肾虚痹证(类风湿性关节炎)病证结合动物模型制作方法研究
发布时间:2018-04-16 05:13
本文选题:痹证 + 类风湿性关节炎 ; 参考:《中国中医科学院》2010年硕士论文
【摘要】: 目的: 探讨肾虚痹证(类风湿性关节炎)病证结合动物模型的制作方法,为研究中医药治疗肾虚痹证(类风湿性关节炎)的作用及其机理提供实验工具,并以期为病证结合动物模型的制作提供进一步的科学依据。 方法: 本课题分为两部分实验来进行研究: 实验一:选用6周龄SPF级SD大鼠80只、Wistar大鼠80只、Lewis大鼠40只,雌雄各半。采用尾根部皮下注射Ⅱ型胶原与不完全弗氏佐剂混合物的方法诱导出关节炎模型,对不同品系、不同性别大鼠在胶原诱导性关节炎(Collagen-induced arthritis, CIA)模型中的发病情况进行比较研究。 实验二:选用6周龄SPF级SD大鼠100只,雌雄各半,除对照组大鼠外,分别切除大鼠的双侧睾丸和卵巢,制作肾虚动物模型。分别在去势1周、2周、3周、4周后,采用尾根部皮下注射Ⅱ型胶原与不完全弗氏佐剂混合物的方法诱导出关节炎模型,从而对不同去势时间(即不同肾虚状态)下,CIA发病情况进行比较研究,并采用ELISA等方法对其机理进行初步探讨。 结果: 实验一: 对三种不同品系大鼠发病情况数据进行动态分析,发现不同种属发病率不同,χ2=72.94,P0.0001;时间与发病率相关,χ2=15.39,P=0.0005,有显著性差异,随着免疫时间的延长,发病率越高。两两比较显示,SD和Wistar大鼠与Lewis大鼠发病率有显著性差异(P均0.0001),但SD与Wistar无显著性差异(P=0.6183)。 重复测量方差分析结果显示:(1)不同品系大鼠AI不同,有显著性差异(P0.0001);组间两两比较结果显示:Lewis比SD、wistar大鼠AI低,有显著性差异(P0.0001),且SD比wistar大鼠AI高,差异亦具有显著性(P=0.045)。(2)性别对AI没有影响(P=0.737)。免疫28d内各时段与其他时间相比均有显著性差异(P0.0001),28d后各时段比无显著性差异(P均0.05)。 显著性检验考虑性别和属种两因素对病理积分的影响,采用双因素方差分析统计,总体方差分析结果显示性别对病理积分影响无显著性(P0.05),种属对病理积分影响有显著性差异(P0.05)。两两比较采用LSD法检验,结果显示,Lewis比SD、Wistar大鼠关节病理积分低,有显著性差异(P0.05),而SD与Wistar大鼠之间无显著性差异。 实验二: 对不同去势时间关节炎发病情况数据进行动态分析,发现对照CIA组大鼠与CIA去势组大鼠,或者不同去势时间CIA大鼠发病率影响没有显著性差异(P=0.9159)。 重复测量方差分析结果显示:(1)性别对AI没有影响(P=0.667);(2)与对照CIA组相比,与去势4周组AI明显升高(P=0.006),与去势1周相比,亦有显著性差异(P=0.014),其余各组之间相比均无显著性差异。 考虑性别和去势时间两因素对病理积分的影响,采用双因素方差分析统计,结果显示性别对病理积分影响无显著性(P0.05),去势时间对病理积分影响有显著性差异(P0.05)。 双因素方差分析统计结果显示性别对CORT影响有显著性(P0.0001),数据显示性别和分组之间有交互作用(P0.05),所以按性别做分层分析可以了解不同性别分组之间的差异。结果显示,去势后,不同性别大鼠对CORT的反应变化相反,即与未去势CIA组比较,雌性去势CIA组大鼠血浆CORT水平明显降低(P0.01,P0.05),除去势1周组外,雄性大鼠CORT均显著升高(P<0.01,P0.05),且与去势时间呈正相关,大鼠去势3周血浆CORT与去势4周相比,无显著性差异。 双因素方差分析统计显示性别对抗Ⅱ型胶原抗体影响无显著性(P=0.384),去势时间对抗Ⅱ型胶原抗体影响有显著性差异(P0.0001),LSD法两两比较显示,随着去势时间增加,抗Ⅱ型胶原抗体明显升高,与对照CIA组比较,去势2、3、4周CIA组大鼠血清抗Ⅱ型胶原抗体水平明显升高(P0.05),去势1、2、3、4周CIA组大鼠血清抗Ⅱ型胶原抗体水平有显著性差异(P0.05)。 双因素方差分析统计结果显示性别对IL-6影响有显著性(P=0.0034),数据显示性别和分组之间无交互作用(P=0.3356),去势时间对IL-6影响有显著性差异(P0.0001),两两比较采用LSD法检验,结果显示,与对照CIA组比较,去势2周、3周、4周CIA组大鼠IL-6水平明显升高(P0.05),去势1周、2周、3周、4周CIA组大鼠血清IL-6水平亦有显著性差异(P0.05),随着去势时间增加,明显升高。 双因素方差分析结果显示性别对IL-10影响有显著性(P0.0001),数据显示性别和分组之间无交互作用(P=0.3356),去势时间对IL-10影响有显著性差异(P0.0001),两两比较采用LSD法检验,结果显示,各组CIA组大鼠血浆IL-10水平随着去势时间增加,明显升高,均有显著性差异(P0.05)。 结论: (1)对于Ⅱ型胶原免疫诱导的大鼠关节炎模型,Wistar和SD大鼠发病率和发病程度均明显高于Lewis大鼠。Wistar和SD大鼠发病率无显著性差异,但发病程度SD大鼠明显高于Wistar大鼠。三种品系大鼠雌雄之间发病率和发病程度无明显差异。因此,在制作痹证(类风湿性关节炎)大鼠模型时,建议首选SD大鼠,其次可以选择Wistar大鼠,雌雄均可。 (2)肾虚在CIA发病过程中起着重要作用,随着去势时间的延长,即肾虚程度加重,CIA有效发病率和发病程度亦随之增加;在去势4周后其有效发病率就可达到95%,且发病程度明显高于同期未去势大鼠。因此,在制作肾虚痹证(类风湿性关节炎)病证结合动物模型时,在大鼠去势4周时就可用CⅡ进行免疫诱导。 (3)根据以上实验结果,并结合前期工作基础,肾虚痹证(类风湿性关节炎)病证结合大鼠模型的具体制作方法如下:选用SD大鼠,鼠龄6周龄,SPF级,雌雄均可。按常规方法切除大鼠的双侧睾丸或卵巢。于去势后4周,取适量Ⅱ型胶原溶液(浓度为2mg/ml)逐滴加入至等容积的不完全弗氏佐剂中,Ⅱ型胶原终浓度为1mg/ml。冰浴中用匀浆器充分乳化,以滴加水中不扩散为度,取乳化后的混合物按0.2ml/只,即200μgCⅡ/只,于尾根部皮下注射。7d后按0.1ml/只,即100μg CⅡ/只,于尾根部皮下加强免疫一次。
[Abstract]:Objective:
Study of kidney deficiency syndrome (rheumatoid arthritis) method for making animal model combining disease with syndrome, for the study of Chinese medicine treatment of kidney deficiency syndrome (rheumatoid arthritis) and provide experimental tools and its mechanism, and to provide further scientific basis for the combination of disease and syndrome animal model.
Method:
This topic is divided into two parts of the experiment to study:
Experiment one: using 6 weeks SPF SD 80 rats, 80 Wistar rats and 40 Lewis rats, half male and half female. The root of tail subcutaneous injection of type II collagen with Freund's incomplete adjuvant mixture induced arthritis model of different strains, in collagen induced arthritis in different gender rats (Collagen-induced arthritis, CIA) incidence model were investigated.
Experiment two: Thirty 6 week old SPF 100 SD rats, male and female, except the rats in the control group, respectively, and the rat testis and ovary, making kidney deficiency animal model. In ovariectomized for 1 weeks, 2 weeks, 3 weeks, 4 weeks, using the method of subcutaneous injection of type II tail root. Collagen with Freund's incomplete adjuvant mixture induced arthritis model, so as to different time (different castration kidney deficiency), CIA incidence of comparative study, and uses the ELISA method to explore its mechanism.
Result:
Experiment 1:
Dynamic analysis on three different strains of rats the incidence data, found that different species at different rates, X 2=72.94, P0.0001; time and incidence, X 2=15.39, P=0.0005, there are significant differences, with prolonging the time of immunization, the higher the incidence. 22 comparison showed that SD and Wistar rats with the onset of Lewis rats was significantly different (P < 0.0001), but no significant difference between SD and Wistar (P=0.6183).
Repeated measurement of variance analysis results showed that: (1) different rat strains of different AI, there were significant differences between the 22 groups (P0.0001); the comparison results show that the Lewis ratio of SD, Wistar in rats with AI low, there was significant difference (P0.0001), SD and AI higher than Wistar rats, the difference is significant a (P=0.045). (2) the gender has no effect on AI (P=0.737). Immune 28d within the time when there were significant differences as compared with other time (P0.0001), each time after 28d, there was no significant difference (P 0.05).
Significant test considering gender and species effects of the two factors on pathological scores, using two factor analysis of variance statistics, variance analysis showed that gender had no significant effect on pathological score (P0.05), a significant difference on the effects of pathological scores (P0.05). 22 compared with the LSD test. The results showed that Lewis SD, joint pathology scores in Wistar rats were low, there were significant differences (P0.05), but there is no significant difference between SD and Wistar rats.
Experiment two:
Dynamic analysis of the incidence of arthritis in different castration time showed that there was no significant difference between the control group CIA and the CIA ovariectomized rats or the CIA rats with different castration time (P=0.9159).
The results of repeated measures analysis showed that: (1) gender did not affect AI (P=0.667); (2) compared with the control group CIA, AI increased significantly (4) compared with the ovariectomized group (P=0.006), and there was a significant difference compared with the 1 week of castration (P=0.014), but there was no significant difference between the other groups.
Considering the two factors of sex and castration time, the influence of pathological score was analyzed by two way ANOVA. The results showed that there was no significant difference in gender between the two groups (P0.05), and the time of castration time had significant difference in pathological score (P0.05).
Two factor analysis of variance statistics showed that the effect of gender on CORT was significant (P0.0001), data indicating the interaction between gender and group (P0.05), so do the differences by gender stratified analysis can understand the different gender groups. The results showed that after castration, different responses to CORT don't change of the rat instead that compared with non castrated CIA group, ovariectomized female rats in CIA group plasma CORT levels were significantly lower (P0.01, P0.05), remove the potential for 1 weeks, CORT rats were significantly increased (P < 0.01, P0.05), and castrated time Cheng Zhengxiang Kwan, CORT and plasma of castrated rats castrated for 3 weeks 4 week compared with no significant difference.
Double factor variance analysis showed that gender statistics against type II collagen antibody have no significant difference (P=0.384), castration time on type II collagen antibody had significant difference (P0.0001), LSD 22 shows with castration time increased, collagen antibody was significantly increased, compared with CIA control group, castrated 2,3,4 weeks the rats in group CIA serum collagen antibody levels were significantly increased (P0.05), 1,2,3,4 CIA group II anti castration week rat serum collagen antibody level had significant difference (P0.05).
Two factor analysis of variance statistics showed that the effect of gender on IL-6 was significant (P=0.0034). The data showed no interaction between gender and group (P=0.3356), the influence of castration time on IL-6 have significant difference (P0.0001 22), compared with the LSD test, the results showed that compared with CIA control group, castrated for 2 weeks 3 weeks, 4 weeks, the level of IL-6 of CIA group rats increased significantly (P0.05), ovariectomized for 1 weeks, 2 weeks, 3 weeks, 4 weeks in the CIA group rats serum IL-6 levels also showed a significant difference (P0.05), with castration time increased, increased significantly.
Double factor variance analysis results showed that the effect of gender on IL-10 was significant (P0.0001). The data showed no interaction between gender and group (P=0.3356), the influence of castration time on IL-10 have significant difference (P0.0001), 22 were compared with LSD test. The results showed that the plasma IL-10 level in CIA group rats with ovariectomy time increased significantly, there were significant differences (P0.05).
Conclusion:
(1) the rat arthritis model induced by collagen, Wistar and SD in rats and the incidence degree were significantly higher than that of.Wistar and SD Lewis rat incidence rate had no significant difference, but the incidence of SD rats was significantly higher than that of Wistar rats. No significant difference between the three strains of large between male and female rats and the incidence degree. Therefore, in the production of arthromyodynia (rheumatoid arthritis) rat model, it is recommended that the preferred SD rats, then you can select the Wistar rats, both male and female.
(2) kidney plays an important role in the pathogenesis of CIA, with the prolonged time of castration, the severity of kidney deficiency, CIA incidence rate and the degree is also increased; in the incidence of castration effectively after 4 weeks can reach 95%, and the incidence was significantly higher than the same period in non ovariectomized rats. Therefore, in making kidney arthralgia (rheumatoid arthritis) animal model combining disease with syndrome, you can use the C II in castrated rats at 4 weeks of immune induction.
(3) according to the above results, and combined with the basis of the preliminary work, kidney deficiency syndrome (rheumatoid arthritis) combination of disease and syndrome specific production methods are as follows: the rat model of SD rats, rats aged 6 weeks of age, SPF level, male and female can. According to conventional methods of resection in rats with bilateral testes or ovaries. 4 weeks after castration, take appropriate amount of type II collagen solution (concentration of 2mg/ml) incomplete Freund's adjuvant was added dropwise to the volume, type II collagen concentration was 1mg/ml. in the ice bath with fully emulsified homogenizer, adding water to the diffusion of emulsified mixture, taken by 0.2ml/. The 200 gC II / only to tail root after hypodermic injection of.7d by 0.1ml/, namely 100 g C II / only to tail root subcutaneous booster immunization.
【学位授予单位】:中国中医科学院
【学位级别】:硕士
【学位授予年份】:2010
【分类号】:R255.6;R-332
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