下调X连锁凋亡抑制蛋白基因表达增强结肠癌细胞对5-氟尿嘧啶敏感性的研究

目的 观察下调X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)基因表达后结肠癌细胞对5-氟尿嘧啶(5-Fu)敏感性的变化.方法 采用脂质体包裹方法将携带靶向干扰XIAP序列的表达载体转染人结肠癌细胞HCT-8和HCT116,观察结肠癌细胞生长活性的变化;应用5-Fu后,观察结肠癌细胞对5-Fu敏感性的变化,并应用蛋白印迹方法检测结肠癌细胞内凋亡相关因子caspase-3的活性变化. 结果抑制XIAP基因表达后,结肠癌细胞HCT-8和HCT116的生长活性受到有效抑制;结肠癌细胞HCT-8对5-Fu的耐药性得到逆转(P＜0.01),HCT116对5-Fu的敏感性得到明显提高(P＜0.05);结肠癌细胞内caspase-3的表达活性提高,5-Fu诱导细胞凋亡的活性得到增强. 结论XIAP的表达是结肠癌细胞HCT-8和HCT116对5-Fu耐药的一个重要机制;抑制XIAP基因表达后能够增强结肠癌细胞HCT-8和HCT116对5-Fu化疗的敏感性.

Abstract：

Objective To investigate sensitivity of colon cancer cells to 5-fluorouracil after downregulation of XIAP gene expression. Method Colon cancer cells HCT-8 and HCT116 were transfected with a short hairpin RNA targeted to XIAP by liposome, cells viability were examined.5-fluorouracil was applied into two kinds of colon cancer cells. Tumor cells sensitiviy to chemotherapeutic drug was evaluated. Caspase-3 activity in tumor cells was examined by Western blot. Result After downregulation of XIAP expression, cell growing viability of these two kinds of colon cancer cells was restricted, HCT-8 resistance to 5-fluorouracil was reversed ( P ＜ 0. 01 ), HCT116 sensitivity to 5-fluorouracil was enhanced (P ＜ 0.05), caspase-3 expression in colon cancer cells was highly activated, apoptosis inducing activity of 5-fluorouracil was increased significantly. Conclusions XIAP expression was a important mechanism in colon cancer cells HCT-8 and HCT 116 resistant to 5-fluorouracil, sensitivity to 5-fluorouracil of HCT-8 and HCT-116 was increased by downregulation of XIAP expression.     

短发夹RNA干扰表达质粒逆转人大肠癌细胞LOVO／5-Fu多药耐药的研究

目的：探讨短发夹RNA（shRNA）沉默多药耐药MDR）基因对大肠癌多药耐药细胞株LO-VO/5-Fu生长的影响。方法：构建靶向MDR1的shRNA干扰质粒转染人大肠癌多药耐药细胞株LO-VO/5-Fu,MTT法检测各组细胞对5-Fu的敏感性,流式细胞术检测细胞周期变化、凋亡情况及P-糖蛋白（P-gp）的表达,Realtime-PCR检测各组细胞MDR1 mRNA表达的变化,Western blot检测各组细胞P-gp表达的变化。结果：与对照组质粒组和未转染组相比,转染含shRNA干扰质粒细胞的实验组IC50明显降低,为（2.304±0.232）μmol/L,且差异有统计学意义（P〈0.05）,敏感性的相对逆转率为73.8%;凋亡率明显升高为（5.767±0.694）%（P〈0.05）;MDR1 mRNA表达明显下调（P〈0.05）;P-gp的表达水平降低（P〈0.05）。结论：靶向MDR1的shRNA干扰质粒有效抑制了MDR1的表达,使P-gp的表达降低,从而增强了LOVO/5-Fu细胞对5-Fu的敏感性,有可能为临床上克服大肠癌化疗过程中出现的耐药提供新的作用靶点和治疗途径。     

RNA干扰逆转人胃癌细胞多药耐药的研究

目的：研究RNA干扰沉默多药耐药（multidrug resistance,MDR）基因对胃癌多药耐药细胞株BGC-823/5-Fu生长的影响。方法：构建靶向MDR1的shRNA干扰质粒转染人胃癌多药耐药细胞株BGC-823/5-FU,噻唑蓝（MTT）法检测耐药细胞对5-Fu的敏感性,实时荧光定量PCR（Real-time-PCR）检测MDR1 mRNA表达的变化,Western blot检测各组细胞P-gp表达的变化,流式细胞术检测细胞周期变化、凋亡情况。结果：与RNAi-control组和normal组相比,RNAi-MDR1组细胞干扰质粒细胞的IC50明显降低,为2.104±0.242（P〈0.05）,敏感性的相对逆转率为76.8%;MDR1 mRNA表达明显下调（P〈0.05）;P-gp的表达水平降低（P〈0.05）;凋亡率明显升高至（5.757±0.684）%。结论：应用RNA干扰有效抑制了MDR1的表达,使P-gp的表达降低,增强了BGC-823/5-Fu细胞对5-Fu的敏感性,为寻找逆转胃癌细胞多药耐药有效方法奠定了基础。     

趋化因子2对乳腺癌细胞MCF-7趋化活性及趋化因子5mRNA表达的影响

目的 观察趋化因子2(CCL2)对MCF-7表达趋化因子5(CCL5)mRNA的影响,并观察CCL2对MCF-7趋化活性的影响.方法 使用不同浓度的CCL2作用于MCF-7细胞,通过实时荧光定量聚合酶链反应(RTFQ-PCR)测定不同时间CCL5 mRNA的表达,并使用趋化小室法检测CCL2作用下MCF-7趋化活性的改变.结果 当外源性CCL2浓度为200μg/L时,MCF-7中CCL5 mRNA相对表达量最大(15.22±2.32)(P＜0.01),随着作用时间的延长,CCL5 mRNA表达量增加;MCF-7的趋化活性与CCL2浓度呈正相关,当CCL2浓度为300μg/L时,穿膜细胞数最多(88.00±11.53)(P＜0.01);MCF-7的趋化活性与CCL2作用时间呈正相关,当CCL2作用时间为30 h时,穿膜细胞数最多(81.00±9.54)(P＜0.05).结论 加入外源性CCL2,MCF-7中CCL2 mRNA表达量增加,MCF-7趋化活性增强.

Abstract：

Objective To observe the effects of chemokine 2 (CCL2) on the expression of chemokine 5 ( CCL5) mRNA and chemotactic activity of MCF-7 cells. Methods MCF-7 cells were treated with different concentrations of CCL2, the expression of CCL5 mRNA was detected by using real-time fluorescence quantitative polymerase chain reaction ( RTFQ-PCR), and the chemotactic activity of MCF-7 was measured by using chemotaxis chamber method. Results When the concentration of exogenous CCL2 was 200 μg/L, the MCF-7 cells expressed the highest CCL5 mRNA (15. 22 ± 2. 3, P ＜0. 01). With the prolongation of CCL2 action time, the expression levels of CCL5 mRNA were increased. There was a positive correlation between the chemotactic activity of MCF-7 cells and the concentration of CCL2. When the concentration of exogenous CCL2 was 300 μg/L, the number of penetrating cells was the greatest (88.00 ±11. 53, P ＜0. 01). With the prolongation of CCL2 action time, the chemotactic activity of MCF-7 cells was enhanced. When the action time was 30 h, the number of penetrating cells was the greatest (81.00 ±9. 54, P ＜ 0.05 ). Conclusion Exogenous CCL2 could increase the expression of CCL5 mRNA and the chemotactic activity of MCF-7 cells.     

WWOX在胆管癌组织中的表达及其对胆管癌细胞生物学行为的影响

目的 研究抑癌基因WWOX表达对胆管癌RBE细胞生长的影响.方法 采用免疫组化方法检测2005年7月至2010年5月54例胆管癌组织、12例正常胆管组织中WWOX蛋白表达水平.将携有WWOX基因的真核表达载体转染胆管癌细胞系RBE细胞(RBE/WWOX组),筛选稳定转染的细胞并扩增培养,以转染空载质粒(RBE/con组)及未经转染(自然生长组)的RBE细胞作为对照.荧光定量RT-PCR和Western Blot法检测WWOX在RBE细胞中的表达情况;噻唑蓝实验检测转染前后各组细胞增殖活性;FCM法检测各组细胞的凋亡;JC-1染色法检测细胞线粒体膜电位;Transwell小室侵袭实验检测各组肿瘤细胞侵袭力;荧光定量RT-PCR和Western Blot法检测胆管癌细胞bcl-2、bax、FasL、caspase-3表达的变化.结果 WWOX在胆管癌中的表达低于正常胆管组织(P＜0.05),蛋白表达的缺失频率为40.7%.建立稳定表达WWOX基因的RBE/WWOX细胞株,mRNA及蛋白表达明显增加.转染后的RBE细胞噻唑蓝吸光度明显下降(P＜0.05).与自然生长组和RBE/con组比较,FCM显示RBE/WWOX组细胞的凋亡率明显增高(P＜0.01),JC-1显示转染后的线粒体膜电位下降(P＜0.01),侵袭实验显示转移至下室滤膜的细胞数明显减少(P＜0.01).荧光定量RT-PCR结果显示bcl-2 mRNA表达是自然生长组的0.12倍,bax、caspase-3 mRNA分别是自然生长组的4.72和2.57倍,FasL mRNA的表达无明显变化;Western Blot法检测发现bcl-2蛋白的表达降低,bax、caspase-3蛋白表达升高,FasL无明显变化.结论 抑癌基因WWOX通过诱导胆管癌细胞凋亡发挥抗肿瘤增殖的作用.

Abstract：

Objective To study the effects of anti-oncogene WWOX on cell growth of cholangiocarcinoma. Methods The expression of WWOX protein was detected with immunohistochemical method-SP in 54 patients with cholangiocarcinoma from July 2005 to May 2010 and 12 samples of normal bile duct tissues. The recombinant WWOX eukaryotic expression plasmid was introduced into RBE cells by liposome-mediated transfection and positive cell clones were selected and amplified. The mRNA and protein expressions in RBE cells stably transfected with WWOX were investigated by quantitative RT-PCR and Western Blot before and after transfection. Cell proliferation was tested by MTT, cell apoptosis was assessed by FCM, the alteration of mitochondria membrane potential (△Ψm) was detected by JC-1 staining method,cell invasion was determined by Transwell chamber assay. The expression change of bcl-2, bax, FasL,caspase-3 mRNA and protein was detected by quantitative RT-PCR and Western Blot. Results The expression of WWOX protein was significantly lower in cholangiocarcinoma than that in normal bile duct tissues and loss of WWOX protein expression was found in 40. 7% of cholangiocarcinoma specimens ( P ＜0.05). RBE cells with stable transfection of WWOX were established. Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western Blot demonstrated that WWOX protein expression was markedly increased. MTT showed that WWOX gene transfection significantly decreased the proliferation of RBE cells ( P ＜ 0. 05 ). FCM analysis showed that the apoptosis rate after transfection was significantly promoted [( 1.1 ± 0. 6 ) % vs. ( 1.7 ± 0. 5 ) % vs. ( 35.2 ± 4. 4 ) %, P ＜ 0. 01], JC-1 staining method indicated that the experimental group was loss of △Ψm [( 12. 6 ± 1.9 ) % vs. ( 13.6 ± 1.8 ) % vs.(48. 7 ± 2. 9 ) %, P ＜ 0. 01], transwell chamber assay showed that the number of transfected cells that passed the transwell membrane was significantly less than those of control groups ( 77 ± 6 vs. 72 ± 8 vs. 48 ±6, P ＜0. 01 ). Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased and the expression of bax, caspase-3 were significantly increased. There was no significant change in the expression of FasI. Conclusion WWOX exerts its antitumor effect against proliferation through inducing cell apoptosis in cholangiocarcinoma.     

短发卡RNA稳定沉默FOXM1对肝癌细胞体外生长的影响

目的 探讨短发夹RNA(short hairpin RNA,shRNA)表达载体稳定沉默叉头框M1(forkhead box M1,FOXM1)基因对人肝癌细胞体外生长的影响.方法 构建针对人类FOXM1mRNA的不同干扰靶点的4个shRNA表达载体,选择干扰效果最佳的表达载体和阴性对照质粒转染人肝癌细胞QGY-7703,用新霉素(G418)筛选稳定转染的克隆.用四甲基偶氮唑盐(MTT)比色法和平板克隆形成实验检测FOXM1基因沉默前后肝癌细胞体外生长能力的变化.用Annexin V-APC/PI双染法检测细胞凋亡.结果 不同人肝癌细胞株中普遍表达FOXM1蛋白.4个shRNA表达载体中,shRNA-1026表达载体的干扰效果最佳,对FOXM1 mRNA和蛋白表达水平的抑制率分别为38.5%和53.2%.用shRNA-1026稳定沉默FOXM1基因后,QGY-7703细胞增殖受到抑制,培养48、72和96 h后,沉默组的吸光值均显著低于对照组(分别t=10.830,3.578,5.734,均P＜0.05);沉默组的克隆形成能力较对照组显著降低(t=5.336,P＜0.05),而细胞凋亡较对照组显著增加(t=6.827.P＜0.05).结论 shRNA表达载体稳定沉默FOXM1基因抑制肝癌细胞的生长.

Abstract：

Objective To evaluate the effect of sustained silencing Forkhead box Ml (F0XM1) gene by short-hairpin RNA (shRNA) expression vector on cell growth of hepatocelluar carcinoma (HCC) in vitro.Methods Four shRNA expression vectors targeting different sequences of human F0XM1 mRNA were constructed.The expression vector with the best interfering effect and the negative control plasmid were used to transfect HCC cell line QGY-7703, stably transfected cell clones were selected by neomycin (G418).Cell growth was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation was assessed by clonogenic assay.Cell apoptosis was detected by double staining with APC conjugated Annexin V and PI.Results F0XM1 protein was detected with different levels in all these studied human cell lines.The expression vector shRNA-1026 exhibited excellent interference effect after transient transfection, which showed 38.5% and 53.2% reduction of FOXM1 mRNA and protein level respectively.The growth of QGY-7703 cells was inhibited after stable inhibition of FOXM1 expression by shRNA-1026, which was indicated by decreased absorbance value of the test group after culture for 48, 72 and 96 h compared to control group (t = 10.830,3.578 and 5.734 respectively, P ＜ 0.05).Stable inhibition of F0XM1 also led to reduced colony formation ( t = 5.336, P ＜ 0.05 ) and increased apoptosis of QGY-7703 cells in comparison to control cells (t = 6.827, P ＜ 0.05 ).Conclusions Stable silencing F0XM1 gene by shRNA suppresses the growth of HCC cells in vitro.     

THE EXPRESSION OF mdr-l,MRP AND LRP GENES IN GASTRIC CARCINOMA AND THEIR CLINICAL SIGNIFICANCE

Objective:To explore the expression of mdr-1, multidrug resistance asso-ciated protein(MRP)and lung resistance protein(LRP) genes in human gastric Gancer andtheir Glinical significance. Methods:The mdr-1 mRNA was assayed by RT-PCR, the MRPand LRP were detected by flow cytometry. Results:The positive rate of mdr-1 mRNA is44.4%(12/27), and the mean MRP and LRP expression rate is 39.1% and 29.9%, respec-tively. The mdr-1 mRNA, MRP or LRP expression is independent of patient histologic type,nodal involvement, and TNM stage. The mdr-1 mRNA expression in patients with serosa in-vasion was 30.0% (6/20),much lower than that without serosa invasion (85.7%).Con-clusion:The multidrug resistance cells are present in primary gastric carcinomas priorto chemotherapy and analysis of mdr-1 gene, MRP, LRP may have guiding significance inthe treatment of gastric carcinoma.     

短发夹RNA干扰质粒与粉防己碱对结肠癌耐药细胞多药耐药性的逆转作用

目的 比较短发夹RNA干扰质粒与粉防已碱对结肠癌LoVo/5-氟尿嘧啶(5-FU)耐药细胞多药耐药性(MDR)的逆转作用.方法 用粉防己碱和靶向MDR1的短发夹RNA干扰质粒分别对结肠癌LoVo/5-FU耐药细胞进行处理,实验分为对照组(空白对照)、粉防己碱组和转染组(短发夹RNA干扰质粒).应用MTT法检测细胞对5-FU的敏感性;流式细胞仪检测细胞周期变化、凋亡情况及P-糖蛋白的阳性表达率;RT-PCR法检测MDR1 mRNA的表达情况;Western blot检测P-糖蛋白的表达情况.多组间的比较采用单因素方差分析,两两比较采用SNK-q检验.结果 (1)细胞对5-FU的敏感性:对照组、粉防己碱组、转染组的药物半数抑制浓度( IC50)分别为(7.3±0.3)、(4.4±0.7)、(2.4±0.4) mmol/L,3组比较,差异有统计学意义(F =65.27,P＜0.05);粉防己碱组与转染组比较,差异有统计学意义(q=6.67,P＜0.05).(2)细胞周期的变化:对照组、粉防己碱组、转染组G1期和S期细胞所占比例分别为38.13％±3.75％、51.36％±2.76％、59.24％±4.31％和20.46％±2.23％、14.32％±1.91％、9.40％±1.65％,3组比较,差异有统计学意义(F =25.23,24.37,P＜0.05);粉防己碱组与转染组比较,差异有统计学意义(q=3.67,4.35,P＜0.05).(3)细胞凋亡情况:对照组、粉防己碱组、转染组细胞凋亡率分别为1.32％±0.47％、3.24％±0.26％、5.88％±0.44％,3组比较,差异有统计学意义(F=99.26,P＜0.05);粉防己碱组与转染组比较,差异有统计学意义(q=11.48,P＜O.05).(4)P-糖蛋白的表达:对照组、粉防己碱组、转染组细胞P-糖蛋白的阳性表达率分别为96.9％±2.3％、61.6％±4.9％、76.6％±3.6％,3组比较,差异有统计学意义(F =67.83,P＜0.05);粉防己碱组与转染组比较,差异有统计学意义(q=6.97,P＜0.05).(5)MDR1 mRNA表达情况:对照组、粉防己碱组、转染组细胞MDR1 mRNA的相对表达量分别为1462±161、570±85、233±81,3组比较,差异有统计学意义(F=90.59,P＜0.05);粉防己碱组与转染组比较,差异有统计学意义(q=5.12,P＜0.05).结论 粉防己碱和靶向MDR1的短发夹RNA干扰质粒均能逆转LoVo/5-FU耐药细胞的MDR,且后者逆转作用更强,逆转机制与抑制P-糖蛋白表达及下调MDR1 mRNA表达有关.     

5-FU对结肠癌转移灶抑制率与耐药相关因子表达关系的实验研究

目的探讨结肠癌原发灶及淋巴结转移灶对5-氟尿嘧啶(5-FU)的体外化疗药敏性及其与多种耐药因子表达的关系。方法对48例结肠癌原发灶、转移淋巴结肿瘤细胞进行体外化疗药敏性实验,并行P-gp,GST-π,p53,survivin和Bcl-2,Bax免疫组化染色。结果5-FU对淋巴结转移灶肿瘤细胞的抑制率高于原发灶(P0.05)。P-gp和Bax在肿瘤淋巴结转移灶中表达程度高于原发灶(均P0.05),Bcl-2在淋巴结转移灶中的表达明显下降(P0.05)。结肠癌原发灶中5-FU药物抑制率与P-gp的表达呈负相关(P0.05);转移淋巴结中药物抑制率与Bax表达呈正相关,与Bcl-2表达呈负相关(均P0.05)。结论影响结肠癌淋巴结转移灶中5-FU药敏性的主要耐药相关因子与原发灶不同,术后辅助化疗及逆转结肠癌的多药耐药性应针对淋巴结转移灶进行。     

β1整合素表达下调对人结肠癌细胞侵袭和耐药的影响

目的 观察β1整合素表达下调对人结肠癌HT-29细胞侵袭和耐药的影响.方法 反义寡核苷酸技术转染HT-29细胞,逆转录-聚合酶链反应(RT-PCR)检测β1整合素表达下凋.Transwell侵袭窒方法检测HT-29体外侵袭力.流式细胞术(FCM)和噻唑蓝(MTT)比色法检测不同剂量的化疗药物[5-氟尿嘧啶(5-Fu)]对各组凋亡率和生长抑制率的影响.结果 实验组HT-29细胞βl整合素mRNA表达显著降低,体外侵袭实验迁移的HT-29细胞数(59±12)明显减少(P＜0.05),在高浓度5-Fu(100、200 mg/L)时HT-29细胞的生长抑制率(36.97%和47.58%)和细胞凋亡率[(15.7l±1.46)%、(27.82±1.58)%]与对照组比较差异有统计学意义(P＜0.05).结论 β1整合素表达下调可降低结肠癌细胞侵袭及化疗药物耐药性.     

缺氧诱导因子-1α参与胃癌多药耐药的机制

目的 探讨缺氧诱导因子(HIF)-1α参与胃癌SGC7901/DDP细胞株多药耐药的机制.方法 采用化疗药物顺氯胺铂(顺铂,DDP)按浓度梯度递增法(0.02～1.00mg/L)诱导胃癌SGC7901细胞株,10个月后成功建立对顺铂稳定耐药并具有MDR、MRP4表型的胃癌SGC7901/DDP细胞株,应用HIF-1α通路抑制剂YC-1(75 μmol/L)抑制HIF-1α在敏感株SGC7901和耐药株SGC7901/DDP中的表达,通过噻唑蓝(MTT)比色法检测药物敏感性,流式细胞术分析细胞凋亡率,逆转录一聚合酶链反应(RT-PCR)和Western blot检测敏感株SGC7901和耐药株SGC7901/DDP中HIF-1α和耐药蛋白P-糖蛋白(P-gp)的表达变化.结果 YC-1抑制HIF-1α表达后,耐药株SGC7901/DDP的药物敏感性显著增加,对DDP的半数抑制浓度(IC50)由(52.175±6.163)mg/L降低为(11.078±3.645)mg/L(P＜0.01).耐药株SGC7901/DDP的细胞凋亡率由1.03%增加为14.26%(P＜0.01).耐药株SGC7901/DDP的P-gP在基因和蛋白表达水平均明显减低,分别由1.846±0.135和2.017±0.102下降至0.814±0.057和0.962 ±0.039(P＜0.01).结论 HIF-1α可通过调节P-gP表达及抗凋亡信号通路参与胃癌顺铂耐药,可成为逆转胃癌耐药的新的靶点.     

裸鼠人肝癌原位移植多药耐药模型的建立及其耐药机制的探讨

目的　在体内建立人肝癌裸小鼠原位移植多药耐药模型,并研究其耐药机理。方法采用人肝癌（BEL-7402）裸小鼠（4～6周龄）原位移植,给予阿霉素(1.5mg/kg体重,每周1次)腹腔注射诱导耐药（共8周）,经四唑蓝(MTT)快速比色法检测原代培养的耐药细胞对抗癌药的敏感性,以流式细胞仪检测癌细胞表面mdr1基因产物P-糖蛋白（P-gp）的表达,并用罗丹明试验观察该蛋白功能。结果　移植瘤组织形态及生物学方面符合人肝癌特征,实验组肝癌细胞表面P-gp表达为(75.45±5.67)%,而对照组表达仅(4.25±1.28)%,差异有非常显著性（P＜0.01）,对阿霉素的耐药倍数提高了16.67倍,对羟基喜树碱具有交叉耐受性（13.67倍）。耐药细胞表面P-gp有较强的药物外排功能。结论　阿霉素较易诱导原位移植于裸小鼠的人肝癌多药耐药性的产生,其耐药倍数与临床肝细胞癌相似。该模型的建立对研究肝癌多药耐药的产生机制以及逆转其多药耐药性具有重要价值。     

携带mda-7基因的复制缺陷性腺病毒联合阿霉素致肝癌细胞HepG2凋亡机制的研究

目的 探讨黑色素瘤分化相关基因-7/白介素-24(MDA-7/IL-24)基因联合阿霉素杀伤肝癌细胞HepG2,并逆转HepG2多药耐药的机制.方法 以人肝癌细胞系HepG2为实验对象,使用MTT法和流式细胞仪比较Ad.mda-7联合阿霉素处理组与阿霉素组、Ad.mda-7组对肝癌细胞HepG2的作用差异.阐明MDA-7/IL-24对多药耐药的逆转作用的机制.实时定量PCR检测MDR-1、STAT-3、BCL-2、BAXmRNA的变化.Western blot检测gp-170、stat3、P-stat-3、PKB、bcl-2、hax蛋白的表达的变化.结果 低浓度的Ad.mda-7联合正常肝细胞半抑制浓度浓度的阿霉素(1.5 mg/L)使得细胞抑制率从阿霉素组的17.46%上升到79.5%,生长抑制逆转4.55倍(P＜0.05).联合化疗组MDR-1mRNA相对表达量从16.49±0.11下降至5.48±0.05.STAT-3 mRNA相对表达量从13.17±0.08上升至21.57±0.11.BCL-2及BAX表达与其他实验组相比较差异均有显著统计学意义(P＜0.05).联合实验组P-170蛋白的表达量较其他组明显降低,PKB蛋白表达量明显降低,磷酸化stat-3蛋白的表达量增加.结论 Ad.mda-7具有逆转肝癌细胞HepG2多药耐药的作用.其在下调MDR-1mRNA表达的同时,通过抑制PKB蛋白和活化STAT-3信号通路的表达降低促进肝癌细胞凋亡.     

死亡相关蛋白激酶在结肠癌耐药中的作用

目的 探讨死亡相关蛋白激酶（DAPK）在结肠癌耐药中的作用.方法 应用免疫组织化学（免疫组化）SP法检测61例结肠癌组织及32例癌旁组织中DAPK的表达.以氟尿嘧啶（5-FU）诱导建立的结肠癌耐药细胞系HCT116/5-FU为模型.通过转染DAPK-siRNA下调DAPK的表达（DAPK-siRNA组）,转染FAM-siRNA（FAM-siRNA组）作为对照;通过过表达载体上调DAPK的表达（DAPK过表达组）.采用实时定量荧光PCR及蛋白质印迹法检测3组的DAPK、多药耐药蛋白（MRP）和P-糖蛋白（P-gp）的mRNA及蛋白表达水平;MTT法及流式细胞法分别测定3组细胞在未经5-FU处理及浓度为8 μg/ml的5-FU处理下的细胞增殖和凋亡情况.结果 DAPK在结肠癌组织中的阳性表达率明显低于癌旁组织[18.0％（11/61）比90.6％（29/32）,P＜0.05].与FAM-siRNA组比较,DAPK-siRNA组细胞中DAPK mRNA水平及蛋白表达水平均明显降低,DAPK过表达组则均显著升高（均P＜0.05）.在5-FU处理下,相比FAM-siRNA组,DAPK过表达组细胞增殖受到明显抑制,细胞凋亡率明显升高（均P＜0.05）;DAPK-siRNA组细胞增殖和细胞凋亡率均无明显变化（均P＞0.05）.与FAM-siRNA组比较,DAPK过表达组两种耐药蛋白的mRNA和蛋白表达水平均明显降低（P＜0.05）,而DAPK-siRNA组与FAM-siRNA组间差异无统计学意义（P＞0.05）.结论 DAPK能够抑制结肠癌耐药细胞的增殖,促进其凋亡,并可能通过抑制MRP和P-gp的mRNA和蛋白表达,来增强结肠癌细胞对药物的敏感性。