不同RNAi对肝癌耐药细胞MDR1基因表达抑制作用比较

[目的]探讨应用RNAi技术抑制肝癌耐药细胞MDR1基因表达的抑制作用以及对P-糖蛋白(p-gp)表达和功能的抑制作用. [方法]根据MDR1基因序列,设计并体外转录合成2条siRNA(small interfering RNA),用脂质体转染将其导入细胞内.应用MTT检测转染后癌细胞生长增殖情况,用MTT法测定细胞对化疗药物阿霉素(ADM)的敏感性,流式细胞仪检测细胞膜表面P-糖蛋白(P-gp)表达和细胞内罗丹明(Rhdamingl23,Rh123)的潴留. [结果]转染sh-MDR1-1和sh-MDR1-2可显著抑制两株耐药细胞MDR1 mRNA和P-gp的表达.细胞内的Rh123稳态积累量均明显增高;第1条序列更能有效的抑制MDR1基因表达. [结论]sh-MDR1-1特异性siRNA可更强的抑制肝癌耐药细胞MDR1基因表达.     

多药耐药基因MDR1在大肠癌的表达及意义

目的探讨多药耐药基因（MDR1基因）在大肠癌组织中的表达及其与临床病理的关系。方法应用RT—PCR方法检测了93例手术切除大肠癌组织中的MDR1 mRNA．结果MDR1 mRNA的阳性率为37.63％，MDR1基因的表达与年龄、性别、肿瘤大小、组织学类型、淋巴结转移及Dukes分期等无关。结论化疗前大肠癌组织中MDR1基因即存在较高的表达率，且独立于病理形态学之外。提示化疗前要区别对待，对MDR1基因表达阳性者应合理选择化疗。     

多药耐药基因MDR1在大肠癌的表达及意义

目的探讨多药耐药基因(MDR1基因)在大肠癌组织中的表达及其与临床病理的关系。方法应用RT-PCR方法检测了93例手术切除大肠癌组织中的MDR1 mRNA.结果MDR1mRNA的阳性率为37.63%,MDR1基因的表达与年龄、性别、肿瘤大小、组织学类型、淋巴结转移及Dukes分期等无关。结论化疗前大肠癌组织中MDR1基因即存在较高的表达率,且独立于病理形态学之外。提示化疗前要区别对待,对MDR1基因表达阳性者应合理选择化疗。     

肺耐药蛋白与肿瘤细胞多药耐药的研究进展

肺耐药蛋白是一种110 KD的蛋白。研究发现肺耐药蛋白就是在人正常组织和肿瘤细胞中广泛分布的穹隆体主蛋白，且具有组织特异性。肺耐药蛋白直接或间接介导了多种肿瘤细胞多药耐药的产生，可作为治疗预后评判的指标．肺耐药蛋白高提示预后不良。     

肺耐药蛋白与肿瘤细胞多药耐药的研究进展

肺耐药蛋白是一种110KD的蛋白。研究发现肺耐药蛋白就是在人正常组织和肿瘤细胞中广泛分布的穹隆体主蛋白,且具有组织特异性。肺耐药蛋白直接或间接介导了多种肿瘤细胞多药耐药的产生,可作为治疗预后评判的指标,肺耐药蛋白高提示预后不良。     

肝细胞癌中热休克蛋白70、多药耐药相关蛋白和增殖核抗原的表达研究

目的探讨热休克蛋白70（HSP70）在肝细胞癌（HCC）中的表达以及与多药耐药相关蛋白（MP．P）和增殖核抗原（PCNA）表达的关系。方法采用免疫组化对44例HCC中HSP70、MAP和PCNA的表达进行了检测。结果44例HCC中有30例可检出HSP70（阳性率6812％）,有24例可检出MRP（阳性率53．6％）,有34例可检出PCNA（阳性率77．3％）。HSP70在分化较好和分化不良的HCC中的阳性率分剐为54．2％和85％,两者相比差异显著（Χ^2=4．5,P〈0．05）。MRP和PCNA与HCC组织分化程度无关（Χ^2=2．4,X22=3．5,P〉0．05）。HSP70表达与MRP和PCNA表达有一致性（Χ^2=24,X22=29．2,P〈0．01）。结论肝细胞癌是HSP70高表达肿瘤．其表达与MRP和PCNA表达密切相关．可能和HCC的耐药和发展有关。     

三氧化二砷对人肝癌多药耐药细胞Bel-7402作用相关机制的研究

目的研究As2O3诱导多药耐药人肝癌细胞Bel-7402／VCR凋亡及相关基因表达的情况。方法MTT法观察细胞生长和对VCR敏感性,应用形态学手段和流式细胞术检测细胞凋亡,应用免疫组织化学研究多药耐药相关基因表达的影响。结果As2O3对Bel-7402／VCR及Bel-7402细胞的生长抑制均呈明显的量效关系;Bel-7402／VCR及Bel-7402凋亡细胞百分比随着As2O3作用时间延长和作用浓度的增加明显增加;As2O3对Bel-7402／VCR及Bel-7402MDR1阳性表达率为81．6％和80．2％。结论As2O3在体外表现出较强的逆转肝癌多药耐药性作用。     

三氧化二砷对人肝癌多药耐药细胞Bel-7402作用相关机制的研究

目的研究As2O3诱导多药耐药人肝癌细胞Bel-7402/VCR凋亡及相关基因表达的情况。方法MTT法观察细胞生长和对VCR敏感性,应用形态学手段和流式细胞术检测细胞凋亡,应用免疫组织化学研究多药耐药相关基因表达的影响。结果As2O3对Bel-7402/VCR及Bel-7402细胞的生长抑制均呈明显的量效关系;Bel-7402/VCR及Bel-7402凋亡细胞百分比随着As2O3作用时间延长和作用浓度的增加明显增加;As2O3对Bel-7402/VCR及Bel-7402MDR1阳性表达率为81.6%和80.2%。结论As2O3在体外表现出较强的逆转肝癌多药耐药性作用。     

肝细胞癌中热休克蛋自70、多药耐药相关蛋白和增殖核抗原的表达研究

目的探讨热休克蛋白70（HSP70）在肝细胞癌（HCC）中的表达以及与多药耐药相关蛋白（MP．P）和增殖核抗原（PCNA）表达的关系。方法采用免疫组化对44例HCC中HSP70、MAP和PCNA的表达进行了检测。结果44例HCC中有30例可检出HSP70（阳性率6812％）,有24例可检出MRP（阳性率53．6％）,有34例可检出PCNA（阳性率77．3％）。HSP70在分化较好和分化不良的HCC中的阳性率分剐为54．2％和85％,两者相比差异显著（Χ^2=4．5,P〈0．05）。MRP和PCNA与HCC组织分化程度无关（Χ^2=2．4,X22=3．5,P〉0．05）。HSP70表达与MRP和PCNA表达有一致性（Χ^2=24,X22=29．2,P〈0．01）。结论肝细胞癌是HSP70高表达肿瘤．其表达与MRP和PCNA表达密切相关．可能和HCC的耐药和发展有关。     

多药耐药及相关蛋白基因抗砷作用

目的 研究多药耐药基因(MDRI)、多药耐药相关蛋白基因(MRP1)在长期砷暴露的细胞获得对砷的耐受过程中的作用,为抗砷机制的研究提供基础依据.方法 采用抑制剂维拉帕米(Verapamil)、MK571,在单一水平和联合水平上阻断MDR1和MRP1基因发挥作用,通过四甲基偶氮噻唑蓝(MTT)法检测细胞的生存率及半数致死量(IC₅₀);通过原子荧光法(AFS)分别检测给予Verapamil、MK571的实验组和对照组在不同时间点抗砷细胞内砷的含量.结果 在2,4,8,16,32,64μmol/L砷浓度下,给予抑制剂的抗砷细胞生存率分别为(73.5±0.02)%,(54.6±0.07)%,(44.2±0.05)%,(20.5±0.09)%,(13.5±0.1)%,(7.6±0.05)%,明显低于同步对照组的生存率(93.5±0.05)%,(71.5±0.02)%,(49.2±0.03)%,(38.8±0.08)%,(37.6±0.06)%,(19.5±0.04)%.Verapamil作用下IC₅₀为8.8μmol/L,MK571作用下IC₅₀为6.22μmol/L;同时给于2种抑制剂时,逆转作用更为明显,IC₅₀为5.23μmol/L;MK571作用下,抗砷细胞内砷含量增加明显,至10 h时砷含量达到最大值1.62 ng,明显高出Verapmil组和对照组,差异有统计学意义(P<0.05).结论 MDR1、MRP1基因在抗砷细胞获得耐药性过程中起重要作用.     

多药耐药蛋白在肝癌中的表达及临床意义

目的 探讨肝癌中多药耐药相关蛋白1(MRP1)、肺耐药蛋白(LRP)、P-糖蛋白(Pgp)、胎盘型谷胱甘肽-s-转移酶π(GST-π)和拓扑异构酶Ⅱ(TOPOⅡ)的表达情况,为肝癌的化疗提供依据.方法 选取肝癌手术切除标本26例,取肝癌组织和癌旁肝组织进行MRP1、LRP、Pgp、GST-π和TOPOⅡ检测,采用图像分析系统对结果进行分析.结果 五种多药耐药蛋白在肝癌组织中表达量比癌旁肝组织中高,差异有统计学意义(P＜0.05).Pgp、TOPOⅡ和GST-π在肝癌组织中表达量与肝外转移差异有统计学意义(P＜0.05).五种多药耐药蛋白的阳性率在分化不良肝癌组织高于分化良好肝癌组织,但只有TOPOⅡ差异有统计学意义(P＜0.05).五种多药耐药相关蛋白在肝癌组织中表达量与肿瘤直径、AFP水平、门静脉癌栓、肝硬化情况和肝功能分级之间差异无统计学意义(P＞0.05).结论 五种多药耐药蛋白在肝癌组织中均有不同程度的高表达,且与肿瘤某些生物学行为有关,联合检测有助于选择合理的化疗药物及判断预后.

Abstract：

Objective To investigate the expression of multidrug resistance protein such as multidrug resistance-associated protein 1 (MRP1),lung-resistance related protein (LRP), P-glycoprotein (Pgp),glutathione s-transferase (GST-π) and topoismerase Ⅱ (TOPO Ⅱ ) in hepatocellular carcinoma (HCC), which would be supplied for the clinical chemotherapy of HCC. Methods Twenty-six cases of HCC who underwent hepatectomy were enrolled and immunohistochemical (IHC) staining was carried out on all specimens for the detection of expression of MRP1,LRP,Pgp,GST-πand TOPO Ⅱ and the data was analyzed by image analysis system. Results The expression of five multidrug resistance protein in HCC tissue were significantly higher than those in adjacent tissue beyond cancer (P ＜0.05). The significant differences were found in the expression of Pgp,TOPO Ⅱ and GST-π between HCC tissue and distant metastasis (P ＜ 0.05 ). The expression of the five multidrug resistance protein in poorly differentiated HCC tissue was higher than that in well-differentiated tissue,while the significant difference was only found in the expression of TOPO Ⅱ (P ＜ 0.05 ). The significant association was not found between the expressions of five multidrug resistance protein in HCC tissue and the size of tumor,AFP, the portal vein tumor thrombus,hepatic cirrhosis and liver function. Conclusions Five multidrug resistance protein overexpression in various degrees in HCC tissue, which relates to some biological behavior of the cancer. Combined detection is of much benefit to the choice of the drug of chemotherapy and to the prediction of prognosis.     

多药耐药相关蛋白及谷胱甘肽-S-转移酶π与人肺癌多药耐药细胞系SPC-A1/TAX耐药性关系研究

目的研究多药耐药相关蛋白(MRP)、谷胱甘肽-S-转移酶π(GST-π)与人肺腺癌多药耐药细胞系SPC-A1/TAX多药耐药性之间的关系。方法采用S-P免疫组化法检测细胞MRP、GST-π的表达;采用MTS法分别检测并比较SPC-A1、SPC-A1/TAX细胞对6种不同化疗药物的敏感性。结果耐药组MRP、GST-π表达均为阳性,亲本组MRP、GST-π表达均为阴性,耐药组与亲本组间差异有统计学意义(P<0.05,P<0.01);SPC-A1/TAX较SPC-A1除对原诱导药物TAX具有耐受性外,对其他5种抗肿瘤药物也表现出不同程度的耐药性。结论MRP、GST-π在SPC-A1/TAX细胞系中表达与该人肺腺癌多药耐药细胞系的MDR具有相关性,是其MDR产生的可能机制。     

Modulation of the multidrug resistance (MDR) phenotype in CEM MDR cells simultaneously exposed to anti HIV-1 protease inhibitors (PI's) and cytotoxic drugs

Vinblastine, vincristine and doxorubicyn are currently used in chemotherapeutic treatments of several malignancies including HIV-1 associated tumours Kaposi's sarcoma (KS) and non-Hodgkin lymphoma (NHL). Hence, AIDS patients also affected by KS and NHL may be simultaneously subjected to highly active antiretroviral therapy (HAART) and cytotoxic drugs to combat HIV-1 infection and cancer aggressiveness. In order to assess if the combination of these therapies may affect cell growth and survival of P-glycoprotein expressing MDR variants of the human CD4+ T-lymphoblastoid CEM cell line, the protease inhibitors (PI's) ritonavir, saquinavir and indinavir were tested in an in vitro assay for their ability to potentiate the vinblastine, vincristine and doxorubicyn cytotoxicity. The results we obtained demonstrated that at the concentration of 10 micrograms/ml, ritonavir and in a lesser extent saquinavir act as MDR reversing agents. By contrast, the PI indinavir at least in the CEM cell system, does not affect the patterns of drug resistance. The level of chemosensitization exerted by ritonavir and saquinavir suggests that these PI's may render P-glycoprotein expressing MDR cells de novo susceptible to the antineoplastic drugs vinblastine, vincristine and doxorubicyn.     

Anti-calmodulin acridone derivatives modulate vinblastine resistance in multidrug resistant (MDR) cancer cells

Multidrug resistance (MDR) is one of the main obstacles limiting the efficacy of chemotherapy treatment of tumors. Parent acridones 1A and 1B were prepared by the Ullmann reaction followed by cyclization and N-alkylation. N-(omega-Chloroalkyl) analogues were subjected to iodide catalyzed nucleophilic substitution reaction with secondary amines to get the compounds 3A-13A and 3B-13B, which enhanced the uptake of vinblastine in KBChR-8-5 cells to a greater extent (2.6-13.1-fold relative to control) than verapamil. The study on the structure-activity relationship revealed that substitution of -H at position C-4 in acridone nucleus by -OCH3 increased the cytotoxic and anti-MDR activities. The ability of acridones to inhibit calmodulin dependent cyclic AMP phosphodiesterase has been determined and the results have shown a strong positive correlation between anti-calmodulin activity and cytotoxicity in KBChR-8-5 cells or anti-MDR activity.     

维利帕尼和多柔比星联合作用对人肝癌耐药细胞株BEL-7404增殖和凋亡的影响

目的检测PARP抑制剂维利帕尼与抗癌药物多柔比星联合应用对肝癌耐药细胞株BEL-7404增殖抑制的作用。方法以BEL-7404为研究对象,常规培养传代,经多柔比星和（或）维利帕尼处理24 h后,通过四甲基偶氮唑盐(MTT) 比色法观察细胞的增殖率变化, 采用流式细胞术Annexin V-FITC/PI双染法分析细胞凋亡水平,通过单细胞凝胶电泳实验评价DNA损伤程度,通过聚丙烯酰胺凝胶电泳（Western blotting）法检测细胞线粒体和胞浆中细胞色素c水平变化。结果多柔比星和维利帕尼联合应用组的细胞增殖率明显低于对照组和多柔比星单独处理组（P<0.01）,细胞凋亡率则显著高于对照组和多柔比星单独处理组（P<0.05）;同时,多柔比星和维利帕尼联合应用组细胞DNA损伤水平较对照组和多柔比星单独处理组亦明显加重（P<0.01）,而细胞色素C在胞浆中的水平则显著高于对照组和多柔比星单独处理组（P<0.01）。结论维利帕尼与多柔比星联合作用可以抑制BEL-7404细胞的增殖,并诱导细胞DNA损伤和凋亡,其机制与胞浆中细胞色素c的表达升高有关。     

GP73表达调控的肝癌细胞模型的筛选和构建

目的 筛选合适的细胞株构建干扰和过表达GP73的肝癌细胞模型,为进一步深入研究GP73调控肝癌的机制提供合适的细胞模型.方法 挑选8株肝癌细胞:Huh-7、Hep3B、MHCC-LM3、MHCC97-L、MHCC97-H、SNU-449、SNU-398、SNU-182,采用qPCR和Western Blot验证GP73...     

Ⅰ类整合子与铜绿假单胞菌多重耐药相关性研究

目的了解Ⅰ类整合子在临床分离的铜绿假单胞菌多重耐药株及敏感株中分布的差异,初步阐明Ⅰ类整合子是否与铜绿假单胞菌多重耐药性形成相关。方法应用聚合酶链反应（PCR）法分别检测临床分离的67株多重耐药铜绿假单胞菌和62株敏感株中Ⅰ类整合子的阳性率,并进一步用PCR证实Ⅰ类整合子阳性菌中整合子相关基因盒的存在。结果铜绿假单胞菌多重耐药株中,32株（47.76%）为Ⅰ类整合子阳性;敏感株中仅1株（1.61%）为Ⅰ类整合子阳性,多重耐药株的Ⅰ类整合子分布显著高于敏感株（χ2=36.02,P〈0.05）。PCR进一步证实多重耐药铜绿假单胞菌携带Ⅰ类整合子基因盒,而敏感株携带空整合子。结论多重耐药铜绿假单胞菌具有较高的Ⅰ类整合子阳性率,Ⅰ类整合子可能与铜绿假单胞菌的多重耐药性相关。     

Prevention of hepatocellular carcinoma by immunization

The evidence for an association between the carrier state of hepatitis B virus infection and hepatocellular carcinoma (liver cell cancer) is now sufficiently strong to justify the use of a vaccine against this infection as a means of preventing this cancer. Effective vaccines are available and have been tested in feasibility studies, and their use in field trials to test their effectiveness against the long-term risk of developing this cancer is now possible. At the present time, only limited quantities of the vaccine derived from human plasma infected by the hepatitis B virus are available, and the development of other types of vaccine is discussed together with the necessary revision of the present requirements for vaccine production. As the studies to assess the prevention of this cancer would require the surveillance of subjects for some years, consideration is also given to the design of field studies with short- and medium-term objectives.