CN101643731B - DKK-1特异性siRNA及其在类风湿关节炎治疗中的应用 - Google Patents
DKK-1特异性siRNA及其在类风湿关节炎治疗中的应用 Download PDFInfo
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Abstract
本发明设计了一对靶向DKK-1的特异性siRNA,可明显下调类风湿患者过度活化的纤维样滑膜细胞DKK-1的表达,抑制细胞增殖、炎性因子分泌及侵袭能力,这对于RA的免疫治疗具有重要意义。
Description
技术领域
本研究涉及DKK-1在类风湿关节炎患者纤维样滑膜细胞中的表达水平、靶向DKK-1的小干扰RNA(siRNA)对细胞功能的抑制作用,以及作用途径。
背景技术
类风湿关节炎(rheumatoid arthritis,RA)是一种以慢性致残性关节炎为特征的自身免疫性疾病,约占总人口的0.5-1%,也是我国最主要的引起青壮年劳动力丧失的原因之一。其病理特点是关节滑膜组织中大量炎症细胞聚集增生,滑膜细胞和血管增生,增生的滑膜组织向关节软骨面生长并侵蚀关节软骨并分泌多种炎性因子和酶类,引起关节软骨及骨的破坏,最后关节畸形强直、功能障碍,导致残疾。目前RA的发病机制并不明确,限制了治疗手段的发展。事实上,越来越多的证据表明具有转化特性的关节滑膜细胞参与关节软骨和骨破坏是类风湿关节炎的特征性表现。
滑膜细胞主要分为A型巨噬细胞样滑膜细胞(Macrophage like synovial cell,MLS)以及B型成纤维样滑膜细胞(Fibroblast like synovial cell,FLS)。在RA患者关节中滑膜细胞大量增生,分泌细胞因子,不仅为滑膜中其他炎症细胞的生存提供“微环境”,而且具有转化特性的滑膜细胞还具有参与关节软骨和骨破坏的RA特征表现,特别是FLS。
Dickkopf-1(DKK-1)是Wnt信号通路的内源性抑制因子,Wnt/DKK-1它不仅参与了生物体的正常发育,在疾病的发生中也起着极为重要的作用,为我们了解疾病的发生机制提供了新的线索,试图寻找到能够抑制RA滑膜细胞骨破坏和炎性因子分泌的方法。
本研究在观察RA患者FLSs中DKK-1表达情况的基础上,进一步探索了抑制DKK-1的表达对RA滑膜成纤维细胞功能的影响,旨在寻找靶向DKK-1的siRNA潜在的治疗价值。
发明内容
为了深入了解DKK-1在RA发病中的调控作用,我们研究了下调细胞DKK-1的表达对RA患者FLS增殖、炎性因子分泌及细胞侵袭能力的影响。实验分两组进行,一组为肿瘤坏死因子-α(TNF-α)活化组,一组为无TNF-α活化组,脂质体转染化学合成的siRNA(序列为正义链5′-GCUCUCAUGGACUAGAAAUDTDT-3′,反义链5′-AUUUCUAGUCCAUGAGAGCDTDT-3′),并以无关序列小RNA为阴性对照。48小时后检测FLS的增殖情况,及细胞上清IL-6、IL-8、MMP2及MMP9的水平。我们发现TNF-α活化组FLSs增殖及细胞因子分泌水平明显增高;而下调DKK-1对两组FLS的增殖、炎性细胞因子的分泌和侵袭能力均有明显的抑制作用。这表明,TNF-α在RA患者FLS的活化过程中起着重要作用,而DKK-1对RA患者过度活化的FLS具有促进作用,这对于RA的免疫治疗具有重要意义。
附图说明
图1.siRNA的筛选及鉴定:6对靶向DKK-1的siRNA转染RA患者FLSs后,DKK-1表达水平显著降低,结果0.2314±0.07、0.106±0.04、0.413±0.103、0.156±0.08、0.187±0.06、0.13±0.05均明显低于阴性对照1,差异具有统计学意义见图1。
图2.siRNA的筛选及鉴定:6对靶向DKK-1的siRNA转染RA患者FLSs后,ELISA法检测细胞上清中DKK-1表达水平,结果1.8076±0.467,1.739±0.464,3.27±0.471,2.494±0.478,1.946±0.432和1.956±0.289(ng/ml)均明显低于阴性对照6.926±0.52ng/ml差异具有统计学意义(p<0.01)。
图3.3H掺入法检测DKK-1对RA-FLS增殖的影响。在无TNF-α组(A)及TNF-a活化组(B),转染siRNA 48小时后,FLS cpm值分别为3009.667±812.98和5028.35±544.75均明显低于阴性对照8412.3±866.91,12648.60±1377,P均<0.01)。
图4.靶向DKK-1的siRNA对RA-FLS IL-8分泌的影响。在无TNF-α组(A)及TNF-α活化组(B),转染siRNA 48小时后,FLS上清IL-8分别为34.056±14.2pg/ml和15.905±3.8ng/ml,均明显低于阴性对照组134.515±33.2pg/ml和24.571±4.7ng/ml,P均<0.01)。
图5.靶向DKK-1的siRNA对RA-FLS IL-6分泌的影响:在无TNF-α组(A)及TNF-α活化组(B),转染siRNA 48小时后,FLS上清IL-6分别为299.08±33.86pg/ml和4.38±0.705ng/ml,均明显低于阴性对照组405.76±76.29pg/ml和6.96±1.53ng/ml,P均<0.05)。
图6.靶向DKK-1的siRNA对RA-FLS MMP2分泌的影响:在无TNF-α组(A)及TNF-α活化组(B),转染siRNA 48小时后,FLS上清MMP2分别为5.592±1.23ng/ml和10.064±2.075ng/ml,均明显低于阴性对照组8.937±1.94ng/ml和14.42±2.53ng/ml,P均<0.05)。
图7.靶向DKK-1的siRNA对RA-FLS MMP9分泌的影响:在无TNF-α组(A)及TNF-α活化组(B),转染siRNA 48小时后,FLS上清MMP9分别为2.039±0.78ng/ml和4.312±1.33ng/ml,均明显低于阴性对照组3.384±1.02ng/ml和7.77±1.73ng/ml,P均<0.05)。
图8.Cell Invasion Assay Kit检测下调RA患者FLSs中DKK-1,对细胞侵袭能力的影响:在无TNF-α组(A)及TNF-a活化组(B),转染siRNA48小时后,FLS OD值分别为0.159±0.04和0.237±0.045均明显低于阴性对照0.246±0.033,0.3055±0.022,P均<0.01)。
具体实施方式
实施例1:siRNA的筛选及鉴定
留取RA患者膝关节置换术后滑膜组织,分离培养滑膜成纤维细胞,使用3代后的细胞进行试验。铺于12孔板中,每孔细胞5×104个,设复孔。48小时后收集细胞,Trizol法提取总RNA,用紫外分光光度仪测A260。DKK-1的相对表达水平用△Ct和2-△△Ct计算,GAPD作为内参。实时定量PCR应用美国ABI公司的Green PCR Master Mix进行操作。ELISA法检测细胞上清中DKK-1表达水平。结果显示设计的6对siRNA转入细胞后,都能明显抑制DKK-1的表达,并筛选出抑制效率高的一对siRNA,见图1和图2。
实施例2:靶向DKK-1的siRNA对RA-FLS增殖能力的影响
分离培养滑膜成纤维细胞,分成两组,一组为无TNF-α活化组,一组为TNF-α活化组(10ng/ml),分别同时行Lipo2000脂质体转染化学合成siRNA,对照选用无关序列siRNA。转染时使用96孔板,每孔细胞5×103,设复孔。48小时后收集细胞,3H掺入法检测成纤维样滑膜细胞增殖情况。结果发现TNF-α活化组FLS增殖更为明显,但转染siRNA后对两组FLS增殖均有显著的抑制作用,见图3。
实施例3:下调DKK-1的表达对RA-FLS分泌炎症因子的影响
使用3代以后的RA患者FLS,5×104/孔接种于12孔板,Lipo2000脂质体转染化学合成的siRNA及阴性对照后,分成TNF-α诱导(10ng/ml)及无TNF-α组,每组均为复孔,ELISA法检测转染48h后FLS上清IL-6、IL-8、MMP2及MMP9的分泌水平。结果表明,TNF-α活化组FLS分泌炎性因子更为明显,但转染siRNA后对两组FLS炎症因子的分泌均有显著的抑制作用,见图4、图5、图6和图7。
实施例4:下调DKK-1的表达对RA-FLS侵袭能力的影响
使用3代以后的RA患者FLS,5×104/孔接种于12孔板,Lipo2000脂质体转染化学合成的siRNA及阴性对照后,分成TNF-α诱导(10ng/ml)及无TNF-α组,每组均为复孔,48小时后,将细胞移入Cell Invasion Assay Kit中,孵育24小时,按说明书操作,检测下调RA患者FLSs中DKK-1对细胞侵袭能力的影响。
Claims (3)
1.一种抑制DKK-1基因表达的siRNA,其序列为:
正义链5′-GCUCUCAUGGACUAGAAAUDTDT-3′
反义链5′-AUUUCUAGUCCAUGAGAGCDTDT-3′
2.权利要求1中的siRNA在治疗类风湿关节炎中的应用。
3.根据权利要求2所述的应用,其特征在于:抑制滑膜细胞功能。
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