【作者】 徐宁；

【导师】 庄俊华；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2011， 博士

【摘要】 类风湿关节炎(rheumatoid arthritis, RA)是一种慢性自身免疫性疾病,早期症状为病患关节红热、肿痛和活动障碍,晚期出现关节僵硬和畸形,并伴有不同程度的骨骼肌萎缩,致残率很高。除病患关节的炎性病变以外,RA还累及心、肺、肾等多种器官,严重时危及生命。类风湿关节炎在全世界成人中的发病率约占1%-2%,总计约有2000万患者,严重威胁着人类健康和生存质量。我国RA的平均发病率接近0.4%,70%的患者2年后致残。因此,探索和阐明RA滑膜增殖的病理生理过程,研究开发有效的治疗方法和药物以期提高类风湿患者的生存率和生存质量意义十分重大。RA的病理表现为炎症反复发作、滑膜组织过度增殖和功能异常、关节软骨和骨质遭受滑膜细胞、血管翳的侵蚀和破坏。近年来磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)信号通路的激活已被认为是细胞抗凋亡并引起异常增殖的重要机制之一。有研究表明该通路在RA中呈过度活化状态,PI3K/Akt信号通路和IL-1, TNF, NF-κB等炎性相关细胞因子联系紧密,可能在RA病理过程中起到重要作用。PTEN和Akt分别是PI3K/Akt信号通路中的负调节因子和效应因子。PTEN是具有蛋白、脂类双特异性的磷酸酶,通过使PIP3去磷酸化抑制PI3K的活化信号向Akt传递,下调PI3K/Akt信号通路活性。PTEN有多种底物,除对PI3K/Akt信号通路的调节外,还广泛地参与抑制细胞运动,调节细胞周期,维持染色体稳定性等。RA中PI3K/Akt信号通路的过度活化可能和PTEN功能降低或者缺失有关。因此,在RA成纤维样滑膜细胞中表达PTEN基因,可以研究PTEN在RA中的功能。类似地通过构建靶向Akt的RNA干扰片段的载体并表达,对Akt进行特异性抑制,可以阻断PI3K/Akt信号通路,研究Akt在RA成纤维样滑膜细胞的抗凋亡、促增殖等调节功能。RNA干扰技术是近年来发现的一种新的诱导基因沉默的技术。当外源性或内源性双链RNA(dsRNA)进入细胞后,能够识别含有其互补序列的mRNA并与之结合,在酶的作用下特异性降解mRNA,从而干扰相应基因表达,导致基因沉默。RNA干扰效应具有高特异性、高稳定性、高效性,已成为研究特定基因功能的重要工具。外源基因或目的基因转移至靶细胞的方法可分为病毒载体系统和非病毒载体系统。非病毒表达系统转移效率低,病毒载体系统具有高效性的特点。常用的病毒载体有慢病毒、逆转录病毒、腺病毒和腺相关病毒载体,其中慢病毒载体对分裂期和非分裂期细胞都可以感染,并具有较小的免疫反应,成为目前研究最多、最理想的基因转移载体之一。本课题应用慢病毒表达系统构建了两个载体,一个用于增强PTEN表达,另一个以RNA干扰的方式敲减Akt的表达。两个载体分别转染RA FLS并观察病毒载体对相应基因表达的作用效果,及其对细胞增殖、迁移能力的影响,研究PTEN和Akt基因在RA病理过程中的作用机制。第一部分类风湿关节炎成纤维样滑膜细胞的培养及PTEN/Akt的表达目的研究RA FLS的增殖和生长特征以及PTEN、Akt和p-Akt蛋白在RA中的表达情况。方法免疫组化检测RA和OA滑膜组织石蜡标本中PTEN和Akt两种蛋白的表达位置和表达量,采用组织块培养法培养RA FLS和OA FLS,流式细胞仪检测RA FLS纯度,通过MTT法检测OA FLS和RA FLS增殖并绘制生长曲线,Western Blot检测两种细胞中PTEN蛋白和Akt、p-Akt蛋白的表达量。结果1、免疫组化结果显示,PTEN蛋白在OA滑膜组织中的表达高于RA滑膜组织,在RA滑膜细胞中的表达集中于细胞核内；Akt蛋白在OA滑膜组织中的表达低于RA滑膜组织,在RA滑膜细胞中的表达也集中于细胞核内。2、从组织块培养出的RA FLS传4代后,免疫荧光染色的细胞经流式细胞仪检测,纯度可达99%以上,可以进行后续试验。3、MTT检测到第4代RA FLS和OA FLS在传代第3天增殖出现明显差异, RA FLS吸光值为0.31, OA FLS吸光值为0.259(p<0.05),以后各个检测点均有统计学意义。4、RA FLS中PTEN蛋白表达量低于OA FLS; Akt和p-Akt蛋白的表达量高于OA FLSO结论PTEN蛋白在RA FLS中的表达低于OA FLS, Akt蛋白在RA FLS滑膜组织中的表达高于OA FLS, RA FLS的增殖速度高于OA FLS。第二部分PTEN慢病毒增强表达载体和靶向Akt基因的shRNA慢病毒表达载体的构建目的构建增强PTEN表达的pLenti6/V5-PTEN慢病毒表达载体；筛选有效的Akt siRNA靶序列,构建靶向Akt的RNA干扰慢病毒表达载体。方法1、获得PTEN全长基因,与表达载体质粒pLenti6/V5定向克隆连接,构建成pLenti6/V5-PTEN表达载体,设pLenti6/V5-NC为阴性对照；根据Akt mRNA设计合成两条干扰序列和一条随机序列作对照,克隆到入门载体pENTRTM/U6,鉴定正确后与pLenti6/BLOCK-iTTM-DEST进行LR重组,构建成靶向Akt的shRNA表达载体pLenti6/shAkt-1、pLenti6/shAkt-2,设pLenti6/shAkt-NC为阴性对照,测序正确的表达载体通过脂质体法转染LOVO细胞检测功能。2、将构建成功的表达载体分别与ViraPowerTM Packaging Mix共同转染状态良好、融合度80%-90%左右的293T细胞,细胞继续培养48h、72h后收取含有病毒的上清液,测病毒滴度,冻存待用。结果1、pLenti6/V5-PTEN转染LOVO细胞,检测到PTEN mRNA和蛋白表达的增加；入门载体pU6/shAkt-1、pU6/shAkt-2,表达载体pLenti6/shAkt-1 pLenti6/shAkt-2分别转染LOVO细胞,检测到Akt mRNA和蛋白表达受到抑制,其中pLenti6/shAkt-2的抑制效果显著。2、将重组质粒pLenti6/V5-PTEN.pLenti6/V5-NC.pLenti6／shAkt-1. pLenti6/shAkt-2.pLenti6/sh NC分别在293T细胞中包装成慢病毒表达载体,测得病毒滴度分别为1.5×107TU/ml、8×106TU/ml、4×106TU/ml.4.3×106TU/ml和7.4×106TU/ml。结论成功构建了增强PTEN表达的慢病毒表达载体pLenti6/V5-PTEN和靶向Akt的RNA干扰慢病毒表达载体pLenti6/shAkt-2。第三部分pLenti6/V5-PTEN和pLenti6/shAkt慢病毒表达载体对RA FLS增殖、迁移及蛋白表达的作用目的研究在PTEN和Akt基因对RA FLS中的功能以及对RA FLS增殖、迁移的影响,分析PTEN和Akt蛋白在RA发病机制中的作用。方法1、将稀释后的pLenti6/V5-PTEN、pLenti6/V5-NC、pLenti6/shAkt-1. pLenti6/shAkt-2、pLenti6/sh NC病毒溶液分别转染RA FLS,设未转染病毒的RA FLS为对照组,通过RT-PCR和Western Blot检测PTEN. Akt mRNA和蛋白表达的变化,以及对p-Ak的表达的调节。2、病毒溶液分别转染RA FLS, MTT染色比较增殖情况,划痕实验检测转染病毒后RA FLS迁移能力的变化,设未转染病毒的RA FLS为对照组。结果1、pLenti6/V5-PTEN转染RA FLS后PTEN mRNA表达量显著提高,24h、48h、72h、96h 4个时段RT-PCR结果显示,与对照组相比PTEN mRNA表达量分别增加了76.02%、63.67%、67.24%和72.83%(p<0.05)。转染pLenti6/shAkt-1和pLenti6/shAkt-2后24h、48h、72h、96h4个时段RT-PCR结果显示,pLenti6/shAkt-2组细胞的Akt各亚型表达显著降低,与对照组相比,Aktl分别降低了76.13%、65.1%、79.12%和70.18%(p<0.05)；Akt2和Akt3与对照组相比也有显著差异(p<0.05).pLenti6/shAkt-1组与对照组相比,Akt各亚型mRNA呈微弱降低,其中Aktl mRNA表达量分别降低14.15%和14.84%(p>0.05)。2. Western Blot结果显示,培养48h后PTEN蛋白的表达量与对照组相比提高107.85%,而p-Akt的含量降低了48%(p<0.05),pLenti6/shAkt-2组Akt蛋白的表达量与对照组相比降低了76.19%(p<0.05),p-Akt的含量降低了58.75%(p<0.05)；pLenti6/shAkt-1组无明显变化。3. pLenti6/V5-PTEN转染RA FLS后24h、48h、72h、96h4个时段,检测吸光值分别降低了4.46%、25.14%、41.84%和52.46%,从48h后RA FLS增殖明显低于对照组,增殖差异具有统计学意义(p<0.05)。pLenti6/shAkt-2转染RA FLS后,从第48h开始实验组与对照组相比吸光值出现明显差异,pLenti6/shAkt-2组4个时段RA FLS吸光值分别降低12.81%,27.5%、44.69%和56.80%。4、划痕实验显示转染pLenti6/V5-PTEN和pLenti6/shAkt-2后细胞迁移能力降低,培养72h后发现2组细胞进入划痕区的细胞明显少于各自的对照组,越过划痕边界的距离也小于对照组。结论pLenti6/V5-PTEN和pLenti6/shAkt-2转染RA FLS后能够显著降低p-Akt的表达,并抑制RA FLS的增殖和迁移。更多还原

【Abstract】 Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease. RA early symptoms usually include red and swelling joints, burning pain and action obstacles. As the disease became severe, the joints appear stiffness and deformed, and show vary degrees of skeletal muscle atrophy, which increase the risk of disability. RA can also produce diffuse inflammation in the heart, lung, kidney and other subcutaneous tissue. In extreme cases RA can be fatal. About 1%～2% of the world’s population is afflicted by RA, approximately 20 million patients were suffering from it. RA seriously threatens the human healthy and living quality. In China the inflicted RA incidence approaches 0.4%, almost 70% of the patients become disability two years after diagnosis. Therefore, illustrating RA synovial proliferation pathology process shows great importance in improving the effectiveness of treatments and drugs usage, so that enhance the survival rate and improve the qualities of life of RA patients.The pseudo-tumoral proliferation of RA fibroblast-like synoviocytes (FLS) is considered to be the major mechanism for the hyperplasic growth of the RA synovium. In recent years, phosphatidyl inositol 3-kinase (phosphatidylinositol 3-kinase, PI3K) signal pathway was shown to be activated in RA synovial tissue, and which has been recognized as one of the important mechanisms to regulate the proliferation and apoptosis of RA FLS, thus play key roles in the pathologic process of RA. Moreover, The activity of PI3K/Akt signaling pathways is correlated with distinct proinflammatory cytokines, including IL-1, TNF, NF-κB, and etc. PTEN is the central negative regulator of PI3K/Akt signal pathway. PTEN acts as protein phosphatase and lipid phosphatase, antagonizing the PI3K/Akt signaling pathway by dephosphorylating phosphoinositides, down regulating its activity. Despite of influencing PI3K/Akt pathway, PTEN also involved in inhibiting cell motility, regulating cell cycle and maintaining chromosome stability, etc. The activation of PI3K/Akt signaling pathways maybe associated with the down expression or dysfunction of PTEN. Therefore, induction of PTEN or inhibition of Akt specifically could block PI3K/Akt signaling pathways, thus contributing to study the function of PI3K/Akt pathway in regulation the proliferation and apoptosis in RA FLS.RNAi is a new technique discovered in recent years and it is useful for studying the function of specific genes. Exogenous or endogenous double-stranded RNA (dsRNA) can bind to its complementary sequence of mRNA in vivo, and then selectively degrades targeted mRNA, interferes specific gene expression and lead to gene silence. Meanwhile, RNAi is high specific, high stable and high efficiency and acts as an important tool in studying the function of specific gene.The Methods to transfer exogenous gene into target cells includes either virus expression vector or non-virus expression vector systems. The transfection efficiency of non-virus expression vector is lower than virus expression vector. In general, virus expression vectors include lentivirus, adenoviruses, retrovirus, etc. Among them the lentivirus expression vectors have an important advantage over other types of expression systems, it can transfect dividing and non-dividing cells and elicit less immune response. In this study we constructed two lentivrus expression vectors (pLenti6/V5-PTEN and pLenti6/shAkt), the former was used to induce the expression of PTEN, while the latter was used to inhibit the expression of Akt. Each vector was packed into lentivirus, and then was used to infect RA FLS individually. After infection, MTT assay and scratch test were performed to study the effect of these vectors to inflect the proliferation and migration of RA FLS, and to further explore the function and mechanism of PTEN and Akt in the pathology of RA. ObjectiveThe purpose of this study was to characterize the proliferation and growth of RA FLS, and to investigate the protein expression levels and location of PTEN, Akt and p-Akt.MethodsParaffin-embedded and formalin-fixed RA synovium were cut into 5μm sections, which were then processed for immunohistochemistry (IHC) to monitor the amount and location of PTEN and Akt.RA/OA FLS were isolated by adheration of synovial tissues obtained from RA patients undergoing total joint replacement surgery or knee synovectomy. After FLS were isolated, flow cytometry was used to define the purity, MTT assay was performed to inspect the proliferation of the cells and the growth curve was drawn according to the data. Western blot were performed to detect the protein expression of PTEN, Akt and p-Akt.Results1. IHC showed that PTEN and Akt mainly located in the nuclear of the cells, partially in the plasma.2. In the 4th generation, the purity of RA FLS reached 99% and were suitable to perform further experiment.3. MTT assay showed significant difference in the proliferation rate of the 4th generation of RA FLS and the 3rd generation of OA FLS. OD value was 0.31 of RA against 0.259 of OA, and each time point showed significance in statistic (p<0.05).4. Compared with OA FLS, the protein expression of PTEN was lower, while Akt and p-Akt were higher In RA FLS.ConclusionThe expression of PTEN in RA FLS was lower than in OA FLS and the expression of Akt in RA FLS was higher than in OA FLS. ObjectiveTo construct pLenti6/V5-PTEN lentivirus expression vector; To screen effective Akt siRNA targeting sequences and construct lentivirus expression vector carrying shRNA targetting Akt.Methods1. Human PTEN complementary DNA was cloned into pLenti6/V5 vector, named pLenti6/V5-PTEN. pLenti6/V5-NC served as the control viruses.2. The siRNA sequences targeted human Akt transcript were designed using the software developed by Invitrogen, Inc. Two candidate sequences in human Akt gene, classified as Akt-1 and Akt-2, were selected for RNA interference (RNAi). The control RNA duplex (named as NC), was non-homologous to any human genome sequences. A scrambled sequence (NC), which is nonhomologous to any human DNA sequence, served as a negative control and was classified as NC. The three sequences were cloned into BLOCK-iTTM U6 RNAi Entry Vector (named as pU6/shAkt-1, pU6/shAkt-2, pU6/sh NC). The entry vector containing the U6 RNAi cassette was used to transfer the U6 RNAi cassette into the lentiviral expression plasmid (named as pLenti6/shAkt-1、pLenti6/shAkt-2 and pLenti6/sh NC) using Gateway(?) Technology. Each construct was sequenced to confirm the right sequence of insert. pLenti6/V5-PTEN、pLenti6/shAkt-1 and pLenti6/shAkt-2 were transfected into LOVO cells to determine their efficiencies.3. The successfully constructed lentivirus expression vectors were transfected into 293T cells with ViraPowerTM Packaging Mix, After culture for 48h and 72h, the supernatant contains virus was collected, aliquoted and then stored at-80℃.Results1. In comparison with the parental LOVO cells, pLenti6/V5-NC transfectants showed a significant increase of PTEN in both mRNA and protein levels. While pU6/shAkt-1, pU6/shAkt-2, pLenti6/shAkt-1、pLenti6/shAkt-2 transfectants showed a significant decrease of Akt in both mRNA and protein levels, indicating the successful knockdown of Akt in these derived clones. Furthermore, there was no difference in OPN expression level between the mock cells and the parental LOVO cells. The siRNA sequence Akt-2 shown a better silencing efficiency than Akt-1.2.The recombinant plasmid of pLenti6/V5-PTEN、pLenti6/V5-NC pLenti6/shAkt-1、pLenti6/shAkt-2 and pLenti6/sh NC were packaged into virus with ViraPowerTM Packaging Mix in 293T cells respectively. The virus titers were 1.5×107TU/ml、8×106TU/ml、4×106TU/ml、4.3×106TU/ml and 7.4×106TU/ml separately.ConclusionBoth pLenti6/V5-PTEN and pLenti6/shAkt-2 letivirus expression vectors were successfully constructed.Part 3 The effects of pLenti6/V5-PTEN and pLenti6/shAkt on RA FLS in proliferation, migration and protein expressionObjectiveThe aim of this study was to examine the function of PTEN and Akt gene in RA FLS, to study the effects of these genes on RA FLS proliferation and mortality, to further elucidate the function and relation of PTEN and Akt in the pathogenesis of RA.Methods1. RA FLS were infected with distinct virus. In various time point after infection, the mRNA and protein were extracted from RA FLS, RT-PCR was used to detect the mRNA expression of PTEN and Akt, while western blotting were performed to analyze the protein expression of PTEN, Akt and p-Akt.2. MTT assay and scratch test were performed to determine the proliferation and mortality of RA FLS after infection.Results1. To elucidate the underlying mechanism for the induction of PTEN or silencing of Akt in RA FLS, we examined the kinetic expression of PTEN and Akt mRNA and protein in RA FLS over a 96 h time course.24h、48h、72h、96h after infection, the pLenti6/V5-PTEN transfectants showed higher PTEN mRNA expression than the NC group, the increment was 76.02%、63.67%、67.24% and 72.83% respectively (p<0.05). The pLenti6/sh Akt-2 transfectants showed significant decrease of the Akt subtypes, the decrement of Aktl was 76.13%、65.1%、79.12% and 70.18%(p<0.05). Moreover, the expressions of Akt2 and Akt3 (the other Akt subtypes) were also significantly decreased. In agreement with Part 2, pLenti6/sh Akt-2 was better than pLenti6/sh Akt-1 to silence the expression of Akt.2. We further examined the effects of PTEN induction and Akt inhibition on the protein expressions.48h after transfected with pLenti6/V5-PTEN, western blot showed that PTEN protein was increased by 107.85%(p<0.05), while p- Akt was decreased by 48%(p<0.05) compared to NC. In pLenti6/sh Akt-2 transfectants, Akt protein decreased by 6.19% and p-Akt decreased by 58.75%(p<0.05). And we found that pLenti6/sh Akt-1 had few influences on the Akt and p-Akt expression.3. MTT assay results displayed that pLenti6/V5-PTEN inhibited the proliferation of RA FLS, the OD value was decreased by 4.46%、25.14%、41.84% and 52.46% at 24h、48h、72h、96h post transfection. And we also found the same effects of pLenti6/sh Akt-2, which showed a decline of OD value by 27.5%、44.69%、56.80% in 24h、48h、72h、96h respectively compared with NC.4. The scratch test showed the mortality of RA FLS was dramatically decreased after infected with pLenti6/V5-PTEN and pLenti6/shAkt-2. Furthermore, In comparison with NC, the transfectants displayed high mortality in both cell counts and invasiveness 72 h after infection.ConclusionpLenti6/V5-PTEN and pLenti6/shAkt-2 lentiviral expression vectors can down-regulate p-Akt protein expression and inhibit RA FLS proliferation and migration.更多还原