子宫内膜癌组织HEEC中肿瘤血管生成相关的差异表达基因的筛选及验证

Angiogenesis related gene expression profiles of human endometrial endothelial cells isolated from tissue of endometrial cancer

目的筛选并验证子宫内膜癌和正常子宫内膜组织人子宫内膜血管内皮细胞（HEEC）中肿瘤血管生成相关的差异表达基因。方法（1）选取山东省肿瘤医院2007年6—11月间收治的5例子宫内膜癌患者的癌组织（内膜癌组1）以及5例因妇科良性病变行子宫切除术患者的正常子宫内膜组织（对照组1）标本，分离两组HEEC，应用基因芯片技术检测子宫内膜癌组织HEEC中差异表达基因（指差异表达倍数〉2或〈0．5者，其中差异表达倍数〉2者为上调基因、差异表达倍数〈0．5者为下调基因），筛选出其中与肿瘤血管生成密切相关的基因。（2）另选取2007年1月-2008年4月间收治的36例子宫内膜癌患者的癌组织（内膜癌组2）以及10例因妇科良性病变行子宫切除术患者的正常子宫内膜组织（对照组2）标本，实时定量PCR技术和免疫组化法验证两组HEEC中差异表达上调基因——内皮细胞特异分子1（ESMl）、基质金属蛋白酶（MMP）10、骨桥蛋白（SPP1）、高移动族框1（HMGBl）mRNA和蛋白的表达。比较实时定量PCR技术和免疫组化法检测结果与基因芯片技术检测结果的一致性。结果（1）基因芯片技术检测显示，从内膜癌组1和对照组1中共筛选出317个差异表达基因，其中191个为上调基因、126个为下调基因。上调基因主要包括与细胞外基质功能、细胞周期调节相关的基因，其中与肿瘤血管生成相关的差异表达基因97个；下调基因主要包括具有潜在抗血管生成和增殖作用的基因，其中与肿瘤血管生成相关的差异表达基因44个。（2）实时定量PCR技术检测显示，内膜癌组2HEEC中ESM1、MMP-10、SPP1、HMGB1 mRNA的表达水平分别为0．898、3．890、1．443、1．881，分别与对照组2（分别为0．112、0．323、0．045、0．414）比较，差异均有统计学意义（P〈0．05）。免疫组化法检测显示，内膜癌组2HEEC中SPP1、MMP-10、ESM1、HMGB1蛋白均呈阳性表达，而对照组2中SPP1、ESM1、HMGB1蛋白均呈阴性表达。实时定量PCR技术和免疫组化法检测结果与基因芯片技术检测结果具有良好的一致性。结论子宫内膜癌和正常子宫内膜组织HEEC中肿瘤血管生成相关基因的表达存在明显差异，这些差异表达基因为子宫内膜癌抗血管生成治疗研究提供了新的靶点。

Objective To identify and verify the different genes expression pattern between human endometrial endothelial cells （HEEC） isolated from endometrial cancer and normal endometrium. Methods Endothelial cells were isolated from 5 patients with endometrial cancer （ endometrial cancer group 1 ） and 5 patients with normal endometria tissue （ control group 1 ） admitted from June to November 2007 in Shandong Cancer Hospital. Global expression patterns of endothelial cells were examined using oligonucleotide microarrays. Tissues from 36 patients with endometrial cancer（ endometrial cancer group 2） and 10 normal endometrial tissues （control group 2） admitted from January 2007 to April 2008 were selected to verify the expression of different genes, in which up-regulated genes including ESM1, MMP-10, SPP1 and HMGB1 were tested by quantitative real-time PCR and immunohistochemistry. Results Microarray analyses revealed 317 genes that exhibited 〉 2-fold or 〈 0. 5 differences were identified （including 191 genes up-regulated and 126 down-regulated）. Pathway analysis showed that these genes involved cell cycle, cell adhesion molecules, and extracellular matrix-receptor interaction were obviously predominant. Of them, 97 up-regulated genes and 44 down-regulated genes were related to angiogenesis. The mRNA expression of ESM1, MMP-10, SPPI and HMGB1 in endometrial cancer group 2 were 0. 898 , 3. 890, 1. 433 and 1. 881, respectively. Positive expression of SPP1, MMP-10, ESM1 and HMGB1 was observed in endometrial cancer group 2. However, the SPP1, ESMI and HMGB1 was negative expressed in control group 2. Conclusion It shows that there are the different angiogenesis related genes between endometrial cancer and normal endothelium, which will provide insights into the anti-angiogenesis therapy for endometrial cancers.