C. Immunization Schedule

免疫程序

根據(jù)小鼠品系和實驗?zāi)康牡牟煌?，誘導(dǎo)高發(fā)病率和嚴(yán)重關(guān)節(jié)炎的方法也各有不同。

1.通過單次免疫（無需加強(qiáng)免疫）誘發(fā)關(guān)節(jié)炎（易感品系）

注射膠原和完全弗式佐劑混合而成的含2mg/ml結(jié)合桿菌乳劑。對于易感品系小鼠DBA/1（H-2q）和B10.RIII(H-2r)注射乳劑后28-35天會引發(fā)關(guān)節(jié)炎。42-56天發(fā)病率達(dá)90-100%。關(guān)節(jié)炎嚴(yán)重程度高，臨床評分達(dá)10-12分 （最高16分）。

注：由于高濃度的結(jié)核桿菌會引起免疫部位嚴(yán)重的炎癥反應(yīng)，因此根據(jù)各研究機(jī)構(gòu)對動物實驗的要求，可選用方案2或方案4。

2.通過加強(qiáng)免疫誘發(fā)關(guān)節(jié)炎（易感品系）

注射膠原和完全佐劑混合而成的含0.5mg/ml結(jié)合桿菌乳.在初次注射后第21天，加強(qiáng)注射膠原和不完全佐劑（不含結(jié)核桿菌）混合而成的乳劑。尾部皮下注射0.1ml乳劑， 避開初次免疫部位。一般在初次免疫注射后28-35天引發(fā)關(guān)節(jié)炎。在第42-56天，發(fā)病率為80-100%，臨床評分可達(dá)8-12分（最高16分）。

3.用高劑量的結(jié)核桿菌完全佐劑加強(qiáng)免疫誘發(fā)關(guān)節(jié)炎（CIA抗性小鼠）

對于一些低反應(yīng)的小鼠，例如：B10 (H-2b), C57BL/6(H-2b), and C57BL/6x129/Sv (H-2b) ，采用以下方案可誘發(fā)關(guān)節(jié)炎。初次免疫，尾部皮下注射0.1ml膠原和完全佐劑混合而成的含2.5mg/ml結(jié)核桿菌的乳劑。在第21天加強(qiáng)免疫，再次注射0.1ml膠原和完全佐劑混合而成的含2.5mg/ml結(jié)核桿菌的乳劑。一般在初次免疫注射后28-35天引發(fā)關(guān)節(jié)炎。在第42-56天，發(fā)病率為50-70% （10）。

注：該方案引起的炎癥反應(yīng)比較嚴(yán)重，根據(jù)各研究機(jī)構(gòu)對動物實驗的要求，可選用膠原抗體（CAIA）誘發(fā)的小鼠關(guān)節(jié)炎模型。Chondrex公司的單克隆抗體合劑（Arthrogen-CIA）和LPS能在CIA抗性的小鼠中誘發(fā)關(guān)節(jié)炎。相關(guān)信息請參考www.chondrex.com或咨詢我們代理商欣博盛生物。

4. 通過LPS加強(qiáng)免疫

LPS和體內(nèi)二型膠原自身抗體水平對于誘發(fā)關(guān)節(jié)炎具有協(xié)同效應(yīng)(13)。此外，在經(jīng)典的CIA動物模型中 ，應(yīng)用LPS(B細(xì)胞分裂素)或MAM(支原體產(chǎn)生的T細(xì)胞分裂素)或SEB(金黃色葡萄球菌產(chǎn)生的T 細(xì)胞分裂素)都會加強(qiáng)關(guān)節(jié)炎發(fā)病率和嚴(yán)重程度（14-16）。因此這些毒素不僅用于引發(fā)和加強(qiáng)關(guān)節(jié)炎水平，而且用于指定關(guān)節(jié)炎發(fā)生時間。

這個方案，需要注射0.1ml二型膠原和含0.5mg/ml的CFA混合乳劑（參考方案2）。在第25-28天或在在關(guān)節(jié)炎發(fā)生前3-5天腹腔注射LPS(25-50μg，溶解在生理鹽水)。關(guān)節(jié)炎將在24-48小時發(fā)生，發(fā)病率90-100%。

注：CFA免疫注射小鼠會在2-4周發(fā)生嚴(yán)重的免疫抑制。所以一些小鼠對LPS（50μg）注射非常敏感。

Chondrex公司建議進(jìn)行正式實驗之前先對來自不同供應(yīng)商的動物進(jìn)行測試。

D. Onset of Arthritis

關(guān)節(jié)炎的發(fā)作

有效的免疫注射后，臨床上明顯的關(guān)節(jié)腫脹癥狀會在3-5周出現(xiàn)。關(guān)節(jié)炎的發(fā)作和發(fā)病率取決于小鼠品系和實驗方案。

EVALUATING ARTHRITIS

關(guān)節(jié)炎評估

A.Scoring

A.臨床評分

關(guān)節(jié)炎可通過臨床評分來定性評估或用測厚儀來測量爪子厚度來評估。這些方法適用于 所有的關(guān)節(jié)炎模型比如經(jīng)典的CIA，佐劑誘導(dǎo)的關(guān)節(jié)炎，單克隆抗體合劑和其他炎癥模型。Chondrex公司提供一下系統(tǒng)（表二）

注：由于小鼠的爪子太小，因此無法像測量大鼠爪子體積那樣通過浸沒法測定小鼠爪子體積。

表二 關(guān)節(jié)炎炎癥程度的臨床評分

B. Serum Analysis

B. 血清分析

高濃度自身二型膠原蛋白IgG抗體水平的產(chǎn)生是誘發(fā)關(guān)節(jié)炎的關(guān)鍵（10, 17）。二型膠原蛋白IgG2a 和 IgG2b抗體亞型對激活補(bǔ)體及隨后的關(guān)節(jié)炎的發(fā)生至關(guān)重要。

Chondrex公司提供小鼠膠原IgG及其各亞型抗體ELISA測試盒（具體信息參考www.chondrex.com或咨詢代理商欣博盛生物）。

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