胆囊癌CD133阳性细胞侵袭机制的实验研究

Study on Mechanism of Invasion of CD133 Positive Population in Gallbladder Cancer

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 population in gallbladder cancer. Methods The invasive abilities of the CD133 positive cells and the CD133 negative cells were detected by Transwell. The CXCR4 mRNA and protein in the CD133 positive cells and the CD133 negative cells were detected by the semi-quanti- tative RT-PCR, Western blot method, and immunofluorescence, respectively. SDF-lct and AMD3100 were respectively used to stimulate/inhibit the GBC-SD cells. The invasive ability and the migration force were detected in the CD133 posi- tive cells and the CD133 negative cells. The expressions CD133 mRNA and protein of the GBC-SD cells were detected by semi-quantitative RT-PCR and Western blot method, respectively. Results @ The number of invasion cells in theCD133 positive cells was significantly more than that in the CD133 negative cells （23.78±8.74 versus 6.56±3.09, P= 0. 000 7）. （~） The fluorescent protein of CXCR4 in the CD133 positive cells was stronger than that in the CD133 negative cells. The expression of CXCR4 mRNA in the CD133 positive cells was significantly higher than that in the CD133 negative cells （0. 642 4±0. 020 4 versus O. 335 9±0. 043 2, P=0. 004）. The expression of CXCR4 protein in the CD133 positive cells was significantly higher than that in the CD133 negative cells （0. 765 0±0. 106 6 versus O. 409 4±0. 019 5, P=0. 013）. In the CD133 positive cells, compared with the control group, the number of invasion cells was signifi- cantly increased in the SDF- 1 ct group （62.89±15.27 versus 23.78 ＋ 8.74, P=-0. 000 6） and decreased in the AMD3100 group （10.33 ± 2.00 versus 23.78 ± 8.74, P=0. 000 2）. In the CD133 negative cells, compared with the control group, the number of invasion cells was not significant change in the SDF-lct group （6. 89＋4. 23 versus 6. 59＋3.09, P=-0. 41） and in the AMD3100 group （6. 11 ± 2. 67 versus 6. 59 ＋ 3.09, P=-0.38）, respectively. （~） In the CD133 positive cells, compared with the control group, the number of migration cells was significantly increased in the SDF-lct group （74. 56＋ 15.80 versus 35.56＋ 10.97, P=0. 000 3） and decreased in the AMD3100 group （12.67±2.40 versus 35.56± 10. 97, P=0. 000 2）. In the CD133 negative cells, compared with the control group, the number of migration cells was not significant change in the SDF-lct group （9.78±2.04 versus 9.56± 1.74, P=0.43） and in the AMD3100 group （9.54± 1.74 versus 9. 56± 1.74, P=0. 42）. In the GBC-SD cells, compared with the control group, the CD133 mRNA was significantly increased in the SDF-lct group （0. 626 5 ±0. 048 7 versus O. 450 04±0. 024 3, P=0. 004） and decreased in the AMD3100 group （0. 359 3 4±0. 047 3 versus O. 450 0 4± 0. 024 3, P=0. 011） ; the CD 133 protein was significantly increased in the SDF-lct group （0. 508 9±0. 020 7 versus O. 440 9±0. 013 0, P=0. 016） and decreased in the AMD3100 group （0. 317 74±0. 013 7 versus O. 440 9±0. 013 0, P=0. 004）. Conclusion The high invasion ability of CD133 positive population in gallbladder cancer might be due to the high expression of CXCR4.