.1ml乳剂， 避开初次免疫部位。一般在初次免疫注射后28-35天引发关节炎。在第42-56天，发病率为80-100%，临床评分可达8-12分（最高16分）。
3.用高剂量的结核杆菌完全佐剂加强免疫诱发关节炎（CIA抗性小鼠）
对于一些低反应的小鼠，例如：B10 (H-2b), C57BL/6(H-2b), and C57BL/6×129/Sv (H-2b) ，采用以下方案可诱发关节炎。初次免疫，尾部皮下注射0.1ml胶原和完全佐剂混合而成的含2.5mg/ml结核杆菌的乳剂。在第21天加强免疫，再次注射0.1ml胶原和完全佐剂混合而成的含2.5mg/ml结核杆菌的乳剂。一般在初次免疫注射后28-35天引发关节炎。在第42-56天，发病率为50-70% （10）。
注：该方案引起的炎症反应比较严重，根据各研究机构对动物实验的要求，可选用胶原抗体（CAIA）诱发的小鼠关节炎模型。Chondrex公司的单克隆抗体合剂（Arthrogen-CIA）和LPS能在CIA抗性的小鼠中诱发关节炎。相关信息请参考www.chondrex.com或咨询我们代理商上海金畔生物。
4. 通过LPS加强免疫
LPS和体内二型胶原自身抗体水平对于诱发关节炎具有协同效应(13)。此外，在经典的CIA动物模型中 ，应用LPS(B细胞分裂素)或MAM(支原体产生的T细胞分裂素)或SEB(金黄色葡萄球菌产生的T 细胞分裂素)都会加强关节炎发病率和严重程度（14-16）。因此这些毒素不仅用于引发和加强关节炎水平，而且用于指定关节炎发生时间。
这个方案，需要注射0.1ml二型胶原和含0.5mg/ml的CFA混合乳剂（参考方案2）。在第25-28天或在在关节炎发生前3-5天腹腔注射LPS(25-50µg，溶解在生理盐水)。关节炎将在24-48小时发生，发病率90-100%。
注：CFA免疫注射小鼠会在2-4周发生严重的免疫抑制。所以一些小鼠对LPS（50µg）注射非常敏感。
Chondrex公司建议进行正式实验之前先对来自不同供应商的动物进行测试。
D. Onset of Arthritis
关节炎的发作
有效的免疫注射后，临床上明显的关节肿胀症状会在3-5周出现。关节炎的发作和发病率取决于小鼠品系和实验方案。
EVALUATING ARTHRITIS
关节炎评估
A.Scoring
A.临床评分
关节炎可通过临床评分来定性评估或用测厚仪来测量爪子厚度来评估。这些方法适用于 所有的关节炎模型比如经典的CIA，佐剂诱导的关节炎，单克隆抗体合剂和其他炎症模型。Chondrex公司提供一下系统（表二）
注：由于小鼠的爪子太小，因此无法像测量大鼠爪子体积那样通过浸没法测定小鼠爪子体积。
表二 关节炎炎症程度的临床评分
|
Score得分
|
Condition发病情况 |
| 0 |
Normal 正常 |
| 1 |
轻度的、踝关节、腕关节发红、肿胀 |
| 2 |
踝关节或腕关节中度发红肿胀 |
| 3 |
爪子严重发红、肿胀，包括指端 |
| 4 |
四肢最大程度发炎，包括多关节 |
B. Serum Analysis
B. 血清分析
高浓度自身二型胶原蛋白IgG抗体水平的产生是诱发关节炎的关键（10, 17）。二型胶原蛋白IgG2a 和 IgG2b抗体亚型对激活补体及随后的关节炎的发生至关重要。
Chondrex公司提供小鼠胶原IgG及其各亚型抗体ELISA测试盒（具体信息参考www.chondrex.com或咨询代理商上海金畔生物）。
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