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.     

2.117肠易激综合征病人5-羟色胺转运体基因多态性分析

目的探讨5-羟色胺转运体(SERT)基因多态性在肠易激综合征中的意义. 方法肠易激综合征(IBS)81例,其中便秘型IBS(C-IBS)30例,腹泻型IBS(D-IBS)32例,交替型IBS(A-IBS)19例,对照组48例.从肠黏膜组织或外周静脉血提取DNA,采用PCR技术对SERT基因的2个多态性位点进行研究.     

经内镜活检对消化道肿瘤MDR1和MRP表达的研究

目的 :探讨消化道不同部位实体肿瘤多药耐药基因 (MDR1)和多药耐药基因相关蛋白 (MRP)的表达情况。方法 :应用RT_PCR反应技术 ,测定71例未进行任何化疗的初次内镜检查患者消化道肿瘤的MDR1和MRP。结果 :MDR1阳性表达率为87 3 % (62/71) ,其中食管癌100 % (12/12)表达 ,胃癌82 5 % (33/40)表达 ,结肠癌89 5 %(17/19)表达。3者比较无显著差异 (χ2=2.662,P>0.05)。MRP阳性表达率为43 7 % (31/71) ,其中食管癌25 0 % (3/12)表达 ,胃癌43 2 % (16/37 ,3例RNA降解 )表达 ,结肠癌66 7 % (12/18 ,1例RNA降解 )表达。3处比较无显著差异 (χ2=5.332,P>0.05)。胃内肿瘤在贲门胃底、胃体、胃角和胃窦随着发生部位的下移 ,MDR1阳性表达率逐渐升高 (50 %～100 %) ,但尚无显著性差异 (χ2=7.754,P>0.05) ,而胃内MRP不同部位的阳性表达不同 ,分别为5/6,1/9,2/10和8/15 ,组间比较有显著性差异 (χ2=9.902,P<0.05) ,其中贲门胃底具有高表达。结论 :(1)未经放化疗的消化道肿瘤MDR1具有高度表达。(2)MRP与MDR1表达并不一致。(3)胃内不同部位肿瘤MDR1表达与肿瘤发生的部位无关。MRP的表达则与肿瘤发生的部位有关。     

腺病毒介导的靶向P85和Akt1短发夹RNA对人胃腺癌细胞生长抑制作用的研究

目的 构建靶向P85和蛋白激酶B1(PKB1/Akt1)的短发夹RNA(shRNA)腺病毒载体,研究其对人胃腺癌细胞SGC-7901生长的抑制效果.方法 构建腺病毒载体rAd5-P+A,体外转染SGC-7901细胞后,以实时定量PCR和Western blot分别检测P85和Akt1的mRNA和蛋白质的表达.以噻唑蓝比色分析法(MTT法)和流式细胞法评价转染后胃癌细胞的增殖活性.构建裸鼠皮下荷瘤模型进一步观察rAd5-P+A对SGC-7901细胞生长的抑制效果,并应用原位末端标记技术(TUNEL法)检测肿瘤细胞的凋亡情况.结果 成功构建的rAd5-P+A重组腺病毒载体转染SGC-7901细胞后可显著抑制p85和Akt1的mRNA表达,而P85和Aktl蛋白表达量在转染48 h、72 h后分别下调57.5%、63.7%和67.8%、75.6%,与空白对照组和通用腺病毒对照(rAd5-HK)组相比,差异具有统计学意义(P=0.005,P=0.003).与空白对照组和rAd5-HK组相比,SGC-7901细胞的增殖活性在rAd5-P+A转染后第2天明显下降(P<0.001),且rAd5-P+A转染组进入S期的细胞数减少了5.9%～7.1%,而进入G0/G1期的细胞增加了12.1%～13.7%.裸鼠皮下荷瘤模型治疗实验也显示,rAd5-P+A可抑制胃癌细胞的生长,诱导细胞的凋亡.结论 腺病毒介导的靶向P85和Akt1的shRNA可抑制人胃腺癌细胞的生长,这可能为胃腺癌靶向性联合基因治疗提供新的策略.     

甲基化调节miR-200b的表达及其对胃癌MGC-803细胞增殖侵袭能力的影响

目的 体外研究基因mi R-200b发生甲基化的不同程度及其表达量的差异对胃癌细胞MGC-803增殖、侵袭、凋亡及对上皮间质转化的影响。方法 以正常胃黏膜上皮细胞GES-1、胃癌细胞MGC-803为研究对象,提取细胞总RNA,逆转录后进行实时荧光定量PCR检测2种细胞系中mi R-200b的表达量;亚硫酸氢钠修饰PCR(BSP)法检测mi R-200b启动子区甲基化水平。用不同浓度5-氮杂胞苷(5′-Aza-Cd R)药物处理MGC-803细胞72 h后,同法检测mi R-200b的表达量及启动子区甲基化水平变化。根据上述实验结果选择合适的药物作用浓度和时间即(10μmol/L,72 h)作为处理组。通过Transwell侵袭实验观测细胞侵袭能力;流式细胞技术检测细胞周期及凋亡的分布情况;Western-blot检测上皮间质转化(EMT)相关指标E-cadherin、N-cadherin、ZEB1、Slug、基质金属蛋白酶(MMP)9等的表达水平。结果 mi R-200b在GES-1中表达量较MGC-803高(P=0.022)。启动子区甲基化水平则与之相反(P=0.034)。药物不同浓度作用后的MGC-803细胞系mi R-200b表达量有随浓度升高而升高的趋势,作用浓度为10μmol/L时mi R-200b启动子区甲基化水平较对照组低(P=0.043)。与对照组相比,处理组细胞晚期凋亡数增多;G0/G1期细胞数显著增多,S期显著减少;穿过Transwell小室细胞数显著减少;Western-blot实验表明E-cad-herin表达量升高,N-cadherin、ZEB1、Slug、MMP9表达量降低。结论 mi R-200b启动子区甲基化程度的不同可以影响其表达量,而mi R-200b表达量的异常又与胃癌MGC-803细胞增殖、侵袭能力密切相关。     

胃镜检查及其术前用药对心电图的影响

胃镜检查及其术前用药对心电图的影响张庆瑜，吴琳，黄侠，杜宝恒，黄象谦本文通过胃镜操作过程中监测心电图的变化，了解心电图变化的频发部位，探讨术前用药对心脏的影响，以期有效预防心脏并发症，提高诊疗效果。材料与方法选取９２例无心肺疾患史的胃镜受检者，随机分...     

组织蛋白酶B在大肠肿瘤中的表达与临床应用

组织蛋白酶B(CB)是存在于人体正常组织的一种半胱氨酸蛋白酶,与肿瘤的侵袭和转移密切相关。新的证据认为CB在癌前状态中同样表达升高,为大肠癌的早期诊断和治疗开辟了新的途径。     

Akt1和Akt2基因转染对人胃黏膜上皮细胞GES-1侵袭和迁移的影响

目的:观察蛋白激酶B1(Akt1)和蛋白激酶B2(Akt2)基因转染人胃黏膜上皮细胞系GES-1后对其侵袭和迁移的影响。方法:采用脂质体法将分别含Akt1和Akt2基因全长cDNA序列的p-LXSN-Akt1(Akt1组)和p-LXSN-Akt2(Akt2组)质粒转染到GES-1细胞,以转染p-LXSN空载质粒的细胞为空载组,以正常未转染细胞为对照组,并经抗生素G418筛选抗性克隆,蛋白印迹(Westernblot)鉴定各组细胞Akt1、Akt2及基质金属蛋白酶(MMP)-2的蛋白表达水平;Transwell法分析转染后细胞的侵袭能力;用免疫荧光法观察转染后GES-1细胞丝状肌动蛋白(F-actin)的变化。结果:Akt1组和Akt2组均获得了稳定表达Akt1和Akt2基因的GES-1细胞模型;2组MMP-2蛋白表达均高于空载组和对照组(P<0.01)。Transwell法显示Akt1和Akt2组GES-1细胞侵袭能力均明显增加(P<0.01)。免疫荧光发现Akt1和Akt2组F-actin的聚集均增加。结论:Akt1、Akt2基因均可以通过增加细胞侵袭力和迁移力,导致GES-1细胞恶性转化。     

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

Objective To observe the inhibitory effects on the invasion of gastric adenocarcinoma SGC7901 cells by small hairpin RNA targeting PIK3R1 and AKT1 in vitro. Methods The recombinant adenovirus vector plasmid expression vector which contained PIK3R1 and AKT1 shRNA was transfected into SGC7901 cells. Real time PCR and Western blot were used to detect the expression of P1K3R1 and AKT1. ELESA was used to measure the change in the MMP-2 and MMP-9 expression. The invasion ability of the tumor ceils was examined by Scarification, Transwell and 3-dimensional matrigel matrix tests. Re-sults rAdS-A-P mediated shRNA targeting PIK3R1 ,and AKT1 dramatically down-regulated their expres-sion in SGC7901 cells. MMP-2 and MMP-9 were downregulated,and TIMP-2 was upregulated. The extra-cellular levels of MMP-2 and MMP-9 were decreased. Scarification test indicated that the invision ability was decreased obviously. Transwell showed that the number of cells invading through the matrigel in con-trol. nonsense sequence, and rAd5-A-P transfection groups was 105.0±4.0,102.5±6.4, and 67.0±3.9 respectively. In the rAdS-A-P transfection group, cells formed only small aggregates as compared with the control and nonsense sequence groups in 3-dimensional matrigel matrix growth test. Conclusion ShRNA targeting PIK3R1 ,and AKTI downregulated significantly their expression in a sequence-specific manner, and inhibited the invasion of gastric adenocarcinoma SGC7901 cells in vitro.     

靶向蛋白激酶B1、环氧合酶2和磷脂酰肌醇-3-激酶调节亚基1 shRNA腺病毒表达载体的构建及其抗胃腺癌增殖作用的研究

-C-A-P转染组细胞贴壁生长能力明显减低,体内实验中肿瘤生长也明显减慢(P<0.01).结论 靶向COX-2,AKT1和PIK3R1的shRNA技术可以序列特异性地抑制SGC7901细胞的COX-2,AKT1和PIK3R1表达,在体内外对SGC7901细胞增殖产生明显抑制作用.