【作者】 颜灿群；

【导师】 黄光英；

【作者基本信息】 华中科技大学 ， 中西医结合临床， 2007， 博士

【摘要】 第一部分Cx43基因敲除小鼠胶原性关节炎模型的建立目的:以牛Ⅱ型胶原(Bovine Collagen typeⅡ, BCⅡ)皮内免疫Cx43(connexin43, Cx43)基因敲除杂合型(Cx43+/-)和野生型(Cx43+/+)小鼠,建立Cx43基因敲除小鼠胶原性关节炎(Collagen-induced Arthritis,CIA)模型。方法:用BCⅡ和完全弗氏佐剂(Complete Freund’s adjuvant, CFA)皮内注射免疫Cx43+/-和Cx43+/+雄性小鼠,观察关节部位的肿胀并进行关节炎指数评分;采用X线摄片、病理组织学等方法进行小鼠病理学特征分析;用ELISA法检测小鼠血清抗BCⅡ胶原抗体水平。结果:与对照组相比,模型组小鼠踝关节与脚趾有明显的肿胀,关节炎指数升高;X线摄片结果和病理组织学分析结果显示,Cx43+/+和Cx43+/- CIA小鼠的关节均呈现典型的关节炎病变的特征;ELISA结果显示:模型组小鼠血清中抗BCⅡ胶原抗体水平较对照组明显高(P<0.05)。结论:本实验成功建立了BCⅡ诱导的Cx43+/+小鼠和Cx43+/-小鼠CIA模型。BCⅡ诱导的Cx43基因敲除小鼠CIA模型的病理学特征与人类RA极为相似,为更好地进行人类RA以及Cx43相关研究提供了一个良好的动物模型。9周龄、Cx43+/+小鼠更易诱导出CIA模型。第二部分针刺治疗对胶原性关节炎Cx43基因敲除小鼠的Th1/Th2细胞亚群的影响目的:观察针刺对Cx43+/+和Cx43+/- CIA小鼠的疗效和脾淋巴细胞中Th细胞亚群分布,探讨Cx43与针刺疗效的相关性及其机制。方法:在初次免疫后第4w开始对CIA小鼠连续针刺治疗3w,观察其关节炎指数,应用三色流式细胞术对CIA组小鼠和对照组小鼠脾淋巴细胞中辅助T细胞(T cell helper, T h)亚群分布进行检测。结果:针刺治疗后,Cx43+/+ CIA小鼠的关节炎指数下降较Cx43+/- CIA小鼠明显(P<0.05)。针刺抑制Cx43+/+ CIA小鼠Th1细胞分泌,降低Th1/Th2比值,对Th2细胞的分泌无明显影响;而针刺对Cx43+/- CIA小鼠Th1和Th2均无明显影响。结论:针刺治疗Cx43+/+ CIA小鼠疗效明显优于Cx43+/- CIA小鼠,提示针刺疗效与Cx43基因相关,其疗效可能是通过调节Th1/Th2使之接近动态平衡而起作用的,针刺疗效与这种调节作用成正相关。第三部分针刺对胶原性关节炎Cx43基因敲除小鼠血清IL-1β和TNF-α的影响目的:观察针刺对Cx43+/+和Cx43+/- CIA小鼠的疗效和外周血血清中IL-1β和TNF-α的水平的影响,探讨Cx43与针刺疗效的相关性及其机制。方法:在初次免疫后第4w开始连续针刺治疗3w,观察关节炎指数,并以酶联免疫吸附实验(ELISA)检测各小鼠外周血血清中IL-1β和TNF-α的水平。结果:针刺治疗后,Cx43+/+ CIA小鼠的关节炎指数下降较Cx43+/- CIA小鼠明显(P<0.05)。针刺显著降低了Cx43+/+ CIA小鼠外周血血清IL-1β和TNF-α的含量(P<0.05),而对Cx43+/- CIA小鼠无明显影响。结论:针刺对Cx43+/+ CIA小鼠疗效明显优于Cx43+/- CIA小鼠,提示针刺疗效与Cx43基因相关,其疗效可能是通过调节外周血血清IL-1β和TNF-α的水平而起作用的,针刺疗效与这种调节作用成正相关。更多还原

【Abstract】 PartⅠEstablishment of Collagen-induced Arthritis model in Cx43 knockout MiceObjectives To establish the animal model with collagen-induced arthritis(CIA) using Cx43 knockout mice.Methods The mice were immunized by intradermal injection of bovine collagen typeⅡ(BCⅡ) emulsified with complete Freund’s adjuvant(CFA). The swelling of feet were observed. The pathological characteristics of the animal model were assayed by photos of X-ray, and histopathological techniques. The level of anti-CⅡantibody in serum was analyzed by enzyme linked immunosorbent assay(ELISA).Results Obvious swelling of the feet was found in the model group compared with control group(P<0.05). Anti-CⅡantibody level was higher in model group than that in control. Besides, typical arthritis pathology was showed by roentgenography and histopathology.Conclusion The model of CIA in Cx43+/+ and Cx43+/- mice is established successfully, which is benifical to investigate pathogenesis and the therapy of human RA and Cx43-related research. The mice aged 9 weeks in Cx43+/+ and Cx43+/- are much easier to development CIA, and the former is much easier to development CIA.PartⅡEffect of Acupuncture on Th1/Th2 balance in Cx43 knockout mice with CIAObjectives To explore the correlation of Cx43 and efficacy of acupuncture, by observing the efficacy of acupuncture on CIA mice genotyped Cx43 +/+ and Cx43+/- , and observing the splenic Th1/Th2 cell subsets.Methods Acupuncture treatment,begun on four weeks after the initial injection of collagen, was given once a day for 3 weeks continuously. The arthritic index was observed. The intracellular cytokine detection was performed with three-color flow cytometry to quantitate the splenic Th1/Th2 cell subsets in mice with CIA induced by BCⅡand the sham-immunized controls.Results The arthritic index decreased significantly in Cx43+/+ mice with CIA compared with the Cx43+/- ones after acupuncture treatment. Acupuncture suppresses the Th1 subset and Th1/Th2 in Cx43+/+ mice. Whereas it has no obvious effect on Th2. In contrast, It has no effect on Th1 and Th2 in Cx43+/- mice with CIA.Conclusion Acupuncture has better efficacy on CIA mice genotyped Cx43+/+ than ones genotyped Cx43+/-. It showed the efficacy of acupuncture is associated with Cx43. Its active mechanism might decrease Th1 and regulate the proportion of Th1/Th2. There is a positive correlation among the effect of acupuncture and the regulation. PartⅢEffect of Acupuncture on IL-1βand TNF-αin Cx43 knockout mice with Collagen-induced ArthritisObjectives To explore the correlation of Cx43 and efficacy of acupuncture by observing the efficacy of acupuncture on CIA mice genotyped Cx43 +/+ and Cx43+/-, and observing the level of IL-1βand TNF-αin serum.Methods Acupuncture treatment,begun on four weeks after the initial injection of collagen, was given once a day for 3 weeks continuously. The arthritic index was observed. ELISA method was used to examine the levels of serum IL-1βand TNF-α.Results The arthritic index decreased significantly in Cx43+/+ mice with CIA compared with the Cx43+/- ones after acupuncture treatment. The level of IL-1βand TNF-αin serum decreased significantly in Cx43+/+ mice with CIA. Whereas there is no obvious change in Cx43+/- mice with CIA.Conclusion Acupuncture has better efficacy on CIA mice genotyped Cx43+/+ than ones genotyped Cx43+/-. It showed the efficacy of acupuncture is associated with Cx43. To decrease level of IL-1βand TNF-αis the possible mechanism of acupuncture to treat RA. There is the positive correlation among the effect of acupuncture and the regulation.更多还原