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目的研究CD133作为结肠癌HT29细胞系干细胞特异性生物标志的可行性,为治疗结肠癌提供新的靶点及思路。方法克隆形成试验于6孔板中按浓度梯度各接种50、100、200个经磁珠分选的CD133+或CD133-结肠癌HT29细胞,培养2~3周至肉眼可见的细胞克隆时计数克隆数并计算克隆形成率;经磁珠分选的CD133+或CD133-HT29细胞接种于BALB/C裸鼠侧腹部皮下,观察裸鼠成瘤情况,绘制裸鼠生长曲线。HE染色观察病理学变化。提取瘤体组织RNA,反转录-聚合酶链反应(RT-PCR)检测CD133表达。成瘤率的比较应用Fisher’s Exact Test。结果克隆形成试验中,CD133+HT29细胞在各接种密度下的克隆形成率分别为65.32%±3.56%、78.01%±3.79%、91.23%±4.23%;CD133-分别为20.05%±2.51%、35.19%±2.94%、44.33%±3.03%;同一接种密度下CD133+克隆形成率明显高于CD133-,差异均有统计学意义(P<0.05)。成瘤试验中,CD133+HT29细胞高剂量组于第4天观察到移植瘤全部形成,HE染色呈典型结肠癌组织,RT-PCR检测到CD133表达。而等剂量的HT29 CD133-细胞至第5周仍无明显移植瘤形成。结论在结肠癌HT29细胞系中CD133可以作为鉴定肿瘤干细胞的特异性生物标志,为后续试验研究奠定基础。
Objective To study the feasibility of using CD133 as a biomarker of stem cells in colon cancer HT29 cells and to provide a new target for the treatment of colon cancer. Methods Clone formation assay 50,100,200 CD133 + or CD133-colon cancer HT29 cells sorted by magnetic beads were inoculated into 6-well plates in concentration gradients, and the number of colonies was counted after culturing for 2 to 3 weeks to the macroscopic cell clone The colony formation rate was calculated. The CD133 + or CD133-HT29 cells sorted by magnetic beads were inoculated subcutaneously in the flank of BALB / C nude mice. The growth of the nude mice was observed. HE staining to observe the pathological changes. Tumor tissue RNA was extracted and CD133 expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). The rate of tumor formation was compared using Fisher’s Exact Test. Results The clonogenic rates of CD133 + HT29 cells at each inoculation density were 65.32% ± 3.56%, 78.01% ± 3.79%, 91.23% ± 4.23% and CD133- were 20.05% ± 2.51%, 35.19 % ± 2.94% and 44.33% ± 3.03%, respectively. The formation rate of CD133 + clones under the same inoculation density was significantly higher than that of CD133-, with statistical significance (P <0.05). In the tumorigenicity test, the allografts were found on the 4th day in the high-dose CD133 + HT29 cell group. The typical colon cancer tissues were stained with HE, and the expression of CD133 was detected by RT-PCR. The same dose of HT29 CD133-cells by the 5th week, there is no significant tumor formation. Conclusion CD133 can be used as a specific biomarker in the identification of cancer stem cells in colon cancer HT29 cell line, which lays the foundation for further research.