【摘要】 目的:应用免疫组化法检测抑癌基因p16,p53及DNA聚合酶δ的辅助蛋白PCNA在增生期血管瘤中的表达,及其对血管内皮细胞的影响。方法:实验选取2001-01/2005-04北京世纪坛医院病理科收集的手术切除皮肤增生期血管瘤组织标本36例作为增生期血管瘤组(患者术前均未经任何辅助性治疗),以痔静脉标本30例作为痔静脉对照组。患者均知情同意。①两组标本切片均采用DAKOEnvisions二步法进行免疫组化处理。切片以体积分数为0.03的过氧化氢消除内源性过氧化物酶,微波法抗原修复,92～95℃于pH6.0枸盐酸缓冲液浸泡10min;室温冷却10min,双蒸水洗涤,pH7.2磷酸盐缓冲液浸泡5min;体积分数为0.1的山羊血清(磷酸盐缓冲液稀释)封闭,去血清,加入Ⅰ抗,37℃浸泡30min;pH7.2磷酸盐缓冲液冲洗;Envision孵育,37℃下放置30min;二氨基联苯胺显色,苏木精复染后,脱水、透明、封固。免疫组化阴性对照以磷酸盐缓冲液代替Ⅰ抗,其他步骤维持不变,以确认试剂质量及控制染色效果。②p16,p53阳性信号为棕黄色,以内皮细胞浆和/或核染成棕黄色为阳性细胞。阴性对照除细胞核染成蓝色外,无棕黄色反应物。血管内皮细胞核内见棕黄色颗粒者为PCNA蛋白染色阳性。采用CMIAS高清晰彩色病理图像分析系统分别对两组切片p53,p16及PCNA蛋白的表达进行图像检测,测定每个视野下p16和p53阳性细胞的平均吸光度以及PCNA蛋白阳性表达指数。结果:实验选取手术切除皮肤增生期血管瘤组织标本36例,痔静脉标本30例,全部进入结果分析。①两组p53阳性表达检测及平均吸光度的比较:增生期血管瘤组内皮细胞核内有较多棕黄色颗粒,p53表达强;痔静脉对照组血管内皮细胞核内无棕黄色颗粒沉积,p53表达极弱。增生期血管瘤组p53阳性表达的平均吸光度明显强于痔静脉对照组(6.423±1.415,1.036±0.131,P<0.05)。②两组p16阳性表达检测及平均吸光度的比较:两组切片组织中除细胞核着色外,部分细胞浆有着色。p16的阳性表达在增生期血管瘤组和痔静脉对照组中基本相同,平均吸光度分别为1.241±0.373和1.206±0.267。③两组PCNA蛋白阳性表达指数的比较:PCNA蛋白阳性表达的细胞光镜下可见核内弥漫分布细小的棕黄色颗粒,这些内皮细胞围成血管腔及团块状,核椭圆,体积较大。增生期血管瘤组PCNA蛋白阳性表达指数明显高于痔静脉对照组(67.40±8.79,38.41±9.89,P<0.01)。结论:抑癌基因p53在增生期血管瘤组织中呈高表达,能够促进血管内皮细胞的增殖,但p16的表达与血管内皮细胞增殖的关系不明显。此外PCNA蛋白可作为增生期血管瘤的可靠标志物。更多还原

【Abstract】 AIM: To investigate the expression of p53, p16 and auxiliary protein for polymerase δ(PCNA)in skin proliferating stage hemangioma and its effect on vascular endothelial cell with immunohistochemical method. METHODS: Totally 36 samples of skin proliferating stage hemangioma which were cut in the Department of Pathology, Beijing Shijitan Hospital from January 2001 to April 2005, serving as skin proliferating stage hemangioma group (Experimental group) (No any adjuvant treatments was given before operation), another 30 samples of hemorrhoidal vein were chosen, serving as control group). Informed consent was obtained from the patients. ① DAKO Envisions two-step method was used on the sample sections of the two groups to perform immunohistochemistry. Sections was soaked in 0.03 volume fraction of H2O2 for eliminating endogenous peroxydase and antigen was recovered with microwave. Then, sections were soaked in the citric acid buffer solution (pH 6.0) at 92-95 ℃ for 10 minutes, and cooled at ambient temperature for 10 minutes, washed with double distilled water, soaked in phosphate buffer solution at pH 7.2 for 5 minutes; Afterwards, enveloped with 0.1 volume fraction of goat serum (diluted by phosphate buffer solution) and serum was removed. Sections were soaked for 30 minutes at 37 ℃ following first antibody was at 37 ℃,washed with phosphate buffer solution (pH7.2), incubated by Envision and stayed at 37 ℃ for 30 minutes, developed with diaminobenzidine, re-stained with haematoxylin followed by dehydrated and cleared as well as enveloped. Phosphate buffer solution replaced first antibody in the immunohistochemical negative control, other procedures were kept for affirming the quality of agent and control dyeing. ② The positive sign of p16 and p53 was brown. The positive cells were stained brown in the cytolymph and/or cell nucleus of endotheliocyte. The cell nucleus of negative control group was blue and there were no any brown signs. Those with brown granules in the nucleus of vascular endothelial cells were PCNA protein positive. Imaging detection was performed on sections p53 and p16 as well as the expression of PCNA protein with high-definition colored pathological image analysis system. The mean absorbance of p16 and p53 positive cells and PCNA protein positive expression index were measured in each visual field. RESULTS: Totally 36 samples in experimental group and 30 samples in control group all entered the stage of result analysis. ① Detection of p53 positive expression and comparison of mean absorbance : There were many brown granules and the expression of p53 was strong in experimental group; There was no brown granules of the nucleus of vascular endothelial cells and the expression of p53 was weak in control group. The mean absorbance of p53 positive expression was obviously stronger in experimental group than in control group (6.423±1.415,1.036±0.131,P < 0.05). ② Detection of p16 positive expression and comparison of mean absorbance : nucleus and some cytoplasm were stained in the tissue sections. The positive expression of p16 was the same between experimental group and control group, and the mean absorbance was 1.241±0.373 and 1.206±0.267 respectively . ③ Comparison of PCNA protein positive expression index between two groups : Under the optical microscope, tiny brown granules distributed in the nucleus were seen. These endothelial cells surrounded vascular cavity with clumping shape, oval nucleus and large volume. PCNA protein positive expression was higher in experimental group than in control group (67.40±8.79,38.41±9.89,P < 0.01=. CONCLUSION: p53 presents high expression in the skin proliferating stage hemangioma, can promote the proliferation of vascular endothelial cells, but the expression of p16 is not closely related to the proliferation of vascular endothelial cells. In addition, PCNA protein can be used as the reliable marker of proliferating stage hemangioma.更多还原