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目的研究ELTD1基因在人结直肠正常上皮细胞和肿瘤细胞之间的表达差异,明确其表达蛋白的细胞定位。方法分别抽提对数生长期的人结直肠癌Lo Vo细胞株和正常上皮细胞NCM460细胞株的总RNA,RT-PCR半定量技术扩增靶基因ELTD1和内参基因B2M(β2微球蛋白)的mRNA,利用ELTD1/B2M光密度比值来表示细胞ELTD1 mRNA的相对表达量;Western blot法检测2种细胞株靶蛋白ELTD1和内参蛋白GAPDH,利用ELTD1/GAPDH比值来表示ELTD1蛋白的相对表达量;间接免疫荧光技术检测Lo Vo细胞ELTD1蛋白的细胞定位。结果 Lo Vo细胞和NCM460细胞的ELTD1/B2M灰度比值分别为0.83±0.07和0.11±0.04,ELTD1/GAPDH灰度比值分别是0.74±0.1和0.12±0.04,差异有统计学意义(P<0.05),荧光显微镜下,可见黄绿色荧光信号定位于细胞膜。结论 ELTD1基因在正常上皮NCM460细胞极低表达,而在人结直肠癌Lo Vo细胞株高表达,ELTD1蛋白定位于细胞膜,ELTD1是一种有助于结直肠癌的早期诊断、靶向治疗、疗效判断及预后的潜在生物标志分子。
Objective To study the expression difference of ELTD1 gene between human normal colorectal epithelial cells and tumor cells and to clarify the cellular localization of the expressed protein. Methods Total RNA was extracted from human colorectal cancer Lo Vo cell line and normal epithelial cell line NCM460 in logarithmic growth phase respectively. Semi-quantitative RT-PCR was used to amplify the target genes ELTD1 and B2M (β2 microglobulin) The relative expression of ELTD1 mRNA was expressed by the ratio of ELTD1 / B2M optical density. The target protein ELTD1 and internal reference protein GAPDH were detected by Western blot and the relative expression of ELTD1 protein by ELTD1 / GAPDH ratio. Immunofluorescence was used to detect the cellular localization of ELTD1 protein in Lo Vo cells. Results The ELTD1 / B2M gray scale ratios of Lo Vo cells and NCM460 cells were 0.83 ± 0.07 and 0.11 ± 0.04, respectively, and the ratios of ELTD1 / GAPDH were 0.74 ± 0.1 and 0.12 ± 0.04, respectively (P <0.05) Fluorescence microscopy, visible yellow-green fluorescence signal located in the cell membrane. Conclusion ELTD1 gene is highly expressed in normal human epithelial cell line NCM460, while it is highly expressed in human colorectal cancer LoVo cell line. ELTD1 protein is localized in the cell membrane. ELTD1 is an early diagnosis, target therapy and therapeutic effect for colorectal cancer Judgment and prognosis of potential biomarkers.