Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

AIM: To investigate whether Toll-like receptor 4 (TLR4) and Nod-like receptor protein 3 (NLRP3) inflammasome were involved in contrast medium (CM)-induced inflammation and injury in renal tubular epithelial cells. METHODS: Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin (IL)-1β and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS: CM decreased the viability of NRK-52E cells (P<0.05). CM also elevated the protein levels of cleaved caspase-3, TLR4, NLRP3, IL-1β and IL-18 (P<0.05). Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treatment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION: TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.     

Screening of lentiviral vectors carrying effective siRNA targeting S1P2 gene

AIM:To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2(S1P2) gene expression in the primarily cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).METHODS:SHR and SD rats (n=5 each) were used for primarily culturing corpus cavernosum smooth muscle cells.The cells were randomly divided into 6 groups:SHR siRNA-1,SHR siRNA-2,SHR siRNA-3,SHR GFP,SHR control (SHR non-transfection group),and SD control (SD rat control group).Each group had 5 samples with 1.0×10⁵ cells of each sample.At 72 h after transfection (MOI=60) with lentiviral vectors carrying S1P2 siRNA into the SHR corpus cavernosum smooth muscle cells,the expression of GFP was observed under fluorescence microscope.The protein expression of S1P2,ROCK1,ROCK2 and eNOS in the corpus cavernosum smooth muscle cells,and the mRNA expression of S1P2,ROCK1 and ROCK2 were determined by by Western blot and RT-PCR.RESULTS:The transfection efficiency of the corpus cavernosum smooth muscle cells in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were>80%.Compared with SHR control group,the mRNA levels and the protein expression of S1P2,ROCK1 and ROCK2 in SHR GFP group showed no remarkable changes,while those in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SD control groups were significantly lower than those in SHR control group (P<0.05).The protein expression of eNOS in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were not significantly changed as compared with SHR control group,but that in SD control group was significantly higher than that in SHR control group.CONCLUSION:Three groups of siRNA lentiviral vectors targeting S1P2 inhibit the expression of S1P2 in the corpus cavernosum smooth muscle cells of SHR,and by silencing the S1P2 expression,the expression of ROCK1 and ROCK2 is inhibited.Among them,siRNA-1 has the highest inhibitory efficiency.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling pathway.     

赤点石斑鱼Toll样受体(TLRs)基因家族的鉴定、进化与表达

为研究赤点石斑鱼Toll样受体(TLRs)的进化、分布及表达模式,本实验基于已公开的赤点石斑鱼基因组和转录组数据,利用Blast、系统进化与共线性等生物信息学方法,分析赤点石斑鱼TLRs基因家族的系统进化、染色体分布及在各组织中的表达模式。结果显示,在赤点石斑鱼中共鉴定出17个TLR基因,这些基因分属于5个亚家族(TLR1、TLR3、TLR5、TLR7和TLR11),分别分布在24条染色体中的11条染色体上;17个TLR基因在赤点石斑鱼不同组织中具有不同的表达模式,然而,位于相同染色体上的TLR基因的不同拷贝则存在相似的表达模式。其中EaTLR18-1与EaTLR18-2均位于9号染色体上,二者的表达模式高度相似,均在心脏中高表达;同位于20号染色体上的EaTLR2-1a与EaTLR2-1b的表达模式也高度相似,均在脑中高表达;EaTLR5M与EaTLR5S同位于14号染色体上,二者不仅具有高度相似的LRR结构域,且在不同组织中的表达水平也高度相似;而位于18号染色体的EaTLR7与EaTLR8和位于4号染色体的EaTLR2-2与EaTLR3,其表达模式却存在明显差异。研究结果可为鱼类...     

猪生长抑素Ⅱ型受体基因(SSTR2)shRNA的构建与筛选

生长激素(GH)是调控动物生长的重要激素,GH分泌水平的高低主要受正性调控因子生长激素释放因子(GRF)和负性调控因子生长抑素(SS)的双向调节。SS通过生长抑素受体(somatostatin receptor,SSTR)发挥作用,减少GH的分泌水平。本研究设计了3条SSTR2的短发夹RNA(shRNA),构建猪(Sus scrofa)SSTR2真核表达质粒(pcDNA3.1(-)-SSTR2)和SSTR2慢病毒RNA干扰质粒(pshRNA-copGFP lentivector,LV-shRNA),并共转染中国仓鼠(Cricetulus griseus)卵巢细胞系(CHO),通过对转染细胞的荧光观察、Real-time PCR和酶联免疫法(ELISA)检测,结果表明,CHO转染细胞中猪SSTR2 mRNA表达水平不同程度的抑制,分别为90.4%、28.3%和86.3%,(P<0.05)。其中,LV-shRNA1表现出最高的沉默效率:转染效率在80%左右时,猪SSTR2mRNA水平沉默效率为90.4%,蛋白质水平降低了33.3%。本研究成功地筛选到了降低猪SSTR2基因表达的shRNA,为利用shRNA技术下调动物基因表达,构建转基因模型提供思路。     

磺胺二甲嘧啶残留检测免疫传感器的感受器部件研制

试验研究了用于检测磺胺二甲基嘧啶(SM2)残留的免疫生物传感器的最关键组成,即感受器部件的制备方法、最佳检测条件,并对所制备感受器部件的性能进行了测试。利用二醋酸纤维素作为载体支架结构,戊二醛为交联剂,己二胺为制孔剂,制备了最佳条件符合标准的载体膜。采用双盲评分法测定其性能,选取性能良好的载体膜包被SM2抗体制成抗体膜。将酶标抗原和SM2标准品联到抗体膜上,应用溶解氧分析仪测定体系中过氧化氢减少量。结果表明,质控膜和测定膜的电流变化值差异显著(P<0.05),感受器部件反应灵敏,最低检测限达5μg·L-1。     

香蕉果实乙烯受体基因克隆及其表达特性

 【目的】分析香蕉果实成熟及贮藏冷害下乙烯受体基因的表达特性,探讨热处理（38℃,3 d）提高果实耐冷性与乙烯受体基因表达的关系。【方法】根据已报道的ETR基因的氨基酸保守序列设计引物,以香蕉果皮总RNA为模板,利用RT-PCR方法克隆香蕉的ETR cDNA,采用Northern杂交分析该基因的表达特征。【结果】香蕉果实在自然成熟进程中,两个基因在果皮和果肉中呈现不同的表达模式：果皮和果肉中Ma-ETR1的表达随果实成熟而逐渐减弱,而Ma-ERS3的表达仅在果实成熟后期略有增强;外源丙烯处理和38℃高温抑制果皮和果肉中Ma-ETR1的表达,却促进了Ma-ERS3的表达;低温促进果皮中Ma-ETR1和果肉中Ma-ERS3的表达,而38℃热处理3 d后则能抑制果皮中受低温诱导的Ma-ETR1表达的增强。【结论】外源丙烯和38℃高温正调控Ma-ERS3表达,负调控Ma-ETR1表达;38℃热处理3 d提高采后香蕉果实耐冷性与抑制低温诱导的果皮中Ma-ETR1表达的增强有关。     

氨肽酶N(APN)与鳞翅目昆虫对Bt抗性的关系

使用Bt Cry毒素防治农业害虫是作物生产上的一个革命性的进步,受体与Bt杀虫蛋白结合能力的改变可能是昆虫对Bt产生抗性的主要原因。氨肽酶N(aminopeptidase N,APN)是一类存在于昆虫中肠内的Bt毒素受体蛋白,通过讨论APN与Bt毒素的结合作用,综述了APN基因变异与鳞翅目昆虫Bt抗性相关的分子机理,并介绍了(Bt)Cry毒素与APN相关的作用方式新模型。     

绵羊MTNR1a基因mRNA表达量的季节性差异

本试验分别于春分、夏至、秋分和冬至4个节气,随机选择体况良好体重相近的蒙古羊8只,外科手术取下丘脑、垂体组织样品,采用RT-PCR方法对其褪黑素受体MTNR1a基因表达进行半定量分析.结果表明:绵羊MTNR1a基因表达存在明显的季节性节律,下丘脑MTNR1a基因表达量在夏至时最低,秋分时最高,春冬季节相近;垂体MTNR1a基因表达量在秋分时最低,夏至时最高,春冬季节也相近.