细胞紧密连接基因claudin-4在子宫内膜癌发病和化疗中的作用

Study of claudin-4 in the diagnosis and treatment of endometrial carcinoma

目的探讨细胞紧密连接基因claudin-4在子宫内膜癌发病和化疗中的作用。方法选择62份子宫内膜样腺癌（EEC）、30份不典型增生子宫内膜和60份正常子宫内膜组织，免疫组化方法和实时荧光定量PCR技术检测claudin-4蛋白、mRNA的表达。90只雌性BALB／c裸鼠腋下注射EEC细胞系Ishikawa，构建人子宫内膜癌裸鼠模型，随机分组进行顺铂（顺铂组）和紫杉醇（紫杉醇组）的腹腔注射化疗（以腹腔注射生理盐水作为对照组），测量化疗前后裸鼠体质量及移植瘤体积的变化，实时荧光定量PCR技术和免疫组化方法检测化疗前后claudin-4mRNA和蛋白表达的变化。结果在62份EEC中，21份claudin-4蛋白的免疫组化染色呈中等强度，41份呈强阳性表达；30份不典型增生子宫内膜中8份呈弱阳性染色，16份显示中等强度染色，6份显示强阳性染色；60份正常子宫内膜中，28份呈弱阳性染色，32份无claudin-4蛋白的表达。实时荧光定量PCR检测显示，claudin-4mRNA在EEC、不典型增生内膜和正常子宫内膜中的表达量分别为170±12、89±15和18±3，claudin-4mRNA在EEC中的表达量高于不典型增生和正常子宫内膜，分别比较，差异均有统计学意义（P〈0．05）。子宫内膜癌裸鼠模型检测发现，紫杉醇组和对照组裸鼠化疗前后移植瘤体积及claudin-4的表达均无显著变化；在顺铂组，化疗前移植瘤体积为（0．73±0．12）cm^3，化疗后明显缩小至（0．51±0．21）cm^3。实时PCR检测显示，顺铂组化疗前、后claudin-4mRNA的表达量分别为273±27和153±35，claudin-4mRNA的表达显著降低。结论claudin-4的过度表达可能与子宫内膜癌的恶性行为有关，claudin-4可作为子宫内膜癌治疗疗效的监测指标。

Objective To clarify the role of claudin4 in endometrial tumorigenesis and explore claudin4 be as potentially useful agent in the treatment of endometrial carcinoma. Methods The expression of claudin-4 in 62 endometrioid endometrial carcinoma （EEC）, 30 atypical hyperplasia endometrial tissue and 60 human normal endometrium was determined using immunohistochemistry and real- time PCR. Ninety female BALB/c mice were transplanted with Ishikawa endometrial cancer ceils, which were divided into three groups with different intraperitoneal treatments with cisplatin, paclitaxel and saline solution. After the observation period, the tumors were extracted and stained with monoclonal antibody against claudin4. The messenger RNA expression of claudin-4 was also detected using real-time PCR. Results Among the EEC samples, 34% （21/62） showed medium staining for claudin4 and 66% （41/62） showed intense staining. In atypical hyperplasia group, 27% （8/30） showed weak staining, 53% （16/30） showed medium staining and 20% （6/30） showed intense staining for claudin-4. Of the normal endometrial tissue, 47% （28/60） showed weak staining and 53% （32/60） showed no staining for claudin4. According to real-time PCR, the relative quantity of claudin4 was 170 ± 12 in EEC group, 89 ± 15 in atypical hyperplasia group and 18 ± 3 in normal endometrium. Compared with those in atypical hyperplasia group and normal endometrium group, the protein and mRNA expression of claudin4 were significantly increased in the group of EEC （ all P 〈 0. 05 ）. In the study of Ishikawa xenografts, no significant changes in tumor volume and claudin-4 expression were shown in paclitaxel group compared with that in the control group. Nevertheless, a significant reduction of the tumor growth and a significant decrease in claudin-4 expression were observed in cisplatin group. After cisplatin treatment, the tumor volume was significantly decreased [ （0. 51±0. 21 ） versus （0. 73 ±0. 12） cm^3], and the mRNA expression of claudin-4 was also significantly decreased （153 ± 35 versus 273 ±27）. Conclusion These results demonstrate that claudin-4 is strongly expressed in EEC, which may be a useful biomarker to monitor the effects of chemotherapy in patients with endometrial carcinoma.