SiRNA干扰靶向抑制酪氨酸蛋白激酶Lck对哮喘小鼠T细胞功能的影响

目的通过siRNA技术靶向抑制哮喘小鼠T细胞非受体酪氨酸蛋白激酶Lck的基因表达,研究Lck特异性siRNA对哮喘小鼠T细胞功能的影响。方法化学法合成小鼠T细胞Lck基因21—23bp的RNA片段,以INTERFERinTMsiRNA Transfection Reagent作为转染试剂,将合成的siRNA片段转染哮喘小鼠脾脏来源的T细胞,作用48h后再与哮喘小鼠骨髓来源的树突状细胞（DC）混合反应48h,收集细胞上清液,ELISA法检测细胞因子IL4、IL-13、IL-2、INF-1;Western Blot法检测T细胞Lck蛋白的含量,判断其表达是否被沉默。结果siRNA干扰组T细胞中几乎检测不到Lck蛋白的表达,且其细胞上清液中IL-4、IL-13的水平（10．19±1．66、12．34±0．79）较非siRNA干扰组（28．06±2．88、27．87±1．61）及对照组（22．07±2．51、20．47±2．37）明显下降,差异有统计学意义（P〈0．01）。结论特定的21—23bp的RNA片段能够以siRNA干扰的方式有效的抑制特定基因的表达,Lck特异性siRNA可以阻断哮喘小鼠T细胞的激活和分化,减少哮喘炎症因子的释放。     

EGFR RNA干扰抑制博莱霉素诱导的小鼠肺纤维化

目的探讨降低表皮生长因子受体（EGFR）在肺内的表达对博莱霉素致小鼠肺纤维化的影响。方法将40只4-6周龄C57BLB／c雄性小鼠按简单随机法分为正常对照组（气管滴入PBS）、纤维化组（气管滴入博莱霉素3mg／kg）、RNAi组（气管滴入博莱霉素3mg／kg＋气管滴入siR-NA20μl）和RNAi阴性对照组（气管滴入博莱霉素3mg／kg＋气管滴人siRNA阴性对照20μl）。实验第10天处死小鼠,收获肺组织,检测羟脯氨酸含量。肺组织切片行HE染色观察肺组织病理改变,采用逆转录一聚合酶链反应（RT-PCR）法检测EGFR的表达。Westernblot检测EGFR、磷酸化EGFR表达。结果RNAi组与纤维化组比较,肺组织EGFRmRNA表达（0．31±0．05 vs 0．75±0．08,P〈0．01）和EGFR蛋白表达（1．53±O．47vs2．56±0．37,P〈0．01）均显著下降;肺病理损伤较纤维化组减轻,肺羟脯氨酸含量显著减少（543．00±25．89vs900．73±31．77,P〈0．01）;磷酸化EGFR蛋白表达亦较纤维化组明显减少（1．78±0．35 vs 2．84±0．51,P〈0．01）。结论EGFRRNAi抑制了EGFR活化,减轻了博莱霉素诱导的肺纤维化改变。     

CD4^＋CD25^＋调节性T细胞对哮喘大鼠气道炎症的影响

目的观察CD4^＋CD25^＋调节性T细胞（CD4^＋CD25^＋Treg）对哮喘大鼠气道炎症的影响。方法将卵白蛋白（OVA）免疫耐受大鼠CD4^＋CD25^＋Treg细胞过继转移给哮喘大鼠,然后观察支气管肺泡灌洗液（BALF）中细胞计数及分类,ELISA检测BALF中IL-4、IL-5和IFN-1及血清OVA特异性IgE含量,HE染色观察肺组织的病理改变。结果与哮喘组比较,过继转移CD4^＋CD25^＋Treg细胞后哮喘大鼠BALF中细胞总数、中性粒细胞和淋巴细胞百分率降低（P〈0．05）,嗜酸性粒细胞（Eos）百分率明显降低（P〈0．01）;BALF中IL4和IL-5含量明显降低,IFN-1含量明显升高,血清OVA特异性IgE含量明显降低（P〈0．05）;气道炎症明显减轻。结论过继转移OVA免疫耐受大鼠CD4^＋CD25^＋Treg细胞可以明显抑制哮喘的慢性气道炎症。     

siRNA沉默人类血红素敏感基因1对膀胱癌T24细胞的影响

目的 观察人类血红素敏感基因1(HRG-1)在膀胱正常组织和膀胱肿瘤组织中的表达情况,通过siRNA干扰技术抑制HRG-1在膀胱癌T24细胞的表达水平,观察其生长增殖的变化.方法 应用免疫组化SP法检测85例膀胱肿瘤(膀胱乳头状瘤25例,低级别膀胱癌30例,高级别膀胱癌30例)和20例膀胱正常组织石蜡标本中HRG-1表达情况,并进行相关临床病理分析.设计针对HRG-1基因的siRNA,转染膀胱癌T24细胞,应用免疫印迹法和RT-PCR分析HRG-1 siRNA的表达,MTT和流式细胞术(FCM)检测其增殖凋亡的变化.结果 正常膀胱组织和膀胱癌组织中HRG-1的表达水平比较差异有统计学意义(P＜0.05);HRG-1的阳性表达与膀胱肿瘤病例分级、临床分期相关(P＜0.05);转染siRNA后,HRG-1表达水平下降,MTT和FCM检测发现,细胞增殖曲线受到抑制,HRG-1-siRNA转染组与空载体转染组及未转染组比较,细胞凋亡比例增加,差异均具有统计学意义(P＜0.01).结论 siRNA-HRG-1处理T24细胞后,细胞的增殖受到抑制,同时凋亡比例增加,提示HRG-1基因可能参与了膀胱癌的发病.     

shRNA沉默VEGF—C基因对大肠癌淋巴管生成和淋巴结转移的影响

目的 观察RNA干扰（RNA interference,RNAi）技术抑制大肠癌细胞株Lovo中VEGF-C基因的表达,探讨抑制shRNA-VEGF-C对人大肠癌细胞Lovo生物学特性的影响.方法 构建表达ShRNA-VEGF-C重组质粒转染Lovo细胞,72 h后用实时RT-PCR（real-time polymerase chain recation）检测VEGF-C mRNA表达;建立Lovo细胞皮下移植瘤裸鼠模型,注射shRNA-VEGF-C,动态观察肿瘤体积并于4周后处死裸鼠称取瘤重、计算局部淋巴结转移率,免疫组化法检测大肠癌组织微淋巴管密度（microlymphatic density,MLD）.结果 转染shRNA-VEGF-C后,Lovo细胞VEGF-C mRNA表达下调;体内实验结果显示,4周后shRNA-VEGF-C组移植瘤体积[（324.9±64.8）mm3]明显小于空质粒组[（553.5±90.1）mm3]和生理盐水对照组[（570.1±85.4）mm3]（P〈0.01）;shRNA-VEGF-C组瘤重[（3.01±0.55）g]也低于空质粒组[（4.65±0.65）g]和生理盐水对照组[（4.75±0.55）g]（P〈0.01）;shRNA-VEGF-C组微淋巴管密度LMVD（15.5±6.90）明显低于HK组（24.18±6.45）和生理盐水对照组（29.59±8.21）（P〈0.01）;shRNA-VEGF-C组局部淋巴结转移率（30.1%）明显低于空质粒组（50.2%）和生理盐水组（53.1%）.结论 shRNA-VEGF-C可影响VEGF-C诱导的淋巴管生成并抑制大肠癌淋巴结转移.     

EGFL7基因沉默对裸鼠胃癌血管生成的影响

目的 探讨类表皮生长因子域7（EGFL7）基因沉默对胃癌裸鼠模型瘤内血管生成的影响.方法 构建EGFL7短发夹状RNA表达质粒并转染SGC-7901细胞（psh EGFL7组）,以空质粒转染为对照组.观察两组皮下种植瘤生长曲线及体积.免疫组织化学法检测抗-CD34、血管内皮生长因子（VEGF）、血小板反应蛋白（TSP1）表达;RT-PCR检测MMP-2和TIMP2表达.结果 psh EGFL7组种植瘤体积为（1.86±0.65）cm^3,微血管密度（MVD）为20.84±6.38,分级为1～2级,对照组体积为（4.86±1.15）cm^3,MVD为39.48±9.01,分级为3～4级,两组种植瘤体积、MVD和分级的差异有统计学意义（P＜0.05）.EGFL7组TSP1蛋白阳性表达,而VEGF蛋白为弱阳性或阴性表达,MMP-2 mRNA表达下调,而TIMP2 mRNA表达上调,与对照组比较差异有统计学意义（P＜0.01）.结论 EGFL7基因沉默可降低胃癌种植瘤血管生成,与调节MMP-2/TIMP2表达,影响VEGF/TSP1平衡有关.     

p21和p53在肾间质纤维化大鼠中表达的研究

目的动态观察p21和p53在单侧输尿管梗阻肾间质纤维化大鼠模型中的表达,以及依那普利对其表达的调节。方法将SD大鼠随机分为假手术组（SOR组）、单侧输尿管结扎组（UUO组）和依那普利治疗组（EnT组）,每组30只。用Masson染色的方法动态观察梗阻侧肾脏病理变化,用RT—PCR的方法检测梗阻肾皮质p21mRNA和p53mRNA的变化;并观察依那普利对肾皮质p21和p53表达的调节。结果在UUO大鼠模型早期,肾皮质p21mRNA和p53mRNA的表达即有明显增高,随着梗阻时间的延长,p21和p53的表达逐渐增强,二者具备相关性;依那普利能明显抑制p21和p53的表达。结论p21和p53在UUO大鼠肾皮质呈高表达,依那普利可抑制其表达。p21可能通过p53参与介导了。肾小管间质纤维化的发病过程。     

Differential activation of host cell signalling pathways through infection with two variants of influenza A/Puerto Rico/8/34 (H1N1) in MDCK cells

In cell culture-based influenza vaccine production, few efforts have been undertaken to characterise virus–host cell interactions in detail. Two influenza virus strains that grew to different virus titres, and differed in virus dynamics, apoptosis induction and proteome changes were observed.     

Silk peptides inhibit adipocyte differentiation through modulation of the Notch pathway in C3H10T1/2 cells

Silk protein is a biocompatible material that has been used in many biotechnological applications and exhibits body fat-lowering effects. Recent studies have shown that silk peptides increase expression of osteogenic markers in osteoblast-like cells. Because osteogenic and adipogenic differentiation from common mesenchymal progenitor cells are inverse processes and often regulated reciprocally, we hypothesized that silk peptides might suppress adipocyte differentiation. We therefore endeavored to evaluate the effects of silk peptides on adipocyte differentiation in C3H10T1/2 cells. We find that silk peptides inhibit lipid accumulation and morphological differentiation in these cells. Molecular studies show that silk peptides block expression of adipocyte-specific genes such as peroxisome proliferator-activated receptor γ and its targets, including aP2, Cd36, CCAAT enhancer binding proteinα. Silk peptides appear to inhibit adipogenesis by suppression of the Notch pathway, repressing the Notch target genes Hes-1 and Hey-1. In addition, these peptides inhibit endogenous Notch activation, as shown by a reduction in generation of Notch intracellular domain. N-[N-(3.5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester, compound E, and WPE-III-31C, which are all known Notch signaling inhibitors, block adipocyte differentiation to an extent similar to silk peptides. Together, our data demonstrate that silk peptides can modulate adipocyte differentiation through inhibition of the Notch signaling and further suggest potential future strategies for treating obesity and its related metabolic diseases.     

Capsaicin attenuates palmitate-induced expression of macrophage inflammatory protein 1 and interleukin 8 by increasing palmitate oxidation and reducing c-Jun activation in THP-1 (human acute monocytic leukemia cell) cells

Capsaicin, a spicy component of hot peppers, has been shown to improve inflammatory disease and obesity. In this study, we tested the hypothesis that the anti-inflammatory activity of capsaicin can be used to improve free fatty acid (FFA)-induced inflammation by reducing gene expression of macrophage inflammatory protein 1 (MIP-1) and interleukin 8 (IL-8) in THP-1 (human acute monocytic leukemia cell) macrophages. To investigate whether capsaicin ameliorates palmitate-induced MIP-1 and IL-8 gene expressions, we treated THP-1 cells with palmitate in the presence or absence of capsaicin and measured MIP-1 and IL-8 by real-time polymerase chain reaction. To elucidate the mechanism by which capsaicin effects on palmitate-induced MIP-1 and IL-8 gene expressions, we performed immunoblotting with stress kinase-related antibodies and measured palmitate oxidation and palmitate oxidation-related gene expression. Palmitate and stearate but not the unsaturated FFA oleate significantly increased MIP-1 and IL-8 expressions in THP-1 macrophages. Treatment with capsaicin or FFA oxidation stimulators inhibited palmitate-induced MIP-1 and IL-8 expressions in THP-1 macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the β-oxidation of palmitate. Furthermore, capsaicin significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38. Our data suggest that the attenuation of palmitate-induced MIP-1 and IL-8 gene expressions by capsaicin is associated with reduced activation of c-Jun N-terminal kinase, c-Jun, and p38 and preserved β-oxidation activity.