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目的研究胆囊癌CD133阳性细胞侵袭能力的产生机制。方法 Transwell法检测CD133阳性细胞和CD133阴性细胞的迁移和侵袭能力。半定量聚合酶链式反应(RT-PCR)法、蛋白免疫印迹法、细胞免疫荧光法分别检测CD133阳性细胞和CD133阴性细胞中CXCR4的表达。分别用SDF-1α、AMD3100作用GBC-SD细胞后,Transwell法检测CD133阳性细胞和CD133阴性细胞的迁移和侵袭能力。半定量RT-PCR法检测GBC-SD细胞中CD133 mRNA表达,蛋白免疫印迹法检测GBC-SD细胞中CD133蛋白表达。结果①CD133阳性细胞中穿膜细胞数明显多于CD133阴性细胞(23.78±8.74比6.56±3.09,P=0.000 7)。②CD133阳性细胞中CXCR4mRNA相对灰度值明显高于CD133阴性细胞(0.642 4±0.020 4比0.335 9±0.043 2,P=0.004);CD133阳性细胞中CXCR4蛋白表达相对灰度值明显高于CD133阴性细胞(0.765 0±0.106 6比0.409 4±0.019 5,P=0.013);CD133阳性细胞中CXCR4荧光蛋白表达明显强于CD133阴性细胞。③细胞侵袭能力:穿膜细胞数量在CD133阳性细胞中,与空白对照组(23.78±8.74)相比,SDF-1α组(62.89±15.27)明显增加(P=0.000 6),AMD3100组(10.33±2.00)明显减少(P=0.000 2);在CD133阴性细胞中,与空白对照组(6.59±3.09)相比,SDF-1α组(6.89±4.23)无明显变化(P=0.41),AMD3100组(6.11±2.67)亦无明显变化(P=0.38)。④细胞迁移能力:迁移细胞数量,在CD133阳性细胞中,与空白对照组(35.56±10.97)相比,SDF-1α组(74.56±15.80)明显增加(P=0.000 3),AMD3100组(12.67±2.40)明显减少(P=0.000 2);在CD133阴性细胞中,与空白对照组(9.56±1.74)相比,SDF-1α组(9.78±2.04)无明显变化(P=0.43),AMD3100组(9.54±1.74)亦无明显变化(P=0.42)。⑤在GBC-SD细胞中CD133 mRNA表达:与空白对照组(0.450 0±0.024 3)相比,SDF-1α组明显增加(0.626 5±0.048 7,P=0.004),AMD3100组(0.359 3±0.047 3)明显下降(P=0.011);CD133蛋白表达:与空白对照组(0.440 9±0.013 0)相比,SDF-1α组(0.508 9±0.020 7)明显增加(P=0.016),而AMD3100组(0.317 7±0.013 7)明显下降(P=0.004)。结论胆囊癌CD133阳性细胞高侵袭能力可能由于高表达CXCR4。
Objective To study the mechanism of invasion of CD133 positive cells in gallbladder carcinoma. Methods Transwell assay was used to detect the migration and invasion of CD133 positive cells and CD133 negative cells. The expression of CXCR4 in CD133 positive cells and CD133 negative cells were detected by semi-quantitative polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence. After treated with SDF-1α and AMD3100 respectively, the migration and invasion ability of CD133-positive cells and CD133-negative cells were detected by Transwell assay. CD133 mRNA expression in GBC-SD cells was detected by semi-quantitative RT-PCR and CD133 protein expression in GBC-SD cells was detected by Western blot. Results ① The number of transmembrane cells in CD133 positive cells was significantly higher than that in CD133 negative cells (23.78 ± 8.74 vs. 6.56 ± 3.09, P = 0.0007). ② The relative gray value of CXCR4 mRNA in CD133 positive cells was significantly higher than that of CD133 negative cells (0.642 4 ± 0.020 4 vs 0.335 9 ± 0.043 2, P = 0.004); the relative gray value of CXCR4 protein expression in CD133 positive cells was significantly higher than that of CD133 negative cells (0.765 ± 0.106 6 vs 0.409 4 ± 0.019 5, P = 0.013). The expression of CXCR4 in CD133 positive cells was significantly stronger than that in CD133 negative cells. (3) Cell invasiveness: The number of transmembrane cells in CD133 positive cells was significantly higher in SDF-1α group (62.89 ± 15.27) than in blank control group (23.78 ± 8.74) (P = 0.0006), and that in AMD3100 group (10.33 ± 2.00) (P = 0.000 2). In CD133-negative cells, there was no significant change in the SDF-1α group (6.89 ± 4.23) compared with the blank control group (6.59 ± 3.09) 6.11 ± 2.67) (P = 0.38). ④ Cell migration ability: The number of migrating cells in CD133 positive cells was significantly higher in SDF-1α group (74.56 ± 15.80) than that in blank control group (35.56 ± 10.97) (P = 0.000 3), and that in AMD3100 group (12.67 ± 2.40) (P = 0.0002). In CD133-negative cells, there was no significant change in SDF-1α group (9.78 ± 2.04) compared with blank control group (9.56 ± 1.74) 9.54 ± 1.74) did not change significantly (P = 0.42). ⑤ CD133 mRNA expression in GBC-SD cells: SDF-1α group increased significantly (0.626 5 ± 0.048 7, P = 0.004) compared with the blank control group 3) (P = 0.011). The expression of CD133 protein in SDF-1α group (0.508 9 ± 0.020 7) was significantly higher than that in the blank control group (0.440 9 ± 0.013 0) (P = 0.016) (0.317 7 ± 0.013 7) decreased significantly (P = 0.004). Conclusion The high invasion ability of CD133 positive cells in gallbladder carcinoma may be due to the high expression of CXCR4.