【作者】 李卓；

【导师】 齐范；

【作者基本信息】 中南大学 ， 泌尿外科， 2012， 博士

【摘要】 第一章膀胱癌组织HER-1/HER-2的表达及其临床意义目的研究膀胱移行细胞癌中HER-1和HER-2的表达情况,探讨HER-1和HER-2的表达与肿瘤的分期、病理分级等临床参数的关系。方法采用免疫组化染色的方法研究56例膀胱移行细胞癌的手术肿瘤标本以及10例正常膀胱组织标本的HER-1和HER-2的表达,并进行分析研究HER-1和HER-2在膀胱肿瘤中的表达是否存在相关性,同时采用统计学分析HER-1和HER-2的表达与与肿瘤分期、病理分级等临床参数之间的关系结果56例膀胱移行细胞癌患者的HER-1表达阳性率为55.4%,明显高于正常膀胱组织中HER-1的阳性率(10%)；HER-2在膀胱肿瘤的表达阳性率为37.5%,也明显高于正常膀胱组织中HER-1的阳性率(0%)。HER-1和HER-2在膀胱癌组织中的表达为正相关性。HER-1在高分期肿瘤(T3-T4)中的阳性率显著高于低分期肿瘤(T1-T2)(67.6%vs.36.4%),在高分级肿瘤(G3)中的阳性率显著高于低分期肿瘤(G1-G2)(72.7%vs.30.4%),在复发者中的阳性率显著高于初发组(75.0%vs.29.2%)。HER-2在高分期肿瘤(T3-T4)中的阳性率显著高于低分期肿瘤(T1-T2)(52.9%vs.13.6%),在复发者中的阳性率显著高于初发组(53.1%vs.16.7%)。结论HER-1和HER-2均在膀胱移行细胞癌中表达异常增加,其两者的表达为正相关性。HER-1的表达与膀胱尿路上皮癌的临床分期、病理分级、肿瘤复发相关。HER-2的表达与膀胱尿路上皮癌的临床分期和肿瘤复发相关。第二章HER-1和HER-2双重酪氨酸激酶抑制剂Afatinib对膀胱癌T24细胞生物学行为的影响目的研究Afatinib对T24细胞增殖、凋亡和侵袭能力的影响。方法采用不同浓度的Afatinib (0,1,5,10,20umol/L)作用T24细胞,用MTT法研究Afatinib对T24细胞的增殖能力的影响；用流式细胞术(FCM)检测Afatinib对人膀胱癌T24细胞凋亡的影响；用细胞粘附实验检测Afatinib对人膀胱癌T24细胞对FN粘附的影响；用Transwell实验研究Afatinib对T24细胞的侵袭能力的影响；同时用western blot检测了侵袭相关蛋白MMP-2与MMP-9表达及凋亡相关蛋白Bcl-2与Bax的表达,通过检查ERK1/2和Akt探讨Afatinib作用于膀胱癌的机制。结果Afatinib对膀胱癌T24细胞的增殖能力有着抑制作用；对T24细胞有着促进凋亡的作用,并且能改变T24细胞Bcl-2与Bax蛋白含量之比；Afatinib对膀胱癌T24细胞的侵袭能力有着抑制的作用,能抑制T24细胞MMP-2及MMP-9蛋白的表达；Afatinib作用于膀胱癌T24细胞后,p-ERK1/2和p-Akt蛋白呈下降趋势,且呈剂量依赖性；而对T24细胞的t-ERK1/2和t-Akt蛋白无影响。结论Afatinib抑制了T24细胞的增殖和侵袭能力,促进了T24细胞的凋亡。第三章Afatinib对膀胱癌T24细胞顺铂化疗敏感性研究目的探讨Afatinib与顺铂联合应用于膀胱癌细胞的协同作用,并对其作用机制及耐药性进行初步探讨。方法先进行膀胱癌T24-ADM细胞的制备,用MTT法观察Afatinib联合顺铂对T24细胞增殖的抑制作用；分别用MTT法检测Afatinib作用前后顺铂对T24-ADM细胞增殖的抑制作用；PCR法检测Afatinib作用前后顺铂对T24-ADM细胞MDR-1基因mRNA的表达水平；用western-blot法检测Afatinib作用前后顺铂对T24-ADM细胞p-gp的表达水平。结果Afatinib和顺铂联合用药的T24细胞的增殖抑制率高于单用顺铂；顺铂对Afatinib作用后的T24-ADM细胞抑制率高于Afatinib作用前；经Afatinib作用后,mdr-1基因的表达和p-gp蛋白的水平都有下调。结论Afatinib与顺铂同时作用膀胱癌T24细胞时,两者在抑制T24细胞增殖上表现出协同作用,Afatinib作用后顺铂对于膀胱T24-ADM细胞的抑制率高于单独用顺铂的抑制率,Afatinib能够有效抑制mdr-1基因的表达和p-gp蛋白的水平。更多还原

【Abstract】 Part one The correlation between expression of HER-1/HER-2in bladder transitional cell carcinoma(BTCC) and clinical significanceAim Explore the situation of expression of HER-1/HER-2in BTCC and evaluate the correlation between HER-1/HER-2with the parameters of BTCC such as tumor stage and grade.Method The expression level of HER-1/HER-2was estimated by Immunohistochemistry technique. And the influence factors were also tested.Result The positive rate of HER-1expression among56patients was55.4%, which was distinct higher than that in normal bladder tissue (10%); the positive rate of HER-2expression among56patients was37.5%, which was distinct higher than that in normal bladder tissue (0%); The expression level of HER-1was obvious greater in high stage group, high grade and relapse group than that in low stage group, low grade group and primary group. The expression level of HER-2was obvious greater in high stage group and relapse group than that in low stage group and primary group.Conclusion The expression of HER-1/HER-2was up-regulated in BTCC, and their expression has the positive correlation. Part two The influence of Afatinib on T24cells’biologic behaviorAim Estimate the influence of Afatinib on T24cells’biologic behavior such as invasion, proliferation and apoptosis.Method The T24cell was grouped depend on the concentration of Afatinib treatment (0,1,5,10,20umol/L). MTT was employed to evaluate the impact of Afatinib to T24proliferation; FCM was employed to evaluate the impact of Afatinib to T24with apoptosis ability; transwell analysis was used to detect the effect of Afatinib to T24invasion. The invasion associated protein MMP-2and MMP-9, the apoptosis associated protein Bcl-2and Bax was detected by western blot. Check the ERK1/2and Akt to explore the mechanism of the Afatinib in bladder cancer.Result The T24cells showed more powerful repression of proliferation and invasion under the treatment of Afatinib. Afatinib could regulate the expression of MMP-2/MMP-9and BAX/Bcl-2proteins. Afatinib could promote the apoptosis ability of T24.Conclusion Afatinib inhibition of T24cell proliferation and invasion and promotes apoptosis in T24cells. Part three Afatinib influencing chemotherapy sensibility that cisplatin treat T24cellAim To research Afatinib influencing chemotherapy sensibility that cisplatin treat T24cellMethod First we prepared the T24-ADM cell, then observation of Afatinib combined with cisplatin on the inhibition of T24by MTT. Using the MTT to test sensibility of cisplatin before and after Afatinib used in T24-ADM cell. Using the PCR to test MDR-1before and after Afatinib used in T24-ADM cell and using the western-blot to test p-gp before and after Afatinib used in T24-ADM cell.Result Combining Afatinib and cisplatin could of inhibit T24cell proliferation better than with cisplatin alone; cisplatin could of inhibit T24cell proliferation better if the Afatinib was used. Afatinib can inhibit the mdr-1gene and P-gp protein.Conclusion Afatinib and cisplatin showed a synergistic effect in the inhibition of T24cell proliferation. Cisplatin could of inhibit T24cell proliferation better if the Afatinib was used. Afatinib can inhibit the mdr-1gene and P-gp protein.更多还原