p50PIK基因重组腺病毒的构建及对Hela细胞的影响

目的 构建携带P50基因的重组腺病毒载体,观察其感染宫颈癌Hela细胞后对p-AKT(Thr308)的影响.方法 以PcDNA3.1-p50PIK质粒为模板,构建带有p50PIK基因的重组腺病毒质粒Ad-p50PIK-GFP,酶切鉴定后经PacI线性化后转染HEK293包装细胞,观察细胞内绿色荧光蛋白(GFP)表达情况,并进行3轮扩增,收获带有目的 片段的腺病毒.将重组腺病毒Ad-p50PIK-GFP感染Hela细胞并通过Western blot技术检测其对p-AKT的影响.结果 将Ad-p50PIK-GFP经Xho I和Kpn I双酶切鉴定和琼脂糖电泳,在1400bp附近有目的 条带,送测序结果与GeneBank报道的序列完全一致,表明重组腺病毒Ad-p50PIK-GFP构建成功;然后将其转染HEK293细胞,绿色荧光表明Ad-p50PIK-GFP在HEK293包装细胞中成功表达;用SDS-PAGE电泳方法检测p50对Akt磷酸化的影响,结果表明高表达蛋白p50明显促进AKT的磷酸化(Thr308).结论 成功构建重组腺病毒Ad-p50PIK,p50在宫颈癌Hela细胞株中的过表达可使p-AKT表达升高.

Abstract：

Objective To construct recombinant adenovirus carrying p50PIK gene and examine the effect on cervix cancer Hela cell lines after transfection with Ad-p50PIK. Methods p50PIK cDNA was amplified by polymerase chain reaction (PCR), with the template PcDNA3. 1-p50PIK, then cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrackCMV-p50 was linearized by PmeI, followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183, then identified by enzyme digestion.After linearized by PacI and transfection into HEK293 cells, recombinant adenovirus Ad-p50PIK were obtained in HEK293 cells then amplified by 3 circles. The green fluorescence in HEK293 cells was observed.Ad-p50PIK was transfected into Hela cells. The phosphorylation of Akt was detected by Western blotting.Results After double restriction enzyme digestion and agarose gel electrophoresis of The Ad-p50PIK-GFP,the 1400 bp purpose band was sequenced, Results was exactly the same with the sequence GeneBank had reported,indicating that the recombinant adenovirus Ad-p50PIK-GFP were successfully constructed; Then transfected into HEK293 cells, green fluorescence shows that Ad-p50PIK-GFP in HEK293 packaging cells successfully expressed; by SDS-PAGE electrophoresis was used to detect p50 on Akt phosphorylation, the Results show that high expression of p50 protein significantly increased AKT's Phosphorylated (Thr308).Conclusion Recombinant adenovirus Ad-p50PIK had been successfully constructed. Overexpression of p50PIK in Hela cell lines can promote phosphorylation of AKT.     

CDK1 siRNA干扰在Taxol诱导细胞凋亡中的作用

目的 研究转染细胞周期依赖性蛋白激酶1(cyclin-dependent kinase 1,CDK1)siRNA、以及转染后进行凋亡刺激对细胞周期和凋亡的影响,探讨CDK1在细胞凋亡中的确切作用,揭示细胞周期与细胞凋亡协调的分子机制.方法 以人宫颈癌细胞株HeLa细胞为研究对象,脂质体转染CDK1 siRNA,转染后48 h加紫杉醇(Taxol) (20 μg/ml)刺激凋亡,Western印迹检测CDK1和抗凋亡蛋白BCL2表达,AnnexinV/PI法检测细胞的凋亡,流式细胞仪分析DNA含量检测细胞周期.结果 转染CDK1 siRNA后,CDK1蛋白的表达下降,细胞周期G2/M期比例增加,细胞凋亡率与对照相比没有明显升高.只加Taxol刺激12 h后细胞凋亡率增加并伴有S期和G2/M期比例增加. 转染CDK1 siRNA后再用Taxol刺激,其细胞凋亡率没有明显改变,G2/M期阻滞效应也没有叠加.BCL2蛋白只在加Taxol刺激组表达下降,与CDK1表达减少没有相关性.结论 siRNA沉默导致的CDK1表达降低只导致细胞周期G2/M期阻滞,没有引起细胞凋亡;CDK1的表达降低对紫杉醇所诱导的细胞周期阻滞和细胞凋亡效应没有明显影响.     

高表达蛋白hSav1对人胚肾HEK293增殖能力的影响

Objective To elucidate the effects of human Salvador 1 (hSav1 ) on cell proliferation of human embryonic kidney cell line HEK293. Methods The plasmid CFP-N1-hSav1 was constructed and transfected into HEK293 cells with lipofectamine 2000. The transfection efficiency was detected by fluorescent microscopy. The effects of hSav1 on cell proliferation were measured by MTT and BrdU incorporation. Results The transfection efficiency was about 70% -80%. The MTT results showed that the inhibition rate of cell proliferation in transfected group at the 12th, 24th, 36th, and 48th h was 2% , 5% , 15% , and 23% , respectively;while that in the control group was 2% , 3% , 2% , and 2% respectively. The BrdU incorporation revealed that the BrdU incorporation rate in transfected group (12. 9 ±5. 3)% was significantly lower than in control group (27.3±3.8)% (P<0.05). Conclusion hSav1 is a newly identified protein that can cause cell proliferation inhibition in HEK293 cells.     

c-myc通过提高结肠癌细胞LoVo的HIF-1α表达促进血管生成

目的 研究癌基因c-myc对人结肠癌细胞LoVo中缺氧诱导因子-1α(HIF-1α)的影响及其对血管生成的作用.方法 构建高表达c-myc的质粒pcDNA3.1-c-myc;将构建好的pcDNA3.1-c-myc质粒转染入LoVo细胞系中,通过Western blot检测c-myc在LoVo细胞中的表达情况.应用real-time PCR和Western blot检测常氧和乏氧状态下LoVo细胞系中HIF-1α、血管内皮生长因子(VEGF)的mRNA水平及HIF-1α的蛋白水平.应用条件培养液培养人脐静脉内皮细胞(HUVECs),观察其形成血管的能力.结果 成功构建pcDNA3.1-c-myc质粒;常氧、乏氧状态下转染pcD-NA3.1-c-myc质粒后,LoVo细胞系中HIF-1α的mRNA水平无变化,VEGF的mRNA水平明显升高,HIF-1α蛋白水平明显增高.高表达c-myc的LoVo细胞上清液培养HUVECs细胞后能增强其成管能力.结论 在LoVo细胞系中高表达c-myc,可以促进细胞HIF-1α和其下游的VEGF表达,从而促进血管生成.     

细胞增殖对DNA损伤敏感度的影响

目的 观察细胞增殖状态对DNA损伤敏感度的影响.方法 相同时间和强度的紫外线(uV)作用于过以及未经过植物m凝素(PHA)刺激的外周血淋巴细胞(PBLs),荧光标记的磷酸化H2AX组蛋白(γ-H2AX)抗体特异性标记细胞核内DNA双链断裂(DSBs)处的,y-H2AX,然后用流式细胞仪定量检测并分析DNA损伤,用Annexin V-FITC/PI检测细胞凋亡.结果 经PHA刺激PBLs细胞进人增殖状态,UV可引起DNA双链断裂,增殖期的PBLs细胞DNA损伤程度明显大于静止期PBLs细胞.结论 受到相同打击后,处于增殖期的淋巴细胞DNA损伤较静止期更为明显.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

胃肠机械吻合与传统吻合术后并发症的比较(附932例报告)

目的　比较胃肠机械吻合与传统手工吻合对术后并发症的影响 ,探讨机械吻合的安全性问题。方法　对我院 1999年 1月至 2 0 0 3年 12月期间收治的 932例行BillrothⅡ式胃肠吻合术患者的资料进行回顾性分析 ,了解其术后并发症的发生情况。结果　行机械吻合的 392例中出现术后并发症 8例 (吻合口漏 7例 ,梗阻 1例 ) ,其发生率为 2 .0 4 % ;而使用传统手工吻合的 5 4 0例中出现术后并发症 4 4例 (吻合口漏 2 8例 ,出血 4例 ,梗阻12例 ) ,其发生率为 8.15 % ,明显高于前者 (P<0 .0 1)。结论　胃肠机械吻合较传统手工吻合更为安全。     

0.45%乳酸钠溶液在十二指肠残端瘘中的应用分析

十二指肠残端瘘目前国内应用较多而且效果较佳的治疗方式是:持续负压吸引 抗感染 营养 生长抑素的综合治疗.我们的研究是在此方案基础上加用0.45%乳酸钠持续冲洗,并与不使用0.45%乳酸钠溶液持续冲洗组相比较,观察其瘘后并发症的演变情况,现报告如下.     

经胃壁造口双腔管进行胃肠减压和空肠营养的设计与临床应用

胃肠道手术后,予以有效的胃肠减压和肠内营养有利于手术后的恢复。传统方法是经双侧鼻腔置入胃肠减压管和空肠营养管,或者经鼻置入胃肠减压管、经小肠造瘘置入肠内营养管,以减轻吻合口张力和供给营养犤1犦。但经鼻置管给患者带来诸多不适和增加感染机会。我们设计了一种集胃肠减压和肠内营养功能于一身的双腔管(硅胶管)应用于临床,现将应用体会报道如下。材料制作及置管方法该双腔管中减压管长80cm,内径8m m;营养管长120cm,内径2m m,见图1A。置管方法:术式不同置管位置不同:(1)近端胃切除胃食管吻合。于胃前壁置入联合双腔管,减压管端置入…     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.