脂多糖对人滑膜细胞增殖过程中MAPK途径作用机制的研究

发布时间：2018-05-20 05:43

本文选题：多糖 + 对人　；参考：《武汉大学》2016年博士论文

【摘要】：背景：滑膜炎是一种常见病,多发病,其病程长,类型多,且容易反复发作,患者出现关节肿痛,活动受限,严重影响日常生活。尤其以细菌性滑膜炎,类风湿性滑膜炎为著,难以治愈。临床以无菌性滑膜炎多见,滑膜炎是关节炎的早期症状,人类关节滑膜为疏松结缔组织,可分为两层：滑膜衬里层和滑膜衬里下层。滑膜衬里层是由具有巨噬细胞样功能的A型细胞及纤维母细胞样的B型细胞组成,而衬里下层是由疏松结缔组织基质构成,与其深层的纤维细胞并无基底膜相隔,直接与纤维结缔组织或脂肪组织相连接,故在关节发生炎症时易蔓延到周围组织,导致滑膜炎的发生。人体中包含多个关节,滑膜炎可发生在全身各个关节之中。其中,膝关节具有特殊的解剖结构,是全身关节中滑膜最多的关节。而且,膝关节容易受到外力等的影响而出现损伤。因此,在临床中,滑膜炎大多以膝关节最为多见。而滑膜炎一旦出现,如果治疗不及时,就迁延成慢性膝关节滑膜炎。患者的膝关节便会逐渐呈现出肿胀和疼痛等症状,长此以往,就会对患者的膝关节功能产生严重的影响,导致功能障碍的出现,严重影响患者的日常生活和工作。滑膜炎是早期骨关节炎(Osteoarthritis, OA)中常见的症状之一,被大家认为是炎症反应。它主要是由炎性因子和促炎性因子导致滑膜的炎症反应和关节软骨破坏的不平衡产生的。在长期的致病因素作用下,导致滑膜增生并逐渐增厚,与此同时纤维层和细胞层被嵌入,血管增生。由于炎性细胞浸润与滑膜成纤维细胞的增生,导致滑膜毛细血管在滑膜和滑液中选择性分布,从而使关节遭到进一步破坏。滑膜炎组织病理学表现为滑膜组织急性期充血,水肿,中性粒细胞和大量炎性细胞浸润,滑膜血管扩张,血浆和细胞外渗产生大量渗出液,滑膜细胞活跃,产生大量粘液素。慢性期病变的滑膜组织表现出明显的血管增生和炎症细胞浸润。在初步的流行病学调查中表明,膝关节骨性关节炎的患病率为60%,56岁及以上的患病率为49%。女性比男性更常见。患有骨关节炎患者更易发生滑膜炎疾病,滑膜炎同时加重骨关节炎的发生。由滑膜细胞因子,蛋白酶和前列腺素分泌的炎性细胞如PGE的活化直接参与关节软骨的病损。滑膜炎是OA软骨改变功能的早期表现,促进血管增生,异常的骨周转率,从而严重损害关节结构。滑膜和软骨在所有的接触口都有不同程度的软骨糜烂,光学显微镜下可以看到,滑膜软骨生长侵蚀。关节软骨的破坏,除了机械原因之外,细胞因子也起着关键的作用。主要是滑膜炎导致的IL-1分泌的增加,和金属蛋白酶的分泌及活性增加,引起软骨破坏。同时滑膜炎也是类风湿性关节炎(Rheumatoid arthritis, RA)主要病理学表现。与其他慢性疾病相比,类风湿关节炎的早期诊断率较低。此外,国外学者发现,类风湿性关节炎患者的预期寿命也相对较短。因为它的高致残率,使社会的经济负担逐年上升,RA已成为必须注意的一个公共卫生问题。但不幸的是,对RA的发病机理,目前的研究还不是很透彻明了,在以往对滑膜炎的发生发展及治疗过程的研究中发现,滑膜组织中存在的三种丝裂原活化蛋白激酶(MAPK)信号转导通路,即细胞外调节蛋白激酶(ERK)信号转导通路,P38丝裂原活化蛋白激酶MAPK信号转导通路,C-jun氨基末端激酶信号转导通路。以上信号转导通路与滑膜炎的发生发展存在密切联系。丝裂原活化蛋白激酶(MAPK)是真核生物信号传递网络的重要组成部分,可以参与到细胞质功能活动以及基因表达调控等多项活动中,并发挥十分重要的作用。是细胞内广泛表达的丝氨酸／苏氨酸的蛋白激酶被磷酸化激活后,参与细胞的生长、发育、分化、凋亡等一系列细胞生理活动,主要包括JNK,ERK,P38三条信号转导通路。研究表明,丝裂原活化蛋白激酶(MAPK)能够影响滑膜炎的发生发展。MAPK通路是一种信号转导酶系统,在受到一定应激刺激之后,细胞内部系统便会利用这一信号通路做出一定的反应。而一旦这一信号通路发生异常变化,导致传导异常,并容易导致滑膜炎的出现。受到一些特定细胞因子或者抗原的影响,MAPK可以通过相应的信号转导系统,进入到细胞核之中,启动一系列行为的出现。但是,在发生异常的增殖之后,滑膜细胞的转录因子便会出现改变。这些转录活化因子的活性均与MAPK信号转导通路相关,受到其调控。在细胞异常增殖的情况下,会导致大量促炎性细胞因子等的分泌。进而导致滑膜细胞发生异常改变,导致其出现增生和肥厚等情况。脂多糖(Lipopolysaccharide, LPS)是革兰氏阴性细菌细胞壁的具体结构。是内毒素的主要成分,脂多糖由核心多糖,O多糖侧链和脂质A组成,脂多糖可以通过丝裂原活化蛋白激酶MAPK信号通路作用于滑膜细胞,在一定条件下,革兰氏阴性菌活菌可以缓慢释放少量LPS,死亡后大量释放LPS,革兰氏阴性细菌感染可引起患者的血浆中的LPS水平显著增加。也可以提高所引起的局部相继感染的LPS水平。根据先前的研究中,在患有革兰氏阴性细菌性阴道炎的妇女宫颈中,可以检测到比较高水平的LPS。并且在研究中发现,LPS可以通过丝裂原活化蛋白激酶MAPK信号通路抑制滑膜细胞的生长,其可以诱导滑膜炎的发生发展。脂多糖浓度与滑膜炎滑液中诱导型一氧化氮合酶表达成正相关,其与炎性因子相互作用加重滑膜炎程度。这一作用是通过滑膜组织中存在的丝裂原活化蛋白激酶(MAPK)转导通路实现的。目的：本研究以滑膜细胞为研究对象,用LPS作用滑膜细胞,寻找到LPS对滑膜细胞生长的MAPK通路的作用机制。以至为进一步研究MAPK通路蛋白在滑膜炎的发生和发展过程中存在的重要作用,以期找到新的治疗靶点,为制定有效治疗滑膜炎方案奠定基础。方法：用不同浓度的LPS作用滑膜细胞,然后用MTT和流式细胞仪检测在不同时间的滑膜细胞的增长情况,找到LPS对滑膜细胞生长的最佳作用时间与浓度。通过用LPS作用于HS细胞,在不同时间收集细胞,提取蛋白,用Western blot的方法检测MAPK通路中的关键蛋白ERK,JNK,p38的磷酸化状况,从而确定其激活蛋白。然后用相应的蛋白酶激动剂作用磷酸化的关键蛋白,检测细胞的生长情况及下游的蛋白活化情况。为了进一步研究LPS的作用机制,将LPS的受体TRL4用RNA干扰的方法进行抑制,然后检测蛋白ERK,JNK,p38的磷酸化状况及细胞的生长情况。结果：(1)用MTT法检测了0,10,20和40ng/ml的LPS作用于人滑膜细胞后于第0,24,48和72h计算生长率,当加入LPS后从24h开始,其生长率逐渐降低。在10ng/ml组中细胞的生长率随着时间的增加而降低,但没有20和40ng/ml组降低明显。在20ng/ml和40ng/ml组中其生长情况相似。采用Annexin V/PI流式细胞分析法进行了凋亡率的定量研究,检测HS细胞0,10ng/ml,20ng/ml,40ng/ml的LPS处理后的凋亡率。不同浓度LPS处理HS细胞后,能诱导HS细胞发生凋亡诱导作用,且凋亡率随LPS浓度的增加而增加,当LPS浓度为20ng/ml时,细胞凋亡率达到极值,值后不再变化。这就表明20ng/ml的LPS在48h时对HS细胞的生长率抑制情况最佳为24.0340.3 1%。(2)在本研究中用总浓度为20ng/ml的LPS作用于HS细胞磷酸化JNK(p-JNK)的条带随着时间的增加表达量越来越低,而非磷酸化的JNK(T-JNK)条带在各个时间的表达基本一致。对于P-ERK和T-ERK的条带在各个时间的表达也基本一致。而p-p38的条带在各个时间都很浅几乎看不清。而T-p38的条带则很均一且很深。这就表明LPS对HS细胞生长的抑制作用是通过抑制磷酸化JNK来实现的。而且在LPS作用40min时已经发挥到极致。为了进一步证明LPS对HS细胞生长的抑制作用是通过JNK通路实现的,采用了JNK的激动剂anisomycin作用于HS细胞,同时加入LPS来证明磷酸化的JNK蛋白情况,以及JNK下游蛋白磷酸化的ATF-2情况。结果表明当加入Anisomycin时,对P-ATF-2/ATF-2的值从一开始到60min均没有明显变化,且表达值较高(P0.05).同时研究了当加入Anisomycin后,细胞的生长情况,细胞的生长速度较快从一开始的85.19%长到了第六天的96.52%,在这六天中细胞始终保持较高的生长速度。(3)提取HS的RNA并进行了充分TLR4的RT-PCR实验,然后将PCR产物进行1%的琼脂糖凝胶电泳,在成像系统中可以看到TLR4的表达条带。表明了TLR4在核酸水平上游表达。为进一步证明TLR4的表达,提取了HS的蛋白,进行了Western blot实验。结果同样表明TLR4在HS细胞中有表达。利用RNA干扰技术将TLR4沉默。同时应用PCR技术和Western blot技术验证了TLR4被成功沉默。同时还筛选出了TLR4敲除的细胞系。用SiRNA的方法将TLR4干扰后,并经筛选后得到稳定的细胞细胞系,然后,用MTT法检测了用20ng/ml的LPS作用于HS细胞后,于第0,24,48和72h检测细胞的生长情况,并计算细胞的生长率,在干扰组中(SiRNA+LPS组)细胞在此期间的生长呈增长趋势,无显著变化(P0.05)。而在对照组中(LPS组)细胞的生长率逐渐下降。TLR4被抑制后HS细胞中ERK,JNK,p38的磷酸化情况,本研究对蛋白进行了灰度值得分析,同时将对应的磷酸化蛋白与非磷酸化蛋白的灰度值相比,而后将所得值进行比较。无论是p-JNK/T-JNK还是p-ERK/T-ERK和P-P38/T-P38的值均未随着时间的增加而变化,且p-JNK/T-JNK的值明显低于TLR4未被抑制时的值。结论：总的来说,我们的结果表明LPS抑制HS的增长主要信号通路是通过MAPK通路中JNK通路途径实现的。为进一步的研究MAPK通路蛋白在滑膜炎的发生和发展过程中存在的重要作用,找到新的治疗靶点,制定有效治疗滑膜炎方案奠定基础。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R686.7

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