腺病毒介导的靶向蛋白激酶B1和环氧合酶2短发夹RNA抑制人胃腺癌细胞的侵袭和转移

目的 构建靶向性蛋白激酶B1(Aktl)和环氧合酶2(COX-2)短发夹RNA(shRNA)腺病毒载体,研究其对SGC-7901人胃腺癌细胞侵袭和转移的抑制效果.方法 构建腺病毒载体pGSadeno-Aktl+COX-2(rAd5-A+C)并体外转染SGC-7901人胃癌细胞株后,用实时定量PCR和蛋白印迹分别检测对照组SGC-7901、空载组rAd5-HK和治疗组rAd5-A+C中Aktl、COX-2 mRNA和蛋白质的表达,其中以对照组SGC-7901、空载组rAd5-HK作为阴性对照.用ELISA法分别检测它们的MMP-2和MMP-9含量;用Transwell法分析它们的侵袭和转移能力.结果 构建的rAd5-A+C重组腺病毒载体在转染SGC-7901细胞48 h后可显著抑制Aktl和COX-2 mRNA的表达,而治疗组rAd5-A+C的Aktl和COX-2蛋白表达量与对照组和空载组相比分别下调68.4%和60.2%(均P<0.01);rAd5-A+C组中MMP-2和MMP-9含量分别为(39.7±1.7)ng/ml(31.3±3.6)ng/ml,明显低于对照组SGC-7901(278.4±15.5)ng/ml(225.4±15.1)ng/ml和卒载组rAd5-HK(275.5±2.1)ng/ml、(226.0±23.3)ng/ml(P=0.01、P=0.021).结论 腺病毒介导的靶向性Aktl和COX-2shRNA可抑制人胃腺癌细胞的侵袭和转移.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

RNA干扰技术靶向P13K/AKT信号通路抵制胃腺癌SGC7901细胞侵袭转移的研究

目的 观察应用RNA干扰(RNAi)技术敲低PIK3R1、AKT1表达后在体外对胃腺癌SGC7901细胞侵袭的抑制作用.方法 将重组腺病毒质粒表达载体rAd5-A-P转染至SGC7901细胞.Realtime PCR和Western blot分别检测转染前后目的 基因mRNA和蛋白的表达水平,酶联免疫吸附试验(ELISA)检测细胞外基质金属蛋白酶(MMP)-2、MMP-9的浓度变化.划痕、Transwell、3-DMatrigel基质生长实验评价肿瘤细胞侵袭能力的变化.结果 rAd5-A-P可以有效抑制PIK3R1、AKT1的表达,MMP-2、MMP-9表达下调,基质金属蛋白酶组织抑制因子(TIMP)-2表达上调,ELISA证实细胞外MM9-2、MMP-9浓度在治疗组明显减低.划痕实验显示rAd5-A-P转染组细胞转移运动能力明显减弱,Transwell穿过细胞数结果:对照组(105.0±4.0)和无义序列组(102.5±6.4),rAd5-A-P转染组(67.0±3.9),3-D的Matrigel基质生长实验显示与对照组和无义序列组比较,rAd5-A-P转染组细胞形成的细胞团块较小.结论 靶向AKT1、PIK3R1的RNAi技术可以序列特异性地抑制PIK3R1、AKT1表达,在体外明显抑制SGC7901细胞的侵袭能力.     

shRNA靶向抑制COX-2基因表达对胃癌细胞SGC-7901增殖的影响

目的 探讨环氧化酶-2(COX-2)短发夹RNA(shRNA)对胃癌细胞SGC-7901的生长和细胞周期的影响.方法 构建COX-2基因的特异性小RNA干扰质粒,构建靶向抑制COX-2基因的重组表达载体pshRNA-COX-2.实验分3组:未转染胃癌细胞组,阴性对照HK组,pshRNA-COX-2转染组.用质脂体lipofectamine~(TM) 2000转染胃癌细胞SGC-7901,RT-PCR和Western blot分别检测COX-2基因mRNA和蛋白表达情况,流式细胞术检测细胞周期,细胞计数检测细胞的生长变化.结果 与阴性对照组相比,pshRNA-COX-2重组表达载体抑制COX-2 mRNA及蛋白表达的抑制率分别为70.1%和43.2%;G_0～G_1期细胞由61.5%上升至70.2%,S期细胞由27.3%下降至21.7%;细胞生长明显减慢.结论 pshRNA-COX-2重组表达载体能显著抑制COX-2的表达,从而抑制胃癌细胞生长.     

蛋白激酶B siRNA 转染对胃癌SGC-7901细胞生长增殖的影响

目的:探讨蛋白激酶B（PKB）基因沉默对人胃癌SGC-7901细胞增殖的作用。 方法:应用基因转染技术将蛋白激酶B家族的Akt2 siRNA转染至胃癌细胞中，采用Western blot、琼脂糖凝胶电泳、流式细胞术MTT等方法观察和检测Akt2 siRNA转染后对转染组细胞生长和增殖的影响。结果:与对照组比较，转染Akt2 siRNA组的胃癌SGC-7901细胞生长、增殖速度明显减缓（P＜0.01）， Akt2 siRNA组较对照组细胞的Akt2蛋白表达水平显著下调（P＜0.01）;转染组出现梯状凋亡条带;细胞周期显示转染组细胞聚集在G2/M期。 结论:应用RNAi使蛋白激酶B基因沉默能使人胃癌SGC－7901细胞的生长受到抑制，胃癌细胞增殖速度减缓，其作用机理可能通过诱导细胞凋亡和抑制PI3/K信号传导通路而实现。Akt2有望成为胃癌基因治疗的新靶点。     

靶向人肝素酶短发卡状RNA表达载体的构建及其沉默作用

目的 构建靶向人肝素酶(HPSE)的短发卡状RNA(shRNA)表达载体,探讨其基因沉默作用.方法 根据人肝素酶mRNA序列设计shRNA,合成两条互补的寡核苷酸链,退火后连接入pGenesil-1载体,转化扩增后进行测序鉴定;重组质粒在阳离子脂质体Lipofectamine 2000的介导下,瞬时转染人胃癌细胞株SGC-7901、人前列腺癌细胞株PC-3、人膀胱癌细胞株EJ,每种细胞分别设空白对照组、空载体pGenesil-Negative转染组、pGenesil-HPSE shRNA转染组3组,逆转录-聚合酶链反应(RT-PCR)及Western blot法检测各种细胞中肝素酶基因表达水平,黏附实验和transwell小室实验检测癌细胞游走、侵袭能力.结果 所构建的shRNA载体插入基因片段与设计序列完全匹配,载体构建成功;重组质粒转染SGC-7901、PC-3、EJ细胞后,肝素酶mRNA表达分别下调78.6%(P<0.01)、82.6%(P<0.01)、81.9%(P<0.01),肝素酶蛋白表达分别下调76.4%(P<0.01)、85.9%(P<0.01)、83.3%(P<0.01),细胞黏附能力分别下降37.7%(P<0.01)、44.6%(P<0.01)、43.8%(P<0.01),细胞侵袭能力分别下降66.8%(P<0.01)、76.6%(P<0.01)、80.5%(P<0.01).结论 成功构建了靶向人肝素酶的shRNA表达载体,沉默肝素酶基因表达后,能抑制多种癌细胞的游走和侵袭能力.     

基因特异性短发卡RNA抑制缺氧再给氧人脐静脉内皮细胞的转化生长因子-β1过表达

目的 构建针对人转化生长因子(TGF)-β1基因的短发卡RNA(shRNA)表达载体并检测其对缺氧再给氧(A/R)损伤后的人脐静脉内皮细胞(HUVEC)株的TGF-β1基因表达的影响.方法 设计、合成并构建针对TGF-β1 mRNA的shRNA表达载体,选择效果好者转染HUVEC株,再进行A/R试验,用免疫组织化学和逆转录-聚合酶链反应(RT-PCR)检测TGF.β1蛋白质和mRNA的表达.结果 优选出的表达载体pT12抑制率达(46.4±3.7)%.接受了MR的T、H、L及C组,TGF-Bβ1蛋白的A值分别为0.252±0.032、0.385±0.037、0.406±0.057及0.419±0.041,高于N组(0.103±0.008,P<0.01),转染r pT12的T组A值又显著低于H、L和C组(P<0.01).A/R试验后T、H、L及c组的TGF-β1 mRNA转录量也多于N组(P<0.05),T组的TGF-β1 mRNA转录量低于作为对照组的H组、L及C组(P<0.05).结论 TGF-β1基凶特异性shRNA表达载体能有效地抑制A/R损伤导致的HUVEC株TGF-β1基因过度表达.     

重组人生长激素对不同生长激素受体表达人胃癌细胞的作用

目的 观察重组人生长激素(rhGH)在体外对生长激素受体(GHR)表达不同的人胃癌细胞株增殖及胰岛素样生长因子-1(IGF-1)表达的影响.方法 选取GHR高表达细胞株SGC-7901和低表达细胞株MKN-45.分组:空白对照组、rhGH组1～3(分别予50、150、250 μg/L rhGH).噻唑蓝(MTT)比色法及流式细胞术分析rhGH对胃癌细胞株增殖的影响,酶联免疫吸附试验(ELISA)法分析细胞上清液的IGF-1蛋白浓度.结果 SGC-7901细胞株rhGH组1～3和空白对照组的生长率分别为126.5%、128.7%、128.0%、100.0%;增殖指数(PI)分别为52.49±0.20、54.27±0.45、57.13±0.21、48.37±0.42;IGF-1浓度分别为228.67±17.01、226.88±30.31、229.03±30.31、200.46±21.33;与空白对照组比较,各浓度rhGH组细胞显著增殖,IGF-1浓度显著升高(P均<0.05),各浓度组间差异无统计学意义(P>0.05).rhGH干预对MKN-45细胞株的生长增殖和IGF-1浓度无明显影响(P>0.05),各浓度组间差异无统计学意义(P>0.05).结论 rhGH在体外促进高表达GHR的胃癌细胞增殖及表达IGF-1,对低表达GHR的胃癌细胞增殖和IGF-1表达无明显影响.     

靶向沉默SPHK1基因诱导人胃癌SGC-7901细胞凋亡

目的探讨用小干扰核酸（SmallinterferingRNA,siRNA）靶向沉默神经鞘氨酸激酶1（sDhingosinekinasel,SPHKl）基因敲除后对胃癌SGC-7901细胞凋亡的影响。方法人工化学合成SPHKlsiRNA和对照siRNA,分别转染胃癌SGC-7901细胞。Westernblot法检测SPHKl、Bcl-2和Bax蛋白的表达;流式细胞术（FlowCytometry,FCM）检测细胞凋亡的变化。结果SPHKlsiRNA转染胃癌SGC-7901细胞后,SPHKl蛋白表达明显下调;与对照组相比,SPHKlsiRNA转染组Bcl一2蛋白表达下调了55％（P〈0．01）,Bax蛋白表达差异无统计学意义,Bcl-2／Bax比值下调54％（P〈0．05）;SPHKlsiRNA转染组早期凋亡细胞明显增多（P〈0．01）,晚期凋亡细胞无明显变化。结论SPHKl特异性siRNA可阻断SGC-7901细胞SPHKl蛋白的表达,并通过影响Bcl-2通路诱导细胞凋亡。     

短发夹RNA沉默血红素氧合酶-1基因表达对胃癌细胞系SGC-7901生物学行为的影响

目的 观察血红素氧合酶-1(HO-1)基因沉默对胃癌细胞系SGC-7901生长、增殖的影响.方法 构建靶向HO-1的短发夹RNA(shRNA-HO-1)干扰质粒转染人胃癌细胞系SGC-7901,逆转录-聚合酶链反应(RT-PCR)、细胞免疫化学分别在mRNA、蛋白质水平检测抑制效果.流式细胞仪和噻唑蓝(MTT)检测HO-1基因沉默后细胞的细胞周期和生长情况.结果 shRNA-HO-1在mRNA、蛋白质水平高效特异地抑制了细胞SGC-7901中HO-1的表达(抑制率分别为62.4%、67.6%);较对照质粒组,HO-1的表达被抑制后,G0/G1期细胞百分比明显减少(52.025±1.638比67.525±1.938,P<0.05),细胞生长受抑制[2.036±0.072比2.783±0.067(72 h A值),P<0.05].结论 shRNA-HO-1可有效抑制胃癌细胞中HO-1的表达;HO-1的表达减少抑制肿瘤细胞生长.