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目的探讨分化抗原9(CD9)在个旧矿粉诱导永生化人支气管上皮细胞(BEAS-2B)恶性转化为肺癌细胞过程中的异常表达和基因突变。方法以BEAS-2B为对照组(20瓶),以个旧矿粉体外诱导BEAS-2B所产生的恶性转化细胞为实验组(20瓶)。采用免疫细胞化学技术检测CD9蛋白在BEAS- 2B和恶性转化细胞中的表达。采用逆转录-聚合酶链反应(RT-PCR)技术检测CD9 mRNA在BEAS-2B和恶性转化细胞中的表达;并对其RT-PCR产物进行DNA序列测定。结果CD9蛋自在BEAS-2B中的表达率(100%,20/20)与其在恶性转化细胞中的表达率(35%,7/20)差异有统计学意义(P<0.01)。CD9 mRNA在BEAS-2B中的表达强度(0.91±0.09)明显高于其在恶性转化细胞中的表达强度(0.34±0.14),差异有统计学意义(P<0.01)。DNA序列测定显示,在BEAS-2B的恶性转化细胞中CD9基因存在2处点突变,在第231位置发生G-→T转换;在第119位置发生T→A转换,而且这2处的替代引起的错译突变均导致了编码氨基酸的改变,第40位氨基酸由谷胺酰胺(Gln,Q)变为组氨酸(His,H);第3位氨基酸由缬氨酸(Val,V)变为天冬氨酸(Asp,D)。结论CD9的表达缺失或下调以及基因突变在个旧矿粉诱导BEAS-2B恶性转化为肺癌细胞的过程中可能发挥重要作用。
Objective To investigate the aberrant expression and gene mutation of differentiation antigen 9 (CD9) during the malignant transformation of immortalized human bronchial epithelial cells (BEAS-2B) into lung cancer cells. Methods BEAS-2B was used as the control group (20 bottles). The experimental group (20 bottles) was induced by BEAS-2B with Gejiu mineral powder in vitro. Immunocytochemistry was used to detect the expression of CD9 protein in BEAS-2B and malignant transformed cells. The expression of CD9 mRNA in BEAS-2B and malignant transformed cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The RT-PCR products were sequenced. Results The positive rate of CD9 expression in BEAS-2B (100%, 20/20) was significantly higher than that in malignant transformed cells (35%, 7/20) (P <0.01). The expression intensity of CD9 mRNA in BEAS-2B was significantly higher than that in malignant transformed cells (0.91 ± 0.09 vs 0.34 ± 0.14, P <0.01). DNA sequence analysis showed that there were two point mutations in the CD9 gene in malignant transformed cells of BEAS-2B, a G- → T transition at position 231, a T → A transition at position 119, and substitutions at these two positions All the amino acid changes from glutamine (Gln, Q) to histidine (His, H); the third amino acid is changed from valine (Val, V) Aspartic acid (Asp, D). Conclusion The deletion or down-regulation of CD9 expression and gene mutation may play an important role in the process of malignant transformation of BEAS-2B into lung cancer cells by Gejiu mineral powder.