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ABC转运蛋白ABCG2在皮肤恶性黑素瘤中的表达及意义

Expression of ABC Transporter ABCG2 in Malignant Melanoma and Its Significance

作者: 专业:皮肤病与性病学 导师:于建斌 年度:2010 学位:硕士  院校: 郑州大学

Keywords

Malignant Melanoma, ABC transporter, ABCG2, MDR, CSC

        ATP结合盒膜转运蛋白(ATP Binding Cassette Transporters),又称ABC转运蛋白,与多药耐药(multidrug resistance,MDR)有关,能够介导多种内、外源性底物分子的跨膜转运。通过ATP水解供能,可以逆浓度将药物泵到细胞外,导致细胞内的药物浓度降低从而产生耐药。皮肤恶性黑素瘤(malignant melanoma,MM)是一种高度恶性的皮肤肿瘤,极易发生转移,其发病率位列于皮肤恶性肿瘤的第三位(约6.8%~20%),并呈现逐年上升趋势,其死亡率占皮肤恶性肿瘤总数的80%左右,亦呈现逐年上升趋势。现有的治疗MM的方法主要是手术、化疗和放疗。鉴于MM本身的生物学性质和行为的特殊性,许多患者就诊时肿瘤已发生局部浸润、淋巴结转移或远端转移。此时,手术治疗的意义就很局限,而化疗或生物治疗常常是治疗此类病人的终末手段。MM的化疗分为单独应用和联合治疗,目前多采用联合治疗。但是,对于MM来说,无论是单独应用还是联合治疗,其远期效果都不理想。肿瘤的MDR是指肿瘤细胞对一种抗肿瘤药物出现耐药性的同时,对其他多种结构不同、作用靶位不同的抗肿瘤药物也有耐药性。可能是化疗难以奏效并导致最终失败的原因。MDR机制包括内在性MDR和获得性MDR两个方面,目前,以ABC转运蛋白家族介导的MDR是最重要、最关键的途径。ABC转运蛋白家族能够介导多种内源性、外源性底物分子的跨膜转运。通过ATP水解供能,可以逆浓度将药物泵到细胞外,导致细胞内的药物浓度降低从而产生耐药。人类ABC转运蛋白家族由A、B、C、D、E、F、G七个亚家族组成,共49个成员,ABCG2为ABC转运蛋白家族重要成员之一。ABCG2是ABC转运蛋白家族成员之一,因最初从多药耐药乳腺癌细胞株中被鉴别出来,故又称为乳腺癌耐药蛋白(breast cancer resistance protein,BCRP),是一种膜转运蛋白,其结构中有1个位于胞质中的核苷酸结合区域(即ATP结合盒),因此可以利用ATP水解供能,逆浓度的转运细胞内的各种物质。ABCG2转运蛋白的底物可以是类脂质、胆汁和各种内、外源性化合物等。鉴于其对各种内、外源性化合物的转运,因此可以参与多种有毒物质的吸收、积累和排泄,并减轻各种潜在有害物质对机体组织的危害性。而这一保护作用的副效应,就是可以使各种有用药物从机体内排除,降低细胞内药物浓度,从而引起耐药作用。ABCG2转运蛋白可以将化疗药物米托蒽醌、阿霉素及柔红霉素等从肿瘤细胞中泵出。ABCG2亦被认为是肿瘤干细胞(cancer stem cells,CSCs)标记物的一员,研究发现其在乳腺癌、肺癌、胶质瘤等多种实体肿瘤及白血病中都高表达。为探讨多药耐药蛋白在皮肤MM中的表达及意义,我们采用免疫组化方法研究ABCG2在30例MM组织、30例皮内痣组织及5例A375细胞系裸鼠移植瘤组织中的表达情况,旨在进一步证实MM中存在多药耐药蛋白ABCG2的表达,为进一步阐明MM中可能存在的化疗药物耐药机制提供实验依据。材料与方法1.采用免疫组化SP法检测30例MM、30例皮内痣及5例A375细胞裸鼠移植瘤组织中ABCG2的表达。2.统计学处理:数据用X±S表示,采用SPSS10.0统计软件包进行数据分析,ABCG2在MM和皮内痣组织中的阳性率的比较用计数资料的χ2检验;ABCG2在MM和皮内痣组织中的阳性细胞率平均值的比较用t检验;Spearman法计算相关系数。检验水准均为α=0.05。结果1. ABCG2在MM、皮内痣及A375细胞裸鼠移植瘤中的表达ABCG2在MM和皮内痣组织中的阳性率分别是56.67%和0.00%,两者比较差异有统计学意义(x 2=1.00,P<0.05),ABCG2在A375细胞裸鼠移植瘤组织中表达稳定,阳性率是100.00%。ABCG2在MM和皮内痣组织中的阳性细胞率平均值分别是30.96%±56.96%和0.00%,两者间比较差异有统计学意义(t=12.104,P<0.05),ABCG2在A375细胞裸鼠移植瘤组织中的阳性细胞率平均值是10.35%±3.99%,表达较为一致。ABCG2阳性细胞在肿瘤组织中呈团块状或散在分布。阳性细胞胞核大而透亮或小而深染。皮内痣中未见阳性表达细胞。2. ABCG2阳性细胞率与患者性别、年龄、病程的关系MM中ABCG2阳性细胞率与患者性别、年龄、病程的相关性无统计学意义(r1=0.105,r2=0.122,r3=0.344,P均>0.05)。结论1.恶性黑素瘤及A375细胞裸鼠移植瘤组织中存在不同比例ABCG2的阳性表达;2.恶性黑素瘤化疗后复发与ABCG2的存在可能有关。
    ATP Binding Cassette Transporters, also known as the ABC transporter protein, which related with the multidrug resistance (MDR), can mediate a variety of endogenous and exogenous substrate molecules transmembrane transport. By ATP hydrolysis for energy, the ATP Binding Cassette Transporters can reverse the concentration of the drug and pump the drug out of the cells, resulting in lower intracellular drug concentration and cause drug resistance.Cutaneous malignant melanoma (MM)is one kind of highly malignant skin tumor, and prone to metastasis, its incidence rate ranked the third of skin cancer (about 6.8%~20%), and showed an upward trend year by year. Its mortality rate accounting for about 80% of the total skin cancer, also shown an upward trend year by year. At present, the major treatments of MM are surgical, chemotherapy and radiotherapy. As the onset of misprision of malignant melanoma and patients lack of the awareness of the disease, when most patients go to the doctor’s, the tumor has entered the middle or late stage and often shifted. At this point, the significance of surgical treatment is very limited, but chemotherapy seems more appropriate and important. The chemotherapy of malignant melanoma is divided into alone and combined apply anticancer drugs, at the moment, the majority method is combination therapy. However, to malignant melanoma, either alone or combination therapy, its long-term effects are far from satisfactory.The multidrug resistance of tumor means that tumor cells emergence of a resistance to one kind of anticancer drugs, at the same time have resistance to a variety of other different structures, roles of different target site anticancer drugs. The multidrug resistance of tumor may be the reason why chemotherapy difficult to work and eventually lead to failure. The mechanisms of multidrug resistance include the intrinsic and acquired multidrug resistance, at present, the most important and most critical way of multidrug resistance is mediated by the ABC transporter protein family. A variety of endogenous and exogenous substrate molecules can across the membrane mediated by the ABC transporter protein family. By ATP hydrolysis for energy, the ABC transporter protein family can reverse the concentration of the drug and pump the drug out of the cells, resulting in lower intracellular drug concentration and cause drug resistance.The human ABC transporter protein family include 49 members and divide into seven sub-families which are A, B, C, D, E, F and G.ABCG2 is one of the most important members of the ABC transporter protein family.ABCG2 is a member of the ABC transporter protein family, as it was originally identified out of from multidrug resistance breast cancer cell lines, so it is also known as breast cancer resistance protein (BCRP). ABCG2 is a membrane transporter protein, its structure has a nucleotide-binding domain (ATP-binding cassette) in the cytoplasm, so it can use ATP hydrolysis energy and pump various substance out of the cells, in spite of reverse the concentration. The substrate of ABCG2 transporter protein can be a class of lipid, bile and various external and endogenous compounds. In view of it can transports various external and endogenous compounds, so it can participate in absorption, accumulation and excretion of a variety of toxic substances, and reduce the hazarda to tissues arouse by variety of potentially harmful substances. The side effect of this protective effect is that it can make a variety of useful substances exclude from the body, and reducing intracellular drug concentration, which led to the role of drug resistance. ABCG2 transporter protein can pump mitoxantrone, doxorubicin, daunorubicin and many other chemotherapeutic drugs out of from the tumor cells. ABCG2 has also been considered to be one member of the cancer stem cells (CSCs) markers, many studies have found it highly expressed in breast cancer, lung cancer, glioma and other solid tumors and leukemia.In order to discuss the expression and significance of multidrug resistance protein in the cutaneous malignant melanoma tissues, we use immunohistochemistry to study the expression of ABCG2 in 30 cases of malignant melanoma tissue,30 cases of intradermal nevus tissue, and 30 cases of transplanted tumor of A375 cells in nude mice tissue. The purpose of the study is to further confirmed malignant melanoma existence the expression of multidrug resistance protein ABCG2, and it also provides a theoretical basis for further clarify the possible mechanisms of malignant melanoma drug resistance.Material and Methods1. The expression of ABCG2 in malignant melanoma, intradermal nevus and transplanted tumor of A375 cells in nude mice tissue were immunohistochemically detected.2. tatistics processing:All the statistical analyses were expressed with X±S and performed by the SPSS10.0 software package. The comparisons of the positive expression rate of ABCG2 in malignant melanoma and intradermal nevus tissue were carried by Chi-square with the counting material. The comparison of the mean of positive cell rate of ABCG2 between malignant melanoma and intradermal nevus tissue was carried by the t-test. Correlation coefficient calculated by Spearman. a=0.05 was considered to be of statistical significance.Results1. Expression of ABCG2 in malignant melanoma, nevus and transplanted tumor of A375 cells in nude miceThe positive expression rate of ABCG2 in malignant melanoma and intradermal nevus tissue were 56.67% and 0.00%, respectively, the difference of ABCG2 positive rate between them was of statistically significant (x2=1.00, P<0.05).In transplanted tumor of A375 cells in nude mice, the positive expression rate of ABCG2 was 100.00%.The mean of positive cell rate of ABCG2 in malignant melanoma and intradermal nevus were 30.96%±56.96% and 0.00%, respectively, there was statistically significant difference between the two groups (t=12.104, P<0.05). In transplanted tumor of A375 cells in nude mice, the mean of positive cell rate of ABCG2 was 10.35%±3.99%, the expression is consistent.The positive expression of ABCG2 cells distributed in a lumpy or scatter pattern with large and transparent,or small and darkly stained nucleus.2. The correlation between the mean of positive cell rate of ABCG2 and sex, age and the course of diseaseIn malignant melanoma no correlation was found between the mean of positive cell rate of ABCG2 and sex, age and the course of disease (r1=0.105, r2=0.122, r3= 0.344, P>0.05).Conclusions1. The expression of ABCG2 is different in malignant melanoma and transplanted tumor of A375 cells in nude mice;2. ABCG2 may relevant with the recrudescence of malignant melanoma after the chemotherapy treatment.
        

ABC转运蛋白ABCG2在皮肤恶性黑素瘤中的表达及意义

摘要4-7
Abstract7-10
英文缩写词索引12-13
ABC转运蛋白ABCG2在皮肤恶性黑素瘤中的表达及意义13-35
    引言13-16
    材料与方法16-20
    结果20-22
    讨论22-27
    结论27-28
    附图28-30
    参考文献30-35
综述部分 多药耐药与恶性黑素瘤研究进展35-54
    参考文献47-54
附录部分54-55
    个人简历54-55
    致谢55
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