靶向Notch1基因siRNA真核表达载体的构建与鉴定

目的：构建并鉴定靶向Notch1基因的siRNA真核表达载体。方法：根据Notch1基因cDNA序列，设计靶向目的基因的3个siRNA靶序列，将其插入U6启动子下游，克隆到真核表达载体pGsensil中，并进行酶切鉴定和DNA测序鉴定。采用脂质体介导法将构建好的3个siRNA表达载体分别转染T24人膀胱癌细胞中，荧光下观测转染效率。RT-PCR检测Notch1基因mRNA在转染前后的表达。结果：转染48h后3个siRNA表达载体均不同程度的抑制了Notch1 mRNA的表达。酶切鉴定及DNA测序结果显示插入片断正确。结论：成功构建的靶向Notch1的siRNA真核表达载体，具有抑制Notch1基因mRNA表达的功能，可能为膀胱癌基因治疗研究提供一个新的方向。     

靶向Notchl基因siRNA真核表达载体的构建与鉴定

目的:构建并鉴定靶向Notchl基因的siRNA真核表达载体.方法:根据Notchl基因cDNA序列,设计靶向目的基因的3个siRNA靶序列,将其插入U6启动子下游,克隆到真核表达载体pGsensil中,并进行酶切鉴定和DNA测序鉴定.采用脂质体介导法将构建好的3个siRNA表达载体分别转染T24人膀胱癌细胞中,荧光下观测转染效率.RT-PCR检测Notchl基因mRNA在转染前后的表达.结果:转染48 h后3个siRNA表达载体均不同程度的抑制了Notchl mRNA的表达.酶切鉴定及DNA测序结果显示插入片断正确.结论:成功构建的靶向Notchl的siRNA真核表达载体,具有抑制Notchl基因mRNA表达的功能,可能为膀胱癌基因治疗研究提供一个新的方向.     

负载双片段survivin短发夹状RNA质粒载体的构建与鉴定

目的:设计构建负载双片段survivin短发夹状RNA(shRNA)的质粒载体,为进行前列腺癌(PCa)基因治疗的研究奠定基础。方法:分别设计2条靶向survivin基因的shRNA,克隆至质粒载体中,再分别提取质粒酶切后回收、连接,构建重组质粒,提取重组质粒酶切鉴定、测序分析。结果:负载不同单片段survivinshRNA的质粒载体分别用EcoRⅠ及SacⅠ酶切鉴定,证实设计的2条单片段survivinshRNA已经分别插入目的质粒中;基因重组后,重组质粒载体用BamHⅠ酶切鉴定,证实双片段survivinshRNA均已插入重组质粒载体中;测序结果证实重组质粒载体插入的双片段survivinshRNA序列均正确无误。结论:负载双片段survivinshRNA的RNA干扰重组质粒载体构建成功。     

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

目的 构建靶向性蛋白激酶B1(PKB1/Akt1)和环氧合酶-2(COX-2)的短发夹RNA(shRNA)腺病毒载体,观察其在人胃癌细胞株SGC-7901中的表达.方法 利用同源重组技术构建重组腺病毒载体pGSadeno-Akt1+COX-2(pGSadeno-A+C),经酶切及测序鉴定后转染人胚肾细胞HEK293包装成为重组rAd5-A+C腺病毒,并测定病毒的滴度.体外转染人胃癌细胞株SGC-7901后,Real-time PCR和蛋白印迹分别检测Akt1和COX-2 mRNA和蛋白质的表达.结果 成功构建rAd5-A+C重组腺病毒,测定包装的病毒滴度为1.0×1010pfu/ml.转染组Akt1和COX-2 mRNA表达明显下调,其△Ct值分别是(12.26±0.05)和(5.41±0.09),比rAd5-HK空载组(10.63±0.02)、(3.75±0.08)和对照组(10.57±0.02)、(3.73±0.08)增高(P<0.01),而空载组和对照组比较ΔCt值无明显变化(P>0.05).同样转染组Akt1和COX-2蛋白表达量与空载组和对照组比较分别下调70.5%和63.7%(P<0.01),空载组和对照组比较Akt1和COX-2蛋白表达差异无统计学意义(P>0.05).结论 靶向性Akt1和COX-2的shRNA腺病毒载体可以特异性抑制Akt1和COX-2的表达,可能成为胃癌靶向性Akt1和COX-2基因治疗的新策略.     

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.     

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.     

STAT3基因短发卡RNA表达质粒的构建及其对胃癌细胞生长和侵袭的抑制作用

目的 构建信号传导及转录活化因子3(STAT 3)基因短发夹RNA(shRNA)表达质粒,探讨STAT 3抑制后对胃癌MKN-45细胞增殖、凋亡和侵袭的影响.方法 根据STAT 3 Mrna 编码序列,设计RNA干扰靶点,构建STAT 3基因的特异性小RNA干扰质粒(psiRNA-H1/STAT 3),使用脂质体转染MKN-45细胞,实验分为对照组、psiRNA-H1转染组和psiRNA-H1/STAT 3转染组.通过RT-PCR和Western blot检测STAT 3特异性小RNA干扰基因对胃癌细胞STAT 3 Mrna和蛋白表达的影响; MTT比色法检测细胞的生长抑制率; 采用流式细胞术检测转染后对MKN-45细胞凋亡的影响; 采用Boyden小室侵袭实验检测转染后MKN-45细胞侵袭力的变化.结果 成功转染重组体的MKN-45细胞中STAT 3 Mrna和蛋白表达明显下降(P＜0.05).psiRNA-H1/STAT 3转染组各时相均明显抑制MKN-45细胞生长,且MKN-45细胞凋亡率为34.26%,相比对照组(3.75%)和psiRNA-H1转染组(4.43%)明显升高(P＜0.01).另外,psiRNA-H1/STAT 3转染组MKN-45细胞侵袭力较另外2组明显减弱(P＜0.01).结论 成功构建了针对STAT 3基因的shRNA表达载体,通过转染MKN-45细胞,在体外能有效抑制人胃癌细胞STAT 3 Mrna和蛋白表达,细胞增殖、侵袭能力减弱并且促进细胞凋亡,为STAT 3基因靶向治疗提供一定的实验依据.     

Ki67基因短发夹状RNA表达载体对肾癌细胞生长的抑制作用

目的研究针对Ki67基因的短发夹状RNA(shRNA)表达载体pSilencer-Ki67对人肾癌细胞生长的抑制作用。方法将pSilencer-Ki67转染人肾癌786-0细胞,24、48、72、120、144 h后分别采用逆转录-聚合酶链反应(RT-PCR)、Western blot技术检测Ki67 mRNA、蛋白表达,噻唑蓝(MTT)比色法检测细胞增殖,原位末端标记法检测细胞凋亡。结果pSilencer-Ki67转染后48 h 786-0细胞Ki67mRNA表达(34．1±1．7)%、蛋白表达(42．6±1．7)%降低最明显,与空质粒对照组[(97．7±0．9)%、(97．3±1．6)%]比较差异均有统计学意义(P＜0．01)。pSilencer-Ki67处理后48 h细胞增殖抑制率(76．7±3．2)%最高,72 h凋亡细胞阳性率(74．3±5．9)%最高,与空质粒对照组[(7．0±0．5)%、(8．4±1．1)%]比较差异有统计学意义(P＜0．01)。pSilencer-Ki67的这些作用可持续144 h。结论pSilencer-Ki67能有效、持续抑制人肾癌786-0细胞生长,有望成为肾癌基因治疗的有效工具。     

hTERT基因短发夹状RNA表达载体对肾癌细胞生长的抑制作用

目的观察针对hTERT基因的短发夹状RNA（shRNA）表达载体pSilencer-hTERT对人肾癌细胞及移植瘤生长的抑制作用。方法（1）pSilencer-hTERT转染人肾癌Kerr-3细胞，1、3、5、7d后逆转录-聚合酶链反应（RT-PCR）、蛋白印迹技术检测hTERTmRNA、蛋白表达，MTT法检测细胞增殖，原位末端标记法检测凋亡。（2）BALB／C-nu裸鼠接种Ketr-3细胞成瘤，瘤内分别注射pSilencer-hTERT（50μg）、空质粒pSilencer1．0-U6（50μg）及等体积（100μg）生理盐水，隔日1次，共7次。治疗结束后第3天处死小鼠，取瘤组织检测肿瘤体积，免疫组织化学染色检测hTERT表达。结果（1）pSilencer-hTERT转染3d后Ketr-3细胞hTERTmRNA表达（43．2±4．4）％、蛋白表达（42．6±5．6）％最低，细胞增殖抑制率（37．3±6．6）％、凋亡细胞阳性率（30．5±4．7）％最高，分别与空质粒对照组[（98．8±4．7）％、（98．0±3．7）％、（3．3±0．9）％、（10．4±2．4）％]比较，差异均有统计学意义（P〈0．01）。（2）pSilencer-hTERT处理组小鼠肿瘤体积（62．4±36．5）mm^3减小，hTERT表达率（65．7±4．7）％降低，分别与空质粒对照组（83．2±38．7）、（90．7±4．2）％比较，差异均有统计学意义（P〈0．05）。结论pSilencer-hTERT能有效、持续抑制人肾癌Ketr-3细胞及裸鼠肾癌移植瘤生长。     

RhoC基因真核表达载体的构建及其表达

目的 为探讨RhoC基因的功能以及其对肝癌的侵袭转移的影响提供实验模型。方法 用限制性内切酶酶切RhoC基因，定向克隆到pcDNA3.1上，利用脂质体将重组载体（pcDNA3.1-RhoC)空载体（pcDNA3.1)转染入HEPG2细胞，潮霉素选择培养，RT－PCR及免疫组化鉴定其稳定表达。结果 构建的真核表达载体pcDNA3.1-RhoC在HEPG2细胞中有稳定表达。结论 本实验为深入研究RhoC基因的功能以及其对肝癌的侵袭转移的影响提供了理想的体外实验模型。     

反义MBD1基因片段真核表达载体的构建及鉴定

目的构建反义MBD1基因片段真核表达载体,为研究MBD1基因功能提供工具.方法根据MBD1基因cDNA序列中编码序列,设计PCR引物,在引物5＇端分别添加Xba Ⅰ和Kpn Ⅰ酶切位点,将PCR片段反向插入真核表达载体pcDNA3.1（＋）的多克隆位点上,构建反义MBD1基因片段真核表达载体,并用PCR、酶切法和DNA测序鉴定.结果PCR鉴定得到322 bp特异性条带,双酶切得到327 bp目的基因片段和5.4kb载体片段,DNA测序说明插入片段序列是正确的.结论采用基因克隆技术,成功构建了反义MBD1基因片段真核表达载体,为研究MBD1基因在DNA甲基化和肿瘤发生中的功能提供了实验工具.     

EndoA基因真核表达载体构建及其对皮肤表皮细胞的作用

瘢痕组织是人体创伤修复过程中的一种自然产物。然而，胚胎皮肤却具有创伤后无瘢痕愈合的能力。近年来研究发现无瘢痕愈合是胚胎皮肤固有的特性。我们通过构建真核表达载体pcDNA3．1／EndoA,并转染小鼠皮肤表皮细胞，研究EndoA对成鼠皮肤表皮细胞的作用。