RNA干扰沉默TREM-2对内毒素所致急性肺损伤小鼠肺泡巨噬细胞炎症反应的影响
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摘要
背景
急性肺损伤(acute lung injury,ALI)是临床常见的呼吸系统危重症之一,感染是导致ALI的常见原因,也是该病的首位高危因素。革兰氏阴性杆菌是目前临床感染中主要的病原菌,易导致ALI,主要是由于其外膜上的脂多糖(Lipopolysaccharide,LPS),又称内毒素(endotoxin)活化炎性细胞释放大量炎症因子所致,故LPS是介导ALI的重要启动子。肺泡巨噬细胞(alveolar macrophage,AM)广泛分布于肺及气道上皮内,是呼吸道的免疫核心细胞,也是LPS的主要效应细胞。AM表面可表达多种识别病原体的受体,如Toll蛋白样受体4(Toll-like receptor 4,TLR4)、髓样细胞表达的激发受体(triggering receptorsexpressed on myeloid cells,TREMs)、清道夫受体(scavenger receptors,SR)等,LPS通过与这些模式识别受体(pattern recognition receptors,PRRs)结合,启动跨膜及细胞内信号转导,是引起过度炎症反应及ALI的主要因素之一。研究这些受体与LPS识别的信号通路及阻断效应,有助于了解ALI的发生机制,对于寻找ALI治疗的新靶位,具有重要的理论与实际意义。
TLR4是LPS靶细胞膜上的跨膜受体,同时也是天然免疫系统主要的病原模式识别受体,可诱导参与炎症反应的基因表达。TLR4被LPS激活后启动的胞内信号转导最终激活核因子-kB(NF-kB),从而诱导AM产生免疫炎性细胞因子,或扩大非特异性防御反应,或诱导特异性免疫反应,清除入侵的病原体。但过度激活的炎症细胞持续产生大量促炎症因子并发生“瀑布样”级联反应,使炎症反应不断扩大甚至失去控制,最终导致自身破坏性的过度炎症反应。
TREM是2000年新发现的一个免疫球蛋白超家族受体,主要作用在于参与天然免疫应答并且能够调节应答的发展时相。人类TREM至少包括TREM-1和TREM-2这两个受体。TREM-1可以应答内源和外源性危险信号,协同和放大TLR4介导的信号通路,加强炎症反应,最终导致失控性炎症反应的发生如ALI、致命性败血症、内毒素休克等。与TREM-1的作用相反,TREM-2抑制炎症介质的释放,是体内重要的抗炎受体。TREM-2与配体结合后,通过TREM-2/DAP12(死亡细胞激活受体的相关蛋白)介导的信号转导通路,引发细胞内一系列蛋白酪氨酸磷酸化反应及酶促反应而激活髓样细胞,参与T细胞应答,是体内中枢神经系统炎症调节的重要通路。如何在肺组织发挥巨噬细胞表面TREM-2受体的抗炎作用,使炎症反应调控在对机体有利而又不至于对组织、器官造成损害的适度范围内,达到防治ALI等感染性疾病的目的是我们研究的重点。TREM-2是否通过调节TLR4的反应强度来调控炎症反应的强弱程度是我们研究的目标。
RNA干扰(RNA interference,RNAi)是新近发展起来的一种封闭基因表达的有效方法,为基因治疗疾病开启了一扇希望之门。RNAi是指体外人工合成的或体内的双链RNA(double strand RNA,dsRNA),在酶的作用下产生大量的小干扰RNA(small interferenceRNA,siRNA),在细胞内特异性将与之同源的mRNA降解成21nt-23nt的小片段,使相应的基因沉默的一种细胞反应过程,属于转录后基因沉默(posttranscriptional gene silencing,PTGS)的一种。由于RNAi具有高度的序列专一性和有效的干扰活力,可以选择性地使特定基因沉默,获得功能丧失或降低突变,而且与传统的基因敲除及反义技术相比,这一技术具有投入少、周期短、操作简单及效率高等优势。近年来已成为研究特定基因功能和信号转导系统上下游分子相互关系的一种强有力的工具,并有可能为功能基因组学、基因治疗学等众多领域带来新的突破。而国内外尚未见通过基因干预技术对小鼠肺泡巨噬细胞TREM-2信号通道进行调控的报道,采用RNAi技术对TREM-2在ALI反应中的作用及其机制进行研究,有助于阐明以RNAi技术为基础的转录后水平进行抗炎治疗的潜在用途、可能性及局限性。目的
本研究拟通过RNAi技术,选择TREM-2为靶点,构建编码小鼠TREM-2基因的shRNA(short hairpin RNA)真核质粒表达载体,从中筛选出能高效干扰TREM-2 mRNA的siRNA序列;将其合成重组慢病毒载体感染小鼠肺泡巨噬细胞,观察其对TREM-2基因mRNA和相应功能蛋白表达的抑制程度;探讨在LPS所诱导小鼠肺泡巨噬细胞炎症反应中TREM-2在转录及翻译水平的表达规律以及与促炎症因子分泌的关系;通过RNAi技术使体外培养的小鼠肺泡巨噬细胞TREM-2基因沉默后予以LPS刺激,检测TREM-2、TLR4和TREM-1受体的表达和促炎症因子的分泌,观察沉默TREM-2表达对LPS炎症反应的影响;在体外研究的基础上,通过气管内感染重组慢病毒载体后,再予以LPS刺激复制ALI动物模型,检测肺组织中TLR4和TREM受体的表达情况,以及肺泡灌洗液炎症因子的分泌水平,探索TREM-2在ALI治疗中的应用前景,为阐明革兰氏阴性杆菌感染的致病机理提供实验基础,也为临床防治这类感染提供新思路。方法
1构建靶向小鼠TREM-2基因shRNA真核质粒表达载体
首先在GenBank上选取小鼠TREM-2mRNA序列,根据siRNA设计原则,设计合成TREM-2-shRNA及无任何干扰效果的阴性shRNA的DNA模板单链,并进行BLAST验证。在两端加入限制性内切酶BamHⅠ、HindⅢ的酶切位点,退火形成双链。质粒pGCsi的BamHⅠ+HindⅢ双酶切线性化,稀释退火片段与线性化pGCsi质粒表达载体经T4DNA连接酶连接,经过PCR鉴定和DNA测序。构建的三个重组质粒分别命名为pTREM-2-shRNA1、pTREM-2-shRNA2和pNeg-shRNA(阴性对照)。
2质粒pTREM-2-shRNA转染小鼠肺泡巨噬细胞筛选干扰TREM-2的最佳siRNA序列
①体外原代培养小鼠AM:采用支气管肺泡灌洗法从C57BL/6小鼠肺内分离AM,并用贴壁法纯化,在含有10%胎牛血清的RPMI 1640培养液中原代培养AM。倒置相差显微镜观察AM的形态和生长特性,并进行AM特异性表型CD68表达的检测鉴定。②将不同比例的质粒pTREM-2-shRNA和转染试剂TransFectin Lipid的复合物,以不同时间转染小鼠AM,因质粒中含有绿色荧光蛋白(GFP)的报告基因,转染后48小时可用荧光显微镜观察荧光表达,并用流式细胞仪FITC的常规检测方法检测转染效率,同时用MTT法检测细胞存活率。根据转染效率优化转染条件,用最佳配比的质粒pTREM-2-shRNA和转染试剂复合物转染AM,分别于转染前及转染后48h,采用实时荧光定量RT-PCR检测TREM-2mRNA表达水平,同时设立未转染组、空质粒组(pCon)、阴性质粒组(pNeg-shRNA)作为对照。
3构建靶向抑制小鼠TREM-2基因的shRNA重组。幔病毒载体
利用Lenti慢病毒包装系统,自构建的pTREM-2-shRNA真核质粒表达载体中酶切出目的基因片段,与慢病毒穿梭质粒pLenti-EGFP-U6相连接,慢病毒包装必需的3个蛋白LP1、LP2、VSV-G分别独立放置在3个质粒上(pLP1、pLP2、pVSV-G)。四种质粒共转染293T细胞中,包装成重组的慢病毒载体Lenti-EGFP-TREM-2-shRNA并扩增、纯化、鉴定。构建的两个重组慢病毒分别命名为Lenti-EGFP-TREM-2-shRNA和Lenti-Neg-shRNA(阴性对照)。
4慢病毒介导的TREM-2-shRNA干扰体外培养小鼠肺泡巨噬细胞TREM-2表达的研究
应用已构建好的Lenti-EGFP-TREM-2-shRNA真核表达载体感染小鼠AM,对感染复数(MOI)、不同配比的慢病毒.感染试剂复合物、感染时间等感染条件进行优化的前提下,用最佳感染条件感染小鼠AM,分别于感染前及感染后72h,采用荧光定量RT-PCR和流式细胞仪检测TREM-2mRNA及蛋白表达水平的变化。同时设立未感染组、空白对照组(Lenti-EGFP)、阴性对照组(Lenti-Neg-shRNA)作为对照。
5小鼠肺泡巨噬细胞TREM-2在内毒素炎症反应中的表达
以不同浓度(10ng/ml、100ng/ml、1000 ng/ml)LPS分别刺激小鼠肺泡巨噬细胞0h、3h、6h、12h、24h,至预定时间后收集细胞,荧光定量PCR、流式细胞仪等方法检测在LPS诱导的炎症反应过程中TREM-2基因在转录及翻译水平的表达,同时通过ELISA法检测细胞培养上清液中促炎症因子的含量。
6慢病毒介导的TREM-2-shRNA对小鼠肺泡巨噬细胞内毒素炎症反应的影响
采用已构建好的Lenti-EGFP-TREM-2-shRNA,选择最佳感染条件将其导入小鼠AM中,于感染后72h再给予100ng/mlLPS刺激3h,收集细胞。通过荧光定量PCR、流式细胞仪等方法检测在LPS诱导的炎症反应过程中TREM-2、TLR4和TREM-1基因在转录及翻译水平的表达,同时通过ELISA法检测细胞培养上清液中促炎症因子TNF-a和IL-10含量的变化。同时设立未感染组、LPS组、空白对照+LPS组(感染Lenti-EGFP后给予LPS)、阴性对照+LPS组(感染Lenti-Neg-shRNA后给予LPS)作为对照。
7慢病毒介导的TREM-2-shRNA对内毒素所致急性肺损伤小鼠炎症反应的影响
健康雄性C57BL/6小鼠用2%戊巴比妥钠(30mg/kg)腹腔注射麻醉后通过气管滴注的方法感染已构建好的Lenti-EGFP-TREM-2-shRNA(1×10~8TU/animal),感染后待苏醒,继续饲养。7d后,麻醉下气管内滴注内毒素(2.5mg/kg溶于50μl无菌盐水中),建立ALI小鼠模型,24h后结束实验。分别进行以下检测:1)留取各组小鼠肺组织标本制成切片,HE染色后用普通光学显微镜观察。2)通过免疫组化和免疫荧光方法观察在LPS诱导的炎症反应过程中肺组织TREM-2、TLR4和TREM-1的表达情况。3)通过荧光定量PCR、Western-Blot等方法检测肺组织TREM-2、TLR4和TREM-1基因在转录及翻译水平的表达。4)运用ELISA法检测支气管肺泡灌洗液中(BALF)下游炎症因子TNF-a和IL-10蛋白的表达水平。同时设立未感染组、生理盐水对照组(气管内滴注无菌生理盐水)、ALI组(气管内滴注LPS)、空白对照+ALI组(气管内滴注Lenti-EGFP后给予LPS)、阴性对照+ALI组(气管内滴注Lenti-Neg-shRNA后给予LPS)作为对照。
结果
1构建编码小鼠TREM-2基因shRNA质粒表达载体经过PCR鉴定和DNA测序,证明构建的靶向抑制小鼠TREM-2基因的shRNA真核表达载体质粒pTREM-2-shRNA1、pTREM-2-shRNA2和pNeg-shRNA无基因突变,构建正确,符合实验要求。
2 pTREM-2-shRNA体外转染小鼠肺泡巨噬细胞筛选干扰TREM-2的最佳siRNA序列
2.1小鼠AM的体外培养和鉴定
原代培养的小鼠AM状态良好,经过免疫组织化学鉴定,证实AM体外模型建立成功。
2.2转染条件的优化
不同比例的质粒DNA与TransFectin Lipid转染试剂的复合物转染小鼠AM(1×10~6/ml),48小时用流式细胞仪检测,当质粒DNA的量为0.5μg,质粒DNA质量与TransFectin Lipid转染试剂体积之比为1:2时,转染效率最高(60.75+4.16)%,与其余组及对照组相比差异具有统计学意义(P＜0.05)。此时细胞生存率为90.5%,能保证较低的细胞毒性,可以作为进一步实验的最佳选择。
2.3 pTREM-2-shRNA对小鼠肺泡巨噬细胞TREM-2基因表达的抑制作用
质粒转染小鼠肺泡巨噬细胞48h,收集细胞进行荧光定量RT-PCR反应,结果显示:pTREM-2-shRNA1组和pTREM-2-shRNA2组TREM-2基因mRNA的表达较转染前,及各对照组明显降低,差异具有统计学意义(P＜0.05),pTREM-2-shRNA1组(TREM-2mRNA表达9.38e+04+1.28e+04,mRNA抑制效率为45.5%)较pTREM-2-shRNA2组(TREM-2mRNA表达1.15e+05+2.19e+04,mRNA抑制效率为33.1%)抑制程度更强(P＜0.05);与未转染组相比,pNeg组及pCon组TREM-2mRNA表达水平无显著性差异(P＞0.05)。因此筛选出siRNA1为特异性干扰小鼠肺泡巨噬细胞TREM-2的基因片段,其核苷酸序列为5'-GAT GCT GGA GAT CTC TGGG-3',位于TREM-2基因序列的573-591,可以进行后期的慢病毒包装。
3构建靶向抑制小鼠TREM-2基因shRNA重组慢病毒载体
经测序、PCR检测和荧光显微镜观察,证实成功构建了能够表达TREM-2-shRNA的重组慢病毒载体并制备出高滴度的重组病毒。
4慢病毒介导的TREM-2-shRNA干扰体外培养小鼠肺泡巨噬细胞TREM-2表达的研究
4.1感染条件的优化
不同数量的慢病毒与Polybrene感染试剂的复合物感染小鼠肺泡巨噬细胞(1×10~6/ml),72小时用流式细胞仪检测,当MOI=20(即慢病毒的数量为2×10~7TU),Polybrene终浓度为6μg/ml时,感染效率最高(87.58+4.02)%,与其余组及对照组相比差异具有统计学意义(P＜0.05)。此时细胞生存率为89.2%,能保证较低的细胞毒性,可以选择MOI=20作为Lenti-shRNA感染小鼠AM的最佳MOI。
4.2 Lenti-EGFP-TREM-2-shRNA对小鼠肺泡巨噬细胞TREM-2基因表达的抑制作用
病毒感染小鼠肺泡巨噬细胞72h,收集细胞进行荧光定量RT-PCR反应和流式细胞仪检测,结果显示:病毒组TREM-2基因mRNA(mRNA表达为3.96E+04+6.50E+03,mRNA抑制率是91.43%)及蛋白的表达(蛋白水平是13.84+2.87,蛋白抑制率是90.79%)较感染前,及各对照组明显降低,差异具有统计学意义(P＜0.05);与未感染组相比,空白对照组及阴性对照组TREM-2基因mRNA和蛋白表达水平无显著性差异(P＞0.05)。5小鼠肺泡巨噬细胞TREM-2在内毒素炎症反应中的表达
LPS(100ng/ml)刺激小鼠AM(0h、3h、6h、12h、24h),收集细胞进行荧光定量RT-PCR反应和流式细胞仪检测,结果显示:TREM-2mRNA及蛋白的表达于3h明显下调,6h降到最低,各组与0h组相比差异均具有显著性(P＜0.05),对照组培养相应时间后表达水平无明显变化(P＞0.05);而以不同浓度LPS(0ng/ml、10ng/ml、100ng/ml、1000 ng/ml)刺激后,TREM-2mRNA及蛋白的表达在100ng/ml开始下降,以1000ng/ml LPS刺激组的表达水平最低,与LPS 0ng/ml组相比差异均具有显著性(P＜0.05),对照组表达水平无明显变化(P＞0.05);LPS(100ng/ml)刺激后,AM上清中的TNF-a和IL-10的分泌量在3h明显升高,以后迅速回降,与0h组相比差异均有显著性(P＜0.05)。
6慢病毒介导的TREM-2-shRNA对小鼠肺泡巨噬细胞内毒素炎症反应的影响
6.1 shRNA对肺泡巨噬细胞TREM-2表达的影响
以Real Time.荧光定量PCR和流式细胞仪检测发现:病毒组TREM-2mRNA(2.69E+04+5.50E+03)和蛋白表达(11.45+1.09)较未感染组、LPS组及各对照组明显减少,差异具有显著性(P＜0.05);而阴性对照组及空白对照组中TREM-2 mRNA和蛋白表达水平与LPS组相比均无显著性差异(P＞0.05)。
6.2 shRNA对肺泡巨噬细胞TLR4表达的影响
病毒组TLR4 mRNA(4.26E+05+2.93E+04)和蛋白表达(140.42+9.53)较未感染组、LPS组及各对照组明显升高,差异有统计学意义(P＜0.05):而阴性对照组及空白对照组中TLR4 mRNA和蛋白表达水平与LPS组相比均无显著性差异(P＞0.05)。
6.3 shRNA对肺泡巨噬细胞TREM-1表达的影响
病毒组TREM-1 mRNA(1.67E+05+9.22E+03)和蛋白表达(93.54+10.33)比未感染组增加,差异具有显著性(P＜0.05),与LPS组、各对照组相比没有显著性差异(P＞0.05)。
6.4 shRNA对肺泡巨噬细胞分泌促炎症因子的影响
以ELISA法检测细胞上清中促炎症因子TNF-a和IL-10的含量,病毒组TNF-a(197.15+11.86)和IL-10(872.38+45.67)含量比未感染组、LPS组及各对照组明显增加,差异均具有显著性(P＜0.05);而阴性对照组及空白对照组中促炎症因子含量与LPS组相比均无显著性差异(P＞0.05)。
7慢病毒介导的TREM-2-shRNA对内毒素所致急性肺损伤小鼠炎症反应的影响
7.1肺病理组织学检查
肉眼观察:ALI模型组小鼠可见双肺明显肿胀,体积增大,重量增加,色泽暗红。肺表面大片出血区,以双下肺更明显,质地十分脆弱。支气管肺泡灌洗液呈洗肉水样,肺组织切面有大量暗红色泡沫状溢出。病毒组双肺病变更为显著。生理盐水对照组小鼠肺形态基本正常。空白对照组和阴性对照组小鼠肺组织改变程度与ALI模型组基本一致。
光镜观察:ALI模型组小鼠肺组织可见肺间质和肺泡广泛的充血、水肿、出血,肺泡腔有较多巨噬细胞浸润,肺泡腔狭窄。病毒干扰组肺部病变广泛,肺结构明显破坏,巨噬细胞浸润增多,肺泡腔逐渐狭窄,肺泡间隔明显增厚。生理盐水对照组小鼠肺组织形态基本正常。空白对照组和阴性对照组小鼠肺组织改变程度与ALI模型组基本一致。
7.2免疫组化和免疫荧光法检测肺组织TREM-2、TLR4和TREM-1的蛋白表达(见正文图)
7.3 shRNA对肺组织TREM-2表达的影响
以Real Time-荧光定量PCR和Western-Blot法检测发现:病毒组TREM-2mRNA(mRNA表达为2.69E+04+5.50E+03,mRNA抑制率是89.9%)和蛋白表达(蛋白水平是11.45+1.09,蛋白抑制率是93.5%)较未感染组、ALI组、各对照组明显减少,差异具有显著性(P＜0.05);而阴性对照组及空白对照组中TREM-2 mRNA和蛋白表达水平与ALI组相比无显著性差异(P＞0.05)。
7.4慢病毒介导的shRNA对肺组织TLR4表达的影响
病毒组TLR4 mRNA(4.47E+05+1.17E+04)和蛋白表达(0.889+0.0657)高于各实验组,差异有统计学意义(P＜0.05);而阴性对照组及空白对照组中TLR4 mRNA和蛋白表达水平与ALI组相比均无显著性差异(P＞0.05)。
7.5慢病毒介导的shRNA对肺组织中TREM-1表达的影响
病毒组TREM-1 mRNA(2.84E+05+2.52E+04)和蛋白表达(0.697+0.0845)比未感染组增加,差异具有显著性(P＜0.05),与ALI组、各对照组相比没有显著性差异(P＞0.05)。
7.6慢病毒介导的shRNA对支气管肺泡灌洗液(BALF)促炎症因子的影响
以ELISA法检测BALF中病毒组促炎症因子TNF-a(139.81+16.28)和IL-10(1281.71+54.75)的含量比未感染组、ALI组、各对照组均升高(P＜0.05),而阴性对照组及空白对照组与ALl组相比均无显著性差异(P＞0.05)。
结论
1成功构建了以小鼠TREM-2基因为靶位的真核质粒表达载体pTREM-2-shRNA。
2通过优化转染条件并联合使用转染试剂安全、有效地对小鼠AM进行基因转染,TREM-2靶向性shRNA转染可以有效降低TREM-2表达水平,且干扰效果具有序列特异性,其中以shRNA-1最明显。
3成功构建了携带TREM-2-shRNA的重组慢病毒载体。
4应用RNAi技术,通过特异性的shRNA重组慢病毒载体在小鼠AM中实现对靶基因mRNA的降解,进而抑制相应功能蛋白的表达。
5选择以100ng/ml LPS刺激3h作为检测时间点,为后续研究构建了实验平台。
6体外实验证明TREM-2靶向性shRNA转染有效沉默了LPS刺激下TREM-2的表达,同时提高了TLR4受体的表达水平,促进TNF-a和IL-10的分泌,加强了TLR4介导的LPS信号转导效能。从反面证实TREM-2具有负向调节炎症的作用,在TLR4信号通路的调节中占有重要地位。
7通过气管内滴注的方法将携带shRNA的慢病毒载体安全有效地在小鼠肺内转染,成功发挥基因沉默作用,有效抑制了ALI小鼠肺组织TREM-2 mRNA及相应功能蛋白的表达,加重了肺组织病变;同时提高了肺组织TLR4受体的表达水平,促进BALF中TNF-a和IL-10的分泌,再次证实TREM-2是体内重要的抗炎受体,在防止过度炎症反应和组织损伤中发挥重要作用,从而为ALI的防治提供潜在的新靶点。
Backgroud
Acute lung injury(ALI),mainly caused by infection,is one of the most common critical illnesses of respiratory system.ALI induced by Gram-negative bacteria infection is due to effects of cytokines from inflammatory cells activated by Lipopolysaccharides(LPS)or endotoxin.Alveolar macrophages(AM)are important for airway defense and also the target cells of LPS.AM have some kinds of pattern recognition receptors such as triggering receptors expressed on myeloid cells(TREMs),Toll-like receptor4(TLR4),and scavenger receptors(SR). It is helpful and important to investigate LPS signaling transduction of AM and the blocking effects to understand the mechanism of ALI and find out a new therapy way.
TLR4,as a trans-membrane receptor in LPS target cells,is the key molecular in LPS signaling pathway.TLR4 expression and its perfect function directly affect the effects of LPS signaling transduction.Otherwise,excessive inflammatory reaction results in septic shock, multiple organ dysfunction syndrome(MODS)even death.
TREM is a newly identified member of the immunoglobulin superfamily of receptors and modulate the innate response either by amplifying or dampening TLR-induced signals,and thus play crucial roles in fine-tuning of the inflammatory response.TREM-1 as an amplifier of TLR4 signaling pathway in the systemic inflammatory response syndrome associated with sepsis.By its capacity to promote inflammation,TREM-1 is an important player in ALI, bacterial infection and sepsis.Unlike TREM-1,which is involved in the amplification of inflammation,TREM-2 is emerging as an important negative regulator of autoimmunity. TREM-2 as an important anti-inflammatory receptor and suggest that modulation of TREM-2 expression or function may be an important way to regulate inflammation.TREM-2 acts a negative regulator by modulating the TLR4 signaling.If the expression of TREM-2mRNA in the alveolar macrophages is reduced,it will results in increased lung injury.This converse result confirms the criticality of TREM-2 in the lung tissue,reveals an anti-inflammatory role in the process,and allows us to direct potential therapies in ALI.
RNA interference(RNAi)is a new genetic interference technology,which specially degrades the corresponding sequence mRNA by the mediate of double-stranded RNA(dsRNA) synthesized in vitro or formed in vivo which are diced up 21-23 nucleotide-long small interfering RNA(siRNA)by the enzyme Dicer to induce the post-transcriptional gene silencing. In recent years,RNAi has been found in organisms from epiphytes to plants,and from invertebrates to mammalian animals.Compared with gene knockout,RNAi is simple,time saving and effective.RNAi has now been widely used in studies of gene function.With the breakthrough of RNAi technology in mammalian cells,more and more studies begin to apply the RNAi in the research of functional genomics and gene therapy.
Objective
In order to investigate the role of TREM-2 signaling pathway in ALI induced by LPS, eukaryotic expression plasmid vector of short hair RNA(shRNA)targetting mice TREM-2 gene would be designed and constructed to establish a rapid screen system for siRNA targeting TREM-2.Then lentivirus vectors was transfected it into mice AM cells to investigate the inhibitory action to TREM-2 mRNA and protein expression level.To investigate the expression pattern of TREM-2 and its relationship with the secretion of proinflammatory factors during AM cell inflammatory response induced by LPS.In vitro siRNAs were administered to the cultured AM cells before LPS stimulation to study the effect of shRNA targeting TREM-2 gene on inflammatory responses induced by LPS.Expression of TLR4,TREM-1,TREM-2 of AM and cytokine proteins were detected.After transfected from respiratory tract,mice were treated with LPS.Expression of TLR4,TREM-1,TREM-2 of pulmonary tissues and cytokine proteins in BALF were detected.
Methods
1 Construction of eukaryotic expression vector for shRNA targeting TREM-2 gene
Firstly,two RNAi sites targeting mice TREM-2 gene in gene bank were selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed.Secondly,two pairs of oligonucleotides fragments were synthesized and annealed to double strands.Thirdly,the double strands were cloned into plasmid vector pGCsi.Finally,the recombinant plasmids were identified by PCR and sequencing analysis.The three kinds of recombinant plasmids were named pTREM-2-shRNA1、pTREM-2-shRNA2 and pNeg-shRNA.
2 Screening siRNA sequence which could suppression TREM-2 expression most effectively in vitro
Alveolar macrophages were isolated and cultured,cell morphous and growth condition were observed by inverted microscope.After transfected with transfection complex comprising different proportion of pTREM-2-shRNA plasmid and TransFectin Lipid reagent for 12h,24h, 48h,72h,96h,transfection efficiency was defected by inverted fluorescence microscope and flow cytometry(FCM)to choose the optimal proportion of pTREM-2-shRNA plasmid and TransFectin Lipid reagent,and the time point which lead to maximal transfection efficiency for further research.After 48h of being transfected with pTREM-2-shRNA,total RNA was extracted from cells.TREM-2mRNA expression level was evaluated by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR).Total RNA of pCon group(alveolar macrophages transfected with p-GCsi),pNeg group(alveolar macrophages transfected with negative shRNA vector),and non transfected group were also evaluated as control.
3 Construction of recombinant lentivirus vectors expressing shRNA targeting mice TREM-2 gene
The mice TREM-2-shRNA segments were obtained from plasmids pTREM-2-shRNA which were constructed at an earlier date by restriction endonucleases digestion and then cloned into the shuttle plasmids pLenti-EGFP-U6 to form recombinant plasmids pLenti-EGFP-TREM -2-shRNA-U6.The recombinant plasmids were co-transfected with genomic plasmids(pLP1、pLP2、pVSV-G)into 293T cells to package recombinant lentivirus which were named Lenti-EGFP-TREM-2-shRNA.Then the recombinant lentivirus were propagated, purified and identified.
4 Study on the inhibition of TREM-2 mRNA and protein expression in mice alveolar macrophages mediated by Lenti-EGFP-TREM-2-shRNA
Mice alveolar macrophages were transfected with complex of the Multiplicity of Infection(MOI),the optimal ratio of lenti to Polybrene and different time.Transfection efficiency was defected by inverted fluorescence microscope and flow cytometry(FCM)to choose the best MOI,the optimal proportion of lenti to Polybrene,and the time point which lead to maximal transfection efficiency for further research.After 72h of being transfected with Lenti-EGFP-TREM-2-shRNA,total RNA was extracted from alveolar macrophages.TREM-2 mRNA expression level was evaluated by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level was evaluated by fluorescence-activated cell sorting(FACS).Total RNA and protein of Con group(AM transfected with Lenti-EGFP)、Neg group(AM transfected with negative shRNA Lentivirus), and non transfected group were also evaluated as control.
5 Expression of TREM-2 in inflammatory responses induced by lipoplysaccharide to alveolar macrophages
Alveolar macrophages were stimulated with the LPS at different concentration(10ng/ml、100ng/ml、1000 ng/ml)for for 0h,3h,6h,12h,24h in vitro.The expression levels of TREM-2 and protein were assayed by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level was evaluated by FACS analysis.Meanwhile,the proinflammatory factors TNF-a,IL-10 in the supernatants of AM were detected by ELISA.
6 Effect of RNA interference on TLR4 signaling pathway in alveolar macrophage of mice induced by iipopolysaccharide
Lenti-EGFP-TREM-2-shRNA were transfected into alveolar macrophage of mice in vitrofor 72h,then the cells were stimulated with the LPS(100ng/ml)for 3h.At 72h post-transfection,the subsequent changes in TREM-2mRNA,TLR4mRNA and TREM-1mRNA were detected by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR) and protein expression level were evaluated by FACS analysis,respectively.ELISA was applied to examine the secretion of proinflammatory factors TNF-a,IL-10 in the supernatants of AM.Total RNA and protein of Con+LPS group(AM transfected with Lenti-EGFP)、Neg +LPS group(AM transfected with negative shRNA Lentivirus),and non transfected group were also evaluated as normal group and LPS group.
7 Experimental studies of Lenti-EGFP-TREM-2-shRNA transfection in vivo before LPS stimulation in mice
After having been anaesthetized by intraperitoneal injection of 2%sodium pentobarbital (30mg/kg body weight),male C57BL/6 mice were instilled intratracheally via a 50μl of microliter syringes with Lenti-EGFP-TREM-2-shRNA(1×10~8TU/animal,50μl total volume)in vivo.Seven days after transfection,mice were anesthetized and challenged with intratracheal instillation of LPS(E.coli 055:B5;Sigma)(2.5 mg/kg dissolved in 50μl of sterile normal saline) intravenously intratracheally to copy ALI models.The animals were killed after 24h.Cut the pulmonary tissues into microscopic section and examined the section under light microscope after HE stain.Immunohistochemistric and immunofluorescence stain of TREM-2、TLR4和TREM-1.Total RNA and protein were extracted from pulmonary tissues.The subsequent changes in TREM-2mRNA,TLR4mRNA and TREM-1mRNA were detected by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level were evaluated by Western-Blot,respectively.ELISA was applied to examine the secretion of proinflammatory factors TNF-a and IL-10 in the bronchoalveolar lavage fluid (BALF).Total examination of Con+LPS group(mice transfected with Lenti-EGFP)、Neg+ LPS group(mice transfected with negative shRNA Lentivirus),and non transfected group were also evaluated as normal group,ALI group and saline control group.
Results
1 Construction of eukaryotic expression vector for shRNA targeting TREM-2 gene
Restriction enzyme digestion and sequencing analysis showed that eukaryotic expression vectors of shRNA targeting TREM-2 gene and negative shRNA had been constructed successfully.Sequencing results revealed that the sequences of the vectors were all right.
2 Transfecting with pTREM-2-shRNA into alveolar macrophages in vitro to choose the right sequence to suppress TREM-2 expression
2.1 The cultured AM cells were in good condition.
2.2 Optimization of the transfection condition of pTREM-2-shRNA to mice alveolar macrophage
After being transfected with different proportion of plasmids to TransFectin Lipid at different time,transfection efficiency was evaluated by fluorescence microscope and FCM,it showed that the optimal transfection condition was the proportion of pTREM-2-shRNA to TransFectin Lipid reagent as 0.5μg:1μl at 48h.At this transfection condition,transfection efficiency was(60.75±4.16)%and cells viability was 90.5%.
2.3 Study on the inhibitory action of pTREM-2-shRNA to TREM-2mRNA expression level in alveolar macrophage
48h after transfection,total RNA were extracted,and TREM-2mRNA was evaluated by FQ-RT-PCR,it showed:Compared with control groups the expression level of TREM-2mRNA of pTREM-2-shRNA1 and pTREM-2-shRNA2 were significantly suppressed(P＜0.05);the interference efficiency ofpTREM-2-shRNA1 group(45.5%)was higher than that ofpTREM-2-shRNA1 group(33.1%)(P＜0.05);There were no significance difference about TREM-2mRNA expression level of pNeg and pCon group before and after transfection(P＞0.05).So the sequence of siRNAI(5'-GAT GCT GGA GAT CTC TGGG-3',19bp,573-591)was the right sequence to suppress TREM-2 expression.
3 Construction of recombinant lentivirus vectors expressing shRNA targeting mice TREM-2 gene
Recombinant lentivirus vectors Lenti-EGFP-TREM-2-shRNA were constructed successfully, which was confirmed by sequencing analysis and PCR and EGFP expression.
4 Inhibition of TREM-2mRNA expression with Lenti-EGFP-TREM-2-shRNA in vitro
4.1 Optimization of the transfection condition of Lenti-EGFP-TREM-2-shRNA to AM
After being transfected with complex of the Multiplicity of Infection(MOI),the optimal ratio of lenti to polybrene and different time,transfection efficiency was evaluated by fluorescence microscope and FCM,it showed that the optimal transfection condition was the Multiplicity of Infection with twenty,and polybrene with 6μg/ml at 72h.At this transfection condition,transfection efficiency was(87.58±4.02)%and cells viability was 89.2%.So MOI=20 is the best MOI.
4.2 Study on the inhibitory action of Lenti-EGFP-TREM-2-shRNA to TREM-2mRNA expression level in alveolar macrophage
72h after transfection,total RNA were extracted,and TREM-2mRNA was evaluated by FQ-RT-PCR,it showed:Compared with control groups the expression level of TREM-2mRNA with Lenti-EGFP-TREM-2-shRNA was significantly suppressed(P＜0.05), the mRNAexpression was 3.96E+04±6.50E+03,and interference efficiency was 91.43%; There were no significance difference about TREM-2mRNA expression level of Lenti-Neg and Lenti-EGFP group before and after transfection(P＞0.05).
TREM-2 protein was evaluated by FACS analysis,it showed:Compared with control groups the expression level of TREM-2 protein with Lenti-EGFP-TREM-2-shRNA was significantly suppressed(P＜0.05),protein expression was 13.84±2.87,and suppression efficiency was 90.79%;There were no significance difference about expression level TREM-2 protein of Lenti-Neg and Lenti-EGFP group before and after transfection(P＞0.05).
5 TREM-2mRNA expression level of alveolar macrophage in response to LPS
After stimulation of LPS(100ng/ml)cells were harvested either before or 3h,6h,12h or 24h.With total RNA were extracted,TREM-2mRNA was evaluated by FQ-RT-PCR and TREM-2 protein was evaluated by FACS analysis.We found:TREM-2mRNA and protein expression were significantly suppressed at 3h(mRNA 2.96E+05±2.72E+04,protein 106.12±9.47,P＜0.05),even to the lowest point at 6h(mRNA 5.20E+04±1.52E+04,protein 39.13±5.65,P＜0.05)compared with Oh group(mRNA 4.26E+05±6.13E+04,protein 147.36±6.05).There were no significance difference about their expression level of control groups before and after stimulation(P＞0.05).
After priming with of LPS by different concentration(0ng/ml、10ng/ml、100ng/ml、1000 ng/ml)cells were harvested at 3h.With total RNA were extracted,TREM-2mRNA was evaluated by FQ-RT-PCR and TREM-2 protein was evaluated by FACS analysis.We found: TREM-2mRNA and protein expression were markedly down-regulated in 100ng/ml group(mRNA 2.26E+05±7.29E+03,protein 106.12±L6.45,P＜0.05),even to the lowest level in 1000 ng/ml group(mRNA 1.10E+05±5.61E+03,protein 75.62±7.32,P＜0.05)compared with normal group(mRNA 4.26E+05±6.33E+04,protein 147.36±7.43).There were no significance difference about their expression level of control group before and after stimulation(P＞0.05).
The LPS(100ng/ml)also induced massive secretion of TNF-a and IL-10,which peaked at 3h(compared with Oh,P＜0.05),then reduced rapidly.
6 Lentivirus-mediated TREM-2-shRNA on mouse alveolar macrophages in LPS inflammatory response
6.1 shRNA on TREM-2 expression of alveolar macrophage
TREM-2mRNA was evaluated by FQ-RT-PCR.In the virus group TREM-2mRNA (2.69E+04±5.50E+03)was significantly lower than that in other groups(P＜0.05).While the control groups were not significantly different compared with the LPS group(P＞0.05). TREM-2 protein(11.45±1.09)was consistent with TREM-2 mRNA to FACS analysis.
6.2 shRNA on alveolar macrophage expression of TLR4
In the virus group TLR4mRNA(4.26E+05±2.93E+04)was significantly higher than that in other groups(P＜0.05).While the control groups were not significantly different compared with the LPS group(P＞0.05).TLR4 protein(140.42±9.53)was consistent with TLR4 mRNA.
6.3 shRNA on the alveolar macrophage TREM-1 expression
TREM-1mRNA(1.67E+05±9.22E+03)in virus group was significantly higher than that in the normal group(P＜0.05),but had no significant difference with other groups(P＞0.05).Protein expression was in line with the TREM-1 mRNA.
6.4 shRNA on pro-inflammatory factor in alveolar macrophage
We examinated pro-inflammatory factors TNF-a and IL-10 levels in cell supernatants with ELISA.TNF-a(197.15±11.86)and IL-10(872.38±45.67)of virus interference group were significantly higher than those in other groups(P＜0.05,respectively);while the negative control group and blank control group had no significantly different with the LPS groups(P＞0.05).
7 Influence of Lentivirus-mediated TREM-2-shRNA on inflammatory response of acute lung injury induced by LPS in mice
7.1 Lung pathological changes by fight microscope
ALI model mice can be seen clearly swollen lungs.The volume increased and weight of lung increased.Large surface area of lung bleeding was very fragile texture.Bronchoalveolar lavage fluid was like water washed meat.Lentiviral group was more significant heavier than the ALI group.
The ALI group could be seen extensive hyperemia,edema,hemorrhage in lung tissue. Alveolar space was narrow and infiltrated by more macrophages.Virus group had been extensive lung lesions.The saline control group of mice lung tissue was basically normal morphology.The blank control group and negative control group changed as same as the ALI group.
7.2 Immunohistochemistry and Immunofluorescence assay(to see photoes)
7.3 shRNA on TREM-2 expression of pulmonary tissues
With FQ-RT-PCR on TREM-2 mRNA,we found that:in virus group of TREM-2mRNA (2.66E+04±4.20E+03)was significantly lower than those in other groups(P＜0.05),the mRNAexpression was 2.66E+04±4.20E+03,and interference efficiency was 89.9%.TREM-2 mRNA in the negative control group and blank control group was no significantly different(P＞0.05).To western-blot detection(β-actin as internal control),TREM-2 protein was consistent decline with TREM-2 mRNA.Protein expression was 0.062±0.0152,and interference efficiency was 93.5%.
7.4 shRNA on pulmonary tissues expression of TLR4
TLR4mRNA in virus group(4.47E+05±1.17E+04)was significantly higher than that in other groups(P＜0.05).The negative control group and blank control group were not significantly different compared with the ALI group(P＞0.05).The protein expression of TLR4 increased with TLR4 mRNA expression in line.
7.5 shRNA on TREM-1 expression in lung
TREM-1 mRNA(2.84E+05±2.52E+04)in virus group was significantly higher than that in normal group(P＜0.05),but had no significant difference with other groups(P＞0.05).Protein expression was in line with the TREM-1 mRNA.
7.6 shRNA on pro-inflammatory factor in BALF
ELISA assay in BALF showed:TNF-a(139.81±16.28)and IL-10(1281.71±54.75)were significantly higher than those in other group(P＜0.05),while the negative control group and blank control group in promoting content of inflammatory cytokines were not significantly different compared with the ALI group(P＞0.05).
Conclusions
1 Eukaryotic expression vector of pTREM-2shRNA was constructed successfully.
2 TransFectin Lipid can transfect pTREM-2-shRNA into cultured AM efficiently via optimize transfection condition.The siRNA could effectively suppress the expression of TREM-2 mRNA after transfected for 48 hours especially shRNA-1.
3 Recombinant lentivirus vectors Lenti-EGFP-TREM-2-shRNA were constructed successfully, which was confirmed by restriction enzyme digestion and PCR and EGFP expression.
4 Transfection of Lenti-EGFP-TREM-2-shRNA into AM could inhibit the expression of TREM-2mRNA,subsequently inhibit the protein expression efficiently.
5 Expression of TREM-2 can be down-regulated by LPS,which may contribute to the secretion of proinflammatory factors such as TNF-a and IL-10.LPS(100ng/ml)and time point at 3h were choosed to our experiment.
6 TREM-2-shRNA transfection can selectively suppress TREM-2 expression,and increase in TLR4-mediated LPS signal transduction efficiency,promote the secretion of pro-inflammatory factors induced by LPS,then amplified the inflammatory response in vitro.It suggested that TREM-2 can inhibit the inflammatory response.
7 With TREM-2-shRNA transfection in vivo,lentivirus vectors could be transfected intratracheally safe and effectively.Lenti-EGFP-TREM-2-shRNA could inhibit the expression of TREM-2 mRNA and protein in lung and also increase the expression of the down-stream cytokines like TNF-a and IL-10.Moreover,lung injury was aggravated in mice.This converse result confirms the criticality of TREM-2 in the inflammatory response,and allows us to direct potential therapies in RNAi technology on ALI.
急性肺损伤(acute lung injury,ALI)是临床常见的呼吸系统危重症之一,感染是导致ALI的常见原因,也是该病的首位高危因素。革兰氏阴性杆菌是目前临床感染中主要的病原菌,易导致ALI,主要是由于其外膜上的脂多糖(Lipopolysaccharide,LPS),又称内毒素(endotoxin)活化炎性细胞释放大量炎症因子所致,故LPS是介导ALI的重要启动子。肺泡巨噬细胞(alveolar macrophage,AM)广泛分布于肺及气道上皮内,是呼吸道的免疫核心细胞,也是LPS的主要效应细胞。AM表面可表达多种识别病原体的受体,如Toll蛋白样受体4(Toll-like receptor 4,TLR4)、髓样细胞表达的激发受体(triggering receptorsexpressed on myeloid cells,TREMs)、清道夫受体(scavenger receptors,SR)等,LPS通过与这些模式识别受体(pattern recognition receptors,PRRs)结合,启动跨膜及细胞内信号转导,是引起过度炎症反应及ALI的主要因素之一。研究这些受体与LPS识别的信号通路及阻断效应,有助于了解ALI的发生机制,对于寻找ALI治疗的新靶位,具有重要的理论与实际意义。
TLR4是LPS靶细胞膜上的跨膜受体,同时也是天然免疫系统主要的病原模式识别受体,可诱导参与炎症反应的基因表达。TLR4被LPS激活后启动的胞内信号转导最终激活核因子-kB(NF-kB),从而诱导AM产生免疫炎性细胞因子,或扩大非特异性防御反应,或诱导特异性免疫反应,清除入侵的病原体。但过度激活的炎症细胞持续产生大量促炎症因子并发生“瀑布样”级联反应,使炎症反应不断扩大甚至失去控制,最终导致自身破坏性的过度炎症反应。
TREM是2000年新发现的一个免疫球蛋白超家族受体,主要作用在于参与天然免疫应答并且能够调节应答的发展时相。人类TREM至少包括TREM-1和TREM-2这两个受体。TREM-1可以应答内源和外源性危险信号,协同和放大TLR4介导的信号通路,加强炎症反应,最终导致失控性炎症反应的发生如ALI、致命性败血症、内毒素休克等。与TREM-1的作用相反,TREM-2抑制炎症介质的释放,是体内重要的抗炎受体。TREM-2与配体结合后,通过TREM-2/DAP12(死亡细胞激活受体的相关蛋白)介导的信号转导通路,引发细胞内一系列蛋白酪氨酸磷酸化反应及酶促反应而激活髓样细胞,参与T细胞应答,是体内中枢神经系统炎症调节的重要通路。如何在肺组织发挥巨噬细胞表面TREM-2受体的抗炎作用,使炎症反应调控在对机体有利而又不至于对组织、器官造成损害的适度范围内,达到防治ALI等感染性疾病的目的是我们研究的重点。TREM-2是否通过调节TLR4的反应强度来调控炎症反应的强弱程度是我们研究的目标。
RNA干扰(RNA interference,RNAi)是新近发展起来的一种封闭基因表达的有效方法,为基因治疗疾病开启了一扇希望之门。RNAi是指体外人工合成的或体内的双链RNA(double strand RNA,dsRNA),在酶的作用下产生大量的小干扰RNA(small interferenceRNA,siRNA),在细胞内特异性将与之同源的mRNA降解成21nt-23nt的小片段,使相应的基因沉默的一种细胞反应过程,属于转录后基因沉默(posttranscriptional gene silencing,PTGS)的一种。由于RNAi具有高度的序列专一性和有效的干扰活力,可以选择性地使特定基因沉默,获得功能丧失或降低突变,而且与传统的基因敲除及反义技术相比,这一技术具有投入少、周期短、操作简单及效率高等优势。近年来已成为研究特定基因功能和信号转导系统上下游分子相互关系的一种强有力的工具,并有可能为功能基因组学、基因治疗学等众多领域带来新的突破。而国内外尚未见通过基因干预技术对小鼠肺泡巨噬细胞TREM-2信号通道进行调控的报道,采用RNAi技术对TREM-2在ALI反应中的作用及其机制进行研究,有助于阐明以RNAi技术为基础的转录后水平进行抗炎治疗的潜在用途、可能性及局限性。目的
本研究拟通过RNAi技术,选择TREM-2为靶点,构建编码小鼠TREM-2基因的shRNA(short hairpin RNA)真核质粒表达载体,从中筛选出能高效干扰TREM-2 mRNA的siRNA序列;将其合成重组慢病毒载体感染小鼠肺泡巨噬细胞,观察其对TREM-2基因mRNA和相应功能蛋白表达的抑制程度;探讨在LPS所诱导小鼠肺泡巨噬细胞炎症反应中TREM-2在转录及翻译水平的表达规律以及与促炎症因子分泌的关系;通过RNAi技术使体外培养的小鼠肺泡巨噬细胞TREM-2基因沉默后予以LPS刺激,检测TREM-2、TLR4和TREM-1受体的表达和促炎症因子的分泌,观察沉默TREM-2表达对LPS炎症反应的影响;在体外研究的基础上,通过气管内感染重组慢病毒载体后,再予以LPS刺激复制ALI动物模型,检测肺组织中TLR4和TREM受体的表达情况,以及肺泡灌洗液炎症因子的分泌水平,探索TREM-2在ALI治疗中的应用前景,为阐明革兰氏阴性杆菌感染的致病机理提供实验基础,也为临床防治这类感染提供新思路。方法
1构建靶向小鼠TREM-2基因shRNA真核质粒表达载体
首先在GenBank上选取小鼠TREM-2mRNA序列,根据siRNA设计原则,设计合成TREM-2-shRNA及无任何干扰效果的阴性shRNA的DNA模板单链,并进行BLAST验证。在两端加入限制性内切酶BamHⅠ、HindⅢ的酶切位点,退火形成双链。质粒pGCsi的BamHⅠ+HindⅢ双酶切线性化,稀释退火片段与线性化pGCsi质粒表达载体经T4DNA连接酶连接,经过PCR鉴定和DNA测序。构建的三个重组质粒分别命名为pTREM-2-shRNA1、pTREM-2-shRNA2和pNeg-shRNA(阴性对照)。
2质粒pTREM-2-shRNA转染小鼠肺泡巨噬细胞筛选干扰TREM-2的最佳siRNA序列
①体外原代培养小鼠AM:采用支气管肺泡灌洗法从C57BL/6小鼠肺内分离AM,并用贴壁法纯化,在含有10%胎牛血清的RPMI 1640培养液中原代培养AM。倒置相差显微镜观察AM的形态和生长特性,并进行AM特异性表型CD68表达的检测鉴定。②将不同比例的质粒pTREM-2-shRNA和转染试剂TransFectin Lipid的复合物,以不同时间转染小鼠AM,因质粒中含有绿色荧光蛋白(GFP)的报告基因,转染后48小时可用荧光显微镜观察荧光表达,并用流式细胞仪FITC的常规检测方法检测转染效率,同时用MTT法检测细胞存活率。根据转染效率优化转染条件,用最佳配比的质粒pTREM-2-shRNA和转染试剂复合物转染AM,分别于转染前及转染后48h,采用实时荧光定量RT-PCR检测TREM-2mRNA表达水平,同时设立未转染组、空质粒组(pCon)、阴性质粒组(pNeg-shRNA)作为对照。
3构建靶向抑制小鼠TREM-2基因的shRNA重组。幔病毒载体
利用Lenti慢病毒包装系统,自构建的pTREM-2-shRNA真核质粒表达载体中酶切出目的基因片段,与慢病毒穿梭质粒pLenti-EGFP-U6相连接,慢病毒包装必需的3个蛋白LP1、LP2、VSV-G分别独立放置在3个质粒上(pLP1、pLP2、pVSV-G)。四种质粒共转染293T细胞中,包装成重组的慢病毒载体Lenti-EGFP-TREM-2-shRNA并扩增、纯化、鉴定。构建的两个重组慢病毒分别命名为Lenti-EGFP-TREM-2-shRNA和Lenti-Neg-shRNA(阴性对照)。
4慢病毒介导的TREM-2-shRNA干扰体外培养小鼠肺泡巨噬细胞TREM-2表达的研究
应用已构建好的Lenti-EGFP-TREM-2-shRNA真核表达载体感染小鼠AM,对感染复数(MOI)、不同配比的慢病毒.感染试剂复合物、感染时间等感染条件进行优化的前提下,用最佳感染条件感染小鼠AM,分别于感染前及感染后72h,采用荧光定量RT-PCR和流式细胞仪检测TREM-2mRNA及蛋白表达水平的变化。同时设立未感染组、空白对照组(Lenti-EGFP)、阴性对照组(Lenti-Neg-shRNA)作为对照。
5小鼠肺泡巨噬细胞TREM-2在内毒素炎症反应中的表达
以不同浓度(10ng/ml、100ng/ml、1000 ng/ml)LPS分别刺激小鼠肺泡巨噬细胞0h、3h、6h、12h、24h,至预定时间后收集细胞,荧光定量PCR、流式细胞仪等方法检测在LPS诱导的炎症反应过程中TREM-2基因在转录及翻译水平的表达,同时通过ELISA法检测细胞培养上清液中促炎症因子的含量。
6慢病毒介导的TREM-2-shRNA对小鼠肺泡巨噬细胞内毒素炎症反应的影响
采用已构建好的Lenti-EGFP-TREM-2-shRNA,选择最佳感染条件将其导入小鼠AM中,于感染后72h再给予100ng/mlLPS刺激3h,收集细胞。通过荧光定量PCR、流式细胞仪等方法检测在LPS诱导的炎症反应过程中TREM-2、TLR4和TREM-1基因在转录及翻译水平的表达,同时通过ELISA法检测细胞培养上清液中促炎症因子TNF-a和IL-10含量的变化。同时设立未感染组、LPS组、空白对照+LPS组(感染Lenti-EGFP后给予LPS)、阴性对照+LPS组(感染Lenti-Neg-shRNA后给予LPS)作为对照。
7慢病毒介导的TREM-2-shRNA对内毒素所致急性肺损伤小鼠炎症反应的影响
健康雄性C57BL/6小鼠用2%戊巴比妥钠(30mg/kg)腹腔注射麻醉后通过气管滴注的方法感染已构建好的Lenti-EGFP-TREM-2-shRNA(1×10~8TU/animal),感染后待苏醒,继续饲养。7d后,麻醉下气管内滴注内毒素(2.5mg/kg溶于50μl无菌盐水中),建立ALI小鼠模型,24h后结束实验。分别进行以下检测:1)留取各组小鼠肺组织标本制成切片,HE染色后用普通光学显微镜观察。2)通过免疫组化和免疫荧光方法观察在LPS诱导的炎症反应过程中肺组织TREM-2、TLR4和TREM-1的表达情况。3)通过荧光定量PCR、Western-Blot等方法检测肺组织TREM-2、TLR4和TREM-1基因在转录及翻译水平的表达。4)运用ELISA法检测支气管肺泡灌洗液中(BALF)下游炎症因子TNF-a和IL-10蛋白的表达水平。同时设立未感染组、生理盐水对照组(气管内滴注无菌生理盐水)、ALI组(气管内滴注LPS)、空白对照+ALI组(气管内滴注Lenti-EGFP后给予LPS)、阴性对照+ALI组(气管内滴注Lenti-Neg-shRNA后给予LPS)作为对照。
结果
1构建编码小鼠TREM-2基因shRNA质粒表达载体经过PCR鉴定和DNA测序,证明构建的靶向抑制小鼠TREM-2基因的shRNA真核表达载体质粒pTREM-2-shRNA1、pTREM-2-shRNA2和pNeg-shRNA无基因突变,构建正确,符合实验要求。
2 pTREM-2-shRNA体外转染小鼠肺泡巨噬细胞筛选干扰TREM-2的最佳siRNA序列
2.1小鼠AM的体外培养和鉴定
原代培养的小鼠AM状态良好,经过免疫组织化学鉴定,证实AM体外模型建立成功。
2.2转染条件的优化
不同比例的质粒DNA与TransFectin Lipid转染试剂的复合物转染小鼠AM(1×10~6/ml),48小时用流式细胞仪检测,当质粒DNA的量为0.5μg,质粒DNA质量与TransFectin Lipid转染试剂体积之比为1:2时,转染效率最高(60.75+4.16)%,与其余组及对照组相比差异具有统计学意义(P＜0.05)。此时细胞生存率为90.5%,能保证较低的细胞毒性,可以作为进一步实验的最佳选择。
2.3 pTREM-2-shRNA对小鼠肺泡巨噬细胞TREM-2基因表达的抑制作用
质粒转染小鼠肺泡巨噬细胞48h,收集细胞进行荧光定量RT-PCR反应,结果显示:pTREM-2-shRNA1组和pTREM-2-shRNA2组TREM-2基因mRNA的表达较转染前,及各对照组明显降低,差异具有统计学意义(P＜0.05),pTREM-2-shRNA1组(TREM-2mRNA表达9.38e+04+1.28e+04,mRNA抑制效率为45.5%)较pTREM-2-shRNA2组(TREM-2mRNA表达1.15e+05+2.19e+04,mRNA抑制效率为33.1%)抑制程度更强(P＜0.05);与未转染组相比,pNeg组及pCon组TREM-2mRNA表达水平无显著性差异(P＞0.05)。因此筛选出siRNA1为特异性干扰小鼠肺泡巨噬细胞TREM-2的基因片段,其核苷酸序列为5'-GAT GCT GGA GAT CTC TGGG-3',位于TREM-2基因序列的573-591,可以进行后期的慢病毒包装。
3构建靶向抑制小鼠TREM-2基因shRNA重组慢病毒载体
经测序、PCR检测和荧光显微镜观察,证实成功构建了能够表达TREM-2-shRNA的重组慢病毒载体并制备出高滴度的重组病毒。
4慢病毒介导的TREM-2-shRNA干扰体外培养小鼠肺泡巨噬细胞TREM-2表达的研究
4.1感染条件的优化
不同数量的慢病毒与Polybrene感染试剂的复合物感染小鼠肺泡巨噬细胞(1×10~6/ml),72小时用流式细胞仪检测,当MOI=20(即慢病毒的数量为2×10~7TU),Polybrene终浓度为6μg/ml时,感染效率最高(87.58+4.02)%,与其余组及对照组相比差异具有统计学意义(P＜0.05)。此时细胞生存率为89.2%,能保证较低的细胞毒性,可以选择MOI=20作为Lenti-shRNA感染小鼠AM的最佳MOI。
4.2 Lenti-EGFP-TREM-2-shRNA对小鼠肺泡巨噬细胞TREM-2基因表达的抑制作用
病毒感染小鼠肺泡巨噬细胞72h,收集细胞进行荧光定量RT-PCR反应和流式细胞仪检测,结果显示:病毒组TREM-2基因mRNA(mRNA表达为3.96E+04+6.50E+03,mRNA抑制率是91.43%)及蛋白的表达(蛋白水平是13.84+2.87,蛋白抑制率是90.79%)较感染前,及各对照组明显降低,差异具有统计学意义(P＜0.05);与未感染组相比,空白对照组及阴性对照组TREM-2基因mRNA和蛋白表达水平无显著性差异(P＞0.05)。5小鼠肺泡巨噬细胞TREM-2在内毒素炎症反应中的表达
LPS(100ng/ml)刺激小鼠AM(0h、3h、6h、12h、24h),收集细胞进行荧光定量RT-PCR反应和流式细胞仪检测,结果显示:TREM-2mRNA及蛋白的表达于3h明显下调,6h降到最低,各组与0h组相比差异均具有显著性(P＜0.05),对照组培养相应时间后表达水平无明显变化(P＞0.05);而以不同浓度LPS(0ng/ml、10ng/ml、100ng/ml、1000 ng/ml)刺激后,TREM-2mRNA及蛋白的表达在100ng/ml开始下降,以1000ng/ml LPS刺激组的表达水平最低,与LPS 0ng/ml组相比差异均具有显著性(P＜0.05),对照组表达水平无明显变化(P＞0.05);LPS(100ng/ml)刺激后,AM上清中的TNF-a和IL-10的分泌量在3h明显升高,以后迅速回降,与0h组相比差异均有显著性(P＜0.05)。
6慢病毒介导的TREM-2-shRNA对小鼠肺泡巨噬细胞内毒素炎症反应的影响
6.1 shRNA对肺泡巨噬细胞TREM-2表达的影响
以Real Time.荧光定量PCR和流式细胞仪检测发现:病毒组TREM-2mRNA(2.69E+04+5.50E+03)和蛋白表达(11.45+1.09)较未感染组、LPS组及各对照组明显减少,差异具有显著性(P＜0.05);而阴性对照组及空白对照组中TREM-2 mRNA和蛋白表达水平与LPS组相比均无显著性差异(P＞0.05)。
6.2 shRNA对肺泡巨噬细胞TLR4表达的影响
病毒组TLR4 mRNA(4.26E+05+2.93E+04)和蛋白表达(140.42+9.53)较未感染组、LPS组及各对照组明显升高,差异有统计学意义(P＜0.05):而阴性对照组及空白对照组中TLR4 mRNA和蛋白表达水平与LPS组相比均无显著性差异(P＞0.05)。
6.3 shRNA对肺泡巨噬细胞TREM-1表达的影响
病毒组TREM-1 mRNA(1.67E+05+9.22E+03)和蛋白表达(93.54+10.33)比未感染组增加,差异具有显著性(P＜0.05),与LPS组、各对照组相比没有显著性差异(P＞0.05)。
6.4 shRNA对肺泡巨噬细胞分泌促炎症因子的影响
以ELISA法检测细胞上清中促炎症因子TNF-a和IL-10的含量,病毒组TNF-a(197.15+11.86)和IL-10(872.38+45.67)含量比未感染组、LPS组及各对照组明显增加,差异均具有显著性(P＜0.05);而阴性对照组及空白对照组中促炎症因子含量与LPS组相比均无显著性差异(P＞0.05)。
7慢病毒介导的TREM-2-shRNA对内毒素所致急性肺损伤小鼠炎症反应的影响
7.1肺病理组织学检查
肉眼观察:ALI模型组小鼠可见双肺明显肿胀,体积增大,重量增加,色泽暗红。肺表面大片出血区,以双下肺更明显,质地十分脆弱。支气管肺泡灌洗液呈洗肉水样,肺组织切面有大量暗红色泡沫状溢出。病毒组双肺病变更为显著。生理盐水对照组小鼠肺形态基本正常。空白对照组和阴性对照组小鼠肺组织改变程度与ALI模型组基本一致。
光镜观察:ALI模型组小鼠肺组织可见肺间质和肺泡广泛的充血、水肿、出血,肺泡腔有较多巨噬细胞浸润,肺泡腔狭窄。病毒干扰组肺部病变广泛,肺结构明显破坏,巨噬细胞浸润增多,肺泡腔逐渐狭窄,肺泡间隔明显增厚。生理盐水对照组小鼠肺组织形态基本正常。空白对照组和阴性对照组小鼠肺组织改变程度与ALI模型组基本一致。
7.2免疫组化和免疫荧光法检测肺组织TREM-2、TLR4和TREM-1的蛋白表达(见正文图)
7.3 shRNA对肺组织TREM-2表达的影响
以Real Time-荧光定量PCR和Western-Blot法检测发现:病毒组TREM-2mRNA(mRNA表达为2.69E+04+5.50E+03,mRNA抑制率是89.9%)和蛋白表达(蛋白水平是11.45+1.09,蛋白抑制率是93.5%)较未感染组、ALI组、各对照组明显减少,差异具有显著性(P＜0.05);而阴性对照组及空白对照组中TREM-2 mRNA和蛋白表达水平与ALI组相比无显著性差异(P＞0.05)。
7.4慢病毒介导的shRNA对肺组织TLR4表达的影响
病毒组TLR4 mRNA(4.47E+05+1.17E+04)和蛋白表达(0.889+0.0657)高于各实验组,差异有统计学意义(P＜0.05);而阴性对照组及空白对照组中TLR4 mRNA和蛋白表达水平与ALI组相比均无显著性差异(P＞0.05)。
7.5慢病毒介导的shRNA对肺组织中TREM-1表达的影响
病毒组TREM-1 mRNA(2.84E+05+2.52E+04)和蛋白表达(0.697+0.0845)比未感染组增加,差异具有显著性(P＜0.05),与ALI组、各对照组相比没有显著性差异(P＞0.05)。
7.6慢病毒介导的shRNA对支气管肺泡灌洗液(BALF)促炎症因子的影响
以ELISA法检测BALF中病毒组促炎症因子TNF-a(139.81+16.28)和IL-10(1281.71+54.75)的含量比未感染组、ALI组、各对照组均升高(P＜0.05),而阴性对照组及空白对照组与ALl组相比均无显著性差异(P＞0.05)。
结论
1成功构建了以小鼠TREM-2基因为靶位的真核质粒表达载体pTREM-2-shRNA。
2通过优化转染条件并联合使用转染试剂安全、有效地对小鼠AM进行基因转染,TREM-2靶向性shRNA转染可以有效降低TREM-2表达水平,且干扰效果具有序列特异性,其中以shRNA-1最明显。
3成功构建了携带TREM-2-shRNA的重组慢病毒载体。
4应用RNAi技术,通过特异性的shRNA重组慢病毒载体在小鼠AM中实现对靶基因mRNA的降解,进而抑制相应功能蛋白的表达。
5选择以100ng/ml LPS刺激3h作为检测时间点,为后续研究构建了实验平台。
6体外实验证明TREM-2靶向性shRNA转染有效沉默了LPS刺激下TREM-2的表达,同时提高了TLR4受体的表达水平,促进TNF-a和IL-10的分泌,加强了TLR4介导的LPS信号转导效能。从反面证实TREM-2具有负向调节炎症的作用,在TLR4信号通路的调节中占有重要地位。
7通过气管内滴注的方法将携带shRNA的慢病毒载体安全有效地在小鼠肺内转染,成功发挥基因沉默作用,有效抑制了ALI小鼠肺组织TREM-2 mRNA及相应功能蛋白的表达,加重了肺组织病变;同时提高了肺组织TLR4受体的表达水平,促进BALF中TNF-a和IL-10的分泌,再次证实TREM-2是体内重要的抗炎受体,在防止过度炎症反应和组织损伤中发挥重要作用,从而为ALI的防治提供潜在的新靶点。
Backgroud
Acute lung injury(ALI),mainly caused by infection,is one of the most common critical illnesses of respiratory system.ALI induced by Gram-negative bacteria infection is due to effects of cytokines from inflammatory cells activated by Lipopolysaccharides(LPS)or endotoxin.Alveolar macrophages(AM)are important for airway defense and also the target cells of LPS.AM have some kinds of pattern recognition receptors such as triggering receptors expressed on myeloid cells(TREMs),Toll-like receptor4(TLR4),and scavenger receptors(SR). It is helpful and important to investigate LPS signaling transduction of AM and the blocking effects to understand the mechanism of ALI and find out a new therapy way.
TLR4,as a trans-membrane receptor in LPS target cells,is the key molecular in LPS signaling pathway.TLR4 expression and its perfect function directly affect the effects of LPS signaling transduction.Otherwise,excessive inflammatory reaction results in septic shock, multiple organ dysfunction syndrome(MODS)even death.
TREM is a newly identified member of the immunoglobulin superfamily of receptors and modulate the innate response either by amplifying or dampening TLR-induced signals,and thus play crucial roles in fine-tuning of the inflammatory response.TREM-1 as an amplifier of TLR4 signaling pathway in the systemic inflammatory response syndrome associated with sepsis.By its capacity to promote inflammation,TREM-1 is an important player in ALI, bacterial infection and sepsis.Unlike TREM-1,which is involved in the amplification of inflammation,TREM-2 is emerging as an important negative regulator of autoimmunity. TREM-2 as an important anti-inflammatory receptor and suggest that modulation of TREM-2 expression or function may be an important way to regulate inflammation.TREM-2 acts a negative regulator by modulating the TLR4 signaling.If the expression of TREM-2mRNA in the alveolar macrophages is reduced,it will results in increased lung injury.This converse result confirms the criticality of TREM-2 in the lung tissue,reveals an anti-inflammatory role in the process,and allows us to direct potential therapies in ALI.
RNA interference(RNAi)is a new genetic interference technology,which specially degrades the corresponding sequence mRNA by the mediate of double-stranded RNA(dsRNA) synthesized in vitro or formed in vivo which are diced up 21-23 nucleotide-long small interfering RNA(siRNA)by the enzyme Dicer to induce the post-transcriptional gene silencing. In recent years,RNAi has been found in organisms from epiphytes to plants,and from invertebrates to mammalian animals.Compared with gene knockout,RNAi is simple,time saving and effective.RNAi has now been widely used in studies of gene function.With the breakthrough of RNAi technology in mammalian cells,more and more studies begin to apply the RNAi in the research of functional genomics and gene therapy.
Objective
In order to investigate the role of TREM-2 signaling pathway in ALI induced by LPS, eukaryotic expression plasmid vector of short hair RNA(shRNA)targetting mice TREM-2 gene would be designed and constructed to establish a rapid screen system for siRNA targeting TREM-2.Then lentivirus vectors was transfected it into mice AM cells to investigate the inhibitory action to TREM-2 mRNA and protein expression level.To investigate the expression pattern of TREM-2 and its relationship with the secretion of proinflammatory factors during AM cell inflammatory response induced by LPS.In vitro siRNAs were administered to the cultured AM cells before LPS stimulation to study the effect of shRNA targeting TREM-2 gene on inflammatory responses induced by LPS.Expression of TLR4,TREM-1,TREM-2 of AM and cytokine proteins were detected.After transfected from respiratory tract,mice were treated with LPS.Expression of TLR4,TREM-1,TREM-2 of pulmonary tissues and cytokine proteins in BALF were detected.
Methods
1 Construction of eukaryotic expression vector for shRNA targeting TREM-2 gene
Firstly,two RNAi sites targeting mice TREM-2 gene in gene bank were selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed.Secondly,two pairs of oligonucleotides fragments were synthesized and annealed to double strands.Thirdly,the double strands were cloned into plasmid vector pGCsi.Finally,the recombinant plasmids were identified by PCR and sequencing analysis.The three kinds of recombinant plasmids were named pTREM-2-shRNA1、pTREM-2-shRNA2 and pNeg-shRNA.
2 Screening siRNA sequence which could suppression TREM-2 expression most effectively in vitro
Alveolar macrophages were isolated and cultured,cell morphous and growth condition were observed by inverted microscope.After transfected with transfection complex comprising different proportion of pTREM-2-shRNA plasmid and TransFectin Lipid reagent for 12h,24h, 48h,72h,96h,transfection efficiency was defected by inverted fluorescence microscope and flow cytometry(FCM)to choose the optimal proportion of pTREM-2-shRNA plasmid and TransFectin Lipid reagent,and the time point which lead to maximal transfection efficiency for further research.After 48h of being transfected with pTREM-2-shRNA,total RNA was extracted from cells.TREM-2mRNA expression level was evaluated by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR).Total RNA of pCon group(alveolar macrophages transfected with p-GCsi),pNeg group(alveolar macrophages transfected with negative shRNA vector),and non transfected group were also evaluated as control.
3 Construction of recombinant lentivirus vectors expressing shRNA targeting mice TREM-2 gene
The mice TREM-2-shRNA segments were obtained from plasmids pTREM-2-shRNA which were constructed at an earlier date by restriction endonucleases digestion and then cloned into the shuttle plasmids pLenti-EGFP-U6 to form recombinant plasmids pLenti-EGFP-TREM -2-shRNA-U6.The recombinant plasmids were co-transfected with genomic plasmids(pLP1、pLP2、pVSV-G)into 293T cells to package recombinant lentivirus which were named Lenti-EGFP-TREM-2-shRNA.Then the recombinant lentivirus were propagated, purified and identified.
4 Study on the inhibition of TREM-2 mRNA and protein expression in mice alveolar macrophages mediated by Lenti-EGFP-TREM-2-shRNA
Mice alveolar macrophages were transfected with complex of the Multiplicity of Infection(MOI),the optimal ratio of lenti to Polybrene and different time.Transfection efficiency was defected by inverted fluorescence microscope and flow cytometry(FCM)to choose the best MOI,the optimal proportion of lenti to Polybrene,and the time point which lead to maximal transfection efficiency for further research.After 72h of being transfected with Lenti-EGFP-TREM-2-shRNA,total RNA was extracted from alveolar macrophages.TREM-2 mRNA expression level was evaluated by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level was evaluated by fluorescence-activated cell sorting(FACS).Total RNA and protein of Con group(AM transfected with Lenti-EGFP)、Neg group(AM transfected with negative shRNA Lentivirus), and non transfected group were also evaluated as control.
5 Expression of TREM-2 in inflammatory responses induced by lipoplysaccharide to alveolar macrophages
Alveolar macrophages were stimulated with the LPS at different concentration(10ng/ml、100ng/ml、1000 ng/ml)for for 0h,3h,6h,12h,24h in vitro.The expression levels of TREM-2 and protein were assayed by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level was evaluated by FACS analysis.Meanwhile,the proinflammatory factors TNF-a,IL-10 in the supernatants of AM were detected by ELISA.
6 Effect of RNA interference on TLR4 signaling pathway in alveolar macrophage of mice induced by iipopolysaccharide
Lenti-EGFP-TREM-2-shRNA were transfected into alveolar macrophage of mice in vitrofor 72h,then the cells were stimulated with the LPS(100ng/ml)for 3h.At 72h post-transfection,the subsequent changes in TREM-2mRNA,TLR4mRNA and TREM-1mRNA were detected by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR) and protein expression level were evaluated by FACS analysis,respectively.ELISA was applied to examine the secretion of proinflammatory factors TNF-a,IL-10 in the supernatants of AM.Total RNA and protein of Con+LPS group(AM transfected with Lenti-EGFP)、Neg +LPS group(AM transfected with negative shRNA Lentivirus),and non transfected group were also evaluated as normal group and LPS group.
7 Experimental studies of Lenti-EGFP-TREM-2-shRNA transfection in vivo before LPS stimulation in mice
After having been anaesthetized by intraperitoneal injection of 2%sodium pentobarbital (30mg/kg body weight),male C57BL/6 mice were instilled intratracheally via a 50μl of microliter syringes with Lenti-EGFP-TREM-2-shRNA(1×10~8TU/animal,50μl total volume)in vivo.Seven days after transfection,mice were anesthetized and challenged with intratracheal instillation of LPS(E.coli 055:B5;Sigma)(2.5 mg/kg dissolved in 50μl of sterile normal saline) intravenously intratracheally to copy ALI models.The animals were killed after 24h.Cut the pulmonary tissues into microscopic section and examined the section under light microscope after HE stain.Immunohistochemistric and immunofluorescence stain of TREM-2、TLR4和TREM-1.Total RNA and protein were extracted from pulmonary tissues.The subsequent changes in TREM-2mRNA,TLR4mRNA and TREM-1mRNA were detected by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level were evaluated by Western-Blot,respectively.ELISA was applied to examine the secretion of proinflammatory factors TNF-a and IL-10 in the bronchoalveolar lavage fluid (BALF).Total examination of Con+LPS group(mice transfected with Lenti-EGFP)、Neg+ LPS group(mice transfected with negative shRNA Lentivirus),and non transfected group were also evaluated as normal group,ALI group and saline control group.
Results
1 Construction of eukaryotic expression vector for shRNA targeting TREM-2 gene
Restriction enzyme digestion and sequencing analysis showed that eukaryotic expression vectors of shRNA targeting TREM-2 gene and negative shRNA had been constructed successfully.Sequencing results revealed that the sequences of the vectors were all right.
2 Transfecting with pTREM-2-shRNA into alveolar macrophages in vitro to choose the right sequence to suppress TREM-2 expression
2.1 The cultured AM cells were in good condition.
2.2 Optimization of the transfection condition of pTREM-2-shRNA to mice alveolar macrophage
After being transfected with different proportion of plasmids to TransFectin Lipid at different time,transfection efficiency was evaluated by fluorescence microscope and FCM,it showed that the optimal transfection condition was the proportion of pTREM-2-shRNA to TransFectin Lipid reagent as 0.5μg:1μl at 48h.At this transfection condition,transfection efficiency was(60.75±4.16)%and cells viability was 90.5%.
2.3 Study on the inhibitory action of pTREM-2-shRNA to TREM-2mRNA expression level in alveolar macrophage
48h after transfection,total RNA were extracted,and TREM-2mRNA was evaluated by FQ-RT-PCR,it showed:Compared with control groups the expression level of TREM-2mRNA of pTREM-2-shRNA1 and pTREM-2-shRNA2 were significantly suppressed(P＜0.05);the interference efficiency ofpTREM-2-shRNA1 group(45.5%)was higher than that ofpTREM-2-shRNA1 group(33.1%)(P＜0.05);There were no significance difference about TREM-2mRNA expression level of pNeg and pCon group before and after transfection(P＞0.05).So the sequence of siRNAI(5'-GAT GCT GGA GAT CTC TGGG-3',19bp,573-591)was the right sequence to suppress TREM-2 expression.
3 Construction of recombinant lentivirus vectors expressing shRNA targeting mice TREM-2 gene
Recombinant lentivirus vectors Lenti-EGFP-TREM-2-shRNA were constructed successfully, which was confirmed by sequencing analysis and PCR and EGFP expression.
4 Inhibition of TREM-2mRNA expression with Lenti-EGFP-TREM-2-shRNA in vitro
4.1 Optimization of the transfection condition of Lenti-EGFP-TREM-2-shRNA to AM
After being transfected with complex of the Multiplicity of Infection(MOI),the optimal ratio of lenti to polybrene and different time,transfection efficiency was evaluated by fluorescence microscope and FCM,it showed that the optimal transfection condition was the Multiplicity of Infection with twenty,and polybrene with 6μg/ml at 72h.At this transfection condition,transfection efficiency was(87.58±4.02)%and cells viability was 89.2%.So MOI=20 is the best MOI.
4.2 Study on the inhibitory action of Lenti-EGFP-TREM-2-shRNA to TREM-2mRNA expression level in alveolar macrophage
72h after transfection,total RNA were extracted,and TREM-2mRNA was evaluated by FQ-RT-PCR,it showed:Compared with control groups the expression level of TREM-2mRNA with Lenti-EGFP-TREM-2-shRNA was significantly suppressed(P＜0.05), the mRNAexpression was 3.96E+04±6.50E+03,and interference efficiency was 91.43%; There were no significance difference about TREM-2mRNA expression level of Lenti-Neg and Lenti-EGFP group before and after transfection(P＞0.05).
TREM-2 protein was evaluated by FACS analysis,it showed:Compared with control groups the expression level of TREM-2 protein with Lenti-EGFP-TREM-2-shRNA was significantly suppressed(P＜0.05),protein expression was 13.84±2.87,and suppression efficiency was 90.79%;There were no significance difference about expression level TREM-2 protein of Lenti-Neg and Lenti-EGFP group before and after transfection(P＞0.05).
5 TREM-2mRNA expression level of alveolar macrophage in response to LPS
After stimulation of LPS(100ng/ml)cells were harvested either before or 3h,6h,12h or 24h.With total RNA were extracted,TREM-2mRNA was evaluated by FQ-RT-PCR and TREM-2 protein was evaluated by FACS analysis.We found:TREM-2mRNA and protein expression were significantly suppressed at 3h(mRNA 2.96E+05±2.72E+04,protein 106.12±9.47,P＜0.05),even to the lowest point at 6h(mRNA 5.20E+04±1.52E+04,protein 39.13±5.65,P＜0.05)compared with Oh group(mRNA 4.26E+05±6.13E+04,protein 147.36±6.05).There were no significance difference about their expression level of control groups before and after stimulation(P＞0.05).
After priming with of LPS by different concentration(0ng/ml、10ng/ml、100ng/ml、1000 ng/ml)cells were harvested at 3h.With total RNA were extracted,TREM-2mRNA was evaluated by FQ-RT-PCR and TREM-2 protein was evaluated by FACS analysis.We found: TREM-2mRNA and protein expression were markedly down-regulated in 100ng/ml group(mRNA 2.26E+05±7.29E+03,protein 106.12±L6.45,P＜0.05),even to the lowest level in 1000 ng/ml group(mRNA 1.10E+05±5.61E+03,protein 75.62±7.32,P＜0.05)compared with normal group(mRNA 4.26E+05±6.33E+04,protein 147.36±7.43).There were no significance difference about their expression level of control group before and after stimulation(P＞0.05).
The LPS(100ng/ml)also induced massive secretion of TNF-a and IL-10,which peaked at 3h(compared with Oh,P＜0.05),then reduced rapidly.
6 Lentivirus-mediated TREM-2-shRNA on mouse alveolar macrophages in LPS inflammatory response
6.1 shRNA on TREM-2 expression of alveolar macrophage
TREM-2mRNA was evaluated by FQ-RT-PCR.In the virus group TREM-2mRNA (2.69E+04±5.50E+03)was significantly lower than that in other groups(P＜0.05).While the control groups were not significantly different compared with the LPS group(P＞0.05). TREM-2 protein(11.45±1.09)was consistent with TREM-2 mRNA to FACS analysis.
6.2 shRNA on alveolar macrophage expression of TLR4
In the virus group TLR4mRNA(4.26E+05±2.93E+04)was significantly higher than that in other groups(P＜0.05).While the control groups were not significantly different compared with the LPS group(P＞0.05).TLR4 protein(140.42±9.53)was consistent with TLR4 mRNA.
6.3 shRNA on the alveolar macrophage TREM-1 expression
TREM-1mRNA(1.67E+05±9.22E+03)in virus group was significantly higher than that in the normal group(P＜0.05),but had no significant difference with other groups(P＞0.05).Protein expression was in line with the TREM-1 mRNA.
6.4 shRNA on pro-inflammatory factor in alveolar macrophage
We examinated pro-inflammatory factors TNF-a and IL-10 levels in cell supernatants with ELISA.TNF-a(197.15±11.86)and IL-10(872.38±45.67)of virus interference group were significantly higher than those in other groups(P＜0.05,respectively);while the negative control group and blank control group had no significantly different with the LPS groups(P＞0.05).
7 Influence of Lentivirus-mediated TREM-2-shRNA on inflammatory response of acute lung injury induced by LPS in mice
7.1 Lung pathological changes by fight microscope
ALI model mice can be seen clearly swollen lungs.The volume increased and weight of lung increased.Large surface area of lung bleeding was very fragile texture.Bronchoalveolar lavage fluid was like water washed meat.Lentiviral group was more significant heavier than the ALI group.
The ALI group could be seen extensive hyperemia,edema,hemorrhage in lung tissue. Alveolar space was narrow and infiltrated by more macrophages.Virus group had been extensive lung lesions.The saline control group of mice lung tissue was basically normal morphology.The blank control group and negative control group changed as same as the ALI group.
7.2 Immunohistochemistry and Immunofluorescence assay(to see photoes)
7.3 shRNA on TREM-2 expression of pulmonary tissues
With FQ-RT-PCR on TREM-2 mRNA,we found that:in virus group of TREM-2mRNA (2.66E+04±4.20E+03)was significantly lower than those in other groups(P＜0.05),the mRNAexpression was 2.66E+04±4.20E+03,and interference efficiency was 89.9%.TREM-2 mRNA in the negative control group and blank control group was no significantly different(P＞0.05).To western-blot detection(β-actin as internal control),TREM-2 protein was consistent decline with TREM-2 mRNA.Protein expression was 0.062±0.0152,and interference efficiency was 93.5%.
7.4 shRNA on pulmonary tissues expression of TLR4
TLR4mRNA in virus group(4.47E+05±1.17E+04)was significantly higher than that in other groups(P＜0.05).The negative control group and blank control group were not significantly different compared with the ALI group(P＞0.05).The protein expression of TLR4 increased with TLR4 mRNA expression in line.
7.5 shRNA on TREM-1 expression in lung
TREM-1 mRNA(2.84E+05±2.52E+04)in virus group was significantly higher than that in normal group(P＜0.05),but had no significant difference with other groups(P＞0.05).Protein expression was in line with the TREM-1 mRNA.
7.6 shRNA on pro-inflammatory factor in BALF
ELISA assay in BALF showed:TNF-a(139.81±16.28)and IL-10(1281.71±54.75)were significantly higher than those in other group(P＜0.05),while the negative control group and blank control group in promoting content of inflammatory cytokines were not significantly different compared with the ALI group(P＞0.05).
Conclusions
1 Eukaryotic expression vector of pTREM-2shRNA was constructed successfully.
2 TransFectin Lipid can transfect pTREM-2-shRNA into cultured AM efficiently via optimize transfection condition.The siRNA could effectively suppress the expression of TREM-2 mRNA after transfected for 48 hours especially shRNA-1.
3 Recombinant lentivirus vectors Lenti-EGFP-TREM-2-shRNA were constructed successfully, which was confirmed by restriction enzyme digestion and PCR and EGFP expression.
4 Transfection of Lenti-EGFP-TREM-2-shRNA into AM could inhibit the expression of TREM-2mRNA,subsequently inhibit the protein expression efficiently.
5 Expression of TREM-2 can be down-regulated by LPS,which may contribute to the secretion of proinflammatory factors such as TNF-a and IL-10.LPS(100ng/ml)and time point at 3h were choosed to our experiment.
6 TREM-2-shRNA transfection can selectively suppress TREM-2 expression,and increase in TLR4-mediated LPS signal transduction efficiency,promote the secretion of pro-inflammatory factors induced by LPS,then amplified the inflammatory response in vitro.It suggested that TREM-2 can inhibit the inflammatory response.
7 With TREM-2-shRNA transfection in vivo,lentivirus vectors could be transfected intratracheally safe and effectively.Lenti-EGFP-TREM-2-shRNA could inhibit the expression of TREM-2 mRNA and protein in lung and also increase the expression of the down-stream cytokines like TNF-a and IL-10.Moreover,lung injury was aggravated in mice.This converse result confirms the criticality of TREM-2 in the inflammatory response,and allows us to direct potential therapies in RNAi technology on ALI.
引文
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[57]Regamey A,Hohl D,Liu JW,et al.The tumor suppressor CYLD interacts with TRIP and regulates negatively nuclear factor kappaB activation by tumor necrosis factor.J ExpMed,2003,198(12):1959-1964.