CD36短发夹RNA干扰重组慢病毒抑制大鼠肺泡巨噬细胞L-TGF-β1活化及其阻抑矽肺纤维化的研究
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摘要
CD36短发夹RNA干扰重组慢病毒抑制大鼠肺泡巨噬细胞L-TGF-β1活化及其阻抑矽肺纤维化的研究
前言
在我国,尘肺是危害工人健康最严重的一类职业病,到2006年底我国累计已发生尘肺病616442例,占我国各类职业病的80%,并每年以8000-9000例的速度增长,其中矽肺患者占了尘肺病例的近80%。虽然我国在控制粉尘的产生和扩散、降低作业场所粉尘浓度、改善劳动环境方面做了大量工作,而且已取得了一定的成绩,但粉尘危害的形势依然严峻,尘肺病的预防和控制工作任重而道远。虽然尘肺病的肺组织已有的纤维化病变难以消除,但肺纤维化是一个慢性、进行性的病理反应过程,探索肺纤维化发病的分子机制以延缓或阻断纤维化发生发展进程的策略,仍具有重要的实际意义。
研究证明,转化生长因子-β1(Transforming growth factor-β1,TGF-β1)在肺纤维化的发生发展中起最关键的作用。肺损伤后肺巨噬细胞受到刺激而被激活,产生大量的TGF-β1;TGF-β1与肺成纤维细胞膜上TGF-β1的受体结合,激活细胞内TGF-β1的Smads家族蛋白信号传导途径;信号传入细胞核内,激活核内转录蛋白,启动胶原蛋白的mRNA转录,合成胶原蛋白,同时抑制胶原蛋白的降解;最终,引起肺纤维化。然而只有活化形式的TGF-β1才能与其受体结合发挥生物学作用。通常情况下,肺巨噬细胞被激活后,首先分泌大量潜在的非活化形式的TGF-β1(Latent-TGF-β1,L-TGF-β1)。这种L-TGF-β1不能与TGF-β1受体结合,因此无生物学活性。但被激活的肺巨噬细胞可合成血小板反应素-1(Thrombospondin-1,TSP-1)和纤溶酶,且分泌到细胞外的TSP-1能与L-TGF-β1特异结合,形成TSP-1/L-TGF-β1蛋白复合物,该蛋白复合物又通过TSP-1与肺巨噬细胞膜上TSP-1受体CD36特异结合而发生空间构象变化,此时邻近的纤溶酶才能作用于该蛋白复合物产生活化的TGF-β1。
RNA干扰技术(RNA interference,RNAi)是一种可靠的能够阻止或抑制基因表达的方法,它主要通过与目的基因mRNA相同序列的21-23nt双链小分子干扰RNA(small interference RNA,siRNA)或发夹状RNA(short hairpin RNA,shRNA)来特异性高效率的抑制目的基因的表达。慢病毒载体作为一种基因转移载体目前被广泛应用,它可感染非分裂细胞,并可将目的基因整合至靶细胞基因组而长期表达。通过慢病毒载体介导的RNAi,使长效抑制目的基因表达成为可能。
据此,我们选择在L-TGF-β1活化过程中发挥了重要作用的膜蛋白受体CD36为靶基因,利用RNAi技术构建大鼠CD36 shRNA慢病毒载体,并在体外、体内进一步观察其阻断L-TGF-β1活化及其阻抑实验性矽肺纤维化发生发展的效果,为研究探讨矽肺纤维化发生发展的分子机制奠定实验基础。
实验方法
一、CD36基因shRNA重组质粒的构建与鉴定
根据大鼠CD36基因的编码序列,设计4条干扰靶序列和1条对照序列,并在两端加入酶切位点。将合成的正反向寡核苷酸链退火形成双链DNA,并连接到酶切后的pGCL-GFP载体上,然后转化至大肠杆菌DH5α感受态菌,经氨苄青霉素抗性筛选,挑取阳性克隆,采用PCR和DNA序列分析进行鉴定。构建的质粒分另命名为pGCL-GFP-CD36-1、pGCL-GFP-CD36-2、pGCL-GFP-CD36-3、pGCL-GFP-CD36-4、pGCL-GFP-CD36-NC。
二、CD36基因真核表达载体的构建与鉴定
利用RT-PCR技术从大鼠肺泡巨噬细胞NR8383细胞中获得CD36的cDNA,将其克隆至真核表达载体pEGFP-N1中,然后转化至大肠杆菌DH5α感受态菌,经卡那霉素抗性筛选,挑取阳性克隆,采用PCR、酶切和DNA序列分析进行鉴定。构建的质粒命名为pEGFP-N1-CD36。
三、western blot检测重组质粒pGCL-GFP-CD36在人胚肾细胞株293T细胞中干扰CD36表达的作用
脂质体介导法将pGCL-GFP-CD36-(1、2、3、4、NC)与pEGFP-N1-CD36按比例分别转染293T细胞中,转染48小时后收集各组细胞。提取总蛋白,进行westernblot检测,筛选重组质粒pGCL-GFP-CD36-(1、2、3、4)中有效干扰CD36表达的靶序列。
四、重组慢病毒Lv-shCD36的包装以及滴度测定
将pGCL-GFP-CD36-(1、2、3、4、NC)重组质粒与pCMV-dR8.74包装质粒、pMD2G包膜蛋白质粒共转染293 T细胞,转染后8小时更换新鲜培养基。继续培养40小时后,收集富含慢病毒颗粒的细胞上清液。采用超速离心法浓缩获得高滴度的病毒。构建的重组慢病毒分别命名为Lv-shCD36-1、Lv-shCD36-2、Lv-shCD36-3、Lv-shCD36-4、Lv-shCD36-NC。分别将病毒感染293T细胞,将病毒培养液稀释8个梯度,然后荧光镜检GFP蛋白的表达水平来测定病毒滴度。
五、real time-PCR和western blot检测重组慢病毒Lv-shCD36在大鼠肺泡巨噬细胞株NR8383细胞中干扰CD36表达的作用
将重组慢病毒Lv-shCD36-(1、2、3、4、NC)分别感染NR8383细胞,8小时更换新鲜培养基,40小时后荧光镜检感染情况,继续培养48小时后收集细胞。分别提取细胞中的总RNA和总蛋白,用于real time-PCR和western blot检测,筛选重组慢病毒Lv-shCD36-(1、2、3、4)中有效干扰CD36表达的靶序列。
六、重组慢病毒Lv-shCD36在激活的NR8383细胞对L-TGF-β1活化的阻抑作用
实验分为博莱霉素(Bleomycin,BLM)组、BLM+Lv-shCD36组、BLM+Lv-shCD36-NC组。各组细胞均用浓度为0.1μg/ml的BLM刺激,18小时后,更换新鲜培养基。继续培养24小时,分别收集培养上清和细胞。采用real time-PCR和western blot方法检测激活的NR8383细胞中CD36的干扰效率。采用貂肺上皮细胞(CCL-64)增殖抑制实验检测培养上清中TGF-β1的活性。
七、大鼠矽肺模型中观察重组慢病毒Lv-shCD36对L-TGF-β1活化及矽肺纤维化的阻抑作用
取96只Wistar大鼠,雌雄各半,随机分为4组,每组24只。生理盐水组,气管内注入0.5mL生理盐水;SiO_2模型组,气管内注入0.5mL含10mg SiO_2的粉尘悬液;SiO_2+Lv-shCD36组,气管内注入0.5mL含10mg SiO_2和5×10~8TU重组慢病毒Lv-shCD36的粉尘悬液;SiO_2+Lv-shCD36-NC组,气管内注入0.5mL含10mgSiO_2和5×10~8TU重组慢病毒Lv-shCD36-NC的粉尘悬液。分别于7、21、28天分批处死动物。测定称重、肺湿重(含主支气管)。肺泡灌洗后,离心并收集上清,分离培养肺泡巨噬细胞2小时,荧光显微镜检测GFP荧光蛋白表达,并采用Realtime-PCR法检测CD36的干扰效率。采用CCL-64细胞增殖抑制实验测定各组肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中TGF-β1的活性。然后剪去主支气管称重,计算肺脏器系数。取右肺进行肺组织羟脯氨酸含量测定,左肺固定,常规石蜡切片,HE和vG染色,显微镜观察肺组织病理学改变。免疫组织化学技术检测肺组织中胶原Ⅰ和胶原Ⅲ型蛋白的表达。
八、统计分析
全部数据采用SPSS13.0统计学软件进行分析,统计结果表示为mean±S.E.M.,采用单因素方差分析比较各组间的统计学差异;当差异有统计学意义时,用SNK法进行组间比较,P＜0.05具有统计学意义。
实验结果
一、成功构建重组质粒pGCL-GFP-CD36
构建的重组质粒pGCL-GFP-CD36-(1、2、3、4、NC)经PCR和DNA测序证实CD36 shRNA表达载体构建成功。
二、成功构建重组质粒pEGFP-N1-CD36
构建的重组质粒pEGFP-N1-CD36经PCR、酶切和DNA测序证实CD36真核表达载体构建成功。
三、293T细胞中重组质粒pGCL-GFP-CD36有效干扰CD36表达的结果
重组质粒转染293T细胞48小时后,荧光显微镜下观察,可见大量绿色荧光表达。western blot结果显示,0.25μg和0.5μg重组质粒pGCL-GFP-CD36-(1、2、3、4)均可显著干扰0.5μg重组质粒pEGFP-N1-CD36在293T细胞中表达,而pGC-GFP-CD36-NC不具有干扰CD36表达的作用。
四、重组慢病毒Lv-shCD36滴度测定结果
Lv-shCD36-(1、2、3、4、NC)感染293T细胞4天后,观察荧光表达情况。荧光细胞数随稀释倍数的增加而减少。数出荧光细胞比例在10%左右的孔中的荧光细胞个数,将得到的数值乘以相应的稀释倍数就得到了病毒原液的滴度值。Lv-shCD36-(1、2、3、4)的病毒滴度约为2×10~9TU/ml,Lv-shCD36-NC的病毒滴度约为3×10~(10)TU/ml。
五、NR8383细胞中重组慢病毒Lv-shCD36有效干扰CD36表达的结果
在重组慢病毒Lv-shCD36-(1、2、3、4、NC)感染的NR8383细胞中,realtime-PCR结果显示,Lv-shCD36-2,Lv-shCD36-3,Lv-shCD36-4均有干扰CD36表达的作用,其中Lv-shCD36-4达到80%的干扰效率,Lv-shCD36-3有73%以上的干扰效率,Lv-shCD36-2达到58%的干扰效率。western blot结果显示,Lv-shCD36-2,Lv-shCD36-3,Lv-shCD36-4均有干扰CD36表达的作用,其中Lv-shCD36-4的干扰效果更为显著。将Lv-shCD36-4用于体外和体内实验。
六、重组慢病毒Lv-shCD36抑制激活的NR8383细胞分泌的L-TGF-β1活化的结果
real time-PCR结果显示,BLM+Lv-shCD36组的CD36 mRNA表达明显低于未处理的NR8383细胞、BLM组和BLM+Lv-shCD36-NC组,且与BLM+Lv-shCD36-NC组相比较,干扰效率可达83%。western blot结果显示,与未处理的NR8383细胞、BLM组和BLM+Lv-shCD36-NC组相比较,BLM+Lv-shCD36组NR8383细胞的CD36蛋白表达明显降低。CCL-64细胞增殖抑制实验结果显示,BLM+Lv-shCD36组培养上清中活化的TGF-β1显著低于BLM组和BLM+Lv-shCD36-NC组(P＜0.05)。
七、重组慢病毒Lv-shCD36在大鼠矽肺模型中抑制BALF中L-TGF-β1的活化以及矽肺纤维化的结果
荧光镜检结果显示重组慢病毒Lv-shCD36和Lv-shCD36-NC成功在体内感染大鼠肺泡巨噬细胞。real time-PCR结果显示,染尘后7天时,SiO_2+Lv-shCD36组的CD36 mRNA表达明显低于生理盐水组、SiO_2模型组和SiO_2+Lv-shCD36-NC组,且与SiO_2+Lv-shCD36-NC组相比较,干扰效率可达46%。CCL-64细胞增殖抑制实验结果显示,染尘后7天时,SiO_2+Lv-shCD36组大鼠BALF中活化的TGF-β1明显低于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05);肺脏器系数测定结果显示,染尘后21天和28天时,SiO_2+Lv-shCD36组大鼠肺脏器系数显著低于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05);肺组织羟脯氨酸含量测定结果显示,染尘后21天和28天时,SiO_2+Lv-shCD36组大鼠肺组织羟脯氨酸含量显著低于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05);肺组织病理结果显示,SiO_2+Lv-shCD36组大鼠肺纤维化程度明显轻于SiO_2模型组和SiO_2+Lv-shCD36-NC组;肺组织内胶原Ⅰ和胶原Ⅲ蛋白免疫组化结果显示,SiO_2+Lv-shCD36组大鼠肺组织内胶原Ⅰ和胶原Ⅲ蛋白表达显著弱于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05)。
结论
1、成功构建大鼠CD36 shRNA表达的重组慢病毒Lv-shCD36,并在大鼠肺泡巨噬细胞NR8383细胞中证明其具有干扰CD36表达的作用。
2、重组慢病毒Lv-shCD36具有抑制激活的大鼠肺泡巨噬细胞NR8383细胞分泌的L-TGF-β1活化作用。
3、重组慢病毒Lv-shCD36能够在大鼠矽肺模型中感染肺泡巨噬细胞,干扰其CD36的表达,并具有抑制肺泡灌洗液中L-TGF-β1活化和阻抑矽肺纤维化的作用。
Preface
In China,pneumoconiosis is one of the most serious occupational diseases which are harming workers' health.Until the end of 2006,there are totally 616442 cases of pneumoconiosis,and it increases 8000 to 9000 every year.Though the government has already took a lot of measures,such as controlling the production and diffusion of dust, decreasing concentration of dust,improving operational environment,which had got some achievements.Because the harm of dust is also rigorous,preventing and controlling of pneumoconiosis are also important.It is difficult to cure the pathological changes of pneumoconiosis,but lung fibrosis is chronic and progressive pathology reactivity.To discuss the molecular mechanism of lung fibrosis also has practical significance for delaying and inhibiting the occurrence and the development of fibrosis.
Investigations suggested that transforming growth factor-β1(TGF-β1)played a critical role in the occurrence and the development of the lung fibrosis.After lung is injured,alveolar macrophage is activated,and it generates much TGF-β1.TGF-β1 is binded with its receptor on membrane of lung fibroblast,and then the smads signal transduction pathway of intracellular has been activated.After the signal gets into nucleus,transcription proteins in nuclear are activated,and the mRNA transcription of collagen protein is started.Then the collagen protein is synthesized,and its degradation is inhibited.Finally lung fibrosis is formed.However,only activated TGF-β1 has bioactivity by binding with its receptor.Usually,activated alveolar macrophages secrete a lot of latent TGF-β1(L-TGF-β1).It hasn't bioactivity because it can't interact with its receptor.However,activated alveolar macrophages also secrete a lot of thrombospondin-1(TSP-1)and plasmin.TSP-1 can interact with L-TGF-β1 to form TSP-1/L-TGF-β1 complex,and the configuration of TSP-1/L-TGF-β1 complex has changed by binding with CD36 that is the receptor of TSP-1 on the membrane of alveolar macrophages.At this time,the plasmin nearby can make the latent TGF-β1 from the latent precursor to the biologically active form.
RNA interference(RNAi)is a kind of reliable gene silencing technique,which could suppress the expression of target gene by using 21-23 nt double small interfering RNA(siRNA)or short hairpin RNA(shRNA).Lentivirus vector is a kind of hopeful transgenic vector which can infect non-dividing cells.The exogenous gene can achieve a sustained expression by the stable integration of lentivirus vector into host cell genome.So the lentvirus-mediated RNA interference will make it possible to silence the target gene persistently.
Accordingly,CD36 plays an important role during the process of L-TGF-β1 activation.In the present study,we chose CD36 as our targeting gene,used RNAi technology to construct recombinant lentivirus with rat CD36 short hairpin RNA.We used it into the experiment in vitro and vivo,to observe the inhibition of L-TGF-β1 activation and silicosis,which may lay a foundation for studies on the molecular mechanism of silicosis.
Methods
1.Construction and identification of rat CD36 shRNA recombinant plasmids
According the sequence of rat CD36 mRNA in the Genbank,four targeting sequences and a negative sequence were designed.The synthesized sense and antisense oligo nucleotides were annealed.Then the double-strand oligo-DNA was linked to the linear plasmid pGCL-GFP.The production of recombinant plasmids afterwards transformed the competence E.coli DH5α.To identify the positive clone,single positive clone was then chosen.After PCR and DNA sequencing confirmation,the recombinant plasmids were named as pGCL-GFP-CD36-1,pGCL-GFP-CD36-2, pGCL-GFP-CD36-3,pGCL-GFP-CD36-4 and pGCL-GFP-CD36-NC.
2.Construction and identification of eukaryotic expression vector containing CD36 of rat
CD36 gene was amplified by RT-PCR from total RNA of normal rat alveolar macrophage NR8383.The reconstructed expression vectors were constructed by enzyme digestion and cloning into eukaryotic expression vector pEGFP-N1.After restriction endonuclease digestion,PCR and DNA sequencing confirmation,the recombinant CD36 expression plasmid was named as pEGFP-N1-CD36.
3.Efficiency analysis of pGCL-GFP-CD36 shRNA plasmids by western blot
pGCL-GFP-CD36-(1、2、3、4、NC)together with pEGFP-N1-CD36 co-transfected 293T cells under Lipofectamine 2000,respectively.Forty-eight hours after co-transfection,the cells were harvested and total proteins were extracted for western analysis.Western blot assay was performed for selecting the targets that could interference the expression of CD36.
4.Production of recombinant lentiviruses and titration
Lentiviruses were produced in 293T cells by transient transfection of three plasmids:transfer vector pGCL-GFP-CD36-(1、2、3、4、NC),the packaging vector pCMV-dR8.74 and the VSV-G expression plasmid pMD2G.The medium was replaced 8 h later,and virus particles released into the medium were harvested 40 h after transfection.The recombinant lentiviruses were named as Lv-shCD36-1,Lv-shCD36-2, Lv-shCD36-3,Lv-shCD36-4 and Lv-shCD36-NC.The virus medium was purified by ultracentrifugation,and viral titration was measured by checking for GFP fluorescence under fluorescence microscope.
5.Efficiency analysis of Lv-shCD36 in NR8383 cells by realtime-PCR and western blot
Lentviruses viral supernatants were incubated with NR8383 cells for 8 h,and then changed the medium.After 40 h incubation,GFP fluorescence was checked under fluorescence microscope.If the efficiency of infection is lager than 50%,then incubated for another 48 h,collected the cells for realtime-PCR and western blot assay.
6.Inhibition of L-TGF-β1 activation in NR8383 cells by Lv-shCD36
The experiment was divided into three groups,which were bleomycin(BLM) group,BLM+Lv-shCD36 group,and BLM+Lv-shCD36-NC group.Firstly,we used NR8383 cells infected with Lv-shCD36 and NR8383 cells infected with Lv-shCD36-NC to plate at a density of 1×10~6 cells/ml in serum-free F12K in 6-well plates.After 2 h at 37℃,cells were given serum-free F12K medium containing 0.1μg/ml concentration of bleomycin,and incubated for 18 h.The cells of each group were changed the medium and incubated for 24 h.And then the cells were collected for detecting the interference effect by real-time PCR and western blot,and the supernatant were collected for TGF-β1 by CCL-64 mink lung epithelial growth inhibition assay.
7.Inhibition of L-TGF-β1 activation and silicosis in rat by Lv-shCD36
Animals were divided randomly into the following four experimental groups(n= 24 per group):(1)saline control group:instilling 0.5 ml sterile physiological saline;(2) SiO_2 group:instilling a suspension of 10mg SiO_2 in a total volume of 0.5 ml sterile physiological saline;(3)SiO_2+Lv-shCD36 group:instilling a mixed suspension of 10mg SiO_2 and 5×10~8 TU Lv-shCD36 in a total volume of 0.5ml sterile physiological saline;(4)SiO_2+Lv-shCD36-NC group:instilling a mixed suspension of 10mg SiO_2 and 5×10~8 TU Lv-shCD36-NC in a total volume of 0.5ml sterile physiological saline. At 7,21 and 28 days post-instillation,8 rats of each group were anesthetized with anesthetic ether,sacrificed by decapitation,and the lungs and hilar lymph nodes were removed.The alveolar lavage fluid was collected for the activation of TGF-β1 assay, and the alveolar macrophages(AMs)were collected for checking GFP fluorescence under fluorescence microscope.The left lobe of lung was fixed with 4% paraformaldehyde,embedded in paraffin,and sectioned at 5μm The tissue sections were stained with hematoxylin and eosin(HE)and van Gieson stains(vG).The right lobe of lung was used to determine the hydroxyproline content.And immunohistochernical examination of collagenⅠandⅢwas performed.
8.Statistical analysis
Results were expressed as mean±S.E.M.All analyses were carried out using the SPSS 13.0 software.Differences among multiple groups were analyzed using one-way analysis of variance(ANOVA)followed by Student-Newman-Keuls(SNK)test when F was significant.P values of less than 0.05 were considered statistically significant.
Results
1.Construction of recombinant plasmids pGCL-GFP-CD36
The results of PCR and DNA sequencing showed that we had successfully constructed the recombinant plasmids pGCL-GFP-CD36-1,-2,-3,-4,-NC.
2.Construction of recombinant plasmid pEGFP-N1-CD36
The results of PCR,enzyme digestion and DNA sequencing showed that we had successfully constructed the recombinant plasmid pEGFP-N1-CD36.
3.CD36 silencing effect detection of pGCL-GFP-CD36 in 293T cells
GFP expression was observed with fluorescent microscope 48 h after pGCL-GFP-CD36-1,-2,-3,-4,-NC plamids co-transfected with pEGFP-N1-CD36 in HEK-293T cells respectively.Results of western blot showed that 0.25μg and 0.5μg pGCL-GFP-CD36-1,-2,-3,-4 could significantly inhibit 0.5μg pEGFP-N1-CD36 plasmids expression in HEK-293T cells,while pGCL-GFP-CD36-NC did not influence the expression of the pEGFP-N1-CD36 plasmid in HEK-293T cells.
4.Results of recombinant lentiviruses titration
The infectious titers of Lv-shCD36-1,-2,-3,-4,-NC were determined by GFP assay under a fluorescent microscope.Fluorescent cells were reduced with the multiple of dilution increased.We counted the number of fluorescent cells at the hole which had 10%fluorescent cells,then multiplied the value with the corresponding dilution to obtain the titrations of recombinant lentiviruses.The titrations of Lv-shCD36-1,-2,-3, -4 were 2×10~9 TU/ml medium,and the titration of Lv-shCD36-NC was 3×10~(10)TU/ml medium.
5.Detection of CD36 silencing effect by Lv-shCD36 in NR8383 cells
NR8383 cells were respectively infected with Lv-shCD36-1,-2,-3,-4,-NC.Then we performed real time-PCR to determine the mRNA level of CD36 in NR8383 cells. The results of real time-PCR showed that the infection of Lv-shCD36 -2,-3,-4 could suppress the CD36 mRNA expression for about 58%,73%,80%compared with infection of Lv-shCD36-NC,respectively.It demonstrated that Lv-shCD36-2,-3,-4 could significantly suppress the CD36 mRNA expression in the NR8383 cells.And the result of western blot showed that the infection of Lv-shCD36-2,-3,-4 could suppress the CD36 protein expression in NR8383 cells compared with Lv-shCD36-NC. According to the results of real-time PCR and western blot assay,the most obvious gene silencing effect was observed when Lv-shCD36-4 was applied.Thus Lv-shCD36-4 was used as Lv-shCD36 in the following research.
6.Inhibition of L-TGF-β1 activation in NR8383 cells by Lv-shCD36
The results of real time-PCR and western blot assay showed that Lv-shCD36 could significantly suppress the CD36 mRNA and protein expression after the cells activated by bleomycin.In the CCL-64 growth inhibition assay,active TGF-β1 and percent of active TGF-β1 in the BLM+Lv-shCD36 group were significantly lower than those in the BLM group and BLM+Lv-shCD36-NC group(P＜0.05).
7.Inhibition of L-TGF-β1 activation in BALF and silicosis of rat by Lv-shCD36
The result of fluorescence assay showed that the AMs of Lv-shCD36 and Lv-shCD36-NC groups were able to observe the expression of fluorescent,which suggested the AMs were infected with Lv-shCD36 and Lv-shCD36-NC successfully. The result of real time-PCR showed that expression of CD36 mRNA in the Lv-shCD36 group was significantly lower than that of the saline control group,SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05)at 7 days after the instillation.The result of CCL-64 growth inhibition assay showed that the percent of active TGF-β1 of BALF in the SiO_2+Lv-shCD36 group was significantly lower than that of SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05)at 7 days after the instillation.Lung coefficient and hydroxyproline content of SiO_2+Lv-shCD36 group were significantly lower than SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05)at 21 and 28 days after the instillation.The result of pathologic examination showed that the degree of lung fibrosis in SiO_2+Lv-shCD36 group was significantly lighter than SiO_2 group and SiO_2+Lv-shCD36-NC group at 21 and 28 days after the instillation.The expression of collagenⅠandⅢin SiO_2+Lv-shCD36 group were significantly lower than SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05).
Conclusion
1.We construct a lentviral vector expressing the short hairpin RNA(shRNA)that is specific to CD36 of rat successfully,named Lv-shCD36.It is demonstrated that Lv-shCD36 could suppress the CD36 expression of NR8383 cells.
2.Lv-shCD36 can inhibit the activation of L-TGF-β1 derived from bleomycin-treated NR8383 cells.
3.Lv-shCD36 can infect the AMs in silicosis model of rat successfully,and it can surppress the expression of CD36.Lv-shCD36 can inhibit the activation of L-TGF-β1 of BALF and the development of silicosis.
前言
在我国,尘肺是危害工人健康最严重的一类职业病,到2006年底我国累计已发生尘肺病616442例,占我国各类职业病的80%,并每年以8000-9000例的速度增长,其中矽肺患者占了尘肺病例的近80%。虽然我国在控制粉尘的产生和扩散、降低作业场所粉尘浓度、改善劳动环境方面做了大量工作,而且已取得了一定的成绩,但粉尘危害的形势依然严峻,尘肺病的预防和控制工作任重而道远。虽然尘肺病的肺组织已有的纤维化病变难以消除,但肺纤维化是一个慢性、进行性的病理反应过程,探索肺纤维化发病的分子机制以延缓或阻断纤维化发生发展进程的策略,仍具有重要的实际意义。
研究证明,转化生长因子-β1(Transforming growth factor-β1,TGF-β1)在肺纤维化的发生发展中起最关键的作用。肺损伤后肺巨噬细胞受到刺激而被激活,产生大量的TGF-β1;TGF-β1与肺成纤维细胞膜上TGF-β1的受体结合,激活细胞内TGF-β1的Smads家族蛋白信号传导途径;信号传入细胞核内,激活核内转录蛋白,启动胶原蛋白的mRNA转录,合成胶原蛋白,同时抑制胶原蛋白的降解;最终,引起肺纤维化。然而只有活化形式的TGF-β1才能与其受体结合发挥生物学作用。通常情况下,肺巨噬细胞被激活后,首先分泌大量潜在的非活化形式的TGF-β1(Latent-TGF-β1,L-TGF-β1)。这种L-TGF-β1不能与TGF-β1受体结合,因此无生物学活性。但被激活的肺巨噬细胞可合成血小板反应素-1(Thrombospondin-1,TSP-1)和纤溶酶,且分泌到细胞外的TSP-1能与L-TGF-β1特异结合,形成TSP-1/L-TGF-β1蛋白复合物,该蛋白复合物又通过TSP-1与肺巨噬细胞膜上TSP-1受体CD36特异结合而发生空间构象变化,此时邻近的纤溶酶才能作用于该蛋白复合物产生活化的TGF-β1。
RNA干扰技术(RNA interference,RNAi)是一种可靠的能够阻止或抑制基因表达的方法,它主要通过与目的基因mRNA相同序列的21-23nt双链小分子干扰RNA(small interference RNA,siRNA)或发夹状RNA(short hairpin RNA,shRNA)来特异性高效率的抑制目的基因的表达。慢病毒载体作为一种基因转移载体目前被广泛应用,它可感染非分裂细胞,并可将目的基因整合至靶细胞基因组而长期表达。通过慢病毒载体介导的RNAi,使长效抑制目的基因表达成为可能。
据此,我们选择在L-TGF-β1活化过程中发挥了重要作用的膜蛋白受体CD36为靶基因,利用RNAi技术构建大鼠CD36 shRNA慢病毒载体,并在体外、体内进一步观察其阻断L-TGF-β1活化及其阻抑实验性矽肺纤维化发生发展的效果,为研究探讨矽肺纤维化发生发展的分子机制奠定实验基础。
实验方法
一、CD36基因shRNA重组质粒的构建与鉴定
根据大鼠CD36基因的编码序列,设计4条干扰靶序列和1条对照序列,并在两端加入酶切位点。将合成的正反向寡核苷酸链退火形成双链DNA,并连接到酶切后的pGCL-GFP载体上,然后转化至大肠杆菌DH5α感受态菌,经氨苄青霉素抗性筛选,挑取阳性克隆,采用PCR和DNA序列分析进行鉴定。构建的质粒分另命名为pGCL-GFP-CD36-1、pGCL-GFP-CD36-2、pGCL-GFP-CD36-3、pGCL-GFP-CD36-4、pGCL-GFP-CD36-NC。
二、CD36基因真核表达载体的构建与鉴定
利用RT-PCR技术从大鼠肺泡巨噬细胞NR8383细胞中获得CD36的cDNA,将其克隆至真核表达载体pEGFP-N1中,然后转化至大肠杆菌DH5α感受态菌,经卡那霉素抗性筛选,挑取阳性克隆,采用PCR、酶切和DNA序列分析进行鉴定。构建的质粒命名为pEGFP-N1-CD36。
三、western blot检测重组质粒pGCL-GFP-CD36在人胚肾细胞株293T细胞中干扰CD36表达的作用
脂质体介导法将pGCL-GFP-CD36-(1、2、3、4、NC)与pEGFP-N1-CD36按比例分别转染293T细胞中,转染48小时后收集各组细胞。提取总蛋白,进行westernblot检测,筛选重组质粒pGCL-GFP-CD36-(1、2、3、4)中有效干扰CD36表达的靶序列。
四、重组慢病毒Lv-shCD36的包装以及滴度测定
将pGCL-GFP-CD36-(1、2、3、4、NC)重组质粒与pCMV-dR8.74包装质粒、pMD2G包膜蛋白质粒共转染293 T细胞,转染后8小时更换新鲜培养基。继续培养40小时后,收集富含慢病毒颗粒的细胞上清液。采用超速离心法浓缩获得高滴度的病毒。构建的重组慢病毒分别命名为Lv-shCD36-1、Lv-shCD36-2、Lv-shCD36-3、Lv-shCD36-4、Lv-shCD36-NC。分别将病毒感染293T细胞,将病毒培养液稀释8个梯度,然后荧光镜检GFP蛋白的表达水平来测定病毒滴度。
五、real time-PCR和western blot检测重组慢病毒Lv-shCD36在大鼠肺泡巨噬细胞株NR8383细胞中干扰CD36表达的作用
将重组慢病毒Lv-shCD36-(1、2、3、4、NC)分别感染NR8383细胞,8小时更换新鲜培养基,40小时后荧光镜检感染情况,继续培养48小时后收集细胞。分别提取细胞中的总RNA和总蛋白,用于real time-PCR和western blot检测,筛选重组慢病毒Lv-shCD36-(1、2、3、4)中有效干扰CD36表达的靶序列。
六、重组慢病毒Lv-shCD36在激活的NR8383细胞对L-TGF-β1活化的阻抑作用
实验分为博莱霉素(Bleomycin,BLM)组、BLM+Lv-shCD36组、BLM+Lv-shCD36-NC组。各组细胞均用浓度为0.1μg/ml的BLM刺激,18小时后,更换新鲜培养基。继续培养24小时,分别收集培养上清和细胞。采用real time-PCR和western blot方法检测激活的NR8383细胞中CD36的干扰效率。采用貂肺上皮细胞(CCL-64)增殖抑制实验检测培养上清中TGF-β1的活性。
七、大鼠矽肺模型中观察重组慢病毒Lv-shCD36对L-TGF-β1活化及矽肺纤维化的阻抑作用
取96只Wistar大鼠,雌雄各半,随机分为4组,每组24只。生理盐水组,气管内注入0.5mL生理盐水;SiO_2模型组,气管内注入0.5mL含10mg SiO_2的粉尘悬液;SiO_2+Lv-shCD36组,气管内注入0.5mL含10mg SiO_2和5×10~8TU重组慢病毒Lv-shCD36的粉尘悬液;SiO_2+Lv-shCD36-NC组,气管内注入0.5mL含10mgSiO_2和5×10~8TU重组慢病毒Lv-shCD36-NC的粉尘悬液。分别于7、21、28天分批处死动物。测定称重、肺湿重(含主支气管)。肺泡灌洗后,离心并收集上清,分离培养肺泡巨噬细胞2小时,荧光显微镜检测GFP荧光蛋白表达,并采用Realtime-PCR法检测CD36的干扰效率。采用CCL-64细胞增殖抑制实验测定各组肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中TGF-β1的活性。然后剪去主支气管称重,计算肺脏器系数。取右肺进行肺组织羟脯氨酸含量测定,左肺固定,常规石蜡切片,HE和vG染色,显微镜观察肺组织病理学改变。免疫组织化学技术检测肺组织中胶原Ⅰ和胶原Ⅲ型蛋白的表达。
八、统计分析
全部数据采用SPSS13.0统计学软件进行分析,统计结果表示为mean±S.E.M.,采用单因素方差分析比较各组间的统计学差异;当差异有统计学意义时,用SNK法进行组间比较,P＜0.05具有统计学意义。
实验结果
一、成功构建重组质粒pGCL-GFP-CD36
构建的重组质粒pGCL-GFP-CD36-(1、2、3、4、NC)经PCR和DNA测序证实CD36 shRNA表达载体构建成功。
二、成功构建重组质粒pEGFP-N1-CD36
构建的重组质粒pEGFP-N1-CD36经PCR、酶切和DNA测序证实CD36真核表达载体构建成功。
三、293T细胞中重组质粒pGCL-GFP-CD36有效干扰CD36表达的结果
重组质粒转染293T细胞48小时后,荧光显微镜下观察,可见大量绿色荧光表达。western blot结果显示,0.25μg和0.5μg重组质粒pGCL-GFP-CD36-(1、2、3、4)均可显著干扰0.5μg重组质粒pEGFP-N1-CD36在293T细胞中表达,而pGC-GFP-CD36-NC不具有干扰CD36表达的作用。
四、重组慢病毒Lv-shCD36滴度测定结果
Lv-shCD36-(1、2、3、4、NC)感染293T细胞4天后,观察荧光表达情况。荧光细胞数随稀释倍数的增加而减少。数出荧光细胞比例在10%左右的孔中的荧光细胞个数,将得到的数值乘以相应的稀释倍数就得到了病毒原液的滴度值。Lv-shCD36-(1、2、3、4)的病毒滴度约为2×10~9TU/ml,Lv-shCD36-NC的病毒滴度约为3×10~(10)TU/ml。
五、NR8383细胞中重组慢病毒Lv-shCD36有效干扰CD36表达的结果
在重组慢病毒Lv-shCD36-(1、2、3、4、NC)感染的NR8383细胞中,realtime-PCR结果显示,Lv-shCD36-2,Lv-shCD36-3,Lv-shCD36-4均有干扰CD36表达的作用,其中Lv-shCD36-4达到80%的干扰效率,Lv-shCD36-3有73%以上的干扰效率,Lv-shCD36-2达到58%的干扰效率。western blot结果显示,Lv-shCD36-2,Lv-shCD36-3,Lv-shCD36-4均有干扰CD36表达的作用,其中Lv-shCD36-4的干扰效果更为显著。将Lv-shCD36-4用于体外和体内实验。
六、重组慢病毒Lv-shCD36抑制激活的NR8383细胞分泌的L-TGF-β1活化的结果
real time-PCR结果显示,BLM+Lv-shCD36组的CD36 mRNA表达明显低于未处理的NR8383细胞、BLM组和BLM+Lv-shCD36-NC组,且与BLM+Lv-shCD36-NC组相比较,干扰效率可达83%。western blot结果显示,与未处理的NR8383细胞、BLM组和BLM+Lv-shCD36-NC组相比较,BLM+Lv-shCD36组NR8383细胞的CD36蛋白表达明显降低。CCL-64细胞增殖抑制实验结果显示,BLM+Lv-shCD36组培养上清中活化的TGF-β1显著低于BLM组和BLM+Lv-shCD36-NC组(P＜0.05)。
七、重组慢病毒Lv-shCD36在大鼠矽肺模型中抑制BALF中L-TGF-β1的活化以及矽肺纤维化的结果
荧光镜检结果显示重组慢病毒Lv-shCD36和Lv-shCD36-NC成功在体内感染大鼠肺泡巨噬细胞。real time-PCR结果显示,染尘后7天时,SiO_2+Lv-shCD36组的CD36 mRNA表达明显低于生理盐水组、SiO_2模型组和SiO_2+Lv-shCD36-NC组,且与SiO_2+Lv-shCD36-NC组相比较,干扰效率可达46%。CCL-64细胞增殖抑制实验结果显示,染尘后7天时,SiO_2+Lv-shCD36组大鼠BALF中活化的TGF-β1明显低于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05);肺脏器系数测定结果显示,染尘后21天和28天时,SiO_2+Lv-shCD36组大鼠肺脏器系数显著低于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05);肺组织羟脯氨酸含量测定结果显示,染尘后21天和28天时,SiO_2+Lv-shCD36组大鼠肺组织羟脯氨酸含量显著低于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05);肺组织病理结果显示,SiO_2+Lv-shCD36组大鼠肺纤维化程度明显轻于SiO_2模型组和SiO_2+Lv-shCD36-NC组;肺组织内胶原Ⅰ和胶原Ⅲ蛋白免疫组化结果显示,SiO_2+Lv-shCD36组大鼠肺组织内胶原Ⅰ和胶原Ⅲ蛋白表达显著弱于SiO_2模型组和SiO_2+Lv-shCD36-NC组(P＜0.05)。
结论
1、成功构建大鼠CD36 shRNA表达的重组慢病毒Lv-shCD36,并在大鼠肺泡巨噬细胞NR8383细胞中证明其具有干扰CD36表达的作用。
2、重组慢病毒Lv-shCD36具有抑制激活的大鼠肺泡巨噬细胞NR8383细胞分泌的L-TGF-β1活化作用。
3、重组慢病毒Lv-shCD36能够在大鼠矽肺模型中感染肺泡巨噬细胞,干扰其CD36的表达,并具有抑制肺泡灌洗液中L-TGF-β1活化和阻抑矽肺纤维化的作用。
Preface
In China,pneumoconiosis is one of the most serious occupational diseases which are harming workers' health.Until the end of 2006,there are totally 616442 cases of pneumoconiosis,and it increases 8000 to 9000 every year.Though the government has already took a lot of measures,such as controlling the production and diffusion of dust, decreasing concentration of dust,improving operational environment,which had got some achievements.Because the harm of dust is also rigorous,preventing and controlling of pneumoconiosis are also important.It is difficult to cure the pathological changes of pneumoconiosis,but lung fibrosis is chronic and progressive pathology reactivity.To discuss the molecular mechanism of lung fibrosis also has practical significance for delaying and inhibiting the occurrence and the development of fibrosis.
Investigations suggested that transforming growth factor-β1(TGF-β1)played a critical role in the occurrence and the development of the lung fibrosis.After lung is injured,alveolar macrophage is activated,and it generates much TGF-β1.TGF-β1 is binded with its receptor on membrane of lung fibroblast,and then the smads signal transduction pathway of intracellular has been activated.After the signal gets into nucleus,transcription proteins in nuclear are activated,and the mRNA transcription of collagen protein is started.Then the collagen protein is synthesized,and its degradation is inhibited.Finally lung fibrosis is formed.However,only activated TGF-β1 has bioactivity by binding with its receptor.Usually,activated alveolar macrophages secrete a lot of latent TGF-β1(L-TGF-β1).It hasn't bioactivity because it can't interact with its receptor.However,activated alveolar macrophages also secrete a lot of thrombospondin-1(TSP-1)and plasmin.TSP-1 can interact with L-TGF-β1 to form TSP-1/L-TGF-β1 complex,and the configuration of TSP-1/L-TGF-β1 complex has changed by binding with CD36 that is the receptor of TSP-1 on the membrane of alveolar macrophages.At this time,the plasmin nearby can make the latent TGF-β1 from the latent precursor to the biologically active form.
RNA interference(RNAi)is a kind of reliable gene silencing technique,which could suppress the expression of target gene by using 21-23 nt double small interfering RNA(siRNA)or short hairpin RNA(shRNA).Lentivirus vector is a kind of hopeful transgenic vector which can infect non-dividing cells.The exogenous gene can achieve a sustained expression by the stable integration of lentivirus vector into host cell genome.So the lentvirus-mediated RNA interference will make it possible to silence the target gene persistently.
Accordingly,CD36 plays an important role during the process of L-TGF-β1 activation.In the present study,we chose CD36 as our targeting gene,used RNAi technology to construct recombinant lentivirus with rat CD36 short hairpin RNA.We used it into the experiment in vitro and vivo,to observe the inhibition of L-TGF-β1 activation and silicosis,which may lay a foundation for studies on the molecular mechanism of silicosis.
Methods
1.Construction and identification of rat CD36 shRNA recombinant plasmids
According the sequence of rat CD36 mRNA in the Genbank,four targeting sequences and a negative sequence were designed.The synthesized sense and antisense oligo nucleotides were annealed.Then the double-strand oligo-DNA was linked to the linear plasmid pGCL-GFP.The production of recombinant plasmids afterwards transformed the competence E.coli DH5α.To identify the positive clone,single positive clone was then chosen.After PCR and DNA sequencing confirmation,the recombinant plasmids were named as pGCL-GFP-CD36-1,pGCL-GFP-CD36-2, pGCL-GFP-CD36-3,pGCL-GFP-CD36-4 and pGCL-GFP-CD36-NC.
2.Construction and identification of eukaryotic expression vector containing CD36 of rat
CD36 gene was amplified by RT-PCR from total RNA of normal rat alveolar macrophage NR8383.The reconstructed expression vectors were constructed by enzyme digestion and cloning into eukaryotic expression vector pEGFP-N1.After restriction endonuclease digestion,PCR and DNA sequencing confirmation,the recombinant CD36 expression plasmid was named as pEGFP-N1-CD36.
3.Efficiency analysis of pGCL-GFP-CD36 shRNA plasmids by western blot
pGCL-GFP-CD36-(1、2、3、4、NC)together with pEGFP-N1-CD36 co-transfected 293T cells under Lipofectamine 2000,respectively.Forty-eight hours after co-transfection,the cells were harvested and total proteins were extracted for western analysis.Western blot assay was performed for selecting the targets that could interference the expression of CD36.
4.Production of recombinant lentiviruses and titration
Lentiviruses were produced in 293T cells by transient transfection of three plasmids:transfer vector pGCL-GFP-CD36-(1、2、3、4、NC),the packaging vector pCMV-dR8.74 and the VSV-G expression plasmid pMD2G.The medium was replaced 8 h later,and virus particles released into the medium were harvested 40 h after transfection.The recombinant lentiviruses were named as Lv-shCD36-1,Lv-shCD36-2, Lv-shCD36-3,Lv-shCD36-4 and Lv-shCD36-NC.The virus medium was purified by ultracentrifugation,and viral titration was measured by checking for GFP fluorescence under fluorescence microscope.
5.Efficiency analysis of Lv-shCD36 in NR8383 cells by realtime-PCR and western blot
Lentviruses viral supernatants were incubated with NR8383 cells for 8 h,and then changed the medium.After 40 h incubation,GFP fluorescence was checked under fluorescence microscope.If the efficiency of infection is lager than 50%,then incubated for another 48 h,collected the cells for realtime-PCR and western blot assay.
6.Inhibition of L-TGF-β1 activation in NR8383 cells by Lv-shCD36
The experiment was divided into three groups,which were bleomycin(BLM) group,BLM+Lv-shCD36 group,and BLM+Lv-shCD36-NC group.Firstly,we used NR8383 cells infected with Lv-shCD36 and NR8383 cells infected with Lv-shCD36-NC to plate at a density of 1×10~6 cells/ml in serum-free F12K in 6-well plates.After 2 h at 37℃,cells were given serum-free F12K medium containing 0.1μg/ml concentration of bleomycin,and incubated for 18 h.The cells of each group were changed the medium and incubated for 24 h.And then the cells were collected for detecting the interference effect by real-time PCR and western blot,and the supernatant were collected for TGF-β1 by CCL-64 mink lung epithelial growth inhibition assay.
7.Inhibition of L-TGF-β1 activation and silicosis in rat by Lv-shCD36
Animals were divided randomly into the following four experimental groups(n= 24 per group):(1)saline control group:instilling 0.5 ml sterile physiological saline;(2) SiO_2 group:instilling a suspension of 10mg SiO_2 in a total volume of 0.5 ml sterile physiological saline;(3)SiO_2+Lv-shCD36 group:instilling a mixed suspension of 10mg SiO_2 and 5×10~8 TU Lv-shCD36 in a total volume of 0.5ml sterile physiological saline;(4)SiO_2+Lv-shCD36-NC group:instilling a mixed suspension of 10mg SiO_2 and 5×10~8 TU Lv-shCD36-NC in a total volume of 0.5ml sterile physiological saline. At 7,21 and 28 days post-instillation,8 rats of each group were anesthetized with anesthetic ether,sacrificed by decapitation,and the lungs and hilar lymph nodes were removed.The alveolar lavage fluid was collected for the activation of TGF-β1 assay, and the alveolar macrophages(AMs)were collected for checking GFP fluorescence under fluorescence microscope.The left lobe of lung was fixed with 4% paraformaldehyde,embedded in paraffin,and sectioned at 5μm The tissue sections were stained with hematoxylin and eosin(HE)and van Gieson stains(vG).The right lobe of lung was used to determine the hydroxyproline content.And immunohistochernical examination of collagenⅠandⅢwas performed.
8.Statistical analysis
Results were expressed as mean±S.E.M.All analyses were carried out using the SPSS 13.0 software.Differences among multiple groups were analyzed using one-way analysis of variance(ANOVA)followed by Student-Newman-Keuls(SNK)test when F was significant.P values of less than 0.05 were considered statistically significant.
Results
1.Construction of recombinant plasmids pGCL-GFP-CD36
The results of PCR and DNA sequencing showed that we had successfully constructed the recombinant plasmids pGCL-GFP-CD36-1,-2,-3,-4,-NC.
2.Construction of recombinant plasmid pEGFP-N1-CD36
The results of PCR,enzyme digestion and DNA sequencing showed that we had successfully constructed the recombinant plasmid pEGFP-N1-CD36.
3.CD36 silencing effect detection of pGCL-GFP-CD36 in 293T cells
GFP expression was observed with fluorescent microscope 48 h after pGCL-GFP-CD36-1,-2,-3,-4,-NC plamids co-transfected with pEGFP-N1-CD36 in HEK-293T cells respectively.Results of western blot showed that 0.25μg and 0.5μg pGCL-GFP-CD36-1,-2,-3,-4 could significantly inhibit 0.5μg pEGFP-N1-CD36 plasmids expression in HEK-293T cells,while pGCL-GFP-CD36-NC did not influence the expression of the pEGFP-N1-CD36 plasmid in HEK-293T cells.
4.Results of recombinant lentiviruses titration
The infectious titers of Lv-shCD36-1,-2,-3,-4,-NC were determined by GFP assay under a fluorescent microscope.Fluorescent cells were reduced with the multiple of dilution increased.We counted the number of fluorescent cells at the hole which had 10%fluorescent cells,then multiplied the value with the corresponding dilution to obtain the titrations of recombinant lentiviruses.The titrations of Lv-shCD36-1,-2,-3, -4 were 2×10~9 TU/ml medium,and the titration of Lv-shCD36-NC was 3×10~(10)TU/ml medium.
5.Detection of CD36 silencing effect by Lv-shCD36 in NR8383 cells
NR8383 cells were respectively infected with Lv-shCD36-1,-2,-3,-4,-NC.Then we performed real time-PCR to determine the mRNA level of CD36 in NR8383 cells. The results of real time-PCR showed that the infection of Lv-shCD36 -2,-3,-4 could suppress the CD36 mRNA expression for about 58%,73%,80%compared with infection of Lv-shCD36-NC,respectively.It demonstrated that Lv-shCD36-2,-3,-4 could significantly suppress the CD36 mRNA expression in the NR8383 cells.And the result of western blot showed that the infection of Lv-shCD36-2,-3,-4 could suppress the CD36 protein expression in NR8383 cells compared with Lv-shCD36-NC. According to the results of real-time PCR and western blot assay,the most obvious gene silencing effect was observed when Lv-shCD36-4 was applied.Thus Lv-shCD36-4 was used as Lv-shCD36 in the following research.
6.Inhibition of L-TGF-β1 activation in NR8383 cells by Lv-shCD36
The results of real time-PCR and western blot assay showed that Lv-shCD36 could significantly suppress the CD36 mRNA and protein expression after the cells activated by bleomycin.In the CCL-64 growth inhibition assay,active TGF-β1 and percent of active TGF-β1 in the BLM+Lv-shCD36 group were significantly lower than those in the BLM group and BLM+Lv-shCD36-NC group(P＜0.05).
7.Inhibition of L-TGF-β1 activation in BALF and silicosis of rat by Lv-shCD36
The result of fluorescence assay showed that the AMs of Lv-shCD36 and Lv-shCD36-NC groups were able to observe the expression of fluorescent,which suggested the AMs were infected with Lv-shCD36 and Lv-shCD36-NC successfully. The result of real time-PCR showed that expression of CD36 mRNA in the Lv-shCD36 group was significantly lower than that of the saline control group,SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05)at 7 days after the instillation.The result of CCL-64 growth inhibition assay showed that the percent of active TGF-β1 of BALF in the SiO_2+Lv-shCD36 group was significantly lower than that of SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05)at 7 days after the instillation.Lung coefficient and hydroxyproline content of SiO_2+Lv-shCD36 group were significantly lower than SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05)at 21 and 28 days after the instillation.The result of pathologic examination showed that the degree of lung fibrosis in SiO_2+Lv-shCD36 group was significantly lighter than SiO_2 group and SiO_2+Lv-shCD36-NC group at 21 and 28 days after the instillation.The expression of collagenⅠandⅢin SiO_2+Lv-shCD36 group were significantly lower than SiO_2 group and SiO_2+Lv-shCD36-NC group(P＜0.05).
Conclusion
1.We construct a lentviral vector expressing the short hairpin RNA(shRNA)that is specific to CD36 of rat successfully,named Lv-shCD36.It is demonstrated that Lv-shCD36 could suppress the CD36 expression of NR8383 cells.
2.Lv-shCD36 can inhibit the activation of L-TGF-β1 derived from bleomycin-treated NR8383 cells.
3.Lv-shCD36 can infect the AMs in silicosis model of rat successfully,and it can surppress the expression of CD36.Lv-shCD36 can inhibit the activation of L-TGF-β1 of BALF and the development of silicosis.
引文
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