【作者】 许险峰；

【导师】 霍霞；

【作者基本信息】 汕头大学 ， 病理学， 2003， 硕士

【摘要】 背景 PTEN/MMAC1/TEP1(以下简称PTEN)是1997年发现的第1个具磷酸酯酶活性的抑癌基因，该基因定位于染色体10q23.3，PTEN分子量为55kD，具有双功能特异性磷酸酶活性，已经发现在许多进展期肿瘤中有其突变和缺失。食管癌是潮汕地区高发肿瘤，以食管鳞癌居多，对其成因许多学者进行了研究，我们通过对PTEN蛋白水平的检测，观察PTEN在正常食管鳞状上皮，Ⅰ～Ⅲ级食管鳞癌组织中是否有表达的改变，从而探讨PTEN与食管癌发生发展之间的关系，这将有利于探讨食管癌发生发展的分子机理，临床上有助于食管癌病理分级诊断及病人预后的估计，将其转染肿瘤细胞或其单克隆蛋白导入治疗还为食管癌的治疗提供了新的方法和策略。 目的 采用免疫组织化学方法对不同病理分级食管鳞癌及癌旁鳞状上皮中PTEN的表达进行检测，观察PTEN在不同分化程度的食管癌中表达的差异，探讨其与食管癌发生、发展的关系；运用流式细胞检测方法和western blot蛋白印迹对人胚食管上皮永生化细胞株SHEE、SHEEMT及食管癌细胞株EC8712中PTEN的表达进行检测，分析在不同分化的细胞株中PTEN的表达是否有差异，判断食管上皮细胞在恶性转化的过程中是否有PTEN缺失现象的发生。 方法 1.免疫组织化学方法 1)食管癌组织选用56例2002年我校附属二院食管癌分级存档标本，采用免疫组织化学SABC染色法观察食管癌、癌旁食管粘膜PTEN蛋白的表达，统计学分析其表达与食管癌病理分级及其他临床病理的联系; 2)细胞株进行人胚食管上皮永生化细胞株SHEE、S}IEEMT及食管癌细胞株EC8712的培养，收集后制成石蜡包埋标本，切片后进行免疫组织化学SABC法检测，结果用HPIAS一 1 000图象分析系统测定其灰度值，分析PTEN在永生化及食管癌细胞株中的表达情况，以及在三种细胞株中表达的差异; 2.流式细胞仪检测取生长状态良好的51正E和SHEEMT、EC8712三种细胞株，消化离心，固定，间接荧光标记后进行流式细胞检测，结果用软件包WinMDI version 2.9分析平均荧光强度，比较PTEN在三种细胞株的表达; 3.Western blof蛋白印迹将铺满整瓶的SHEE、SHEEMT和EC8712细胞株进行蛋白提取，浓度测定，调整浓度后用研触stem blot蛋白印迹方法半定量检测食管上皮永生化细胞SHEE和SHEEMT、EC8712细胞株中PTEN蛋白的表达，实验结果用Bio一Rad公司的Quantity one一4，2，3分析软件进行蛋白条带的光密度分析，进一步检测抑癌基因PTEN在来源于食管鳞状上皮的不同分化程度的细胞株中表达是否有区别。 结果 1.免疫组织化学 1)食管癌组织PTEN蛋白在正常组织弥漫均一表达，主要定位于细胞浆内，偶见核表达，呈棕黄色颗粒;在癌组织中，阳性表达于细胞浆和细胞核均可见，呈散在分布，有的癌巢有表达，有的汕头大学医学院硕士学位论文则无。在56例石腊包埋标本中，有32例边缘有正常食管鳞状上皮组织，其中PTEN弱阳性表达7例(2 1 .88%)，阳性表达22例 (68.75%)，3例无表达(9 38%)，56例食管鳞癌组织内有9例阳性 (16.07%)，20例弱阳性(35.71%)，其余27例PTEN无表达 (48.22%)，其中I级鳞癌表达率(78.57%)高于11级(60%)和111级(27.27%)，统计分析I一111级鳞癌表达率有区别(P<0.05)。PTEN蛋白在正常鳞状上皮组织，各级食管鳞癌的表达率依次降低，差异有统计学意义(P<0.05)，在食管鳞癌中的表达率明显低于癌旁食管鳞状上皮粘膜，有统计学意义(P<0.05); 2)细胞株三种细胞株均有PTEN的表达，定位于胞核和胞浆，但强弱不一，SHEE细胞株PTEN蛋白几乎均呈强阳性或阳性表达，而SHEEMT细胞株仅少数细胞强表达，EC8712细胞株均为弱表达，灰度值分析三种细胞株PTEN蛋白表达强弱顺序为SHEE>S HEEMT>EC8712，三者差异有统计学意义(P<0.05); 2.流式细胞仪检测人胚食管上皮永生化细胞株SHEE、s匪EMT及食管癌细胞株Ec8712 PTEN蛋白平均荧光表达强度依次降低，峰值依次左移，阳性、强阳性细胞所占比例依次减少 (P<0 .05)。表明三种细胞株随恶性程度的增强，PTEN蛋白表达量依次降低; 3.、vestern blot蛋白印迹由Western blot蛋白印迹条带可见，SHEE细胞株的条带明显比SHEEMT、EC8712细胞株的条带深，用Quantity one一4.2.3分析软件分析得出平均光密度值，三种细胞株平均光密度值顺序依次为SHEE>SHEEMT>EC8712，统计学分析有差异(P<0.05)，光密度曲线分析可见两种永生化细胞株峰值较癌细胞株高，SHEE细胞株峰值又较51正EMT细胞株高，与上述免疫组织细胞化学及流式细胞仪检测结果趋势基本一致。结论汕头大学医学院硕士学位论文 1 .PTEN蛋白在食管鳞癌及癌旁鳞状上皮组织、人胚食管上皮永生化细胞株51…正E、SHEEMT和食管癌细胞株EC8712中定位于胞核和胞浆。 2.PTEN在SHEE、Sr正EMT和EC8712三种均来源于食管鳞状上皮但分化程度不同的细胞株中表达有所差别，高分化SHEE细胞株表达水平最高，低分化EC8712细胞株表达最低，表明PTEN在三种细胞株的表达与其分化程度正更多还原

【Abstract】 Backround The recently identified PTEN/MMAC1/TEP1 gene is the first tumor suppressor which has sequence homology with dual-specificity phosphatase. PTEN is a multifunctional protein endowed with a phosphatase activity capable of dephosphosphorylating both tyrosinephosphate, serine/threonine phosphate residues on proteins and phospholipids of the phosphatidy linositol pathway[1-3]. PTEN appears to be mutated at considerable frequency in several types of human tumors, including those from brain, breast, endometrium, and prostate[4-6]. PTEN play an important role in pathogenesis of tumor, tumor cell invasion and metastasis. In this experiment, we investigated the expression of PTEN in oesophagus carcinoma tissue, normal oesophageal epithelium tissue, three types of cell line-SHEE (an immortalized human fetal esophageal epithelial cell line induced by HPV 18 E6E7 AAV, the cell line has propagated continuously for more than 50 passages, the phenotype keeps the characteristics of primary epithelial cells), SHEEMT (a malignantly transformed immortalized esophageal epithelial cell line) and EC8712 (poor-differentiated esophageal squamous carcinoma cell line).Objective The research aims to detect expression of PTEN in esophageal squamous cell carcinomas with the SABC immunohistochemistry method to study the relationship between its expression with pathological grade of esophagus cancer, and three typesof cell line on different differentiation phase of esophageal squamous epithelial cell were examined the PTEN expression by means of cytometry and Western blot methods.Methods1. Immunohistochemistry The expression level of PTEN was detected with the SABC immunohistochemistry method in 56 cases of paraffin-embedded oesophageal squamous cell carcinoma tissues, with 32 samples paired with normal tissue. The relationship between the geneexpression and clinicopathological features was analyzed. Furthermore, three types of cell line, including SHEE, SHEEMT and EC8712 were cultivated and were also investigated by SABC immunohistochemistry method;2. Flow cytometry Three types of cell line were quantitatively examined by flow cytometry methods for the oncogene PTEN expression. Using goat anti-mouse IgG-FITC labeling and flow cytometry, we analyzed distributive image of PTEN content in cell of immortalized esophageal epithelium and esophageal carcinoma cell and compared its expression with WinMDI 2.9 software;3. Western blot Three types of cell line were cultured and were quantitatively examined by western blot methods for the oncogene PTEN expression. The OD of PTEN expression in cell of immortalized esophageal epithelium and esophageal carcinoma cell was analyzed and compared with Quantityone-4, 2, 3 software.Results1. Immunohistochemistry Positive staining was observed in the cytoplasma as well as in the nucleus. Normal esophageal squamousepithelial cell uniformly showed strong PTEN expression, while in cancer tissue, the positive expression was variable. Of the 56 carcinoma of esophagus cases, there 32 cases paired with normal squamous epithelium. No PTEN expression was detected in 3 out of 32 normal cases, 29 cases were positive for PTEN protein (90.63%). Of the 56 esophageal carcinoma cases, 27 lacked PTEN protein (48.22%). The positive rates of PTEN expression decreased progressively from nomal oesophageal epithelium (29/32) to oesophagus cancer (29/56), with significant difference (P<0.05). The expression level of grade I was higher than that of grade II, III(P<0.05), but the level of grade I was similar with grade II (P>0.05), the expression of PTEN in carcinoma had correlation with pathological stage (P<0.05). PTEN expression also show correlation with other factors as chorion invasion, but no correlation with lymphocytic infiltration; The immunohistochemistry result of cell lines showed the positive signals were found in cytoplasma and nucleus. The SHEE cell line expressed at higher level than SHEEMT cell line and更多还原