【作者】 季德才；

【导师】 李泽良；

【作者基本信息】 中国医科大学 ， 外科学， 2010， 硕士

【摘要】 目的抗上皮细胞生长抑制因子(Antiproliferative Factor, APF)是一种由9个氨基酸与半乳糖相连的小分子量、热稳定的唾液酸糖多肽,它含有Frizzled 8的跨膜片段,主要存在于间质性膀胱炎患者的尿液中,具有强烈的抗膀胱上皮细胞生长的作用,同时发现其具有抗膀胱肿瘤细胞生长的作用,尤其对T-24细胞(膀胱癌细胞)。AP-1在肿瘤形成及发展过程中,通过促进细胞增殖、抑制分化、促进肿瘤细胞的侵袭和转移等过程发挥作用。APF作用于正常膀胱上皮细胞后,其c-jun的水平明显下降,从而导致活性蛋白-1(Activator Protein-1,AP-1)的整体水平下降,其活性降低,AP-1调节靶基因的表达,包括细胞增殖、分化、炎症、宿主反应和恶变,而AP-1的活性降低则影响了抑癌基因P53及细胞周期调控基因P21(P21WAF1/CIP1)基因蛋白的表达,从而使细胞生长及增殖受到抑制,细胞则出现凋亡或死亡。HB-EGF在多种组织和细胞中表达,如血管内皮细胞、骨骼肌细胞、膀胱上皮细胞、角质细胞和肿瘤细胞等,对多种细胞具有促分裂增殖的作用。HB-EGF对膀胱肿瘤细胞具有较强的促其增生的作用,而APF具有强烈抑制HB-EGF功能和分泌的作用,从而抑制膀胱肿瘤细胞生长及增生。通过四唑盐比色法(3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide, MTT)检测APF对膀胱癌细胞生长及增殖影响；通过Western blot方法检测不同浓度的有活性APF和无活性APF对膀胱癌细胞中c-jun与HB-EGF的影响,从而进一步探讨APF对膀胱癌细胞生长及增殖的影响。方法培养膀胱癌细胞T-24细胞系,将有活性APF及无活性APF取不同浓度分为实验组和对照组,作用于膀胱癌T-24细胞。通过MTT用酶联免疫检测仪OD490m处测量各组的吸光值来检测膀胱癌细胞增殖率；通过Western blot方法得到c-jun与HB-EGF的电泳图像,根据电泳图象所得条带的宽度和亮度进行半定量分析来检测APF对膀胱癌T-24细胞中c-jun与HB-EGF蛋白表达的影响。结果MTT得出结果用酶联免疫检测仪OD490nm处测量各孔的吸光值,显示随着有活性APF浓度的增加,OD490nm处吸光值明显减低,其吸光值减低有统计学意义(P<0.05)；而加入无活性APF吸光值无明显变化,与实验对照组比较无统计学意义(P>0.05)。根据电泳图象所得条带的宽度和亮度进行半定量分析,显示随着有活性APF浓度增加,c-jun与HB-EGF表达逐渐减少,不同有活性APF浓度组之间差异有统计学意义(P<0.05),无活性APF组与有活性APF0μg组之间无统计学意义(P>0.05)。结论APF可能影响AP-1中的c-jun的表达,c-jun直接作用于细胞桥接使细胞生长停滞,APF作用细胞后,其c-jun的水平明显下降,从而导致AP-1的整体水平下降,其活性降低,则影响了抑癌基因P53及细胞周期调控基因P21基因蛋白的表达,从而抑制了膀胱癌细胞的生长及增殖,同时也能影响HB-EGF的表达,且随着有活性APF浓度的增加,对两者的抑制作用增强,从而增强对膀胱癌细胞生长及增殖的抑制作用。而无活性的APF并不能影响c-jun与HB-EGF的表达,也对细胞的生长无抑制作用。提示APF对治疗膀胱癌患者具有重要的价值,是治疗膀胱癌的一条新途径。更多还原

【Abstract】 ObjectiveAntiproliferative Factor (APF) is a by nine amino acids and galactose linked to the small molecular weight, heat-stable polypeptide of the sialic acid sugar, it contains the Frizzled 8 transmembrane segments, mainly in interstitial the urine of patients with bladder inflammation, with a strong anti-bladder epithelial cell growth, while anti-bladder cancer was found with a role in cell growth, especially for T-24 cells (bladder cancer). AP-1 in tumor formation and development process, by promoting cell proliferation, inhibit differentiation and promote invasion and metastasis of tumor cells play a role in such processes。APF role in normal bladder epithelial cells, its levels of c-jun decreased, resulting in activated protein-1 (Activator Protein-1, AP-1) decline in the overall level of its activity decreased, AP-1 regulation of target genes expression, including cell proliferation, differentiation, inflammation, host response and malignant transformation, while the AP-1 activity reduces the impact of the tumor suppressor gene P53 and cell cycle control gene P21 (P21WAF1/CIP1) gene expression, so that cell growth and proliferation was inhibited, the cells appeared apoptosis or death. HB-EGF was expressed in a variety of tissues and cells, such as vascular endothelial cells, skeletal muscle cells, bladder epithelial cells, keratinocytes and tumor cells, and so on, on a variety of cells and promote proliferation of the role of. HB-EGF in bladder tumor cells has a strong role in promoting their proliferation, while the APF has a strong inhibiting HB-EGF function and the role of secretion and thus inhibit bladder cancer cell growth and proliferation. The influence of APF on bladder cancer cell growth and proliferation by MTT. By Western blot, were found in different concentrations of APF activity and no activity of APF on bladder transitional cell cancer cells in vitro. To explore the role of APF in bladder transitional cells (T-24) cell line c-jun and HB-EGF protein expression and its significance. MethodCultured bladder cancer cell T-24 cell line, there will be a non-active activity of APF and APF to take different concentrations were divided into experimental and control groups, effect on T-24 cells. Enzyme unite immunodetection equipment measure the extinction value of each group in OD 490nm by MTT, and to detect the cell growth rate of bladder tumor. Detected by Western blot,the influence of the different concentration of active and inactive APF on c-jun and HB-EGF in bladder cancer cells to further explore the APF on bladder cancer cell growth and proliferation.ResultThe result which is gotten by MTT measure the extinction value of each hole in OD 490nm by enzyme unite immunodetection equipment. It shows that to follow the increasing of APF density, the extinction value in OD 490nm degrade obviously, and the degrade of entinction value has statistical significance (P＜0.05); But the extinction value of adding incompetence APF has not marked change, and it has not statistical significance compare to experiment control group (P>0.05). According to electrophoretic bands derived from the width of the image and brightness of the semi-quantitative analysis shows that with the increase in the concentration with APF activity, c-jun and HB-EGF expression gradually decreased APF activity of different concentrations of a difference between groups was statistically significant (P<0.05), a non-active APF group and the APF Oμg no statistical significance between groups (P> 0.05).ConclusionAPF may affect the AP-1 in the c-jun expression, c-jun direct role in cell growth arrest bridging the cells, APF role of the cells, the c-jun levels significantly decreased, resulting in the overall level of AP-1 decline in, its activity decreased, then the impact of the tumor suppressor gene P53 and cell cycle regulation of gene expression of P21 protein, which inhibits bladder cancer cell growth and proliferation, but also may affect the expression of HB-EGF and APF activity as there is concentration increased, inhibition of cell growth and proliferation are also gradually. Without APF activity and not affect the expression of c-jun and HB-EGF, but also no inhibitory effect on cell growth. Tip APF for the treatment of bladder cancer patients has important value in the treatment of bladder cancer, a new way.更多还原