P-糖蛋白表达与活性的药物调节及其基因疫苗的免疫效应
摘要
化学治疗（chemotherapy）是肿瘤主要治疗方法之一。在临床应用中可能有2个问题影响疗效：化疗药物的毒副作用如骨髓抑制和心肌毒性等以及肿瘤治疗过程中发生的耐药现象。近年来国内外学者对P-糖蛋白等亲脂性药物转运体蛋白的表达，活性以及药物调节进行了大量研究，为克服肿瘤化疗药物毒性及耐受问题提供了可能性。
P糖蛋白（p-gp）为跨膜糖蛋白，属于ATP结合匣转运体超家族。编码基因称mdr1。完整p-gp分子含1246个氨基酸残基；N端和C端构成跨膜-胞外区（分别6次跨膜）；肽链中部的氨基酸序列构成ATP结合功能区，可结合及水解ATP，为逆浓度梯度转运底物的跨膜运输提供能量。几乎全部涉及肿瘤细胞与p-gp表达的文献都指出：通过p-gp转运的底物具有结构和药理作用多样性的特点；然而近年已有研究表明p-gp对亲脂性分子选择性结合，结合位点可能位于跨膜区。而其转运底物也不仅限于抗肿瘤药物，异博定（VRPL）、类固醇等非极性分子都能与p-gp结合而被其转运。
在多种组织细胞中，p-gp组成型表达。这表明p-gp在生理状态下也承担物质转运过程。应激性刺激等细胞内外环境的改变也可成为p-gp表达的诱导因素。肿瘤细胞长期接受化疗药物刺激，p-gp的转录和翻译明显提高，这是发生所谓多药耐受（MDR）的原因。此外，细胞内[Ca2+]增加也是诱导包括mdr-1在内的多个活化基因表达的信号。毒毛旋花子甙K（STPK）通过抑制Na+/K+ATP酶活性而升高细胞内[Ca2+]，可刺激mdr1的转录。
本论文的研究内容有：（1）以K562、MCF-7和SMM7721细胞作为反
应细胞，分析VRPL和STPK的细胞增殖调节作用以及对p-gp活性和表达的影响；（2）通过STPK诱导mdr-1表达，克隆mdr-1编码序列并构建基因疫苗；（3）以mdr1基因疫苗免疫小鼠，观察特异性血清抗体滴度以及心、肝、肾组织的形态性。为p-gp生物学活性研究的深入以及p-gp的调节性药物在临床上用于纠正p-gp相关的毒性和耐药性奠定基础。
STPK和VRPL对K562细胞化疗药物敏感性的调节作用
采用MTT标记细胞增殖反应，观察STPK和VRPL对传代肿瘤细胞系体外增殖的影响以及对柔红霉素（ADR）敏感性的调节作用。
本研究对传统MTT分析法做了两点修改：其一，细胞培养中加入MTT后离心弃去培养的上清部分；其二，仅加入异丙醇溶解MTT代谢后生成的甲臢 沉淀，实验结果显示第一项修改可以大大减低由于培养液颜色改变产生的非特异性背景；第一和第二项修改则明显提高甲臢 的溶解性。增加了MTT试验的度量区间（OD570值域随细胞密度不同达到0~2.0），因此增加了该方法测量细胞增殖的敏感性。细胞密度的可测量区间2×104~20×104。
1、STPK和VPRL的细胞毒性：在K562、MCF-7和SMMC 7721 3种传代细胞系的培养中分别加入0.022~1.4μg/ml STPK或0.25~10μg/ml VRPL，培养时间48hrs，然后加入MTT，检测细胞的增殖反应是否因不同浓度药物的存在而表现浓度相关的抑制作用或称细胞毒作用。STPK 0.022~0.35μg/ml，K562细胞的增殖无明显改变；两种贴壁生长的传代细胞系在0.088~0.35μg/ml浓度范围由细胞增殖表现不同程度抑制（30~40%），但无药物浓度相关性。镜下细胞贴壁明显减少。STPK 0.70μg/ml，K562细胞增殖抑制率达29.6%，MCF-7和SMMC7721则分别为60.6%和52.3%；而药物浓度升至1.4μg/ml时，相应增至约70%。这说明STPK在0.70μg/ml以上时，3种细胞呈现细胞毒反应。VRPL 0.25~2.5μg/ml，K562细胞增殖与对照（药物浓度=0）相比无变化，但MCF-7和SSMC7721的OD570下降明显，药物浓度超过1μg/ml，细胞增殖超过50%。因此得出结论：这两种药物对非粘附生长的传代细胞体外增殖影响较小，但对粘附生长的肿瘤细
胞，则由于干扰细胞的贴壁而对增殖产生较大的抑制作用。
2、STPK和VRPL改变K562细胞对ADR的敏感性：ADR是白血病化疗治疗的常用药物，在K562细胞培养中加入倍比稀释的ADR，最大和最小浓度分别是4μg/ml和0.0625μg/ml。6复孔平行测量培养24小时的细胞增殖。对实验结果，采用统计学分析系统软件包（Statistic Analyses System, SAS）进行药物浓度单因素线性相关的F检验。OD570测量显示：ADR 2-4μg/ml加入1×105个细胞的培养中，测量值与未加细胞的空白对照相同（0.07±0.04）。ADR 0.065μg/ml对K562细胞增殖无影响，抑制率（％）等于0。ADR 0.0625—2μg/ml，OD570和细胞增殖抑制率与药物浓度的自然对数之间线性相关（r=0.987, CN=9.728, F=422.7）。
在分析ADR敏感性之前，K562细胞培养中加入STPK或VRPL（终浓度分别为0.25和2.5μg/ml），37℃ 5%CO2孵育48hrs。进行ADR敏感性测定。SAS双因素（对照及两种药物3个分组；每组中0~2μg/mlADR 7种药物浓度）；线性相关的Anova检验显示未加药物预培养之对照与两种药物分别预处理三组间差别明显：组间平均平方（MS）1.0347，误差平均平方0.00514，F=201.3；无药物预培养对照ADR IC50 0.21μg/ml，STPK预培养的K562细胞ADR IC50 0.37μg/ml, VRPL预培养者ADR IC50 0.14μg/ml。结论（1）STPK或VRPL预培养对K562细胞增殖无刺激或抑制作用。（2）STPK预培养降低细胞对ADR的敏感性。（3）VRPL则增加细胞
Chemotherapy is one of the major regimens for treatment of cancerous diseases including solid carcinomas and leukemia, the efficacy of which have been limited by two obstacles, that is, the toxic reactions such as cardiac lesions by doxorubicin (ADR) and the multidrug resistance (MDR) required during chemotherapy. It has been indicated that both the problems might be solved by modulation of drug redistribution in cells and tissues through regulation of the transportor of hydrophobic molecules, p-glycoprotein (p-gp).
P-gp, a member of the superfamily of ATP-binding cassette (ABC) transportor, is a transmembrane (TM) protein encoded by the gene designated as mdr1. Intact p-gp consists of 1226 aa residues, with its N- and C-terminals across cell membrane. The middle segment of p-gp conform functional domain binding and hydrolysing ATP to provide energy for the trans-concentration movement of transported substrates. It has been recognized that the substrates of p-gp are featured by diversities of their structures and bioactivities, but recently it has been suggested that p-gp bind and transport hydrophobic molecules selectively by the binding sites located in TM domain and that not only anti-tumor agents but also cholesteroids be conveyed by p-gp. And verepamil, a blockor for Ca2+ channel, may be also one of the substrates of p-gp.
P-gp expresses constitutively in many tissues and cells, which indicates its
physiological action needed for maintaining the substantial metabolisms of the cells. On the other hand, p-gp expression can be regulated by some stimuli raised by the changes of cell environment such as stress. It is widely realized that over-expression of p-gp in tumor cells results from the long-term exposure to anti-tumor drugs and during chemotherapeutic episodes. In other way, elevation of cytosol [Ca2+] will induce activation of many genes including mdr1.
The purpose of the research is focused on artificial modulation of the expression and activity of p-gp with so-called cardiotonic drugs, verapamil (VRPL) and strophan K (STPK). By inducting expression of mdr1, the gene vaccines specific for p-gp are developed and the in vivo immunoresponses induced with the vaccine are further examined.
The modulation of chemosensitivity of K562 cells by STPK and VRPL
MTT assay was used to detect the in vitro cell proliferation, which was considered as an analyzing system for cellular toxicity of both STPK and VRPL; for the chemosensitivity of the cells to ADR as well as modulating effects of the two cardiotonic agents. Some modifications were made in the study. The samples were centrifuged to remove the supernatants completely after incubation of the cells with MTT and isopropenol was added without HCl to dissolve the formazan precipitant. These procedures (1) greatly decreased the non-specific background, indicated by OD570 almost zero in the cell-free controls and (2) increased the test sensitivity. As seen in the study, OD570 values reached up to 2.0 when 2X105 of the cells were present in the wells. The detectable range for the number of living cells was among 2-20 X 104.
1. Cellular toxicity of STPK and VRPL to the cultured tumor cell lines: different amount of STPK (0.022-1.4μg/ml)or VRPL (0.25-25μg/ml) were respectively added into 105 cell cultures of 3 tumor cell lines, K562 cells, MCF-7 cells and SMMC 7721 cells. The cultivation was maintained for 48hrs to
examine cell growth in presence or absence of the drugs. The results showed that proliferation of K562 cells was not altered compared with the control when the concentrations of STPK were less than 0.7μg/ml or those of VRPL beneath 5μg/ml, the OD570 of which were 1.06-1.18. MCF-7 cells and SMMC 7721 cells grew only when they adhere. In the culture with either STPK or VRPL, the cells detached, as the result, proliferation of the cells decreased obviously.
2. The effect of STPK and VRPL on ADR sensitivity of K562 cells: 105 of K562 cells were inoculated and cultured with 0.0625 to 4μg/ml of ADR for 24 hr
P糖蛋白（p-gp）为跨膜糖蛋白，属于ATP结合匣转运体超家族。编码基因称mdr1。完整p-gp分子含1246个氨基酸残基；N端和C端构成跨膜-胞外区（分别6次跨膜）；肽链中部的氨基酸序列构成ATP结合功能区，可结合及水解ATP，为逆浓度梯度转运底物的跨膜运输提供能量。几乎全部涉及肿瘤细胞与p-gp表达的文献都指出：通过p-gp转运的底物具有结构和药理作用多样性的特点；然而近年已有研究表明p-gp对亲脂性分子选择性结合，结合位点可能位于跨膜区。而其转运底物也不仅限于抗肿瘤药物，异博定（VRPL）、类固醇等非极性分子都能与p-gp结合而被其转运。
在多种组织细胞中，p-gp组成型表达。这表明p-gp在生理状态下也承担物质转运过程。应激性刺激等细胞内外环境的改变也可成为p-gp表达的诱导因素。肿瘤细胞长期接受化疗药物刺激，p-gp的转录和翻译明显提高，这是发生所谓多药耐受（MDR）的原因。此外，细胞内[Ca2+]增加也是诱导包括mdr-1在内的多个活化基因表达的信号。毒毛旋花子甙K（STPK）通过抑制Na+/K+ATP酶活性而升高细胞内[Ca2+]，可刺激mdr1的转录。
本论文的研究内容有：（1）以K562、MCF-7和SMM7721细胞作为反
应细胞，分析VRPL和STPK的细胞增殖调节作用以及对p-gp活性和表达的影响；（2）通过STPK诱导mdr-1表达，克隆mdr-1编码序列并构建基因疫苗；（3）以mdr1基因疫苗免疫小鼠，观察特异性血清抗体滴度以及心、肝、肾组织的形态性。为p-gp生物学活性研究的深入以及p-gp的调节性药物在临床上用于纠正p-gp相关的毒性和耐药性奠定基础。
STPK和VRPL对K562细胞化疗药物敏感性的调节作用
采用MTT标记细胞增殖反应，观察STPK和VRPL对传代肿瘤细胞系体外增殖的影响以及对柔红霉素（ADR）敏感性的调节作用。
本研究对传统MTT分析法做了两点修改：其一，细胞培养中加入MTT后离心弃去培养的上清部分；其二，仅加入异丙醇溶解MTT代谢后生成的甲臢 沉淀，实验结果显示第一项修改可以大大减低由于培养液颜色改变产生的非特异性背景；第一和第二项修改则明显提高甲臢 的溶解性。增加了MTT试验的度量区间（OD570值域随细胞密度不同达到0~2.0），因此增加了该方法测量细胞增殖的敏感性。细胞密度的可测量区间2×104~20×104。
1、STPK和VPRL的细胞毒性：在K562、MCF-7和SMMC 7721 3种传代细胞系的培养中分别加入0.022~1.4μg/ml STPK或0.25~10μg/ml VRPL，培养时间48hrs，然后加入MTT，检测细胞的增殖反应是否因不同浓度药物的存在而表现浓度相关的抑制作用或称细胞毒作用。STPK 0.022~0.35μg/ml，K562细胞的增殖无明显改变；两种贴壁生长的传代细胞系在0.088~0.35μg/ml浓度范围由细胞增殖表现不同程度抑制（30~40%），但无药物浓度相关性。镜下细胞贴壁明显减少。STPK 0.70μg/ml，K562细胞增殖抑制率达29.6%，MCF-7和SMMC7721则分别为60.6%和52.3%；而药物浓度升至1.4μg/ml时，相应增至约70%。这说明STPK在0.70μg/ml以上时，3种细胞呈现细胞毒反应。VRPL 0.25~2.5μg/ml，K562细胞增殖与对照（药物浓度=0）相比无变化，但MCF-7和SSMC7721的OD570下降明显，药物浓度超过1μg/ml，细胞增殖超过50%。因此得出结论：这两种药物对非粘附生长的传代细胞体外增殖影响较小，但对粘附生长的肿瘤细
胞，则由于干扰细胞的贴壁而对增殖产生较大的抑制作用。
2、STPK和VRPL改变K562细胞对ADR的敏感性：ADR是白血病化疗治疗的常用药物，在K562细胞培养中加入倍比稀释的ADR，最大和最小浓度分别是4μg/ml和0.0625μg/ml。6复孔平行测量培养24小时的细胞增殖。对实验结果，采用统计学分析系统软件包（Statistic Analyses System, SAS）进行药物浓度单因素线性相关的F检验。OD570测量显示：ADR 2-4μg/ml加入1×105个细胞的培养中，测量值与未加细胞的空白对照相同（0.07±0.04）。ADR 0.065μg/ml对K562细胞增殖无影响，抑制率（％）等于0。ADR 0.0625—2μg/ml，OD570和细胞增殖抑制率与药物浓度的自然对数之间线性相关（r=0.987, CN=9.728, F=422.7）。
在分析ADR敏感性之前，K562细胞培养中加入STPK或VRPL（终浓度分别为0.25和2.5μg/ml），37℃ 5%CO2孵育48hrs。进行ADR敏感性测定。SAS双因素（对照及两种药物3个分组；每组中0~2μg/mlADR 7种药物浓度）；线性相关的Anova检验显示未加药物预培养之对照与两种药物分别预处理三组间差别明显：组间平均平方（MS）1.0347，误差平均平方0.00514，F=201.3；无药物预培养对照ADR IC50 0.21μg/ml，STPK预培养的K562细胞ADR IC50 0.37μg/ml, VRPL预培养者ADR IC50 0.14μg/ml。结论（1）STPK或VRPL预培养对K562细胞增殖无刺激或抑制作用。（2）STPK预培养降低细胞对ADR的敏感性。（3）VRPL则增加细胞
Chemotherapy is one of the major regimens for treatment of cancerous diseases including solid carcinomas and leukemia, the efficacy of which have been limited by two obstacles, that is, the toxic reactions such as cardiac lesions by doxorubicin (ADR) and the multidrug resistance (MDR) required during chemotherapy. It has been indicated that both the problems might be solved by modulation of drug redistribution in cells and tissues through regulation of the transportor of hydrophobic molecules, p-glycoprotein (p-gp).
P-gp, a member of the superfamily of ATP-binding cassette (ABC) transportor, is a transmembrane (TM) protein encoded by the gene designated as mdr1. Intact p-gp consists of 1226 aa residues, with its N- and C-terminals across cell membrane. The middle segment of p-gp conform functional domain binding and hydrolysing ATP to provide energy for the trans-concentration movement of transported substrates. It has been recognized that the substrates of p-gp are featured by diversities of their structures and bioactivities, but recently it has been suggested that p-gp bind and transport hydrophobic molecules selectively by the binding sites located in TM domain and that not only anti-tumor agents but also cholesteroids be conveyed by p-gp. And verepamil, a blockor for Ca2+ channel, may be also one of the substrates of p-gp.
P-gp expresses constitutively in many tissues and cells, which indicates its
physiological action needed for maintaining the substantial metabolisms of the cells. On the other hand, p-gp expression can be regulated by some stimuli raised by the changes of cell environment such as stress. It is widely realized that over-expression of p-gp in tumor cells results from the long-term exposure to anti-tumor drugs and during chemotherapeutic episodes. In other way, elevation of cytosol [Ca2+] will induce activation of many genes including mdr1.
The purpose of the research is focused on artificial modulation of the expression and activity of p-gp with so-called cardiotonic drugs, verapamil (VRPL) and strophan K (STPK). By inducting expression of mdr1, the gene vaccines specific for p-gp are developed and the in vivo immunoresponses induced with the vaccine are further examined.
The modulation of chemosensitivity of K562 cells by STPK and VRPL
MTT assay was used to detect the in vitro cell proliferation, which was considered as an analyzing system for cellular toxicity of both STPK and VRPL; for the chemosensitivity of the cells to ADR as well as modulating effects of the two cardiotonic agents. Some modifications were made in the study. The samples were centrifuged to remove the supernatants completely after incubation of the cells with MTT and isopropenol was added without HCl to dissolve the formazan precipitant. These procedures (1) greatly decreased the non-specific background, indicated by OD570 almost zero in the cell-free controls and (2) increased the test sensitivity. As seen in the study, OD570 values reached up to 2.0 when 2X105 of the cells were present in the wells. The detectable range for the number of living cells was among 2-20 X 104.
1. Cellular toxicity of STPK and VRPL to the cultured tumor cell lines: different amount of STPK (0.022-1.4μg/ml)or VRPL (0.25-25μg/ml) were respectively added into 105 cell cultures of 3 tumor cell lines, K562 cells, MCF-7 cells and SMMC 7721 cells. The cultivation was maintained for 48hrs to
examine cell growth in presence or absence of the drugs. The results showed that proliferation of K562 cells was not altered compared with the control when the concentrations of STPK were less than 0.7μg/ml or those of VRPL beneath 5μg/ml, the OD570 of which were 1.06-1.18. MCF-7 cells and SMMC 7721 cells grew only when they adhere. In the culture with either STPK or VRPL, the cells detached, as the result, proliferation of the cells decreased obviously.
2. The effect of STPK and VRPL on ADR sensitivity of K562 cells: 105 of K562 cells were inoculated and cultured with 0.0625 to 4μg/ml of ADR for 24 hr
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Hibino H, tani K, Ikebuchi K, et al. The common marmoset as a target preclinical primate model for cytokine and gene therapy studies. Blood 1999, 93(9):2839-2848.
Bunting KD, Galipeau J, topham D, et al. Transduction of murine bone marrow cells with an MDR1 vector enables ex vivo stem cell expansion, but
these expanded grafts cause a myeloproliferative syndrome in transplanted mice. Blood 1998, 92:2269-2279.
Srour EF, Abonour R, Cornetta K, et al. State-of-the-art-review: ex vivo expansion of hematopoietic stem and progenitor cells.are we there yet? J Hematother 1999, 8:93-102.
Tephanie E. Sellers, John F. Tisdale, Brian, et al. The effect of multidrug- resistance 1 gene versus neo transduction on ex vivo and in vivo expansion of rhesus macaque hematopoietic repopulating cells. Blood 2001, 97(6): 1888-1891.
Dell’Acqua G, Polishchuck R, Fallon JT, et al. Cardiac resistance to adriamycin in transgenic mice expressing a rat alpha-cardiac myosin heavy chain/human multiple drug resistance 1 fusion gene. Hum Gene Ther 1999 10(8):1269-1279.
Marianne Z. Metz, Linda Matsumoto, James H. Doroshow, et al. Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene. Virology 1996, 217:230-241.
Robert A. sokolic, Gilda F. Linton, Gottesman MM, et al. A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression. Blood 1996, 87(1):42-50.
Iwata M, Nunoi H, Matsuda I, et al. Drug-selected complete restoration of superoxide generation in Epstein-Barrvirus-transformed B cells from p47phox-deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human miltidrug-resistance gene (MDR1) and the p47phox gene. Hum Genet 1998, 103(4):419-423.
Kleiman SE, Pastan I, Gottesman MM, et al. Characterization of an MDR1 retroviral bicistronic vector for correction of X-linked severe combined immunodeficiency. Gene Ther 1998,5(5):671-676.