本文選題：痹證 + 類風(fēng)濕性關(guān)節(jié)炎��； 參考：《中國中醫(yī)科學(xué)院》2010年碩士論文

【摘要】： 目的： 探討腎虛痹證(類風(fēng)濕性關(guān)節(jié)炎)病證結(jié)合動物模型的制作方法,為研究中醫(yī)藥治療腎虛痹證(類風(fēng)濕性關(guān)節(jié)炎)的作用及其機理提供實驗工具,并以期為病證結(jié)合動物模型的制作提供進一步的科學(xué)依據(jù)。 方法： 本課題分為兩部分實驗來進行研究： 實驗一：選用6周齡SPF級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型膠原與不完全弗氏佐劑混合物的方法誘導(dǎo)出關(guān)節(jié)炎模型,對不同品系、不同性別大鼠在膠原誘導(dǎo)性關(guān)節(jié)炎(Collagen-induced arthritis, CIA)模型中的發(fā)病情況進行比較研究。 實驗二：選用6周齡SPF級SD大鼠100只,雌雄各半,除對照組大鼠外,分別切除大鼠的雙側(cè)睪丸和卵巢,制作腎虛動物模型。分別在去勢1周、2周、3周、4周后,采用尾根部皮下注射Ⅱ型膠原與不完全弗氏佐劑混合物的方法誘導(dǎo)出關(guān)節(jié)炎模型,從而對不同去勢時間(即不同腎虛狀態(tài))下,CIA發(fā)病情況進行比較研究,并采用ELISA等方法對其機理進行初步探討。 結(jié)果： 實驗一： 對三種不同品系大鼠發(fā)病情況數(shù)據(jù)進行動態(tài)分析,發(fā)現(xiàn)不同種屬發(fā)病率不同,χ2=72.94,P0.0001；時間與發(fā)病率相關(guān),χ2=15.39,P=0.0005,有顯著性差異,隨著免疫時間的延長,發(fā)病率越高。兩兩比較顯示,SD和Wistar大鼠與Lewis大鼠發(fā)病率有顯著性差異(P均0.0001),但SD與Wistar無顯著性差異(P=0.6183)。 重復(fù)測量方差分析結(jié)果顯示：(1)不同品系大鼠AI不同,有顯著性差異(P0.0001)；組間兩兩比較結(jié)果顯示：Lewis比SD、wistar大鼠AI低,有顯著性差異(P0.0001),且SD比wistar大鼠AI高,差異亦具有顯著性(P=0.045)。(2)性別對AI沒有影響(P=0.737)。免疫28d內(nèi)各時段與其他時間相比均有顯著性差異(P0.0001),28d后各時段比無顯著性差異(P均0.05)。 顯著性檢驗考慮性別和屬種兩因素對病理積分的影響,采用雙因素方差分析統(tǒng)計,總體方差分析結(jié)果顯示性別對病理積分影響無顯著性(P0.05),種屬對病理積分影響有顯著性差異(P0.05)。兩兩比較采用LSD法檢驗,結(jié)果顯示,Lewis比SD、Wistar大鼠關(guān)節(jié)病理積分低,有顯著性差異(P0.05),而SD與Wistar大鼠之間無顯著性差異。 實驗二: 對不同去勢時間關(guān)節(jié)炎發(fā)病情況數(shù)據(jù)進行動態(tài)分析,發(fā)現(xiàn)對照CIA組大鼠與CIA去勢組大鼠,或者不同去勢時間CIA大鼠發(fā)病率影響沒有顯著性差異(P=0.9159)。 重復(fù)測量方差分析結(jié)果顯示：(1)性別對AI沒有影響(P=0.667)；(2)與對照CIA組相比,與去勢4周組AI明顯升高(P=0.006),與去勢1周相比,亦有顯著性差異(P=0.014),其余各組之間相比均無顯著性差異。 考慮性別和去勢時間兩因素對病理積分的影響,采用雙因素方差分析統(tǒng)計,結(jié)果顯示性別對病理積分影響無顯著性(P0.05),去勢時間對病理積分影響有顯著性差異(P0.05)。 雙因素方差分析統(tǒng)計結(jié)果顯示性別對CORT影響有顯著性(P0.0001),數(shù)據(jù)顯示性別和分組之間有交互作用(P0.05),所以按性別做分層分析可以了解不同性別分組之間的差異。結(jié)果顯示,去勢后,不同性別大鼠對CORT的反應(yīng)變化相反,即與未去勢CIA組比較,雌性去勢CIA組大鼠血漿CORT水平明顯降低(P0.01,P0.05),除去勢1周組外,雄性大鼠CORT均顯著升高(P＜0.01,P0.05),且與去勢時間呈正相關(guān),大鼠去勢3周血漿CORT與去勢4周相比,無顯著性差異。 雙因素方差分析統(tǒng)計顯示性別對抗Ⅱ型膠原抗體影響無顯著性(P=0.384),去勢時間對抗Ⅱ型膠原抗體影響有顯著性差異(P0.0001),LSD法兩兩比較顯示,隨著去勢時間增加,抗Ⅱ型膠原抗體明顯升高,與對照CIA組比較,去勢2、3、4周CIA組大鼠血清抗Ⅱ型膠原抗體水平明顯升高(P0.05),去勢1、2、3、4周CIA組大鼠血清抗Ⅱ型膠原抗體水平有顯著性差異(P0.05)。 雙因素方差分析統(tǒng)計結(jié)果顯示性別對IL-6影響有顯著性(P=0.0034),數(shù)據(jù)顯示性別和分組之間無交互作用(P=0.3356),去勢時間對IL-6影響有顯著性差異(P0.0001),兩兩比較采用LSD法檢驗,結(jié)果顯示,與對照CIA組比較,去勢2周、3周、4周CIA組大鼠IL-6水平明顯升高(P0.05),去勢1周、2周、3周、4周CIA組大鼠血清IL-6水平亦有顯著性差異(P0.05),隨著去勢時間增加,明顯升高。 雙因素方差分析結(jié)果顯示性別對IL-10影響有顯著性(P0.0001),數(shù)據(jù)顯示性別和分組之間無交互作用(P=0.3356),去勢時間對IL-10影響有顯著性差異(P0.0001),兩兩比較采用LSD法檢驗,結(jié)果顯示,各組CIA組大鼠血漿IL-10水平隨著去勢時間增加,明顯升高,均有顯著性差異(P0.05)。 結(jié)論： (1)對于Ⅱ型膠原免疫誘導(dǎo)的大鼠關(guān)節(jié)炎模型,Wistar和SD大鼠發(fā)病率和發(fā)病程度均明顯高于Lewis大鼠。Wistar和SD大鼠發(fā)病率無顯著性差異,但發(fā)病程度SD大鼠明顯高于Wistar大鼠。三種品系大鼠雌雄之間發(fā)病率和發(fā)病程度無明顯差異。因此,在制作痹證(類風(fēng)濕性關(guān)節(jié)炎)大鼠模型時,建議首選SD大鼠,其次可以選擇Wistar大鼠,雌雄均可。 (2)腎虛在CIA發(fā)病過程中起著重要作用,隨著去勢時間的延長,即腎虛程度加重,CIA有效發(fā)病率和發(fā)病程度亦隨之增加；在去勢4周后其有效發(fā)病率就可達(dá)到95%,且發(fā)病程度明顯高于同期未去勢大鼠。因此,在制作腎虛痹證(類風(fēng)濕性關(guān)節(jié)炎)病證結(jié)合動物模型時,在大鼠去勢4周時就可用CⅡ進行免疫誘導(dǎo)。 (3)根據(jù)以上實驗結(jié)果,并結(jié)合前期工作基礎(chǔ),腎虛痹證(類風(fēng)濕性關(guān)節(jié)炎)病證結(jié)合大鼠模型的具體制作方法如下：選用SD大鼠,鼠齡6周齡,SPF級,雌雄均可。按常規(guī)方法切除大鼠的雙側(cè)睪丸或卵巢。于去勢后4周,取適量Ⅱ型膠原溶液(濃度為2mg/ml)逐滴加入至等容積的不完全弗氏佐劑中,Ⅱ型膠原終濃度為1mg/ml。冰浴中用勻漿器充分乳化,以滴加水中不擴散為度,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,于尾根部皮下加強免疫一次。
[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.

【學(xué)位授予單位】：中國中醫(yī)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R255.6;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 樓宇舫;;從腎論治話痹證[J];浙江中醫(yī)藥大學(xué)學(xué)報;2007年03期

2 張吉;針?biāo)幉⒂弥委煶Ｒ娒庖咝约膊”宰C146例臨床觀察[J];中國醫(yī)藥學(xué)報;2004年11期

3 高崗;楊根金;婁子洋;;腎虛證大鼠尿液的核磁共振譜代謝組學(xué)研究[J];第二軍醫(yī)大學(xué)學(xué)報;2009年05期

4 李芬,吳轟,鄧軍衛(wèi),游學(xué)科,魏啟幼,范松青,朱亞輝,盧光t;以轉(zhuǎn)基因動物研究類風(fēng)濕關(guān)節(jié)炎的易感基因[J];中華風(fēng)濕病學(xué)雜志;1999年02期

5 申秀云;;益腎壯骨祛風(fēng)除濕法防治類風(fēng)濕性關(guān)節(jié)炎的實驗研究[J];福建中醫(yī)學(xué)院學(xué)報;2009年01期

6 張相宏;;類風(fēng)濕性關(guān)節(jié)炎的中醫(yī)發(fā)病機制探討[J];甘肅中醫(yī);2007年04期

7 王開峰;辨證分型治療類風(fēng)濕性關(guān)節(jié)炎62例[J];廣西中醫(yī)藥;2000年03期

8 梅小斌;類風(fēng)濕關(guān)節(jié)炎腎損害[J];國外醫(yī)學(xué).泌尿系統(tǒng)分冊;2002年02期

9 焦樹德;再談\*痹的辨證論治[J];河北中醫(yī);2004年11期

10 何永生,方瑯;類風(fēng)濕性關(guān)節(jié)炎的中醫(yī)病因探討[J];河南中醫(yī);2003年05期

，

本文編號：1757443