本文選題：類風(fēng)濕性關(guān)節(jié)炎 + 京尼平苷酸��； 參考：《河北醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis,RA)是一種以累及周圍關(guān)節(jié)為主的多系統(tǒng)慢性自身免疫性疾病,以滑膜組織炎性增生、關(guān)節(jié)軟骨進(jìn)行性破壞為特征。該病遍布世界各地,各個(gè)種族均有發(fā)病。RA的傳統(tǒng)治療藥物主要包括非甾體類抗炎藥、抗風(fēng)濕藥、糖皮質(zhì)激素、植物藥、腫瘤壞死因子α拮抗劑等幾大類。目前臨床上應(yīng)用的藥物主要用于控制和緩解RA癥狀,且存在較多的不良反應(yīng),所以尋找高效低毒治療RA的新藥是今后研究的重要方向。我們前期研究發(fā)現(xiàn),中藥梔子具有顯著抗炎鎮(zhèn)痛作用,梔子浸膏及其單體京尼平苷酸明顯抑制RA大鼠足腫脹,提示該藥物對(duì)治療RA有較好的作用前景。其中京尼平苷酸(geniposidic acid,GA),又名梔子苷酸,是一種環(huán)烯醚萜類化合物。MAPK信號(hào)轉(zhuǎn)導(dǎo)通路包括ERK1/2、JNK1/2,P38共5條途徑,是真核生物信號(hào)傳遞網(wǎng)絡(luò)中的重要途徑之一,在基因表達(dá)調(diào)控和細(xì)胞質(zhì)功能活動(dòng)中發(fā)揮關(guān)鍵作用,而信號(hào)轉(zhuǎn)導(dǎo)途徑的活化是類風(fēng)濕性關(guān)節(jié)炎慢性滑膜炎的典型特征。因此我們猜測(cè),京尼平苷酸治療類風(fēng)濕性關(guān)節(jié)炎可能與此途徑有關(guān)。但京尼平苷酸如何作用于RA滑膜細(xì)胞、并進(jìn)一步影響細(xì)胞內(nèi)的MAPK信號(hào)轉(zhuǎn)導(dǎo)通路,從而調(diào)節(jié)炎癥介質(zhì)分泌和功能蛋白的表達(dá),目前并不清楚,尚需揭示其作用機(jī)制,為進(jìn)一步將其篩選為有效藥物提供理論依據(jù)。方法:本實(shí)驗(yàn)主要分為四個(gè)部分:第一部分實(shí)驗(yàn):用弗氏完全佐劑建立大鼠佐劑型關(guān)節(jié)炎(AA)模型并分離培養(yǎng)成纖維樣滑膜細(xì)胞。據(jù)文獻(xiàn)報(bào)道FLS在體外培養(yǎng)后能夠穩(wěn)定增生。我們?nèi)〉?代成纖維樣滑膜細(xì)胞進(jìn)行免疫細(xì)胞化學(xué)鑒定;第二部分實(shí)驗(yàn)分為5組:對(duì)照組(1640培養(yǎng)基,不含藥物),京尼平苷酸高(終濃度為1×10-5 molL-1)、中(終濃度為1×10-6 molL-1)、低(終濃度為1×10-7 molL-1)給藥組和甲氨蝶呤(methotrexate,MTX)陽(yáng)性對(duì)照藥組(終濃度為1×10-6 molL-1)。給予藥物干預(yù)后,檢測(cè)相關(guān)指標(biāo)。分別在加入藥物24小時(shí)、48小時(shí)和72小時(shí)用MTT法檢測(cè)細(xì)胞增殖情況,用Hoechst33342和PI雙染熒光顯微鏡觀察給予不同濃度GA后凋亡和壞死的成纖維樣滑膜細(xì)胞的細(xì)胞形態(tài)學(xué)改變。第三部分試驗(yàn):研究京尼平苷酸對(duì)細(xì)胞上清液細(xì)胞因子的影響。分別用ELISA法測(cè)定給藥后細(xì)胞上清液中IL-1β、TNF-α、IL-10的含量。通過(guò)檢測(cè)各類細(xì)胞因子含量的變化探討京尼平苷酸對(duì)類風(fēng)濕性關(guān)節(jié)炎的治療作用機(jī)制;第四部分實(shí)驗(yàn):研究京尼平苷酸對(duì)成纖維樣滑膜細(xì)胞的Ras-MAPKS途徑的影響。用RT-PCR法檢測(cè)JNK、ERK、P38基因m RNA表達(dá),用western blotting檢測(cè)磷酸化的JNK、ERK、P38蛋白表達(dá)的影響,從細(xì)胞膜信號(hào)傳導(dǎo)途徑探討京尼平苷酸在RA中的作用機(jī)制。結(jié)果:第一部分實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)AA模型建立成功:大鼠在用弗氏完全佐劑注射后第14天開(kāi)始出現(xiàn)足部紅腫。炎癥出現(xiàn)的高峰期是在免疫后的第25天。大鼠逐漸開(kāi)始出現(xiàn)活動(dòng)障礙,并可見(jiàn)足部、尾根部“關(guān)節(jié)結(jié)節(jié)”。將關(guān)節(jié)炎指數(shù)3分以上的大鼠脫臼處死,取大鼠病變部位關(guān)節(jié)滑膜組織進(jìn)行分離與培養(yǎng)。培養(yǎng)后的第3代成纖維樣滑膜細(xì)胞進(jìn)行免疫細(xì)胞化學(xué)染色,陽(yáng)性細(xì)胞數(shù)量達(dá)到95%以上,符合實(shí)驗(yàn)要求,原代細(xì)胞培養(yǎng)理想。第二部分實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),京尼平苷酸高濃度組(10-5 molL-1)能夠抑制滑膜細(xì)胞增殖,較之模型對(duì)照組比較明顯降低(P0.05);京尼平苷酸高濃度組(10-5 molL-1)、京尼平苷酸中濃度組(10-6 molL-1)能夠促進(jìn)滑膜細(xì)胞凋亡,較之模型對(duì)照組比較明顯降低(P0.05)。第三部分試驗(yàn)細(xì)胞因子的檢測(cè)發(fā)現(xiàn):加京尼平苷酸藥物后,治療組細(xì)胞上清液中京尼平苷酸高濃度組(10-5 molL-1),京尼平苷酸中濃度組(10-6 molL-1)中的IL-1β、TNF-α的含量較之模型對(duì)照組比較明顯降低(P0.05);京尼平苷酸高濃度組(10-5 molL-1)中的IL-10的含量明顯上調(diào)。第四部分試驗(yàn)由RT-PCR結(jié)果可以看出:京尼平苷酸高濃度組(10-5 molL-1),京尼平苷酸中濃度組(10-6 molL-1)能夠抑制JNK、ERK、P38基因m RNA表達(dá)。由western blotting結(jié)果可以看出:京尼平苷酸高濃度組(10-5 molL-1),京尼平苷酸中濃度組(10-6 molL-1)能夠抑制磷酸化JNK、ERK、P38蛋白的表達(dá)。結(jié)論:1足夠量濃度的京尼平苷酸能夠抑制成纖維樣滑膜細(xì)胞的增生,促進(jìn)成纖維樣滑膜細(xì)胞的凋亡。2足夠量濃度的京尼平苷酸對(duì)于成纖維樣滑膜細(xì)胞的上清液中IL-1β、TNF-α的含量起到抑制分泌作用,對(duì)成纖維樣滑膜細(xì)胞的上清液中IL-10的含量起到刺激分泌的作用。3足夠量濃度的京尼平苷酸能抑制成纖維樣滑膜細(xì)胞中磷酸化的JNK、ERK、P38-MAPKs蛋白及m RNA的表達(dá)。綜合以上四部分實(shí)驗(yàn)結(jié)果,我們發(fā)現(xiàn)京尼平苷酸對(duì)于AA有很好的治療作用,可以明顯抑制成纖維樣滑膜細(xì)胞的增殖,促進(jìn)成纖維樣滑膜細(xì)胞的凋亡。抑制成纖維樣滑膜細(xì)胞釋放炎性因子IL-1β、TNF-α,促進(jìn)保護(hù)性因子IL-10的釋放。同時(shí)還可以抑制AA大鼠成纖維樣滑膜細(xì)胞中的JNK、ERK、P38-MAPKs信號(hào)通路的激活。
[Abstract]:Objective: rheumatoid arthritis (RA) is a multi system chronic autoimmune disease involving the surrounding joints, characterized by inflammatory hyperplasia of the synovial tissue and the progressive destruction of articular cartilage. The disease is all over the world, and the traditional therapeutic drugs for the pathogenesis of.RA, including non steroidal anti-inflammatory drugs, are all around the world. Antirheumatic drugs, glucocorticoids, plant drugs, tumor necrosis factor alpha antagonists, and other major categories. The current clinical drugs are mainly used to control and alleviate RA symptoms, and there are many adverse reactions. So it is an important direction for future research to find new drugs with high efficiency and low toxicity for the treatment of RA. Anti inflammatory and analgesic effects, gardenia extract and mono geniposide acid obviously inhibit foot swelling in RA rats, suggesting that the drug has a good role in the treatment of RA. Among them, geniposide acid (geniposidic acid, GA), also known as geniposide, is a.MAPK signal transduction pathway including ERK1/2, JNK1/2 and P38, which is a kind of enidoterpenoids. One of the important pathways in the eukaryotic signal transmission network plays a key role in the regulation of gene expression and cytoplasmic function, and the activation of signal transduction pathway is a typical characteristic of chronic synovitis in rheumatoid arthritis. Therefore, we suspect that geniposide may be related to this pathway in the treatment of rheumatoid arthritis. How niping glycoside acts on RA synovial cells and further affects the MAPK signal transduction pathway in the cells, thus regulating the secretion of inflammatory mediators and the expression of functional proteins, is not clear at present. It is still necessary to reveal its mechanism of action and provide a theoretical basis for further screening of its effective drugs. Methods: this experiment is divided into four parts: the first part: A part of the experiment: the model of rat adjuvant arthritis (AA) was established with Freund's complete adjuvant and fibrous synovial cells were isolated and cultured. It was reported that FLS could be stable after culture in vitro. We took third generations of fibroblast like synovial cells for immunocytochemical identification; the second part of the experiment was divided into 5 groups: the control group (1640 medium, no) Drugs), geniposide acid high (final concentration is 1 x 10-5 mol? L-1), medium (final concentration is 1 * 10-6 mol? L-1), low (final concentration is 1 * 10-7 mol? L-1) administration group and methotrexate (methotrexate, MTX) positive control group (terminal concentration is 1 * 10-6 mol? L-1). Give drug drying prognosis, detection of related indicators, 24 hours, 48 hours and 72, respectively. The cell proliferation was detected by MTT method. The morphological changes of fibroid synovial cells with apoptosis and necrosis after different concentrations of GA were observed by Hoechst33342 and PI double staining fluorescence microscope. The third part experiment: To study the effect of geniposide on cytokine of cell supernatant. The cells were measured by ELISA method respectively. The content of IL-1 beta, TNF- a, IL-10 in the clear liquid. The therapeutic mechanism of Geniposide in the treatment of rheumatoid arthritis was explored by detecting the changes in the content of various cytokines. The fourth part of the experiment was to study the effect of geniposide on the Ras-MAPKS pathway of fibroid synovial cells. JNK, ERK, P38 gene m RNA expression was detected by RT-PCR, and West was used. Ern blotting detected the effects of phosphorylated JNK, ERK, P38 protein expression, and explored the mechanism of Geniposide acid in RA from cell membrane signal transduction pathway. Results: the first part of the experiment found that the AA model was established successfully: the rats began to appear foot swelling at fourteenth days after the injection of the Freund complete adjuvant. The peak period of the inflammation appeared at the peak period. After twenty-fifth days of immunization, the rats gradually began to appear activity disorder, and the foot and tail nodules were seen. The rats were dislocated and killed in the rats with the arthritis index more than 3 points. The synovial tissue of the lesion parts of the rats was isolated and cultured. The cultured third generation fibroblast like cells were stained with immunocytochemical staining and positive cells. The number reached more than 95%, which was in line with the experimental requirements and the primary cell culture was ideal. The second part of the experiment found that the high concentration group (10-5 mol? L-1) could inhibit the proliferation of synovial cells (P0.05) compared with the model control group (10-5 mol? L-1), and the concentration group of Geniposide (10-6 mol? L-1). The apoptosis of synovial cells was significantly reduced (P0.05) compared with the model control group (P0.05). After the test of cytokine, the content of IL-1 beta in the concentration group (10-6 mol? L-1) in the concentration group (10-6 mol? L-1) in the cell supernatant of the treatment group was compared with the model of IL-1 beta, and the content of TNF- a was more than that of the model. The control group was significantly lower (P0.05); the content of IL-10 in the high concentration group of Geniposide (10-5 mol? L-1) was obviously up-regulated. The fourth part of the test showed that the high concentration group of Geniposide acid (10-5 mol? L-1) and the concentration group of Geniposide (10-6 mol? L-1) could inhibit JNK, ERK, P38 gene expression. Ng results showed that the high concentration group of Geniposide (10-5 mol? L-1), the concentration group of Geniposide (10-6 mol? L-1) could inhibit the expression of phosphorylated JNK, ERK, P38 protein. Conclusion: 1 concentration of Geniposide can inhibit the proliferation of fibroid synovial cells and promote the concentration of apoptotic.2 in fibroid synovial cells. Geniposide acid inhibits the secretion of IL-1 beta, TNF- a in the supernatant of fibroid synovial cells and stimulates the secretion of IL-10 in the supernatant of fibroid synovial cells..3 sufficient concentration of Geniposide can inhibit the phosphorylation of JNK, ERK, P38-MAPKs protein in fibroblast like synovial cells. M RNA expression. Combined with the four parts of the experimental results, we found that geniposide has a good therapeutic effect on AA, which can obviously inhibit the proliferation of fibroid synovial cells and promote the apoptosis of fibroid synovial cells. Inhibition of fibroid synovial cells release inflammatory factor IL-1 beta, TNF- alpha, and promote the release of protective factor IL-10. It can also inhibit the activation of JNK, ERK and P38-MAPKs signaling pathways in fibroblast like synovial cells of AA rats.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.22

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相關(guān)博士學(xué)位論文 前1條

1 李芯;雷公藤治療類風(fēng)濕關(guān)節(jié)炎的療效及安全性評(píng)估[D];北京協(xié)和醫(yī)學(xué)院;2012年

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