shRNA下调Tspan8基因对肝癌细胞侵袭与转移的影响

目的 探讨Tspan 8基因对人肝癌细胞侵袭与转移的影响.方法 应用RT-PCR与Western印迹检测不同转移潜能肝癌细胞及组织中Tspan 8的表达;应用shRNA-Tspan 8(LV/GFP/Tspan 8)下调Tspan 8表达;MTT法检测细胞增殖变化;Western印迹检测基质金属蛋白酶12表达改变;最后应用Transwell检测细胞侵袭能力变化.结果 高表达Tspan 8见于高转移潜能的肝癌细胞,Tspan 8在肝癌组织中表达明显高于相应癌旁组织.感染LV/GFP/Tspan 8、LV/GFP后,LV/GFP/Tspan 8感染肝癌细胞的Tspan 8表达显著下调(P＜0.01);下调Tspan 8后,细胞增殖反应变化不明显(24 h,48 h与72 h,P＞0.05);但MMP-12表达抑制明显(P＜0.01),细胞侵袭能力明显下降.结论 下调Tspan 8基因的表达对肝癌侵袭与转移能力有明显抑制.     

上调基因-4对结肠癌细胞增殖的影响

目的 探讨上调基因-4(URG-4)对结肠癌细胞增殖的影响.方法 筛选高表达URG-4的结肠癌细胞.构建URG-4基因siRNA的逆转录病毒载体及阴性对照,经PT67细胞包装后,获得可表达人URG-4基因siRNA逆转录病毒及阴性对照.RT-PCR和Western blot法检测MKN45、SW480、LoVo、HCT116、HT29细胞中URG-4 mRNA和蛋白表达水平,筛选稳定转染细胞株.分别使用重组病毒(干扰组)、原始病毒(阴性对照组)和等量PBS(空白对照组)转染结肠癌LoVo细胞.MTT法检测各组结肠癌LoVo细胞生长情况.计量资料比较采用单因素方差分析和t检验.结果 测序结果证实表达siRNA的逆转录病毒成功构建.URG-4 mRNA在MKN45、SW480、LoVo、HCT116和HT29细胞中的相对表达量分别为0.58±0.02、0.63 +0.03、0.81±0.01、1.01±0.02和0.91±0.04;URG-4蛋白相对表达量分别为0.73±0.02、0.85士0.03、1.42 +0.01、0.80 +0.03和0.80+0.04.结肠癌LoVo细胞高表达URG-4.干扰组中URG-4 mRNA表达为0.55±0.03,显著低于阴性对照组的1.15±0.02和空白对照组的1.15±0.01(t=-5.179,-9.285,P＜0.05).干扰组URG-4 mRNA的抑制率为52.6％.干扰组中URG-4蛋白表达为0.82 +0.05,显著低于阴性对照组的1.46士0.07和空白对照组的1.54 ±0.04(t=-4.239,-3.704,P＜0.05).干扰组URG-4蛋白的抑制率为43.6％.各组结肠癌LoVo细胞呈指数生长.与阴性对照组比较,第3～6天干扰组细胞增殖受到明显抑制,差异有统计学意义(t=-6.436,-6.045,-6.434,-4.285,P＜0.05).结论 干扰URG-4表达能够抑制LoVo细胞的生长.     

RNA干扰降低环氧化酶2基因表达对人胆管癌QBC939细胞的体外作用

目的研究小干扰RNA（siRNA）降低环氧化酶2（COX-2）基因对胆管癌QBC939细胞凋亡及凋亡蛋白caspase-3蛋白表达的影响。方法采用RNA干扰的方法,将胆管癌细胞株QBC939细胞分为4组：COX-2 siRNA干预组、阴性对照siRNA组、空脂质体、空白对照组。将COX-2 siRNA转染入QBC939细胞;流式细胞仪检测siRNA降低COX-2基因表达对凋亡的作用,WesternBlot法检测siRNA降低COX-2基因表达对caspase-3表达变化的影响。结果 RNA干扰后QBC939细胞COX-2表达下降至空白对照组的42%,流式细胞仪检测结果显示COX-2 siRNA干预组细胞的凋亡率以及凋亡蛋白caspase-3的表达明显高于其他3个对照组。结论 RNA干扰可抑制细胞COX-2蛋白的表达,促使胆管癌QBC939细胞的凋亡,caspase-3途径可能是其调控通路之     

CXCR4 RNA干扰序列筛选及对结肠癌细胞SW480生长的影响

目的筛选细胞表面趋化因子受体（CXCR4） RNA干扰高效序列,探讨其对SW480细胞生长的影响。方法采用RT-PCR和Western blot方法选择最有效的CXCR4 RNA干扰序列,流式细胞术分析转染该序列后SW480细胞生长情况。结果 sihCXCR4-3序列可使CXCR4 RNA相对含量仅为SW480组的5.4%,CXCR4蛋白相对表达量为18.95%。sihCXCR4-3组细胞数[（0.92±0.06）×105]低于SW480细胞组[（1.38±0.04）×105,P=0.0050]及NCsihCXCR4组[（1.28±0.05）×105,P=0.0256];相对SW480组和NCsihCXCR4组,sihCXCR4-3组的抑制率分别为33.03%及28.00%。sihCXCR4-3组G1期百分率明显低于SW480细胞组[（59.5±0.57）%vs.（74.11±0.54）%,P=0.0167];S期百分率高于SW480组[（23.85±0.24）%vs.（14.62±0.46）%,P=0.0305]和NCsihRNA组[（17.63±0.48）%,P=0.0013]。结论 sihCXCR4-3序列可高效沉默CXCR4基因表达,抑制SW480细胞生长。     

Induction of Alloimmune Tolerance in Heart Transplantation Through Gene Silencing of TLR Adaptors

Toll‐like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.     

靶向Plk1基因的shRNA对肝癌BEL-7402细胞株生物学行为及化疗增敏的影响

目的 利用RNA干扰技术(RNAi),研究表达Polo-like kinase 1 (Plk1)的短发夹RNA (shRNA)载体对肝癌细胞株BEL-7402生物学行为及对化疗药物长春新碱敏感性的影响. 方法 根据靶基因的不同区域构建针对Plk1mRNA的两组干扰质粒shRNA-Plk1:Plk1siRNA-251,Plk1siRNA-1396以及阴性对照质粒Plk1siRNA-hk,利用电穿孔法转染入肝癌细胞株BEL-7402,用长春新碱处理细胞后,用MTT法检测四组细胞的生长抑制率,计算IC50值,流式细胞仪检测各组细胞凋亡. 结果 半定量RT-PCR检测Plk1siRNA-251,Plk1siRNA-1396组Plk1mRNA、Cdc25CmRNA表达较Plk1siRNA-hk转染组与空白对照组明显下降(P＜0.01),p53mRNA表达增加.长春新碱处理后,Plk1siRNA-251组和Plk1siRNA-1396组与BEL7402组之间的生长抑制率有显著性差异(P＜0.01).5 μg/ml浓度长春新碱作用24h后,Plk1siRNA-251组和Plk1siRNA-1396组凋亡指数分别为19.09％ ±3.97％、19.80％ ±5.47％明显高于Plk1siRNA-hk组和BEL7402组(P＜0.01).结论 成功筛选出两条能特异而高效抑制Plk1基因表达的shRNA,可以显著抑制Plk1 mRNA、Cdc25CmRNA及Plkl蛋白的表达,并使p53mRNA表达增高,并明显抑制细胞增殖,能增加长春新碱的化疗敏感性,从而为进一步研究该基因的功能和作用机制提供了新的手段.     

RNA干扰RelB基因对小鼠RM-1前列腺癌细胞株放射敏感性的影响及其机制

目的:探讨RNA干扰RelB基因对鼠RM-1前列腺癌细胞株放射敏感性的影响及其机制。方法:利用靶向RelB基因的慢病毒载体(pLentilox-sh-RelB),脂质体介导转染RM-1细胞后进行放射(2,4,6,和8Gy剂量)处理培养,分别采用RT-PCR、Western印迹法及流式细胞术等方法检测RelB的mRNA和蛋白表达,锰超氧化物歧化酶(Mn-SOD)活力及细胞放射敏感性的变化。结果:转染后放射处理培养,siRelB-RM-1组RelB的mRNA和蛋白表达水平明显低于siVector-RM-1组和nontrans-RM-1组(P<0.05);放射处理培养后siRelB-RM-1组细胞凋亡百分率明显高于siVector-RM-1组和nontrans-RM-1组(P<0.05);培养后siRelB-RM-1组细胞Mn-SOD活力下降,放射增敏比为5.13。结论:RNA干扰RelB基因可增强鼠RM-1前列腺癌细胞株放射后的放射敏感性,其机制可能与RNA干扰RelB基因,抑制细胞增殖,降低Mn-SOD活力和诱导凋亡有关。     

靶向Wnt10b的小干扰RNA抑制胚胎皮肤毛囊发育的初步研究

目的 利用小干扰RNA(siRNA)抑制皮肤Wnt10b基因的表达,观察Wnt10b基因沉默能否抑制毛囊的发育并探讨其潜在机制.方法 化学合成siRNA-Wnt10b,将siRNA转染体外培养的胎鼠背部皮肤,荧光定量PCR检测转染后不同时间段皮肤组织Wnt10b和p-连环蛋白(β-catenin) mRNA的表达,Western blot检测转染后72 h皮肤组织的Wnt10b和β-catenin蛋白含量.将转染后72 h的皮肤组织石蜡包埋、切片,HE染色,镜下观察各组毛囊发育情况并做统计学处理.结果 siRNA-Wnt10b转染后的24、48 h,胎鼠背部皮肤Wnt10b mRNA的表达呈不同程度下降;但β-catenin mRNA表达未随Wnt10b mRNA水平的起伏而明显波动;转染后72 h,Wnt10b蛋白和β-catenin的蛋白表达同时减少,新形成毛囊数量也随之减少(t=3.254,P=0.002).结论 在体外器官培养的环境中,siRNA-Wnt10b能够抑制胎鼠背部皮肤组织Wnt10b蛋白的表达,减少新形成毛囊的数量;Wnt10b可能只在蛋白水平上调控细胞β-catenin的表达.     

Associations between pain and current smoking status among cancer patients

There is growing empirical and clinical interest in purported associations between smoking and the aggravation of cancer symptoms and treatment side effects, such as pain. Both pain and smoking are highly prevalent among persons with cancer, and there is recent evidence to suggest that cancer patients who continue to smoke despite their diagnosis experience greater pain than nonsmokers. Accordingly, the main goal of this cross-sectional study was to examine associations between multiple levels of smoking status and several pain-related outcomes among a sample of 224 cancer patients about to begin chemotherapy. Patients completed self-report measures of pain severity, pain-related distress, and pain-related interference, as well as a demographics questionnaire. Results indicated that persons who continued to smoke despite being diagnosed with cancer reported more severe pain than never smokers, F (2, 215) = 3.47, p < .05. Current smokers also reported greater interference from pain than either former or never smokers, F (2, 215) = 5.61, p < .01. Among former smokers, an inverse relation between pain severity and the number of years since quitting smoking was observed, r (104) = −.26, p < .01. These data suggest that continued smoking despite a cancer diagnosis is associated with greater pain severity and interference from pain; however, future research is warranted to determine the directionality of this relationship.