TRAF6基因沉默对内毒素炎症反应的抑制效应研究
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摘要
第一部分内毒素刺激对TRAF6基因表达的影响
目的：观察内毒素刺激对小鼠RAW264.7巨噬细胞肿瘤坏死因子受体相关因子6(TNF receptor-associated factor-6, TRAF6)基因表达的影响,阐明内毒素刺激与TRAF6基因表达的关系。
方法：RAW264.7细胞分为LPS组、地塞米松组、姜黄素组、实验对照组和正常对照组。LPS组以终浓度为100μg/L LPS刺激；地塞米松组以终浓度为0.5 mg/L地塞米松预处理8 h,姜黄素组以终浓度为20μmol/L姜黄素预处理8 h；实验对照组用含DMSO(浓度<0.1%)的DMEM培养液培养,正常对照组作为实验对照不予以任何处理。LPS刺激2 h、4 h、8 h和16 h后,免疫细胞化学染色检测TRAF6蛋白表达,realtime-PCR检测TRAF6 mRNA表达；LPS刺激8 h时Western blot检测TRAF6蛋白,在mRNA水平和蛋白质水平检测内毒素刺激对TRAF6基因表达的影响。
结果：小鼠RAW264.7细胞中TRAF6 mRNA的正常表达量相对较低,当LPS刺激4 h时,TRAF6 mRNA表达明显增加；随着时间延长其表达进一步增加,至16 h时,TRAF6 mRNA表达达到高峰。用地塞米松和姜黄素干预后,TRAF6 mRNA表达量均低于同一时间LPS组(P<0.05),但始终高于正常水平。同一时间点地塞米松组与姜黄素组比较无明显差异(P>0.05),实验对照组细胞TRAF6 mRNA表达水平较正常对照组无明显变化(P>0.05)。Western blot检测TRAF6蛋白表达,结果与TRAF6 mRNA结果相似。
结论：TRAF6 mRNA及TRAF6蛋白表达与LPS刺激有明确的时效关系。
第二部分TRAF6 siRNA表达质粒的构建和筛选
实验一TRAF6 siRNA表达质粒的构建与鉴定
目的：构建针对小鼠TRAF6基因的短发夹RNA (short hairpin RNA, shRNA)真核表达载体,为下一步的细胞转染奠定基础。
方法：在NCBI的Nucleotide库中检索小鼠TRAF6 mRNA序列,应用Invitrogen公司的在线设计软件BLOCK-iTTM RNAi Designer,针对小鼠TRAF6 mRNA设计筛选4条siRNA序列。体外合成编码短发夹RNA序列的DNA寡核苷酸单链,经退火成互补双链,将相应的双链DNA被插入pGCsi-U6/GFP/Hygro质粒中,构建重组质粒pGCsi-TRAF6-shRNA1、2、3、4。将重组质粒转化进E.coli DH5a,最后以PCR法分析鉴定阳性重组质粒并进行DNA测序。在紫外分光光度计上OD260和OD280读取吸光度值,检测质粒DNA的浓度。
结果：siRNA寡核苷酸单链成功退火,合成双链DNA模板,凝胶电泳可见,59 bpMark处呈现清晰条带。靶向TRAF6 mRNA的4种重组质粒载体pGCsi-TRAF6-shRNA,经酶切和DNA测序分析,shRNA编码序列与设计的片段完全一致,证实重组质粒载体构建成功。4种重组质粒DNA OD260/OD280比值均在1.8～2.0之间,质粒DNA质量良好,符合实验要求,为下一步体外、体内实验奠定了基础。
结论：靶向TRAF6基因的重组质粒载体pGCsi-TRAF6-shRNA构建成功,为研究TRAF6对内毒素炎症反应的调控作用奠定基础。
实验二TRAF6基因沉默优化序列的筛选和确认
目的：将TRAF6基因特异性重组质粒载体pGCsi-TRAF6-shRNA转染RAW 264.7细胞,检测TRAF6基因在mRNA和蛋白质水平的表达情况,体外筛选干扰小鼠RAW264.7细胞TRAF6基因的最佳重组质粒载体pGCsi-TRAF6-shRNA。
方法：使用不同比例的DNA质粒／脂质体复合物,将重组质粒转染至RAW 264.7细胞,荧光显微镜观察转染效果。重组质粒转染细胞48 h后,应用MTT法检测转染重组质粒pGCsi-TRAF6-shRNA后细胞活性的变化,realtime-PCR和Western blot检测转染细胞TRAF6基因的mRNA和蛋白质表达,体外筛选出对TRAF6基因表达具有最佳抑制效果的重组质粒。
结果：荧光显微镜分析转染效率显示,DNA(g)/Trans Fectin(L)为2：5时重组质粒转染效率最高。MTT检测结果显示,重组质粒pGCsi-TRAF6-shRNA1、2能显著抑制RAW264.7细胞的增殖活性,与正常对照组相比有显著差异(P<0.01)。realtime-PCR结果显示,pGCsi-TRAF6-shRNA1、2组细胞TRAF6 mRNA表达水平显著降低。其中,pGCsi-TRAF6-shRNA 1对RAW264.7细胞TRAF6基因表达的抑制作用最为显著,抑制率为65.25%,与正常对照组相比具有明显差异(P<0.05)。Western blot检测结果显示,pGCsi-TRAF6-shRNA1对靶基因蛋白表达抑制作用最明显,TRAF6基因蛋白表达抑制率为60.07%,与正常对照组相比差异显著(P<0.05)。
结论：pGCsi-TRAF6-shRNA1真核表达载体可在体外高效地抑制TTRAF6基因表达,为进一步研究TRAF6基因沉默对内毒素炎症反应的影响奠定基础。
第三部分TRAF6 siRNA对RAW264.7细胞内毒素炎症的影响
目的体外研究TRAF6沉默基因对RAW 264.7细胞内毒素炎症反应的影响。
方法实验分为5组：(A)pTRAF6-shRNA1(即pGCsi-TRAF6-shRNA1)组,质粒pTRAF6-shRNA1+LPS; (B)阳性对照组,姜黄素(20μmol/L)+LPS; (C)阴性对照组,空白质粒±LPS；(D)空白对照组,仅予100μg/L LPS刺激；(E)正常对照组,正常培养不予任何处理。LPS刺激后0 h、4 h、8 h和16 h,收集细胞上清,ELISA法测定上清液中TNF-α、IL-1β、TGF-β1。LPS刺激24 h后,收集细胞,realtime PCR方法检测TRAF6、IL-6、COX-2 mRNA, Western blot检测细胞核内NF-κB p65蛋白表达。
结果100μg/L LPS刺激各组RAW264.7细胞后,细胞上清液中TNF-α、IL-1β、TGF-β1表达量都明显增加,与正常对照组相比差异显著(P<0.01),其中TNF-α、IL-1β在8 h内达高峰,TGF-β1在16 h达高峰,说明LPS刺激可引起细胞TNF-α、IL-1β、TGF-β1分泌。将pTRAF6-shRNA1转染RAW264.7细胞后,TNF-α、IL-1β、TGF-β1增长率均明显低于阴性对照组和空白对照组(P<0.01)。Realtime PCR检测结果显示,pTRAF6-shRNA1组IL-6、COX-2 mRNA表达明显下调,与正常对照组比较有显著差异。Western blot结果表明,pTRAF6-shRNA1组细胞核内NF-κB p65蛋白表达明显减少。
结论重组质粒pTRAF6-shRNA可能通过抑制LPS诱导的NF-κB p65核转位,降低相关炎症细胞因子和介质的分泌,抑制RAW264.7细胞内毒素炎症反应。
第四部分TRAF6siRNA对急性肝衰竭内毒素炎症小鼠的保护作用
实验一不同转染方式对GFP表达质粒在小鼠肝脏的表达影响
目的：研究经流体力学注射、门静脉和腹腔常规注射3种不同途径注射GFP表达质粒后,目的基因在小鼠肝脏的表达情况及其转基因效率。
方法：将裸质粒或脂质体包裹的质粒DNA分别应用流体力学注射、门静脉及腹腔常规注射法注入同种异体小鼠体内,48 h后分别取血和肝组织,常规生化方法检测血清谷丙转氨酶(ALT)和总胆红素(TB),HE染色检测肝组织病理变化,新鲜组织冰冻切片观察3种转染途径对质粒DNA在小鼠肝脏的表达影响。
结果：注射48 h后,腹腔常规注射组与正常组比较,ALT和TB的升高幅度较小,与流体力学注射组比较该差异无统计学意义(P>0.05),且各组内脂质体复合物组与裸质粒组比较差异无统计学意义(P>0.05)。常规石蜡切片HE染色显示,HI组及PV组小鼠肝细胞轻度水肿,各组内裸质粒组和脂质体复合物组之间无明显差异。流体力学注射组及门静脉常规注射组均可见大量绿色荧光蛋白表达,两组的荧光表达量差异无统计学意义(P>0.05),腹腔注射组小鼠的肝脏仅见少量的绿色荧光表达,但三组内脂质体／质粒DNA复合物组绿色荧光表达量均明显高于裸质粒组(P<0.05)。
结论：应用流体力学注射及门静脉常规注射脂质体／质粒DNA复合物途径,目的基因均在小鼠肝脏高效表达,两种途径无明显差异,流体力学注射可广泛用于肝靶向性的活体基因转染。
实验二TRAF6siRNA对急性肝衰竭内毒素炎症小鼠的保护作用
目的：研究TRAF6沉默基因对LPS/D-GalN诱导的急性肝衰竭内毒素炎症小鼠的作用及其可能的机制。
方法：BALB/c小鼠随机分为正常对照组、急性肝衰竭(ALF)模型组、阳性对照(姜黄素)组、阴性对照(空白质粒)组和RNAi(pTRAF6-shRNA1)组。采用流体力学注射法将pTRAF6-shRNA1表达质粒转染小鼠,24 h后重复注射一次。第二次注射后24 h,予LPS(20μg/kg)联合D-氨基半乳糖(D-GalN,600 mg/kg)制做急性肝衰竭内毒素炎症模型。观察小鼠存活率,并于造模后16 h,采用常规生化方法血清ALT、AST,免疫组化染色检测iNOS和NF-κB p65在肝细胞的表达；Realtime-PCR检测TRAF6、IL-6、COX-2及NF-κB p65 mRNA的表达；Western blot检测总蛋白和细胞核内NF-κB p65的表达；造模后4、8、16 h,ELISA检测血清TNF-α、IL-1β及TGF-β1水平变化。
结果：荧光显微镜观察显示真核表达质粒pTRAF6-shRNA1成功转染进入小鼠肝组织。pTRAF6-shRNA1可明显抑制小鼠肝组织TRAF6 mRNA和TRAF6蛋白表达(P<0.01),其中TRAF6 mRNA表达抑制率为60.13%, TRAF6蛋白抑制率为52.08%,与正常小鼠比较均有显著性差异(P<0.01)。LPS/D-GalN腹腔注射16 h后,小鼠血清ALT、AST水平及肝组织病理学检测显示造模成功。RNAi (pTRAF6-shRNA1)组小鼠72h的存活率为49.3%,高于其它组(姜黄素组为36.2%). pTRAF6-shRNA1可明显降低小鼠血清ALT和AST水平,与ALF模型组比较有显著意义(P<0.01)。Realtime-PCR结果显示RNAi组小鼠IL-6、COX-2及NF-κB p65 mRNA表达下降,与ALF模型组比较有显著意义(P<0.01)。ELISA检测显示RNAi组小鼠血浆TNF-α、IL-1β及TGF-β1水平上调,但始终明显低于同时间点ALF模型组,TNF-α、IL-1p于8 h达到峰值,TGF-β1于16 h达到峰值。免疫组化染色显示RNAi组iNOS蛋白表达较ALF模型组降低。姜黄素组与RNAi组比较无显著差异(P>0.05),姜黄素有降低转氨酶,对阻止TNF-α、IL-1β、IL-6、COX-2及iNOS升高有一定作用。Western blot技术显示ALF模型组小鼠肝组织中总蛋白和胞核NF-κB p65表达均增加；pTRAF6-shRNA1组小鼠肝组织总蛋白NF-κB p65表达水平显著高于正常组小鼠总蛋白NF-κB p65水平(P<0.01),但较ALF模型组比较无明显变化(P>0.05),胞核NF-κB p65明显降低,与ALF模型组比较有显著性差异(P<0.05)。结果提示TRAF6基因沉默,不仅能降低细胞NF-κB p65表达水平,还能明显抑制NF-κB p65核转位。
结论：pTRAF6-shRNA1可能通过下调NF-κB p65水平,抑制炎症相关细胞因子和炎性介质表达水平,从而减轻内毒素／半乳糖诱导的小鼠急性肝损伤。
Part I Effects of LPS on Expression of TRAF6 gene in RAW264.7 cells
Objective:To study the effects of LPS on the expression of TRAF6 mRNA in RAW264.7 cells.
Methods:RAW264.7 cells were divided into five groups, lipopolysaccharide (LPS) group, dexamethasone (DM) group, curcumin (Cur) group, experiment control group and normal control group. Cells in LPS group were added LPS (100μg/L) in cell culture. Cells in Cur group or DM group were pretreated with dexamethasone (0.5 mg/L) or curcumin (20μmol/L) for 8 hours. Cells in experiment control group were treated with DMEM culture media with DMSO (<0.1%). No treatment was done for the normal control group. The expression of TRAF6 was determined by immunocytochemical staining and realtime-PCR at 2,4,8 and 16 hours after LPS stimulation. TRAF6 protein expression was determined by Western blotting at 8 h after LPS stimulation. In order to elucidate the relation between LPS and expression of TRAF6 gene, our studies revealed the effects of LPS on expression of TRAF6 gene both at the mRNA and protein levels.
Results:The expressed mRNA of TRAF6 gene was detected by realtime-PCR. There was the low expression of TRAF6 mRNA in RAW264.7 cells. When RAW264.7 cells were continually stimulated with LPS (100μg/L), the number of expression increased markedly, and peaked at 16h. In RAW264.7 cells, the expressions of TRAF6 mRNA were much lower than those in LPS group at the corresponding time points (P<0.05). Howere, the expression of TRAF6 mRNA was still higher than normal level. Expressions of TRAF6 mRNA in DM group shown no significant difference as compared with those in Cur group at the corresponding time points (P>0.05). Levels of TRAF6 mRNA were unobviously changed in the experiment and normal control group (P>0.05). The expressed protein of TRAF6 gene was detected with Western blot. The statistics show that there are a lot of similarities between realtime-PCR and Western blotting/Immunocytochemical staining.
Conclusions:There are tight relations between LPS and expression of TRAF6 gene.
Part II The Construction and Screening of TRAF6-shRNA expressing Plasmid The first experiment:
Construction and identification of expression plasmid for mouse TRAF6
Objective:To construct the eukaryotic expression plasmid expressing shRNA targeting TNF receptor-associated factor-6 (TRAF6) gene as a tool for following experiments.
Methods:TRAF6 mRNA sequence of mouse was retrieved in nucleotide library of NCBI. According to guidelines for shRNA design, four pairs of oligos for hairpin RNA which targeted mice TRAF6 gene were chemically synthesized. Coding sequences of short hairpin RNA DNA single-strands were synthesized in vitro. The annealed oligos were inserted into the down stream of U6 promoter of linearized pGCsi-U6/GFP/Hygro vector to construct RNA interference (RNAi) plasmid (pGCsi-TRAF6-shRNA) respectively.
Results:The shRNA expression plasmid was constructed, and the inserted sequence was confirmed by restrict endonuclease digestion and DNA sequencing.
Conclusions:The siRNA plasmid targeting mice TRAF6 gene was successfully constructeded and can be applied to study the function of TRAF6 on LPS-induced inflammatory reaction in vitro and in vivo.
The second experiment:
Identification and Screening of TRAF6 Gene silencing optimization sequences
Objective:To select TRAF6 (TNF receptor-associated factor-6) shRNA (short hairpin RNA) eukaryotic expression plasmid that can inhibit TRAF6 gene expression in Raw 264.7 cells.
Methods:To get most effective and optimal dosage pTRAF6-shRNA, the four vectors were transfected into Raw 264.7 cell with different ratio between plasmid (g) and Trans Fectin (L), the expression of fluorescence and efficiency of transfection were detected by fluorescence microscopy. Forty-eight hours after pTRAF6-shRNA transfection with the best ratio of plasmid (g) and Trans Fectin (L), proliferation of transfected cells was measured with MTT assay, and the levels of TRAF6 mRNA and TRAF6 protein were detected by realtime-PCR and Western blot, respectively.
Results:The ratio of 2:5 between plasmid (g) and Trans Fectin (L) were considered as the most ratio. The results of MTT in transfected cells were significantly decreased compared with that of. The results of realtime-PCR revealed that pTRAF6-shRNA1 can markedly inhibit the expression of TRAF6 mRNA (the inhibition rate was 75.25%), comparing with the normal control group (P<0.05). The results of Western blotting indicated that pTRAF6-shRNAl can markedly inhibit the expression of TRAF6 protein (the inhibition rate was 70.07%), comparing with the normal control group (P<0.05).
Conclusions:The construct of pTRAF6shRNA1 successfully interfered in the TRAF6 gene expression in vitro, and it can be applied to study the function of TRAF6 on LPS-induced inflammatory reaction.
PartⅢInfluence of TRAF6 siRNA on LPS-induced inflammation in vitro
Objective:To investigate the influences of silencing TRAF6 gene by RNA interference (RNAi) on LPS-induced inflammation in vitro.
Methods:Cells were divided into five groups:(A) pTRAF6-shRNA1 (pGCsi-TRAF6-shRNA1)group, pTRAF6-shRNA1+LPS; (B) positive control group, curcumin (20μmol/L) +LPS; (C) negative control group, blank plasmid+LPS; (D) blank control group, stimulated with LPS(100μg/L); (E) normal control group, maintained in DMEM without any intervention. siRNA recombinant expression vector targeting TRAF6 gene was transfected into RAW264.7. The effect of siRNA recombinant expression vector was detected by Realtime PCR and Western blot. The cells were cultured for 48h after the second pTRAF6-shRNA transfection, and stimulated by LPS (100μg/L), and then supernatants were harvested after 0h,4h,8h and 16h. TNF-α, IL-1βand TGF-β1 were assayed by ELISA. TRAF6, IL-6, COX-2 mRNA were detected by real-time PCR, and intranuclear NF-κB P65 protein level were examined by Western blotting.
Results:ELISA indicated that the secretions of TNF-α, IL-1βand TGF-β1 were markedly higher than those of normal cells (P<0.01) at 4h,8h and 16h after LPS stimulation, and peaked in a short time, TNF-α, IL-1βat 8h, TGF-β1 at 16h respectively. After LPS stimulation with TRAF6 gene knocked down, the rates of increase of those cytokines were significantly inhibited than that in single LPS stimulation (P<0.01). The experiment indicated that TRAF6-shRNA could down regulate the expression of IL-6, COX-2 mRNA. In the same way, it could significantly inhibit the activation of NF-κB.
Conclusions:pTRAF6-shRNA1 inhibited inflammatory reaction which stimulated by LPS resulting from inflammatory cytokines and mediators suppression through NF-κB levels down-regulation in RAW264.7 cell.
PartⅣTNF Receptor-associated Factor 6 Gene Silencing Ameliorates Acute Liver Failure in Mice
The first experiment:
Comparison of Three Nonviral Transfection Methods for GFP Plasmid Expression in Liver of Mice
Objective:To compare the transfection efficiency and the expression intensity of a green fluorescent protein (GFP) reporter gene in liver of mice by using three nonviral transfection methods, i.e., hydrodynamic injection, portal vein injection and peritoneal injection.
Methods:The naked GFP plasmid or liposome encapsulated plasmid were injected via the tail vein or portal vein or abdominal cavity of different mice with homogeneity. The blood and liver were harvested 48 h after injection, and the contents of alanine aminotransferase (ALT) and total bilirubin (TB) were detected in serum. HE staining was used to observe the pathological changes of liver tissues. Meanwhile, the expression intensity of GFP was evaluated by fluorescence microscopy.
Results:The levels of ALT and TB in peritoneal injection (PI) group were little higher than that in normal group at 48h after injection, and there is no significant difference comparing with hydrodynamic injection (HI) group (P>0.05). Furthermore, there was no statistically significant difference between liposome-DNA compound and naked plasmid DNA in same group (P>0.05). HE staining of liver indicated that dropsy in hepatocytes could be seen in HI and portal vein (PV) groups. Of the three transfection methods employed, hydrodynamic gene delivery and portal vein injection conferred the stronger expression of GFP with similar transfection efficiency (P>0.05). However, after encapsulated by liposome, the expression levels of GFP are significantly higher than that of naked plasmid DNA (P<0.05).
Conclusions:High level gene expression in mouse liver can be achieved by hydrodynamic injection or portal vein injection of liposome encapsulated plasmid DNA, and hepatic delivery of foreign gene can be accomplished by hydrodynamics-based injection.
The second experiment:
TNF Receptor-associated Factor 6 Gene Silencing Ameliorates Acute Liver Failure in Mice
Objective:To investigate the influence and protective mechanism of tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) gene silencing on inflamation in LPS/D-galactosamine (D-GalN)-induced acute liver failure (ALF) mice.
Methods:BALB/c mice were divided into five groups, normal control group, ALF model group, positive control (curcumin, Cur) group, negative control (blank plasmid) group and RNAi (pTRAF6-shRNA1) group. Specific expressing plasmid pTRAF6-shRNAl was delivered into BALB/c mice repeatedly by hydrodynamics-based gene transfection. LPS (20μg/kg) plus D-GalN (600 mg/kg) were injected intraperitoneally into mice after the first injection of plasmid DNA for 48 hours. The survival of mice was examined and blood samples were collected from the retro-orbital plexus each mouse, at 4, 8 and 16h after LPS/D-GalN injection. Serum [TNF-a, interleukin (IL)-1β, transforming growth factor (TGF)-β1] were measured by ELISA kits respectively. Sixteen hours after the injection of LPS/D-GalN, serum were gathered from the eyeball to measure aminotransferase (ALT) and aspartate aminotransferase (AST) using a colorimetric analyzer. TRAF6, IL-6, cyclooxygenease (COX)-2 and NF-κB p65 mRNA levels were detected by realtime-PCR. Expression features of inducible nitric oxide synthase (iNOS) and NF-κB in liver tissue were detected in immunohistochemistry. NF-κB p65 in the nuclear extracts were assessed by Western blotting.
Results:Results bright green fluorescence of the transfected cells in mice liver sections can be observed under fluorescent microscopesafter transfection. The recombinant plasmid pTRAF6-shRNA1 could be expressed in liver tissues of mice. Our results show that TRAF6 mRNA expression was remarkably reduced by 60.13% in mice liver during the experiment period. Concomitantly, pTRAF6-shRNA1 decreased serum transaminases levels and increased survival of mice. The amounts of mRNA of interleukin (IL)-6 and cyclooxygenease-2(COX-2), the productions of inflammatory cytokines [TNF-α, IL-1βand transforming growth factor (TGF)-β1], and the expression of inducible nitric oxide synthase (iNOS) in liver were significantly lower in pTRAF6-shRNAl group than that in pNi-shRNA control group. Moreover, NF-κB p65 levels in the total cellular and the nuclear extracts were effectively inhibited by pTRAF6-shRNA1. This may due to the inhibition of NF-κB activity specified with decreased expression of inflammatory cytokines and inflammatory mediators.
Conclusions:pTRAF6-shRNA1 inhibited inflammation resulting from inflammatory cytokines and mediators suppression through levels down-regulation in LPS/D-GalN-stimulated mice. pTRAF6-shRNA1 successful alleviated LPS/D-GalN induced acute liver injury in vivo, which has implications for our understanding of the role of TRAF6 gene silencing in LPS/D-GalN-mediated liver injury and for the development of new therapies for human ALF.
目的：观察内毒素刺激对小鼠RAW264.7巨噬细胞肿瘤坏死因子受体相关因子6(TNF receptor-associated factor-6, TRAF6)基因表达的影响,阐明内毒素刺激与TRAF6基因表达的关系。
方法：RAW264.7细胞分为LPS组、地塞米松组、姜黄素组、实验对照组和正常对照组。LPS组以终浓度为100μg/L LPS刺激；地塞米松组以终浓度为0.5 mg/L地塞米松预处理8 h,姜黄素组以终浓度为20μmol/L姜黄素预处理8 h；实验对照组用含DMSO(浓度<0.1%)的DMEM培养液培养,正常对照组作为实验对照不予以任何处理。LPS刺激2 h、4 h、8 h和16 h后,免疫细胞化学染色检测TRAF6蛋白表达,realtime-PCR检测TRAF6 mRNA表达；LPS刺激8 h时Western blot检测TRAF6蛋白,在mRNA水平和蛋白质水平检测内毒素刺激对TRAF6基因表达的影响。
结果：小鼠RAW264.7细胞中TRAF6 mRNA的正常表达量相对较低,当LPS刺激4 h时,TRAF6 mRNA表达明显增加；随着时间延长其表达进一步增加,至16 h时,TRAF6 mRNA表达达到高峰。用地塞米松和姜黄素干预后,TRAF6 mRNA表达量均低于同一时间LPS组(P<0.05),但始终高于正常水平。同一时间点地塞米松组与姜黄素组比较无明显差异(P>0.05),实验对照组细胞TRAF6 mRNA表达水平较正常对照组无明显变化(P>0.05)。Western blot检测TRAF6蛋白表达,结果与TRAF6 mRNA结果相似。
结论：TRAF6 mRNA及TRAF6蛋白表达与LPS刺激有明确的时效关系。
第二部分TRAF6 siRNA表达质粒的构建和筛选
实验一TRAF6 siRNA表达质粒的构建与鉴定
目的：构建针对小鼠TRAF6基因的短发夹RNA (short hairpin RNA, shRNA)真核表达载体,为下一步的细胞转染奠定基础。
方法：在NCBI的Nucleotide库中检索小鼠TRAF6 mRNA序列,应用Invitrogen公司的在线设计软件BLOCK-iTTM RNAi Designer,针对小鼠TRAF6 mRNA设计筛选4条siRNA序列。体外合成编码短发夹RNA序列的DNA寡核苷酸单链,经退火成互补双链,将相应的双链DNA被插入pGCsi-U6/GFP/Hygro质粒中,构建重组质粒pGCsi-TRAF6-shRNA1、2、3、4。将重组质粒转化进E.coli DH5a,最后以PCR法分析鉴定阳性重组质粒并进行DNA测序。在紫外分光光度计上OD260和OD280读取吸光度值,检测质粒DNA的浓度。
结果：siRNA寡核苷酸单链成功退火,合成双链DNA模板,凝胶电泳可见,59 bpMark处呈现清晰条带。靶向TRAF6 mRNA的4种重组质粒载体pGCsi-TRAF6-shRNA,经酶切和DNA测序分析,shRNA编码序列与设计的片段完全一致,证实重组质粒载体构建成功。4种重组质粒DNA OD260/OD280比值均在1.8～2.0之间,质粒DNA质量良好,符合实验要求,为下一步体外、体内实验奠定了基础。
结论：靶向TRAF6基因的重组质粒载体pGCsi-TRAF6-shRNA构建成功,为研究TRAF6对内毒素炎症反应的调控作用奠定基础。
实验二TRAF6基因沉默优化序列的筛选和确认
目的：将TRAF6基因特异性重组质粒载体pGCsi-TRAF6-shRNA转染RAW 264.7细胞,检测TRAF6基因在mRNA和蛋白质水平的表达情况,体外筛选干扰小鼠RAW264.7细胞TRAF6基因的最佳重组质粒载体pGCsi-TRAF6-shRNA。
方法：使用不同比例的DNA质粒／脂质体复合物,将重组质粒转染至RAW 264.7细胞,荧光显微镜观察转染效果。重组质粒转染细胞48 h后,应用MTT法检测转染重组质粒pGCsi-TRAF6-shRNA后细胞活性的变化,realtime-PCR和Western blot检测转染细胞TRAF6基因的mRNA和蛋白质表达,体外筛选出对TRAF6基因表达具有最佳抑制效果的重组质粒。
结果：荧光显微镜分析转染效率显示,DNA(g)/Trans Fectin(L)为2：5时重组质粒转染效率最高。MTT检测结果显示,重组质粒pGCsi-TRAF6-shRNA1、2能显著抑制RAW264.7细胞的增殖活性,与正常对照组相比有显著差异(P<0.01)。realtime-PCR结果显示,pGCsi-TRAF6-shRNA1、2组细胞TRAF6 mRNA表达水平显著降低。其中,pGCsi-TRAF6-shRNA 1对RAW264.7细胞TRAF6基因表达的抑制作用最为显著,抑制率为65.25%,与正常对照组相比具有明显差异(P<0.05)。Western blot检测结果显示,pGCsi-TRAF6-shRNA1对靶基因蛋白表达抑制作用最明显,TRAF6基因蛋白表达抑制率为60.07%,与正常对照组相比差异显著(P<0.05)。
结论：pGCsi-TRAF6-shRNA1真核表达载体可在体外高效地抑制TTRAF6基因表达,为进一步研究TRAF6基因沉默对内毒素炎症反应的影响奠定基础。
第三部分TRAF6 siRNA对RAW264.7细胞内毒素炎症的影响
目的体外研究TRAF6沉默基因对RAW 264.7细胞内毒素炎症反应的影响。
方法实验分为5组：(A)pTRAF6-shRNA1(即pGCsi-TRAF6-shRNA1)组,质粒pTRAF6-shRNA1+LPS; (B)阳性对照组,姜黄素(20μmol/L)+LPS; (C)阴性对照组,空白质粒±LPS；(D)空白对照组,仅予100μg/L LPS刺激；(E)正常对照组,正常培养不予任何处理。LPS刺激后0 h、4 h、8 h和16 h,收集细胞上清,ELISA法测定上清液中TNF-α、IL-1β、TGF-β1。LPS刺激24 h后,收集细胞,realtime PCR方法检测TRAF6、IL-6、COX-2 mRNA, Western blot检测细胞核内NF-κB p65蛋白表达。
结果100μg/L LPS刺激各组RAW264.7细胞后,细胞上清液中TNF-α、IL-1β、TGF-β1表达量都明显增加,与正常对照组相比差异显著(P<0.01),其中TNF-α、IL-1β在8 h内达高峰,TGF-β1在16 h达高峰,说明LPS刺激可引起细胞TNF-α、IL-1β、TGF-β1分泌。将pTRAF6-shRNA1转染RAW264.7细胞后,TNF-α、IL-1β、TGF-β1增长率均明显低于阴性对照组和空白对照组(P<0.01)。Realtime PCR检测结果显示,pTRAF6-shRNA1组IL-6、COX-2 mRNA表达明显下调,与正常对照组比较有显著差异。Western blot结果表明,pTRAF6-shRNA1组细胞核内NF-κB p65蛋白表达明显减少。
结论重组质粒pTRAF6-shRNA可能通过抑制LPS诱导的NF-κB p65核转位,降低相关炎症细胞因子和介质的分泌,抑制RAW264.7细胞内毒素炎症反应。
第四部分TRAF6siRNA对急性肝衰竭内毒素炎症小鼠的保护作用
实验一不同转染方式对GFP表达质粒在小鼠肝脏的表达影响
目的：研究经流体力学注射、门静脉和腹腔常规注射3种不同途径注射GFP表达质粒后,目的基因在小鼠肝脏的表达情况及其转基因效率。
方法：将裸质粒或脂质体包裹的质粒DNA分别应用流体力学注射、门静脉及腹腔常规注射法注入同种异体小鼠体内,48 h后分别取血和肝组织,常规生化方法检测血清谷丙转氨酶(ALT)和总胆红素(TB),HE染色检测肝组织病理变化,新鲜组织冰冻切片观察3种转染途径对质粒DNA在小鼠肝脏的表达影响。
结果：注射48 h后,腹腔常规注射组与正常组比较,ALT和TB的升高幅度较小,与流体力学注射组比较该差异无统计学意义(P>0.05),且各组内脂质体复合物组与裸质粒组比较差异无统计学意义(P>0.05)。常规石蜡切片HE染色显示,HI组及PV组小鼠肝细胞轻度水肿,各组内裸质粒组和脂质体复合物组之间无明显差异。流体力学注射组及门静脉常规注射组均可见大量绿色荧光蛋白表达,两组的荧光表达量差异无统计学意义(P>0.05),腹腔注射组小鼠的肝脏仅见少量的绿色荧光表达,但三组内脂质体／质粒DNA复合物组绿色荧光表达量均明显高于裸质粒组(P<0.05)。
结论：应用流体力学注射及门静脉常规注射脂质体／质粒DNA复合物途径,目的基因均在小鼠肝脏高效表达,两种途径无明显差异,流体力学注射可广泛用于肝靶向性的活体基因转染。
实验二TRAF6siRNA对急性肝衰竭内毒素炎症小鼠的保护作用
目的：研究TRAF6沉默基因对LPS/D-GalN诱导的急性肝衰竭内毒素炎症小鼠的作用及其可能的机制。
方法：BALB/c小鼠随机分为正常对照组、急性肝衰竭(ALF)模型组、阳性对照(姜黄素)组、阴性对照(空白质粒)组和RNAi(pTRAF6-shRNA1)组。采用流体力学注射法将pTRAF6-shRNA1表达质粒转染小鼠,24 h后重复注射一次。第二次注射后24 h,予LPS(20μg/kg)联合D-氨基半乳糖(D-GalN,600 mg/kg)制做急性肝衰竭内毒素炎症模型。观察小鼠存活率,并于造模后16 h,采用常规生化方法血清ALT、AST,免疫组化染色检测iNOS和NF-κB p65在肝细胞的表达；Realtime-PCR检测TRAF6、IL-6、COX-2及NF-κB p65 mRNA的表达；Western blot检测总蛋白和细胞核内NF-κB p65的表达；造模后4、8、16 h,ELISA检测血清TNF-α、IL-1β及TGF-β1水平变化。
结果：荧光显微镜观察显示真核表达质粒pTRAF6-shRNA1成功转染进入小鼠肝组织。pTRAF6-shRNA1可明显抑制小鼠肝组织TRAF6 mRNA和TRAF6蛋白表达(P<0.01),其中TRAF6 mRNA表达抑制率为60.13%, TRAF6蛋白抑制率为52.08%,与正常小鼠比较均有显著性差异(P<0.01)。LPS/D-GalN腹腔注射16 h后,小鼠血清ALT、AST水平及肝组织病理学检测显示造模成功。RNAi (pTRAF6-shRNA1)组小鼠72h的存活率为49.3%,高于其它组(姜黄素组为36.2%). pTRAF6-shRNA1可明显降低小鼠血清ALT和AST水平,与ALF模型组比较有显著意义(P<0.01)。Realtime-PCR结果显示RNAi组小鼠IL-6、COX-2及NF-κB p65 mRNA表达下降,与ALF模型组比较有显著意义(P<0.01)。ELISA检测显示RNAi组小鼠血浆TNF-α、IL-1β及TGF-β1水平上调,但始终明显低于同时间点ALF模型组,TNF-α、IL-1p于8 h达到峰值,TGF-β1于16 h达到峰值。免疫组化染色显示RNAi组iNOS蛋白表达较ALF模型组降低。姜黄素组与RNAi组比较无显著差异(P>0.05),姜黄素有降低转氨酶,对阻止TNF-α、IL-1β、IL-6、COX-2及iNOS升高有一定作用。Western blot技术显示ALF模型组小鼠肝组织中总蛋白和胞核NF-κB p65表达均增加；pTRAF6-shRNA1组小鼠肝组织总蛋白NF-κB p65表达水平显著高于正常组小鼠总蛋白NF-κB p65水平(P<0.01),但较ALF模型组比较无明显变化(P>0.05),胞核NF-κB p65明显降低,与ALF模型组比较有显著性差异(P<0.05)。结果提示TRAF6基因沉默,不仅能降低细胞NF-κB p65表达水平,还能明显抑制NF-κB p65核转位。
结论：pTRAF6-shRNA1可能通过下调NF-κB p65水平,抑制炎症相关细胞因子和炎性介质表达水平,从而减轻内毒素／半乳糖诱导的小鼠急性肝损伤。
Part I Effects of LPS on Expression of TRAF6 gene in RAW264.7 cells
Objective:To study the effects of LPS on the expression of TRAF6 mRNA in RAW264.7 cells.
Methods:RAW264.7 cells were divided into five groups, lipopolysaccharide (LPS) group, dexamethasone (DM) group, curcumin (Cur) group, experiment control group and normal control group. Cells in LPS group were added LPS (100μg/L) in cell culture. Cells in Cur group or DM group were pretreated with dexamethasone (0.5 mg/L) or curcumin (20μmol/L) for 8 hours. Cells in experiment control group were treated with DMEM culture media with DMSO (<0.1%). No treatment was done for the normal control group. The expression of TRAF6 was determined by immunocytochemical staining and realtime-PCR at 2,4,8 and 16 hours after LPS stimulation. TRAF6 protein expression was determined by Western blotting at 8 h after LPS stimulation. In order to elucidate the relation between LPS and expression of TRAF6 gene, our studies revealed the effects of LPS on expression of TRAF6 gene both at the mRNA and protein levels.
Results:The expressed mRNA of TRAF6 gene was detected by realtime-PCR. There was the low expression of TRAF6 mRNA in RAW264.7 cells. When RAW264.7 cells were continually stimulated with LPS (100μg/L), the number of expression increased markedly, and peaked at 16h. In RAW264.7 cells, the expressions of TRAF6 mRNA were much lower than those in LPS group at the corresponding time points (P<0.05). Howere, the expression of TRAF6 mRNA was still higher than normal level. Expressions of TRAF6 mRNA in DM group shown no significant difference as compared with those in Cur group at the corresponding time points (P>0.05). Levels of TRAF6 mRNA were unobviously changed in the experiment and normal control group (P>0.05). The expressed protein of TRAF6 gene was detected with Western blot. The statistics show that there are a lot of similarities between realtime-PCR and Western blotting/Immunocytochemical staining.
Conclusions:There are tight relations between LPS and expression of TRAF6 gene.
Part II The Construction and Screening of TRAF6-shRNA expressing Plasmid The first experiment:
Construction and identification of expression plasmid for mouse TRAF6
Objective:To construct the eukaryotic expression plasmid expressing shRNA targeting TNF receptor-associated factor-6 (TRAF6) gene as a tool for following experiments.
Methods:TRAF6 mRNA sequence of mouse was retrieved in nucleotide library of NCBI. According to guidelines for shRNA design, four pairs of oligos for hairpin RNA which targeted mice TRAF6 gene were chemically synthesized. Coding sequences of short hairpin RNA DNA single-strands were synthesized in vitro. The annealed oligos were inserted into the down stream of U6 promoter of linearized pGCsi-U6/GFP/Hygro vector to construct RNA interference (RNAi) plasmid (pGCsi-TRAF6-shRNA) respectively.
Results:The shRNA expression plasmid was constructed, and the inserted sequence was confirmed by restrict endonuclease digestion and DNA sequencing.
Conclusions:The siRNA plasmid targeting mice TRAF6 gene was successfully constructeded and can be applied to study the function of TRAF6 on LPS-induced inflammatory reaction in vitro and in vivo.
The second experiment:
Identification and Screening of TRAF6 Gene silencing optimization sequences
Objective:To select TRAF6 (TNF receptor-associated factor-6) shRNA (short hairpin RNA) eukaryotic expression plasmid that can inhibit TRAF6 gene expression in Raw 264.7 cells.
Methods:To get most effective and optimal dosage pTRAF6-shRNA, the four vectors were transfected into Raw 264.7 cell with different ratio between plasmid (g) and Trans Fectin (L), the expression of fluorescence and efficiency of transfection were detected by fluorescence microscopy. Forty-eight hours after pTRAF6-shRNA transfection with the best ratio of plasmid (g) and Trans Fectin (L), proliferation of transfected cells was measured with MTT assay, and the levels of TRAF6 mRNA and TRAF6 protein were detected by realtime-PCR and Western blot, respectively.
Results:The ratio of 2:5 between plasmid (g) and Trans Fectin (L) were considered as the most ratio. The results of MTT in transfected cells were significantly decreased compared with that of. The results of realtime-PCR revealed that pTRAF6-shRNA1 can markedly inhibit the expression of TRAF6 mRNA (the inhibition rate was 75.25%), comparing with the normal control group (P<0.05). The results of Western blotting indicated that pTRAF6-shRNAl can markedly inhibit the expression of TRAF6 protein (the inhibition rate was 70.07%), comparing with the normal control group (P<0.05).
Conclusions:The construct of pTRAF6shRNA1 successfully interfered in the TRAF6 gene expression in vitro, and it can be applied to study the function of TRAF6 on LPS-induced inflammatory reaction.
PartⅢInfluence of TRAF6 siRNA on LPS-induced inflammation in vitro
Objective:To investigate the influences of silencing TRAF6 gene by RNA interference (RNAi) on LPS-induced inflammation in vitro.
Methods:Cells were divided into five groups:(A) pTRAF6-shRNA1 (pGCsi-TRAF6-shRNA1)group, pTRAF6-shRNA1+LPS; (B) positive control group, curcumin (20μmol/L) +LPS; (C) negative control group, blank plasmid+LPS; (D) blank control group, stimulated with LPS(100μg/L); (E) normal control group, maintained in DMEM without any intervention. siRNA recombinant expression vector targeting TRAF6 gene was transfected into RAW264.7. The effect of siRNA recombinant expression vector was detected by Realtime PCR and Western blot. The cells were cultured for 48h after the second pTRAF6-shRNA transfection, and stimulated by LPS (100μg/L), and then supernatants were harvested after 0h,4h,8h and 16h. TNF-α, IL-1βand TGF-β1 were assayed by ELISA. TRAF6, IL-6, COX-2 mRNA were detected by real-time PCR, and intranuclear NF-κB P65 protein level were examined by Western blotting.
Results:ELISA indicated that the secretions of TNF-α, IL-1βand TGF-β1 were markedly higher than those of normal cells (P<0.01) at 4h,8h and 16h after LPS stimulation, and peaked in a short time, TNF-α, IL-1βat 8h, TGF-β1 at 16h respectively. After LPS stimulation with TRAF6 gene knocked down, the rates of increase of those cytokines were significantly inhibited than that in single LPS stimulation (P<0.01). The experiment indicated that TRAF6-shRNA could down regulate the expression of IL-6, COX-2 mRNA. In the same way, it could significantly inhibit the activation of NF-κB.
Conclusions:pTRAF6-shRNA1 inhibited inflammatory reaction which stimulated by LPS resulting from inflammatory cytokines and mediators suppression through NF-κB levels down-regulation in RAW264.7 cell.
PartⅣTNF Receptor-associated Factor 6 Gene Silencing Ameliorates Acute Liver Failure in Mice
The first experiment:
Comparison of Three Nonviral Transfection Methods for GFP Plasmid Expression in Liver of Mice
Objective:To compare the transfection efficiency and the expression intensity of a green fluorescent protein (GFP) reporter gene in liver of mice by using three nonviral transfection methods, i.e., hydrodynamic injection, portal vein injection and peritoneal injection.
Methods:The naked GFP plasmid or liposome encapsulated plasmid were injected via the tail vein or portal vein or abdominal cavity of different mice with homogeneity. The blood and liver were harvested 48 h after injection, and the contents of alanine aminotransferase (ALT) and total bilirubin (TB) were detected in serum. HE staining was used to observe the pathological changes of liver tissues. Meanwhile, the expression intensity of GFP was evaluated by fluorescence microscopy.
Results:The levels of ALT and TB in peritoneal injection (PI) group were little higher than that in normal group at 48h after injection, and there is no significant difference comparing with hydrodynamic injection (HI) group (P>0.05). Furthermore, there was no statistically significant difference between liposome-DNA compound and naked plasmid DNA in same group (P>0.05). HE staining of liver indicated that dropsy in hepatocytes could be seen in HI and portal vein (PV) groups. Of the three transfection methods employed, hydrodynamic gene delivery and portal vein injection conferred the stronger expression of GFP with similar transfection efficiency (P>0.05). However, after encapsulated by liposome, the expression levels of GFP are significantly higher than that of naked plasmid DNA (P<0.05).
Conclusions:High level gene expression in mouse liver can be achieved by hydrodynamic injection or portal vein injection of liposome encapsulated plasmid DNA, and hepatic delivery of foreign gene can be accomplished by hydrodynamics-based injection.
The second experiment:
TNF Receptor-associated Factor 6 Gene Silencing Ameliorates Acute Liver Failure in Mice
Objective:To investigate the influence and protective mechanism of tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) gene silencing on inflamation in LPS/D-galactosamine (D-GalN)-induced acute liver failure (ALF) mice.
Methods:BALB/c mice were divided into five groups, normal control group, ALF model group, positive control (curcumin, Cur) group, negative control (blank plasmid) group and RNAi (pTRAF6-shRNA1) group. Specific expressing plasmid pTRAF6-shRNAl was delivered into BALB/c mice repeatedly by hydrodynamics-based gene transfection. LPS (20μg/kg) plus D-GalN (600 mg/kg) were injected intraperitoneally into mice after the first injection of plasmid DNA for 48 hours. The survival of mice was examined and blood samples were collected from the retro-orbital plexus each mouse, at 4, 8 and 16h after LPS/D-GalN injection. Serum [TNF-a, interleukin (IL)-1β, transforming growth factor (TGF)-β1] were measured by ELISA kits respectively. Sixteen hours after the injection of LPS/D-GalN, serum were gathered from the eyeball to measure aminotransferase (ALT) and aspartate aminotransferase (AST) using a colorimetric analyzer. TRAF6, IL-6, cyclooxygenease (COX)-2 and NF-κB p65 mRNA levels were detected by realtime-PCR. Expression features of inducible nitric oxide synthase (iNOS) and NF-κB in liver tissue were detected in immunohistochemistry. NF-κB p65 in the nuclear extracts were assessed by Western blotting.
Results:Results bright green fluorescence of the transfected cells in mice liver sections can be observed under fluorescent microscopesafter transfection. The recombinant plasmid pTRAF6-shRNA1 could be expressed in liver tissues of mice. Our results show that TRAF6 mRNA expression was remarkably reduced by 60.13% in mice liver during the experiment period. Concomitantly, pTRAF6-shRNA1 decreased serum transaminases levels and increased survival of mice. The amounts of mRNA of interleukin (IL)-6 and cyclooxygenease-2(COX-2), the productions of inflammatory cytokines [TNF-α, IL-1βand transforming growth factor (TGF)-β1], and the expression of inducible nitric oxide synthase (iNOS) in liver were significantly lower in pTRAF6-shRNAl group than that in pNi-shRNA control group. Moreover, NF-κB p65 levels in the total cellular and the nuclear extracts were effectively inhibited by pTRAF6-shRNA1. This may due to the inhibition of NF-κB activity specified with decreased expression of inflammatory cytokines and inflammatory mediators.
Conclusions:pTRAF6-shRNA1 inhibited inflammation resulting from inflammatory cytokines and mediators suppression through levels down-regulation in LPS/D-GalN-stimulated mice. pTRAF6-shRNA1 successful alleviated LPS/D-GalN induced acute liver injury in vivo, which has implications for our understanding of the role of TRAF6 gene silencing in LPS/D-GalN-mediated liver injury and for the development of new therapies for human ALF.
引文
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