本文選題：多糖 + 對(duì)人�。� 參考：《武漢大學(xué)》2016年博士論文

【摘要】：背景：滑膜炎是一種常見病,多發(fā)病,其病程長(zhǎng),類型多,且容易反復(fù)發(fā)作,患者出現(xiàn)關(guān)節(jié)腫痛,活動(dòng)受限,嚴(yán)重影響日常生活。尤其以細(xì)菌性滑膜炎,類風(fēng)濕性滑膜炎為著,難以治愈。臨床以無菌性滑膜炎多見,滑膜炎是關(guān)節(jié)炎的早期癥狀,人類關(guān)節(jié)滑膜為疏松結(jié)締組織,可分為兩層：滑膜襯里層和滑膜襯里下層�；ひr里層是由具有巨噬細(xì)胞樣功能的A型細(xì)胞及纖維母細(xì)胞樣的B型細(xì)胞組成,而襯里下層是由疏松結(jié)締組織基質(zhì)構(gòu)成,與其深層的纖維細(xì)胞并無基底膜相隔,直接與纖維結(jié)締組織或脂肪組織相連接,故在關(guān)節(jié)發(fā)生炎癥時(shí)易蔓延到周圍組織,導(dǎo)致滑膜炎的發(fā)生。人體中包含多個(gè)關(guān)節(jié),滑膜炎可發(fā)生在全身各個(gè)關(guān)節(jié)之中。其中,膝關(guān)節(jié)具有特殊的解剖結(jié)構(gòu),是全身關(guān)節(jié)中滑膜最多的關(guān)節(jié)。而且,膝關(guān)節(jié)容易受到外力等的影響而出現(xiàn)損傷。因此,在臨床中,滑膜炎大多以膝關(guān)節(jié)最為多見。而滑膜炎一旦出現(xiàn),如果治療不及時(shí),就遷延成慢性膝關(guān)節(jié)滑膜炎�；颊叩南リP(guān)節(jié)便會(huì)逐漸呈現(xiàn)出腫脹和疼痛等癥狀,長(zhǎng)此以往,就會(huì)對(duì)患者的膝關(guān)節(jié)功能產(chǎn)生嚴(yán)重的影響,導(dǎo)致功能障礙的出現(xiàn),嚴(yán)重影響患者的日常生活和工作�；ぱ资窃缙诠顷P(guān)節(jié)炎(Osteoarthritis, OA)中常見的癥狀之一,被大家認(rèn)為是炎癥反應(yīng)。它主要是由炎性因子和促炎性因子導(dǎo)致滑膜的炎癥反應(yīng)和關(guān)節(jié)軟骨破壞的不平衡產(chǎn)生的。在長(zhǎng)期的致病因素作用下,導(dǎo)致滑膜增生并逐漸增厚,與此同時(shí)纖維層和細(xì)胞層被嵌入,血管增生。由于炎性細(xì)胞浸潤(rùn)與滑膜成纖維細(xì)胞的增生,導(dǎo)致滑膜毛細(xì)血管在滑膜和滑液中選擇性分布,從而使關(guān)節(jié)遭到進(jìn)一步破壞�；ぱ捉M織病理學(xué)表現(xiàn)為滑膜組織急性期充血,水腫,中性粒細(xì)胞和大量炎性細(xì)胞浸潤(rùn),滑膜血管擴(kuò)張,血漿和細(xì)胞外滲產(chǎn)生大量滲出液,滑膜細(xì)胞活躍,產(chǎn)生大量粘液素。慢性期病變的滑膜組織表現(xiàn)出明顯的血管增生和炎癥細(xì)胞浸潤(rùn)。在初步的流行病學(xué)調(diào)查中表明,膝關(guān)節(jié)骨性關(guān)節(jié)炎的患病率為60%,56歲及以上的患病率為49%。女性比男性更常見�；加泄顷P(guān)節(jié)炎患者更易發(fā)生滑膜炎疾病,滑膜炎同時(shí)加重骨關(guān)節(jié)炎的發(fā)生。由滑膜細(xì)胞因子,蛋白酶和前列腺素分泌的炎性細(xì)胞如PGE的活化直接參與關(guān)節(jié)軟骨的病損�；ぱ资荗A軟骨改變功能的早期表現(xiàn),促進(jìn)血管增生,異常的骨周轉(zhuǎn)率,從而嚴(yán)重?fù)p害關(guān)節(jié)結(jié)構(gòu)�；ず蛙浌窃谒械慕佑|口都有不同程度的軟骨糜爛,光學(xué)顯微鏡下可以看到,滑膜軟骨生長(zhǎng)侵蝕。關(guān)節(jié)軟骨的破壞,除了機(jī)械原因之外,細(xì)胞因子也起著關(guān)鍵的作用。主要是滑膜炎導(dǎo)致的IL-1分泌的增加,和金屬蛋白酶的分泌及活性增加,引起軟骨破壞。同時(shí)滑膜炎也是類風(fēng)濕性關(guān)節(jié)炎(Rheumatoid arthritis, RA)主要病理學(xué)表現(xiàn)。與其他慢性疾病相比,類風(fēng)濕關(guān)節(jié)炎的早期診斷率較低。此外,國外學(xué)者發(fā)現(xiàn),類風(fēng)濕性關(guān)節(jié)炎患者的預(yù)期壽命也相對(duì)較短。因?yàn)樗母咧職埪?使社會(huì)的經(jīng)濟(jì)負(fù)擔(dān)逐年上升,RA已成為必須注意的一個(gè)公共衛(wèi)生問題。但不幸的是,對(duì)RA的發(fā)病機(jī)理,目前的研究還不是很透徹明了,在以往對(duì)滑膜炎的發(fā)生發(fā)展及治療過程的研究中發(fā)現(xiàn),滑膜組織中存在的三種絲裂原活化蛋白激酶(MAPK)信號(hào)轉(zhuǎn)導(dǎo)通路,即細(xì)胞外調(diào)節(jié)蛋白激酶(ERK)信號(hào)轉(zhuǎn)導(dǎo)通路,P38絲裂原活化蛋白激酶MAPK信號(hào)轉(zhuǎn)導(dǎo)通路,C-jun氨基末端激酶信號(hào)轉(zhuǎn)導(dǎo)通路。以上信號(hào)轉(zhuǎn)導(dǎo)通路與滑膜炎的發(fā)生發(fā)展存在密切聯(lián)系。絲裂原活化蛋白激酶(MAPK)是真核生物信號(hào)傳遞網(wǎng)絡(luò)的重要組成部分,可以參與到細(xì)胞質(zhì)功能活動(dòng)以及基因表達(dá)調(diào)控等多項(xiàng)活動(dòng)中,并發(fā)揮十分重要的作用。是細(xì)胞內(nèi)廣泛表達(dá)的絲氨酸／蘇氨酸的蛋白激酶被磷酸化激活后,參與細(xì)胞的生長(zhǎng)、發(fā)育、分化、凋亡等一系列細(xì)胞生理活動(dòng),主要包括JNK,ERK,P38三條信號(hào)轉(zhuǎn)導(dǎo)通路。研究表明,絲裂原活化蛋白激酶(MAPK)能夠影響滑膜炎的發(fā)生發(fā)展。MAPK通路是一種信號(hào)轉(zhuǎn)導(dǎo)酶系統(tǒng),在受到一定應(yīng)激刺激之后,細(xì)胞內(nèi)部系統(tǒng)便會(huì)利用這一信號(hào)通路做出一定的反應(yīng)。而一旦這一信號(hào)通路發(fā)生異常變化,導(dǎo)致傳導(dǎo)異常,并容易導(dǎo)致滑膜炎的出現(xiàn)。受到一些特定細(xì)胞因子或者抗原的影響,MAPK可以通過相應(yīng)的信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng),進(jìn)入到細(xì)胞核之中,啟動(dòng)一系列行為的出現(xiàn)。但是,在發(fā)生異常的增殖之后,滑膜細(xì)胞的轉(zhuǎn)錄因子便會(huì)出現(xiàn)改變。這些轉(zhuǎn)錄活化因子的活性均與MAPK信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān),受到其調(diào)控。在細(xì)胞異常增殖的情況下,會(huì)導(dǎo)致大量促炎性細(xì)胞因子等的分泌。進(jìn)而導(dǎo)致滑膜細(xì)胞發(fā)生異常改變,導(dǎo)致其出現(xiàn)增生和肥厚等情況。脂多糖(Lipopolysaccharide, LPS)是革蘭氏陰性細(xì)菌細(xì)胞壁的具體結(jié)構(gòu)。是內(nèi)毒素的主要成分,脂多糖由核心多糖,O多糖側(cè)鏈和脂質(zhì)A組成,脂多糖可以通過絲裂原活化蛋白激酶MAPK信號(hào)通路作用于滑膜細(xì)胞,在一定條件下,革蘭氏陰性菌活菌可以緩慢釋放少量LPS,死亡后大量釋放LPS,革蘭氏陰性細(xì)菌感染可引起患者的血漿中的LPS水平顯著增加。也可以提高所引起的局部相繼感染的LPS水平。根據(jù)先前的研究中,在患有革蘭氏陰性細(xì)菌性陰道炎的婦女宮頸中,可以檢測(cè)到比較高水平的LPS。并且在研究中發(fā)現(xiàn),LPS可以通過絲裂原活化蛋白激酶MAPK信號(hào)通路抑制滑膜細(xì)胞的生長(zhǎng),其可以誘導(dǎo)滑膜炎的發(fā)生發(fā)展。脂多糖濃度與滑膜炎滑液中誘導(dǎo)型一氧化氮合酶表達(dá)成正相關(guān),其與炎性因子相互作用加重滑膜炎程度。這一作用是通過滑膜組織中存在的絲裂原活化蛋白激酶(MAPK)轉(zhuǎn)導(dǎo)通路實(shí)現(xiàn)的。目的：本研究以滑膜細(xì)胞為研究對(duì)象,用LPS作用滑膜細(xì)胞,尋找到LPS對(duì)滑膜細(xì)胞生長(zhǎng)的MAPK通路的作用機(jī)制。以至為進(jìn)一步研究MAPK通路蛋白在滑膜炎的發(fā)生和發(fā)展過程中存在的重要作用,以期找到新的治療靶點(diǎn),為制定有效治療滑膜炎方案奠定基礎(chǔ)。方法：用不同濃度的LPS作用滑膜細(xì)胞,然后用MTT和流式細(xì)胞儀檢測(cè)在不同時(shí)間的滑膜細(xì)胞的增長(zhǎng)情況,找到LPS對(duì)滑膜細(xì)胞生長(zhǎng)的最佳作用時(shí)間與濃度。通過用LPS作用于HS細(xì)胞,在不同時(shí)間收集細(xì)胞,提取蛋白,用Western blot的方法檢測(cè)MAPK通路中的關(guān)鍵蛋白ERK,JNK,p38的磷酸化狀況,從而確定其激活蛋白。然后用相應(yīng)的蛋白酶激動(dòng)劑作用磷酸化的關(guān)鍵蛋白,檢測(cè)細(xì)胞的生長(zhǎng)情況及下游的蛋白活化情況。為了進(jìn)一步研究LPS的作用機(jī)制,將LPS的受體TRL4用RNA干擾的方法進(jìn)行抑制,然后檢測(cè)蛋白ERK,JNK,p38的磷酸化狀況及細(xì)胞的生長(zhǎng)情況。結(jié)果：(1)用MTT法檢測(cè)了0,10,20和40ng/ml的LPS作用于人滑膜細(xì)胞后于第0,24,48和72h計(jì)算生長(zhǎng)率,當(dāng)加入LPS后從24h開始,其生長(zhǎng)率逐漸降低。在10ng/ml組中細(xì)胞的生長(zhǎng)率隨著時(shí)間的增加而降低,但沒有20和40ng/ml組降低明顯。在20ng/ml和40ng/ml組中其生長(zhǎng)情況相似。采用Annexin V/PI流式細(xì)胞分析法進(jìn)行了凋亡率的定量研究,檢測(cè)HS細(xì)胞0,10ng/ml,20ng/ml,40ng/ml的LPS處理后的凋亡率。不同濃度LPS處理HS細(xì)胞后,能誘導(dǎo)HS細(xì)胞發(fā)生凋亡誘導(dǎo)作用,且凋亡率隨LPS濃度的增加而增加,當(dāng)LPS濃度為20ng/ml時(shí),細(xì)胞凋亡率達(dá)到極值,值后不再變化。這就表明20ng/ml的LPS在48h時(shí)對(duì)HS細(xì)胞的生長(zhǎng)率抑制情況最佳為24.0340.3 1%。(2)在本研究中用總濃度為20ng/ml的LPS作用于HS細(xì)胞磷酸化JNK(p-JNK)的條帶隨著時(shí)間的增加表達(dá)量越來越低,而非磷酸化的JNK(T-JNK)條帶在各個(gè)時(shí)間的表達(dá)基本一致。對(duì)于P-ERK和T-ERK的條帶在各個(gè)時(shí)間的表達(dá)也基本一致。而p-p38的條帶在各個(gè)時(shí)間都很淺幾乎看不清。而T-p38的條帶則很均一且很深。這就表明LPS對(duì)HS細(xì)胞生長(zhǎng)的抑制作用是通過抑制磷酸化JNK來實(shí)現(xiàn)的。而且在LPS作用40min時(shí)已經(jīng)發(fā)揮到極致。為了進(jìn)一步證明LPS對(duì)HS細(xì)胞生長(zhǎng)的抑制作用是通過JNK通路實(shí)現(xiàn)的,采用了JNK的激動(dòng)劑anisomycin作用于HS細(xì)胞,同時(shí)加入LPS來證明磷酸化的JNK蛋白情況,以及JNK下游蛋白磷酸化的ATF-2情況。結(jié)果表明當(dāng)加入Anisomycin時(shí),對(duì)P-ATF-2/ATF-2的值從一開始到60min均沒有明顯變化,且表達(dá)值較高(P0.05).同時(shí)研究了當(dāng)加入Anisomycin后,細(xì)胞的生長(zhǎng)情況,細(xì)胞的生長(zhǎng)速度較快從一開始的85.19%長(zhǎng)到了第六天的96.52%,在這六天中細(xì)胞始終保持較高的生長(zhǎng)速度。(3)提取HS的RNA并進(jìn)行了充分TLR4的RT-PCR實(shí)驗(yàn),然后將PCR產(chǎn)物進(jìn)行1%的瓊脂糖凝膠電泳,在成像系統(tǒng)中可以看到TLR4的表達(dá)條帶。表明了TLR4在核酸水平上游表達(dá)。為進(jìn)一步證明TLR4的表達(dá),提取了HS的蛋白,進(jìn)行了Western blot實(shí)驗(yàn)。結(jié)果同樣表明TLR4在HS細(xì)胞中有表達(dá)。利用RNA干擾技術(shù)將TLR4沉默。同時(shí)應(yīng)用PCR技術(shù)和Western blot技術(shù)驗(yàn)證了TLR4被成功沉默。同時(shí)還篩選出了TLR4敲除的細(xì)胞系。用SiRNA的方法將TLR4干擾后,并經(jīng)篩選后得到穩(wěn)定的細(xì)胞細(xì)胞系,然后,用MTT法檢測(cè)了用20ng/ml的LPS作用于HS細(xì)胞后,于第0,24,48和72h檢測(cè)細(xì)胞的生長(zhǎng)情況,并計(jì)算細(xì)胞的生長(zhǎng)率,在干擾組中(SiRNA+LPS組)細(xì)胞在此期間的生長(zhǎng)呈增長(zhǎng)趨勢(shì),無顯著變化(P0.05)。而在對(duì)照組中(LPS組)細(xì)胞的生長(zhǎng)率逐漸下降。TLR4被抑制后HS細(xì)胞中ERK,JNK,p38的磷酸化情況,本研究對(duì)蛋白進(jìn)行了灰度值得分析,同時(shí)將對(duì)應(yīng)的磷酸化蛋白與非磷酸化蛋白的灰度值相比,而后將所得值進(jìn)行比較。無論是p-JNK/T-JNK還是p-ERK/T-ERK和P-P38/T-P38的值均未隨著時(shí)間的增加而變化,且p-JNK/T-JNK的值明顯低于TLR4未被抑制時(shí)的值。結(jié)論：總的來說,我們的結(jié)果表明LPS抑制HS的增長(zhǎng)主要信號(hào)通路是通過MAPK通路中JNK通路途徑實(shí)現(xiàn)的。為進(jìn)一步的研究MAPK通路蛋白在滑膜炎的發(fā)生和發(fā)展過程中存在的重要作用,找到新的治療靶點(diǎn),制定有效治療滑膜炎方案奠定基礎(chǔ)。
[Abstract]:Background: synovitis is a common disease, frequently occurring disease, long course of disease, many types, and easy to relapse. The patients have joint swelling and pain, and their activities are limited, which seriously affect daily life. Especially, bacterial synovitis and rheumatoid synovitis are difficult to cure. Clinical symptoms of non bacterial synovitis are common, synovitis is an early symptom of arthritis. The synovium of the joint synovium is a loose connective tissue, which can be divided into two layers: the synovial lining and the underlining of the synovial lining. The synovial lining is composed of A type cells with macrophage like function and B cells like fibroblast like cells, and the underlayer of the lining is composed of loose connective tissue matrix and no basement membrane separated from its deep fibrous cells. Connecting with fibrous connective tissue or adipose tissue, it is easy to spread to the surrounding tissue during the joint inflammation, leading to the occurrence of synovitis. The body contains multiple joints and synovitis can occur among the various joints of the body. Among them, the knee joint has a special anatomical structure, the most synovial joint in the joint. In clinical, synovitis is most common in the knee joint. Once the synovitis occurs, if the treatment is not timely, the synovitis will be extended to chronic knee synovitis. The patient's knee joint will gradually show swelling and pain and other symptoms, in the long run, the knee joint will be on the patient's knee joint. Function has a serious effect that causes the appearance of dysfunction, which seriously affects the daily life and work of the patient. Synovitis is one of the common symptoms in Osteoarthritis (OA). It is considered to be an inflammatory reaction. It is mainly caused by inflammatory factors and proinflammatory factors leading to the inflammation of the synovial membrane and the destruction of the articular cartilage. The imbalance is produced. The synovial hyperplasia and thickening of the synovial membrane and the proliferation of the blood vessels are caused by the long-term pathogenic factors. The infiltration of inflammatory cells and the proliferation of the synovial fibroblasts can lead to the selective distribution of the synovial capillaries in the synovium and synovial fluid, thus causing the joint to be further broken. The histopathology of synovitis is characterized by hyperemia, edema, neutrophils and infiltration of inflammatory cells in the acute phase of synovitis, the dilatation of the synovial vessels, a large amount of exudation in plasma and cell exudation, the active synovial cells and a large number of mucin. The synovial tissue of chronic phase lesions shows obvious vascular proliferation and inflammatory cell infiltration. In a preliminary epidemiological survey, the prevalence of osteoarthritis of the knee is 60%, the prevalence rate of 56 years old and above is more common in 49%. women than in men. Patients with osteoarthritis are more likely to have synovitis, and synovitis aggravates osteoarthritis. The inflammatory properties of synovial cell factor, protease and prostaglandin secretion are more frequent. The activation of the cells, such as PGE, is directly involved in the lesion of articular cartilage. Synovitis is an early manifestation of the function of OA cartilage change, promoting vascular proliferation, abnormal bone turnover, and serious damage to the joint structure. Synovial and cartilage at all contacts have varying degrees of cartilage erosion. Under optical microscope, the synovial cartilage can be seen to invade cartilage. In addition to mechanical causes, cytokine also plays a key role in the destruction of articular cartilage. It is mainly the increase of IL-1 secretion caused by synovitis, and the increase of the secretion and activity of metalloproteinase, causing cartilage destruction. Meanwhile, synovitis is also the main pathological manifestation of Rheumatoid arthritis (RA). The early diagnosis rate of rheumatoid arthritis is lower than that of sexual diseases. In addition, foreign scholars have found that the life expectancy of patients with rheumatoid arthritis is also relatively short. Because of its high disability rate, the economic burden of the society is increasing year by year, and RA has become a public health problem that must be paid attention to. Unfortunately, the pathogenesis of RA is now at present. The study is not clear. In the previous study of the development and treatment of synovitis, three kinds of mitogen activated protein kinase (MAPK) signal transduction pathway in synovial tissue, namely, extracellular regulated protein kinase (ERK) signal transduction pathway, P38 mitogen activated protein kinase MAPK signal transduction pathway, C-jun The signal transduction pathway of the amino terminal kinase. The signal transduction pathway is closely related to the development of synovitis. Mitogen activated protein kinase (MAPK) is an important part of the eukaryotic signaling network. It can be involved in many activities, such as cytoplasmic functional activity and gene expression regulation, and plays a very important role. A series of cell physiological activities such as growth, development, differentiation and apoptosis are involved in cell growth, development, differentiation, and apoptosis, which mainly include JNK, ERK, and P38 three signal transduction pathways. The study shows that mitogen activated protein kinase (MAPK) can affect the occurrence and development of synovitis.MAP The K pathway is a signal transduction enzyme system. After a certain stress stimulation, the internal system of the cell can use this signal pathway to make a certain response. Once this signal pathway changes, it causes abnormal conduction and causes the occurrence of synovitis. It is affected by some specific cytokines or antigens, MAPK The transcriptional factors of the synovial cells change after the abnormal proliferation, and the activity of these activators is related to the MAPK signal transduction pathway and is regulated by its regulation. In the case of abnormal cell proliferation, the activity of the transcription activator is regulated by the signal transduction pathway. Lipopolysaccharide (LPS) is the specific structure of the cell wall of Gram-negative bacteria. It is the main component of the endotoxin, and the lipopolysaccharide is composed of the core polysaccharide, the O polysaccharide side chain and the lipid A. Lipopolysaccharide can act on the synovial cells through the mitogen activated protein kinase MAPK signaling pathway. Under certain conditions, Gram-negative bacteria can release a small amount of LPS slowly and release a large amount of LPS after death. The gram negative bacterial infection can cause a significant increase in the level of LPS in the plasma of the patient. The LPS level of infection. According to previous studies, a higher level of LPS. can be detected in the cervix of women with gram-negative bacterial vaginitis and in the study, LPS can inhibit the growth of synoviocytes through the mitogen activated protein kinase MAPK signaling pathway, which can induce the development of synovitis. The concentration of polysaccharides is positively related to the expression of inducible nitric oxide synthase in synovitis and synovitis, and its interaction with inflammatory factors aggravates the degree of synovitis. This effect is achieved through the mitogen activated protein kinase (MAPK) transduction pathway in the synovial tissue. Objective: This study was based on synovial cells and used LPS as a synovial membrane. Cells, looking for the mechanism of the action of LPS on the MAPK pathway of synovial cell growth, so as to further study the important role of MAPK pathway protein in the occurrence and development of synovitis, in order to find new therapeutic targets and lay the foundation for the formulation of effective treatment scheme for synovitis. Methods: Using different concentrations of LPS in synovial cells, Then MTT and flow cytometry were used to detect the growth of synovial cells at different time and to find the best time and concentration of LPS on the growth of synovial cells. By using LPS to act on HS cells, the cells were collected at different time and the protein was extracted. The key protein ERK, JNK, p38 in the MAPK pathway was detected by Western blot. In order to determine the activation protein, then use the corresponding protease agonist to act as the key protein of phosphorylation, detect the cell growth and the downstream protein activation. In order to further study the mechanism of the action of LPS, the LPS receptor TRL4 is suppressed by RNA interference, and then the phosphorylation status of protein ERK, JNK, p38 is detected. Results: (1) the growth rate of 0,10,20 and 40ng/ml LPS in human synovial cells was measured in 0,24,48 and 72h by MTT method. When LPS was added to LPS, the growth rate decreased gradually. In the 10ng/ml group, the growth rate of cells decreased with the increase of time, but no 20 and 40ng/ml groups decreased obviously. In 2 The growth of 0ng/ml and 40ng/ml group was similar. The apoptosis rate was quantified by Annexin V/PI flow cytometry. The apoptotic rate after LPS treatment of 0,10ng/ml, 20ng/ml, 40ng/ml in HS cells was detected. The apoptosis induction of HS cells could be induced by the treatment of HS cells with different concentrations of LPS, and the apoptosis rate increased with the increase of concentration. Adding, when the concentration of LPS is 20ng/ml, the cell apoptosis rate reaches the extreme value and no longer changes. This indicates that the optimal growth rate of 20ng/ml LPS at 48h is 24.0340.3 1%. (2) in this study, LPS with the total concentration of 20ng/ml is used in HS cell phosphorylation JNK. The low, non phosphorylated JNK (T-JNK) bands were basically the same at all time. The bands of the P-ERK and T-ERK bands were almost identical at all time. And the bands of p-p38 were very shallow at every time. And the bands of T-p38 were very homogeneous and very deep. This shows that the inhibition of LPS to HS cell growth is by inhibiting the growth of HS cells. In order to further prove that the inhibitory effect of LPS on HS cell growth is realized by the JNK pathway, the activator anisomycin of JNK acts on HS cells, and LPS is added to prove the JNK protein of phosphorylation, and the phosphorylation of downstream JNK downstream proteins in order to further prove that the inhibitory effect of LPS on HS cell growth is achieved. The results showed that when Anisomycin was added, the value of P-ATF-2/ATF-2 was not significantly changed from the beginning to 60min, and the expression value was higher (P0.05). At the same time, the growth of cells was studied when Anisomycin was added, and the growth rate of the cells was faster from 85.19% to 96.52% in sixth days, and the cells were all the time in these six days. Keep high growth speed. (3) the RNA of HS was extracted and the RT-PCR experiment of full TLR4 was carried out. Then the PCR product was carried out by 1% agarose gel electrophoresis, and the expression strip of TLR4 could be seen in the imaging system. The expression of TLR4 in the upstream of nucleic acid was shown. To further prove the expression of TLR4, the HS protein was extracted, and Western blot was carried out. The results also showed that TLR4 was expressed in HS cells. TLR4 was silenced using RNA interference technique. Meanwhile, PCR and Western blot techniques were used to verify that TLR4 was successfully silenced. At the same time, TLR4 knockout cell lines were screened. TLR4 interfered with SiRNA, and stable cell lines were obtained after screening, and then, MTT method was used. The growth of cells in 0,24,48 and 72h cells was detected after the action of 20ng/ml's LPS on HS cells, and the growth rate of cells was calculated. The growth of cells in the interference group (group SiRNA+LPS) showed a growing trend in this period, without significant change (P0.05). The growth rate of the cells in the control group (LPS group) decreased gradually after the.TLR4 was suppressed by HS cells. The phosphorylation of ERK, JNK and p38 in this study is worth analyzing in this study. At the same time, the corresponding phosphorylated protein is compared with the gray value of the non phosphorylated protein, and then the values are compared. Both the values of p-JNK/T-JNK and p-ERK/T-ERK and P-P38/T-P38 have not changed with the time, and the value of p-JNK/T-JNK. It is significantly lower than the value of TLR4 that is not suppressed. Conclusion: Overall, our results show that the main signal pathway of the growth of LPS inhibition HS is achieved through the JNK pathway in the MAPK pathway. To further study the important role of MAPK pathway protein in the occurrence and development of synovitis, find new therapeutic targets and formulate a new therapeutic target. It lays the foundation for the scheme of effective treatment of synovitis.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R686.7

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