腺样囊性癌多药耐药机制的实验研究
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摘要
目的
建立腺样囊性癌细胞耐药模型,分析多药耐药相关标志p53、gstpl、mdrl、bcl-2、mrp和lrp的表达,并了解P53、谷胱甘肽-S-转移酶(GST-π)、P糖蛋白(P170)、Bcl-2、多药耐药相关蛋白(MRP)和肺耐药蛋白(LRP)在眼眶腺样囊性癌组织中的关系与临床意义,为阐明腺样囊性癌的多药耐药机制、指导临床制定化疗方案、改善预后及逆转耐药提供理论依据及临床参考。
方法
1.免疫组化法检测51例眼眶腺样囊性癌临床标本和10例正常眼眶组织中P53、GST-π、P170、Bcl-2、MRP和LRP的表达状况。
2.以长春新碱(VCR)为诱导剂,通过浓度递增间断刺激法对人腺样囊性癌细胞系(ACC)进行体外诱导耐药,建立耐VCR的腺样囊性癌细胞系ACC/VCR。
3.细胞计数法绘制细胞生长曲线,流式细胞术检测凋亡,噻唑蓝(MTT)比色法检测细胞对化疗药物敏感性,RT-PCR法定量分析细胞中多药耐药相关基因的表达水平,免疫组化法检测细胞中P53、GST-π、P170和Bcl-2的表达情况。
结果
1.P53、GST-π、P170和Bcl-2在眼眶腺样囊性癌组织中的表达阳性率分别为52.94%、100%、82.35%和64.71%,显著高于眼眶正常组织。实性型腺样囊性癌中P53和P170的表达率显著高于其在腺样-管状型中的表达率;P53和Bcl-2在复发组中的表达率显著高于其在初发组
OBJECTIVESIn this present study, we establish an in vitro resistance model of adenoid cystic carcinoma, detect six multidrug resistance-associated Markers: p53, gstpl, mdrl, bcl-2, mrp and 1rp, studied the expression and clinical significances of the P53, P-glycoprotein (P170), glutathione S-transferase (GST-π), Bcl-2, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in orbital adenoid cystic carcinoma. These can provide theoretical information and clinical referance for elucidating the mechanisms of multidrug resistance (MDR), improving the chemotherapy scheme, evaluating the prognosis and reserving the MDR in orbital adenoid cystic carcinoma.METHODS1. The expression of P53, GST-π, P170, Bcl-2, MRP and LRP in 51 cases of orbital adenoid cystic carcinoma and 10 cases of normal orbital tissues were detected by immunohistochemisry analysis.2. The resistance cell line, ACC/VCR, was induced in the ACC cell line in vitro by progressive concentrations of vincristine(VCR), a drug of choice in the treatment of adenoid cystic carcinoma.3. The growth curve ,drug sensitivity in the parental and resistant cell lines were investigated by cell counting, flow cytometry and MTT assay. The expression of p53, gstp1, mdrl and bcl-2 gene were detected by RT-PCR while P53, GST- π , P170 and Bcl-2 protein were
detected by immunohistochemisry analysis.4. The expression of P53> GST- n , P170 and Bcl-2 in 51 cases of orbital adenoid cystic carcinoma and 10 cases of normal orbital tissues were detected by immunohistochemisry analysis.RESULTS1. P53^ GST- it> P170 and Bcl-2 expression percentage were 52. 94%> 1009L 82. 35% and 64. 71% respectively in orbital adenoid cystic carcinoma and were higher than in normal orbital tissues. The positive rate of P53 and P170 expression in adenoid-tubular ACC was higher than in solid ACC. The positive rate of P53 and Bcl-2 expression in recurrent tumor group was higher than in primary tumor group. Significant correlation was found between the expression of P53 and Bcl-2.2. ACC/VCR cell line was cross-resistant to vincristine (VCR), adriamycine (ADM) and pingyangmycin (PYM), but not resistant to Cyclophosphamide (CTX)> fluorouracil (5-FU) and cisplatin (DDP). There was no significant difference in the doubling time and morphological changes between ACC cell line and ACC/VCR cell line.3. In ACC cell line, the expressions of p53 and gstpl mRNA were lower and mdrl and bcl-2 mRNA were undetectable while P53 and GST- n were positive and P170 and Bcl-2 were negative. In ACC/VCR cell line, the expressions of p53x gstpK mdrl and bcl-2 mRNA were relatively higher while P53, GST-n. P170 and Bcl-2 were positive.CONCLUSIONS1. The overexpressions of P53>GST-n ^ PI70 and Bcl-2 were participated
建立腺样囊性癌细胞耐药模型,分析多药耐药相关标志p53、gstpl、mdrl、bcl-2、mrp和lrp的表达,并了解P53、谷胱甘肽-S-转移酶(GST-π)、P糖蛋白(P170)、Bcl-2、多药耐药相关蛋白(MRP)和肺耐药蛋白(LRP)在眼眶腺样囊性癌组织中的关系与临床意义,为阐明腺样囊性癌的多药耐药机制、指导临床制定化疗方案、改善预后及逆转耐药提供理论依据及临床参考。
方法
1.免疫组化法检测51例眼眶腺样囊性癌临床标本和10例正常眼眶组织中P53、GST-π、P170、Bcl-2、MRP和LRP的表达状况。
2.以长春新碱(VCR)为诱导剂,通过浓度递增间断刺激法对人腺样囊性癌细胞系(ACC)进行体外诱导耐药,建立耐VCR的腺样囊性癌细胞系ACC/VCR。
3.细胞计数法绘制细胞生长曲线,流式细胞术检测凋亡,噻唑蓝(MTT)比色法检测细胞对化疗药物敏感性,RT-PCR法定量分析细胞中多药耐药相关基因的表达水平,免疫组化法检测细胞中P53、GST-π、P170和Bcl-2的表达情况。
结果
1.P53、GST-π、P170和Bcl-2在眼眶腺样囊性癌组织中的表达阳性率分别为52.94%、100%、82.35%和64.71%,显著高于眼眶正常组织。实性型腺样囊性癌中P53和P170的表达率显著高于其在腺样-管状型中的表达率;P53和Bcl-2在复发组中的表达率显著高于其在初发组
OBJECTIVESIn this present study, we establish an in vitro resistance model of adenoid cystic carcinoma, detect six multidrug resistance-associated Markers: p53, gstpl, mdrl, bcl-2, mrp and 1rp, studied the expression and clinical significances of the P53, P-glycoprotein (P170), glutathione S-transferase (GST-π), Bcl-2, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in orbital adenoid cystic carcinoma. These can provide theoretical information and clinical referance for elucidating the mechanisms of multidrug resistance (MDR), improving the chemotherapy scheme, evaluating the prognosis and reserving the MDR in orbital adenoid cystic carcinoma.METHODS1. The expression of P53, GST-π, P170, Bcl-2, MRP and LRP in 51 cases of orbital adenoid cystic carcinoma and 10 cases of normal orbital tissues were detected by immunohistochemisry analysis.2. The resistance cell line, ACC/VCR, was induced in the ACC cell line in vitro by progressive concentrations of vincristine(VCR), a drug of choice in the treatment of adenoid cystic carcinoma.3. The growth curve ,drug sensitivity in the parental and resistant cell lines were investigated by cell counting, flow cytometry and MTT assay. The expression of p53, gstp1, mdrl and bcl-2 gene were detected by RT-PCR while P53, GST- π , P170 and Bcl-2 protein were
detected by immunohistochemisry analysis.4. The expression of P53> GST- n , P170 and Bcl-2 in 51 cases of orbital adenoid cystic carcinoma and 10 cases of normal orbital tissues were detected by immunohistochemisry analysis.RESULTS1. P53^ GST- it> P170 and Bcl-2 expression percentage were 52. 94%> 1009L 82. 35% and 64. 71% respectively in orbital adenoid cystic carcinoma and were higher than in normal orbital tissues. The positive rate of P53 and P170 expression in adenoid-tubular ACC was higher than in solid ACC. The positive rate of P53 and Bcl-2 expression in recurrent tumor group was higher than in primary tumor group. Significant correlation was found between the expression of P53 and Bcl-2.2. ACC/VCR cell line was cross-resistant to vincristine (VCR), adriamycine (ADM) and pingyangmycin (PYM), but not resistant to Cyclophosphamide (CTX)> fluorouracil (5-FU) and cisplatin (DDP). There was no significant difference in the doubling time and morphological changes between ACC cell line and ACC/VCR cell line.3. In ACC cell line, the expressions of p53 and gstpl mRNA were lower and mdrl and bcl-2 mRNA were undetectable while P53 and GST- n were positive and P170 and Bcl-2 were negative. In ACC/VCR cell line, the expressions of p53x gstpK mdrl and bcl-2 mRNA were relatively higher while P53, GST-n. P170 and Bcl-2 were positive.CONCLUSIONS1. The overexpressions of P53>GST-n ^ PI70 and Bcl-2 were participated
引文
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