PTEN对乳腺癌多药耐药MCF-7/ADR细胞凋亡的促进作用及机制

目的观察野生型PTEN基因促进乳腺癌多药耐药细胞MCF-7/ADR凋亡的作用并探讨其机制。方法采用脂质体介导法将真核表达载体pEGFP-C1-PTEN及突变载体pEGFP-C1-PTEN-C124S转染人乳腺癌多药耐药MCF-7/ADR细胞。MTT法观察细胞对阿霉素的敏感性和耐药倍数,流式细胞技术检测细胞凋亡率,Western blot检测Bcl-2和Caspase-3蛋白。结果转染组的凋亡率为36.86%,高于对照组（3.75%）和C124S组（4.00%）（P均〈0.05）。转染组对阿霉素的耐药倍数显著降低（t=4.77,P〈0.05）,其半数致死剂量IC50值为对照组的26.1%。转染后细胞内Bcl-2表达降低,且检测到Caspase-3活性裂解片段。结论PTEN基因可能通过下调Bcl-2表达及活化Caspase-3来促进乳腺癌多药耐药MCF-7/ADR细胞凋亡,增加其对阿霉素的药敏性。     

靶向survivin的siRNA降低人乳腺癌MCF-7~（Adr）耐药细胞株对阿霉素耐受效应的研究

目的探讨靶向构建survivin的小干扰性RNA（siRNA）能否逆转人乳腺癌MCF-7Adr耐药细胞株对阿霉素的耐药性。方法将已构建含siRNAsurvivin真核载体PcDNA3-siRNA通过脂质体转染入MCF-7Adr细胞内,通过RT-PCR及Western blot半定量检测MCF-7Adr转染前后survivin mRNA蛋白表达量的变化,MTT法检测不同浓度阿霉素对MCF-7Adr转染前后增殖抑制率的影响。结果以survivin为靶向的siRNA能明显降低survivin基因在MCF-7Adr中的表达及MCF-7Adr对阿霉素的耐药性。结论靶向survivin的siRNA能有效沉默survivin基因在MCF-7Adr中的表达,并能逆转MCF-7Adr对阿霉素的耐药性,为将来在乳腺癌耐药治疗过程中选择基因治疗提供一定的实验依据。     

RNA干扰对胃癌耐药细胞MDR1及P-gP表达的影响

目的 寻找逆转胃癌细胞多药耐药的有效方法.方法 根据多药耐药基因1(MDR1)的基因序列,设计并体外转录合成2条siRNA,用脂质体转染将其导入人胃癌多药耐药BGC-823细胞内.应用MTT法检测转染后癌细胞生长增殖情况及对阿霉素(ADM)的敏感性,流式细胞仪检测细胞膜表面P-糖蛋白(P-gp)表达和细胞内Rh123的潴留情况.结果 转染sh-MDR1-1和sh-MDR1-2后,BGC-823细胞对ADM的IC50均降低,敏感性提高;BGC-823细胞MDR1 mRNA和P-gP表达均显著降低,细胞内Rh123稳态积累量均明显增高;上述效果均以转染sh-MDR1-1序列为著.结论 sh-MDR1-1特异性siRNA可抑制BGC-823细胞MDR1表达,此为寻找逆转胃癌细胞多药耐药有效方法奠定了基础.     

PTEN的表达及其在阿霉素诱导胃癌细胞凋亡中的意义

目的 研究阿霉素干预胃癌细胞后PTEN的表达及其在阿霉素诱导胃癌细胞凋亡中的意义.方法 (1)阿霉索干预胃癌细胞BGC-823后以四甲基偶氮唑盐比色法(MTT法)和流式细胞法检测细胞存活率及凋亡率,并检测VFEN的mRNA和蛋白水平.(2)构建胃癌裸鼠异位种植瘤,采用原位末端标记(TUNEL)法检测异位种植瘤中胃癌细胞的凋亡情况,并用RT-PCR和Western blot法检测PTEN mRNA和蛋白的表达水平.(3)以PTEN特异性小干扰RNA(siRNA)转染BGC-823细胞,并以阿霉素进行干预,检测BGC-823细胞的存活率和凋亡率以及PTEN蛋白表达水平.结果 (1)阿霉素干预后,BGC-823细胞的生存率呈时间依赖性降低.(2)阿霉素能够有效诱导BGC-823细胞凋亡.(3)阿霉素在BGC-823细胞中可时间依赖性地促进PTEN的mRNA和蛋白水平的升高.裸鼠异位种植瘤试验中,阿霉素干预组的凋亡率[(28.11±1.05)%]明显高于对照组[(2.78±1.63)%];阿霉素干预组瘤体组织中PTEN mRNA和蛋白水平亦高于对照组(0.5667±0.0043比0.2217±0.0063,0.14±0.26比0.04±0.15,P值均<0.05).(4)转染与未转染[WEN siRNA的胃癌细胞以阿霉素干预后,PTEN siRNA转染组的PTEN蛋白表达水平明显低于对照组(P<0.0001),且PTEN siRNA转染组[(10.35±1.04)%]凋亡率明显小于未转染组[(31.37±3.58)%],P<0.05.结论 阿霉素干预胃癌细胞后可以抑制其生长,诱导细胞凋亡,PTEN表达水平的升高可能是其机制之一.     

MUC5AC特异性siRNA对胆管癌细胞株HCCC-9810增殖及凋亡的影响

目的:探讨黏蛋白5AC特异性siRNA对人肝内胆管癌细胞珠HCCC-9810体外增殖及凋亡能力的影响.方法:设计合成三对特异性siRNA, 构建了三个可在哺乳动物细胞中表达siRNA的表达质粒, pRNAT-U6.1/Neo-MUC5AC-siRNA1/2/应用脂质体转染技术分别将三个稳定表达的质粒和对照质粒( 空质粒对照) 转染HCCC-9810, 应用有荧光标记的非特异性小分子的siRNA检测转染效率, RT-PCR检测黏蛋白5AC基因mRNA水平;SABC免疫组化染色技术检测黏蛋白5AC的表达;MTT检测细胞生长增殖情况;流式细胞仪分析细胞凋亡.结果:基因测序表明成功构建质粒;转染后荧光蛋白表达率28.57%;RT-PCR结果表明在mRNA水平, 三个质粒都可抑制黏蛋白5AC基因的表达, 并可使黏蛋白5AC的表达下降,MTT结果对细胞的生长有一定的抑制作用,流式细胞仪分析结果使肿瘤细胞凋亡增加.结论:构建pRNAT-U6.1/Neo-MUC5ACsiRNA1/2/3质粒明显抑制了黏蛋白5AC基因在mRNA水平及蛋白的表达, 并可抑制肿瘤细胞的生长增殖和诱导其凋亡.     

靶向mdr1不同位点的siRNA对两种耐药细胞MDR的逆转效果

目的:探讨靶向mdr1 4个不同位点的siRNAs对胃癌耐药细胞SGC7901/VCR和人红白血病耐药细胞K562/A02的作用效果.方法:设计并体外转录合成4条靶向mdr1的siRNAs(mdr1si326、mdr1si1513、mdr1si2631及mdr1si3071), 分别转染SGC7901/VCR细胞和K562/A02细胞, 用RT-PCR和免疫组织化学检测mdr1 mRNA与P-gp的表达, 流式细胞仪检测细胞内阿霉素(ADR)的蓄积, MTT法检测细胞对阿霉素的敏感性.结果:4条s iRNAs对人胃癌细胞SGC7901/VCR mdr1介导的MDR逆转效果由高到低依次为mdr1si326、mdr1si2631、mdr1si3071及mdr1si1513; 对人红白血病细胞K562/A02md r1介导的MDR逆转效果由高到低依次为mdr1si326、mdr1si2631、mdr1si3071及mdr1si1513.结论:4条siRNAs对SGC7901/VCR和K562/A02两种耐药细胞作用效果趋势相似.     

Cyclin G_2表达载体的构建及其对乳腺癌MCF-7细胞的作用

目的构建包含Cyclin G2基因的真核表达载体,探讨其对乳腺癌MCF-7细胞体外增殖的作用。方法运用RT-PCR和基因克隆技术构建pDsRed2-Cyclin G2重组质粒,脂质体法转染至MCF-7细胞中,荧光显微镜下观察其蛋白的表达及定位;AnnexinⅤ-FITC染色流式细胞术检测细胞凋亡率。结果成功构建了pDsRed2-Cyclin G2融合表达质粒,转染MCF-7细胞后散在分布于细胞质和细胞,核转染重组质粒的MCF-7细胞凋亡率明显增高（P〈0.01）。结论 Cyclin G2重组质粒转染至MCF-7细胞后能有效促进细胞凋亡。     

沉默LAIR-1表达对乳腺癌细胞增殖和侵袭功能的影响

目的探讨白细胞相关免疫球蛋白样受体(LAIR)-1在乳腺癌组织中的表达及其对乳腺癌细胞增殖和侵袭能力的影响。方法采用RT-PCR方法检测乳腺癌细胞系和组织标本中LAIR-1 mRNA的表达情况。采用电穿孔转染法,将LAIR-1 siRNA转染MCF-7细胞,通过Western印迹检测LAIR-1在MCF-7细胞中的表达情况。通过四甲基偶氮唑蓝比色(MTT)增殖实验检测LAIR-1对MCF-7细胞增殖的影响。通过伤Boyden小室检测LAIR-1对MCF-7细胞侵袭能力的影响。结果同癌旁组织相比,乳腺癌组织中LAIR-1 mRNA的表达量显著降低(P0.01);且同非转移性乳腺癌组织相比,转移性乳腺癌组织中LAIR-1 mRNA的表达量亦显著降低(P0.01)。转染LAIR-1 siRNA可显著抑制MCF-7细胞中LAIR-1的表达。抑制LAIR-1表达后,MCF-7细胞的增殖和侵袭能力均显著升高(P0.01)。结论 LAIR-1具有抑制乳腺癌细胞增殖和侵袭的作用,乳腺癌组织中LAIR-1的低表达可能与乳腺癌的发生发展相关。     

血清糖皮质激素调节蛋白激酶3过表达对乳腺癌细胞凋亡的影响

目的探讨血清糖皮质激素调节蛋白激酶3(SGK3)基因过表达对乳腺癌细胞凋亡的影响。方法构建pEGFP-N1-SGK3重组质粒,用载体质粒pEGFP-N1和重组质粒pEGFP-N1-SGK3转染乳腺癌MCF7细胞;MTT法观察转染细胞的增殖情况,流式细胞术分析细胞凋亡情况,RT-PCR检测凋亡相关基因表达。结果利用pEGFP-N1-SGK3质粒转染乳腺癌MCF7细胞,建立表达SGK3蛋白的细胞系;通过细胞增殖实验发现,SGK3的过表达可促进MCF7细胞增殖;流式细胞术分析显示,外源性SGK3可抑制MCF7细胞凋亡的发生,并影响凋亡相关基因bad、bcl-xl的表达。结论 SGK3的过表达可抑制乳腺癌MCF7细胞凋亡。     

靶向抑制乳腺癌细胞GLS1基因表达对癌细胞凋亡的影响

目的探讨抑制乳腺癌细胞谷氨酰胺酶(GLS) 1基因表达对癌细胞凋亡的影响。方法参照Lipofectamine~(TM) 2000说明将合成的GLS1 siRNA及无干扰作用的siRNA转染MCF-7细胞,分别为GLS1-siRNA组和阴性对照组,并设置空白对照组,转染48 h后,Western印迹检测GLS1、凋亡相关蛋白survivin、p53及磷脂酰肌醇-3激酶(PI3K)/丝氨酸苏氨酸激酶(AKT)信号通路PI3K和磷酸化(p-AKT)的蛋白表达; 3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测GLS1-siR-NA转染24、48和72 h的细胞活力;流式细胞仪检测GLS1-siRNA转染48 h的细胞凋亡率。结果转染GLS1-siRNA的MCF-7细胞GLS1蛋白表达显著低于空白对照组(P<0. 05);与空白对照组比较,GLS1-siRNA组细胞在转染24、48和72 h的细胞活力均显著降低,细胞凋亡率显著升高,survivin、PI3K和p-AKT蛋白表达均显著降低,p53蛋白表达显著升高(P<0. 05)。结论抑制GLS1基因在乳腺癌中的表达可降低细胞活力,促进细胞凋亡及下调PI3K/AKT信号通路,其中诱导细胞凋亡的方式是下调survivin表达和上调p53表达。     

Influence of cytokines onmdr1 expression in human colon carcinoma cell lines: Increased cytoxicity of MDR relevant drugs

We investigated the effects of tumor necrosis factor (TNF) , interferon (IFN) and interleukin-2 (IL-2) on themdr1 gene expression in four human colon carcinoma cell lines (LoVo, HT 115, SW 480, and LS 174T) at different times (8. 24, 48, and 72h). We found no significant changes inmdr1 expression after 8h and 24h of cytokine treatment in all four lines. After 48h and 72h, however, a marked reduction ofmdr1 expression in LoVo, HT 115, and SW 480 cells and an unaffected expression in LS 174T cells was observed. We examined whether the cytokine-mediated reduction ofmdr1 expression correlates to the multidrug resistance (MDR) phenotype. In those cell lines showing a decreasedmdr1 expression after a long-term cytokine pretreatment we found a dramatic enhancement of cytotoxicity of the MDR relevant drugs vincristine and doxorubicin, whereas LS 174T cells remained resistant. By contrast, the simultaneous application of cytokines and cytostatics caused no additive or synergistic effects. We conclude that in certain colon carcinoma cell lines a decreasedmdr1 expression caused by prolonged cytokine pretreatment correlates with an enhanced cytotoxicity of drugs susceptible to MDR as an MDR-overcoming effect.Abbreviations MDR multidrug resistance - TNF tumor necrosis factor - IFN interferon - IL-2 interleukin-2 - Vin vincristine - Dox doxorubicin - IC₅₀ inhibition concentration     

原发性肝癌中MDR1基因表达研究

探讨MDR1基因编码的P-糖蛋白（Ppg)在肝癌（HCC）及癌周正常肝组织中的表达及其临床意义。用免疫组化SABC法，检测78例肝癌及60例癌旁组织中Pgp表达，分析肝癌中Pgp表达程度和临床病理特征。（1）Pgp表达主要位于肝癌，胆管上皮细胞膜上，少量位于胞质内；Pgp表达量，在治疗前癌组织中平均为26％，而癌旁组织平均为56％以上，治疗后接近癌旁组织。（2）Pgp表达量与肝癌分化程度有关，分化程度越高，其表达量越高。（3）术后1，3，5年，Pgp高表达组生存率略高于低表达组。     

Genetic transfer of non-P-glycoprotein-mediated multidrug resistance (MDR) in somatic cell fusion: dissection of a compound MDR phenotype.

A non-P-glycoprotein-mediated mechanism of multidrug resistance (non-Pgp MDR) has been identified in doxorubicin-selected sublines of the human non-small cell lung carcinoma cell line SW-1573. These sublines are cross-resistant to daunorubicin, VP16-213, Vinca alkaloids, colchicine, gramicidin D, and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). They accumulate less drug than the parental cells and their resistance is not due to the MDR1-encoded P-glycoprotein, as the resistant cell lines have lost the low amount of MDR1 mRNA detectable in parental cells. Here we show that the resistant cell lines also contain less topoisomerase II mRNA and enzyme activity than the parental cells. This might contribute to the resistance of these lines to drugs interacting with topoisomerase II, such as doxorubicin, daunorubicin, and VP16-213, but cannot account for the resistance to the other drugs. We have tested whether all properties of the non-Pgp MDR cell lines cosegregate in somatic cell fusions between lethally gamma-irradiated, resistant donor cells and drug-sensitive acceptor cells. Whereas a MDR phenotype with reduced drug accumulation and the loss of MDR1 P-glycoprotein mRNA were cotransferred to the acceptor cells, the decrease in topoisomerase II gene expression was not. We conclude that the MDR phenotype, the reduced drug accumulation, and the loss of MDR1 P-glycoprotein mRNA are genetically linked. They might be due to a single dominant mutation, which does not cause the alteration in topoisomerase II.     

MDR1 and MRP expression in chronic B-cell lymphoproliferative disorders

The role of the MDR1 and MRP genes in drug resistance in patients with chronic lymphocytic leukaemia (CLL)/non-Hodgkin's lymphoma (NHL) is unclear. We hypothesized that any relationship between levels of expression and exposure to P-glycoprotein (P-gp) transportable drugs may become evident by using a measure of gene expression that combined the number of positive cells and the degree of positivity. 68 CLL/NHL patients were analysed using flow cytometry with MDR1 and MRP specific antibodies and were divided into subgroups, untreated ( n  = 31), treated with non P-gp transportable drugs ( n  = 26), those treated with low total doses of P-gp transportable drugs ( n  = 6) and patients treated with high total doses of P-gp transportable drugs ( n  = 5). The group exposed to high doses of P-gp transportable drugs had higher levels of MDR1 expression when compared to all other groups ( P  < 0.05, ANOVA). A positive correlation between the level of MDR1 expression and the cumulative dose of P-gp transportable drugs was demonstrated ( P  = 0.02). MRP expression was higher in those patients exposed to high doses of P-gp transportable drugs when compared to all other groups ( P  < 0.05, ANOVA), although only a trend towards a linear dose correlation effect could be established ( P  = 0.08). We concluded that MDR1 and MRP are involved in drug resistance but only in patients treated with P-gp transportable drugs.     

耐多药结核病——预防和控制的关键步骤

<正>耐多药结核病(multidrug-resistant tuberculosis,MDR-TB)被定义为,由至少对异烟肼(isoniazid)和利福平(rifampicirn)治疗耐药的结核分枝杆菌(Mycobacterium tuberculosis)引起的疾病。广泛耐药结核病(extensively drug-resistant,tuberculosis,XDR-TB)是指导疾病的多重耐药菌株还对使用任