miR-10a-5p敲低通过靶向THBS2抑制胃癌细胞的生长和转移

背景在胃癌(gastric cancer, GC)的发病和进展中,已经发现许多miRNAs可发挥抑癌或促癌的作用.但miRNAs数量众多,在GC中仍有众多miRNAs的作用并不明确,因此,继续筛选具有影响GC细胞生长和转移的miRNAs仍十分必要.目的探究miR-10a-5p对GC细胞生长和转移的影响及其机制.方法采用实时荧光定量PCR(quantitative real-time PCR,RT-q PCR)检测GC组织和GC细胞(MGC-803和AGS)中miR-10a-5p表达;miR-10a-5p-inhibitor转染入MGC-803和AGS细胞,CCK-8实验,集落形成实验和Transwell实验分别检测细胞的增殖,集落形成,迁移和侵袭.采用Western-blot和RT-q PCR检测GC组织和GC细胞(MGC-803和AGS)中THBS2表达;采用Target Scan在线软件筛选miR-10a-5p的潜在靶基因THBS2,并进一步验证. NC+pc DNA-Con, miR-10a-5pinhibitor+pc DNA-Con和miR-10a-5p-inhibitor+pc DNATHBS2共转染入MGC-803细胞,CCK-8实验,集落形成实验和Transwell实验分别检测各组细胞的增殖,集落形成,迁移和侵袭.结果miR-10a-5p在GC组织,MGC-803和AGS细胞中表达异常上调; miR-10a-5p敲低显著降低了MGC-803和AGS细胞的增殖,集落形成和侵袭.THBS2m RNA和蛋白水平在GC组织、MGC-803和AGS细胞中表达均异常下调; THBS2是miR-10a-5p的靶基因;过表达THBS2能逆转miR-10a-5p敲低对MGC-803细胞增殖,集落形成,迁移和侵袭的抑制作用.结论miR-10a-5p敲低通过靶基因THBS2来抑制GC细胞的生长和转移.     

Med19基因沉默对胃癌细胞增殖和细胞周期的影响

目的 研究Med19基因在胃癌MGC-803细胞中沉默后对细胞增殖和周期的影响.方法 应用shRNA慢病毒载体感染胃癌MGC-803细胞沉默Med19基因,通过MTT和克隆形成实验观察Med19基因在胃癌细胞增殖中的作用,并用流式细胞术验证抑制Med19基因对细胞周期的影响.结果 构建的shRNA慢病毒载体感染MGC-803细胞后细胞增殖能力显著降低、细胞克隆形成能力明显减弱,同时,细胞周期阻滞于G1期.结论 Med19基因沉默后胃癌细胞的增殖能力和细胞周期均受到显著影响,提示Med19在肿瘤形成过程中具有重要作用.     

miR-1284过表达对胃癌细胞中EIF4A1的影响

目的通过验证miR-1284可能的靶基因,从而探讨miR-1284影响胃癌发生发展可能的分子作用机制。方法采用生物信息学软件预测miR-1284可能的靶基因。以慢病毒介导miR-1284过表达转染胃癌MGC-803细胞为miR-1284组(LV-miR-1284组),转染空载体慢病毒载体的细胞为阴性对照组,未转染慢病毒载体的细胞为空白对照组。运用荧光定量PCR检测各组EIF4A1基因的表达,Western blot法检测各组EIF4A1蛋白的表达,通过双荧光素酶对EIF4A1进行靶基因验证。结果与阴性对照组和空白对照组比较,miR-1284组的EIF4A1基因和蛋白的表达量下降,双荧光素酶示miR-1284能与靶基因EIF4A1的3'UTR的结合。结论 miR-1284通过作用其靶基因EIF4A1发挥生物功能学作用。     

慢病毒介导的E2F-1沉默对人胃癌细胞MGC803中药人参黄芪复方药物敏感性的影响

目的:利用重组慢病毒介导的E2F-1基因沉默载体感染人胃癌MGC-803细胞,观察其对中药人参黄芪复方药物敏感性的影响.方法:将胃癌MGC-803细胞接种于培养板中分为4组:正常组(正常培养基)、空白对照组(中药培养基)、阴性对照LV-RNAi组(中药培养基+感染空载体慢病毒颗粒2.0×109TU/mL)、E2F-1-RNAi-LV组(中药培养基+感染E2F-1基因重组慢病毒颗粒2.0×109TU/mL),分别采用半定量逆转录-聚合酶链反应(RT-PCR)和Western blot检测E2F-1mRNA及蛋白表达;MTT方法检测细胞的增殖抑制率;Giemsa染色检测细胞的克隆形成;流式细胞仪检测细胞的凋亡;细胞划痕实验检测细胞迁移能力.结果:E2F-1基因沉默蛋白及mRNA表达水平均明显下降,差异有统计学意义(0.384±0.036vs0.883±0.051和0.923±0.049,0.220±0.056vs0.729±0.021和0.712±0.037,P<0.05).中药人参黄芪复方能抑制人胃癌MGC-803细胞生长、诱导胃癌细胞凋亡、抑制细胞克隆形成及迁移;与空白对照组及阴性对照组相比较,E2F-1基因沉默组MGC-803细胞增殖抑制率、细胞凋亡率显著提高,其细胞克隆形成、细胞迁移面积显著降低,差异均有统计学意义(0.789±0.013vs0.484±0.060和0.454±0.006,0.383±0.027vs0.114±0.038和0.089±0.051,0.024±0.003vs0.036±0.004和0.052±0.002,0.553±0.038vs0.897±0.020和0.928±0.051,P<0.05).结论:重组慢病毒载体介导的E2F-1基因沉默能有效增强中药人参黄芪复方对人胃癌MGC-803细胞的药物敏感性.     

RNA干扰沉默CLIC4基因对胃癌细胞增殖和侵袭的影响

目的 探讨氯离子胞内通道(CLIC)4对胃癌细胞增殖和侵袭的影响.方法 采用实时荧光定量聚合酶链反应(RT-PCR)检测人胃癌细胞SGC-7901和MGC-803中CLIC4的表达.应用Lipofectamine2000将CLIC4 siRNA转染到SGC-7901细胞和MGC-803细胞中,分为siRNA1组、siR...     

eIF4A1基因沉默对胃癌SGC-7901细胞生物学行为影响机制的探讨

目的探讨沉默eIF4A1基因对胃癌细胞增殖、凋亡、迁移的细胞生物学行为影响的机制。方法以eIF4A1沉默重组慢病毒颗粒(LV-EIF4A1-RNAi组)感染人胃癌SGC-7901为eIF4A1沉默组(eIF4A1-RNAi组),感染阴性对照慢病毒颗粒(CON077)的胃癌细胞为阴性对照组(CON077组),空白对照组(control组)不作任何处理。以每个副孔初始含4×10~3个细胞,CCK-8检测各组细胞增殖能力;流式细胞仪检测各组5×10~6个细胞凋亡和1×10~6个细胞周期分布;Transwell实验检测各组1×10~5个细胞迁移和侵袭能力;实时荧光定量PCR和Western blot检测各组细胞eIF4A1和AKT基因的mRNA和蛋白表达水平。结果 LV-EIF4A1-RNAi(44683-1)组eIF4A1基因的表达量最低为(0.31±0.04),因此选择该组为eIF4A1沉默组(eIF4A1-RNAi)。与CON077组和control组相比,eIF4A1-RNAi组细胞增殖活力明显降低,细胞凋亡率提高,细胞周期阻滞于S期,G1/G2期细胞减少,细胞的迁移、侵袭能力下降,eIF4A1和AKT基因的mRNA和蛋白表达明显降低,差异均有统计学意义(P0.05);CON077组与control组比较,上述指标差异均无统计学意义(P0.05)。结论慢病毒介导eIF4A1基因沉默能抑制人胃癌细胞株SGC-7901的增殖和迁移、促进细胞凋亡、阻滞细胞周期于S期,其机制可能与AKT表达下调有关。     

过表达CD44v6基因对人大肠癌SW480细胞侵袭迁移能力的影响

目的:研究CD44v6基因过表达对SW480细胞侵袭和迁移能力的影响.方法:慢病毒介导的CD44v6过表达细胞(CD44v6组)和空载体对照细胞(NC组)由前期实验构建.采用荧光显微镜观察增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)表达、实时荧光定量PCR检测CD44v6 mRNA表达水平、免疫荧光检测Flag标签蛋白三种方法重新鉴定过表达细胞模型;CCK-8法检测细胞增殖活性;划痕试验和Transwell试验检测细胞侵袭和迁移能力.结果:绿色荧光蛋白观察显示细胞转染效率近100%;实时荧光定量PCR显示CD44v6组细胞CD44v6 mRNA表达水平较对照组.显著升高(P0.001);Flag标签蛋白免疫荧光染色显示过表达CD44v6蛋白主要定位于细胞膜.CCK-8结果显示2组细胞增殖无明显差异;划痕试验结果显示CD44v6组细胞划痕愈合指数较对照组显著增高(P0.05);Transwell试验结果显示CD44v6组细胞迁移和侵袭相关指数均较对照组显著增高(均P0.05),且CD44v6抗体处理后,CD44v6组细胞迁移和侵袭相关指数均较前显著减低(均P0.05).结论:CD44v6基因过表达能显著增强SW480细胞侵袭和迁移能力.     

藏红花素通过调控微管蛋白进而影响胃癌细胞MGC-803的增殖、侵袭和凋亡

目的 探讨藏红花素(Crocin)是否通过调控微管蛋白进而影响人胃癌细胞MGC-803的增殖、侵袭和凋亡,以期为胃癌的治疗提供新的思路。方法 使用藏红花素处理MGC-803细胞,并用顺铂作为阳性对照药,观察藏红花素对MGC-803细胞生物学的影响。采用MTT分析细胞活性,流式细胞术分析细胞凋亡,Transwell检测细胞的侵袭能力,划痕实验检测细胞的迁移能力。最后通过蛋白免疫印迹分析藏红花素作用的潜在机制。结果 藏红花素使MGC-803细胞活力、迁移和侵袭能力明显下降,凋亡增加。进一步研究发现,藏红花素对MGC-803细胞的抑制能力与微管蛋白作用密切相关,藏红花素通过降低MGC-803细胞中的微管蛋白表达进而影响其增殖、侵袭和凋亡。结论 藏红花素能够显著抑制MGC-803细胞生长、迁移、侵袭能力,并能促进MGC-803胃癌细胞的凋亡反应。     

RNAi沉默CD14基因对人胃癌细胞生物学行为的影响

目的以胃癌SGC-7901细胞为研究对象,在细胞水平观察沉默CD14基因对胃癌细胞增殖、凋亡、周期及下游靶因子TNF-α、IL-8的影响,探讨CD14基因在胃癌中的发病地位。方法重组CD14shRNA慢病毒为载体转染胃癌SGC-7901细胞。实验分3组,正常组、慢病毒阴性对照组(NCshRNA)、CD14沉默组(CD14shRNA)。采用荧光显微镜观察转染效率,确定最佳转染时间;Western blotting检测CD14蛋白的沉默效率;MTT法检测细胞增殖,流式细胞仪检测细胞周期及凋亡,Real-time PCR技术检测TNF-α、IL-8表达。结果成功转染慢病毒CD14shRNA的SGC-7901人胃腺癌细胞可表达绿色荧光蛋白,测得在病毒MOI=10时,转染72 h后,慢病毒的转染效率最佳。转染后24 h,正常组细胞增殖率为218.18%,阴性对照组为216.27%,CD14shRNA组为211.63%,各组间相比,差异无统计学意义(P0.05);转染后48 h、72 h,正常组细胞增殖率为427.49%、479.22%,阴性对照组为417.84%、473.37%,CD14shRNA组为345.54%、362.64%,经比较,CD14沉默后细胞增殖率下降(P0.05)。CD14shRNA组细胞早期凋亡、晚期凋亡及总凋亡率均高于正常组,差异有统计学意义(P0.05)。CD14shRNA组细胞G1期42.57%、S期38.65%、G2期13.22%,正常组细胞G1期35.91%、S期52.60%、G2期11.49%,差异有统计学意义(P0.05)。Western blotting结果显示,在转染胃癌细胞后,CD14shRNA慢病毒载体组细胞CD14蛋白表达相对于正常组及阴性对照组明显减少(P0.05);Real-time PCR结果显示,CD14shRNA转染胃癌细胞后,TNF-α、IL-8 mRNA表达较正常组、阴性对照组明显下降。结论 CD14基因沉默后,胃癌细胞增殖能力减弱,细胞周期阻滞在S期,细胞合成减少,凋亡率增加,TNF-α、IL-8表达下降,这说明CD14基因在胃癌细胞增殖、发展过程中占据重要地位。     

miR-133靶向JAK2抑制胃癌细胞增殖、迁移和侵袭

目的研究miR-133对胃癌(gastric cancer, GC)细胞增殖、迁移和侵袭的影响,并探讨其作用机制。方法运用qRT-PCR法检测组织和细胞中miR-133、JAK2的mR NA表达;将miR-133组(转染miR-133 mimics)、miR-NC组(未转染细胞)、miR-133 inhibitors组(转染miR-133 inhibitors)、inhibitors-NC组(转染inhibitors)、JAK2WT(载体psiCHECK2-JAK2-32 MUT(载体pUT和miR-133共转染)、miUTRWT和miR-133共转染)、JAKsiCHECK2-JAK2-3UTR MR-133+JAK2组(miR-133 mimics和JAK2共转染)、miR-133+Vector组(miR-133 mimics和pc-DNA 3。1共转染)均用脂质体法转染至AGS、MGC-803细胞;用Western blot检测细胞中JAK2的蛋白表达; MTT法检测细胞增殖;Transwell法检测细胞迁移和侵袭;双荧光素酶报告基因检测实验检测细胞荧光素酶活性。结果与人癌旁组织、人正常胃黏膜细胞GES-1相比, GC组织、GC细胞AGS、MGC-803中miR-133均低表达,JAK2均高表达;且过表达miR-133、沉默JAK2均可抑制GC细胞增殖、迁移和侵袭; JAK2为miR-133的靶点,且回补JAK2可逆转miR-133对GC细胞增殖、迁移和侵袭的抑制作用。结论miR-133可抑制GC细胞增殖、迁移和侵袭,其可能与靶向JAK2有关,将可为GC的临床诊断治疗提供新靶点。     

Effects of therapeutic paracentesis on systemic and hepatic hemodynamics and on renal and hormonal function

Thirteen patients with cirrhosis and tense ascites (six with and seven without peripheral edema) underwent 4- to 15-liter paracentesis without intravenous "colloid" replacement. Cardiac output increased from 6.6 +/- 0.7 liters per min at baseline to 8.2 +/- 0.7 liters per min (p less than 0.003) 1 hr after large-volume paracentesis completion and fell to 7.5 +/- 0.69 liters per min (p less than 0.05 vs. baseline, p less than 0.02 vs. 1 hr) 24 hr after large-volume paracentesis completion. There was no change in mean arterial pressure or mean pulmonary artery pressure. Central venous pressure fell from 9.1 +/- 0.8 mm Hg at baseline to 8.6 +/- 1.4 mm Hg 1 hr post-large-volume paracentesis to 6.8 +/- 1.0 mm Hg (p less than 0.005 vs. baseline, p less than 0.02 vs. 1 hr value) at 24 hr, and pulmonary capillary wedge pressure fell from 13.1 +/- 0.9 to 11.1 +/- 1.3 mm Hg 1 hr after large-volume paracentesis and to 9.89 +/- 1.2 (p less than 0.01 vs. baseline, p less than 0.03 vs. 1 hr after large-volume paracentesis) at 24 hr. Heart rate fell from 90 +/- 3.0 to 85 +/- 2.9 beats per min (p less than 0.01) 1 hr after large-volume paracentesis completion, but increased to 89 +/- 2.5 beats per min (p less than 0.02 vs. 1 hr after large-volume paracentesis) at 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

锌、铜对染砷小鼠肝及肾毒作用的干预研究

为观察补充锌、铜对染砷小鼠的肝、肾脏组织超氧化物歧化酶 (SOD)水平、丙二醛 (MDA)含量并初步探讨其作用机制 ,对染砷小鼠同步进行锌、铜干预试验 ,同时作肝脏、肾脏组织SOD活性及MDA含量测定和病理学观察。干预后 ,与阳性对照组相比 ,锌干预组肝脏、肾脏SOD活性升高 ,MDA含量下降 (P <0 .0 1) ,锌加铜组仅肾脏SOD活性升高 (P <0 .0 1) ,铜组肝、肾脏SOD活性及MDA含量均无显著性差异 (P >0 .0 5 )。病理学观察可见 ,锌干预组效果最好 ,其次为锌加铜组 ,而铜组无干预效果。提示补充一定剂量锌对染砷小鼠的脂质过氧化程度、抗氧化能力以及组织病理改变均有一定的干预效应。铜在该剂量下与锌起拮抗作用。     

Effect of particle size on quartz-induced hemolysis and on lung inflammation and fibrosis

To assess the role of crystal size in biologic responses, we quantitated red blood cell lysis and lung inflammation and fibrosis in the mouse using 4 alpha-quartz preparations with average diameters of 1, 5, 7.8, and 11.2 microns. When compared on the basis of identical crystal surface areas, the 1-micron fraction was more hemolytic than the other 3 fractions. The three larger fractions had equivalent membranolytic activities. After 6 weeks of postintratracheal instillation of the crystals into mice, the 1-micron-diameter crystal fraction increased wet lung weights by 1.25 x that of saline controls, while a 1.75 x increase was found for the three larger crystal fractions. A similar response was found when evaluating fibrosis development by determining lung hydroxyproline levels. Measurement of the percentage of the crystal dose remaining in the lungs revealed that the biologic differences observed were not due to a difference in the clearance of the smaller crystal fraction. Thus, larger crystals of alpha-quartz produce a greater degree of inflammation and fibrosis when instilled into the lung than those of 1 micron diameter, even though the smaller crystals are more membranolytic in vitro and appear to be cleared from the lung at the same rate as the larger crystals.     

Effects of trenbolone acetate and testosterone on growth and on plasma concentrations of corticosterone and ACTH in rats

The effects of trenbolone acetate (TBA) on growth and on plasma concentrations of corticosterone were examined in male and female rats. At 5 weeks of age, rats were injected with TBA (0.8 mg/kg) dissolved in peanut oil, or with oil alone, daily for 10 days. In female rats, TBA caused an increase in weight gain (20-38%), a reduction in adrenal weight (19%) and a reduction in plasma concentrations of corticosterone (55%). In contrast, TBA-treated male rats showed no significant increase in weight gain, no significant change in adrenal weight and no reduction in plasma concentrations of corticosterone. The mechanism by which adrenal activity was suppressed in TBA-treated female rats was examined and the response compared with that to testosterone. Female rats (8 weeks old) were injected daily either with oil vehicle, TBA (0.8 mg/kg) or testosterone propionate (0.8 mg/kg). Testosterone increased weight gain (24%), but the growth response to TBA treatment was significantly greater (97%). A reduction in plasma concentrations of corticosterone (45%) was again observed in response to TBA. However, testosterone increased plasma concentrations of corticosterone (52%) above those of control values. Neither androgen affected plasma concentrations of ACTH. Finally, the effects of TBA were examined in 6-week-old female rats, to characterize further the apparent age-related increase in responsiveness. The growth response of 6-week-old rats (60-74%) was intermediate between that seen in 5- and 8-week-old animals. It is concluded that part of the anabolic activity of TBA may be related to a reduction in circulating concentrations of corticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of adiponectin and ghrelin on incident glucose intolerance and on weight change

Objectives Adiponectin and ghrelin are associated with adiposity and type 2 diabetes in several studies. We sought to prospectively determine the interaction of adiponectin and ghrelin in the development of adiposity and hyperglycaemia. Design Prospective observational study. Participants 393 community‐dwelling Afro‐Jamaicans (mean age 47 ± 13 years; BMI 27·3 ± 6·3 kg/m²; 63% women) without glucose intolerance at baseline. Measurements Anthropometry, fasting plasma glucose, 2‐h plasma glucose, insulin resistance (HOMA‐IR), adiponectin and ghrelin concentrations were measured at baseline and 4·1 ± 0·9 years later. Multivariate analyses were used to explore the associations of HOMA‐IR, adiponectin and ghrelin with weight change and glycaemia. Results The mean weight change was 2·6 ± 5·5 kg. There were 114 incident cases of impaired glucose tolerance (IGT) and 35 cases of diabetes mellitus. Adiponectin was positively correlated with age and female sex (P‐values < 0·01). After adjusting for age and sex, adiponectin and ghrelin were significantly correlated with weight at baseline and follow‐up. However, they were not associated with weight change even after further adjustment for baseline weight. Adiponectin, but not ghrelin, was associated with 2‐h glucose concentrations at follow‐up even after adjusting for age, sex, HOMA‐IR and BMI (P = 0·04). In the fully adjusted logistic regression model, adiponectin predicted incident IGT (OR 0·93; 95% CI: 0·87–0·99) and attenuated the effect of BMI on incident IGT. Conclusions These longitudinal data show that adiponectin and ghrelin may not be causally involved in the development of obesity. However, adiponectin is independently associated with decreased risk of incident IGT.