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目的 了解环磷酰胺 (CP)和塞替哌 (TEPA)诱导永生化人支气管上皮细胞 (BEAS 2B)的恶性转化株内BEAS CP和BEAS TE细胞p16和p15基因的突变情况。方法 采用聚合酶链反应 (PCR)、聚合酶链反应 单链构象多态性分析 (PCR SSCP)及DNA序列分析技术。结果 BEAS CP细胞p15基因的外显子 1和外显子 2都出现了异常带型。BEAS TE细胞p16基因的外显子 1及外显子 2a出现了异常带型和迁移率滞后现象 ;p15基因外显子 1的 2条单链带之间出现异常条带。DNA序列分析发现BEAS CP细胞的p15基因外显子 1的 182位有C的插入 ,2 0 6位有G的插入 ;外显子 2的第 30 8位密码子有C→T(TCC→TTC)转换 (Ser→Phe) ,第 32 7位密码子有T→A(AAT→AAA)颠换 (Asn→Lys)。BEAS TE细胞的p16基因外显子 2a的第 12 5位密码子有G→A(CGC→CAC)转换 (Arg→His)。结论 p15基因在CP诱导BEAS 2B发生恶性转化的过程中发挥了重要作用。在TEPA诱导BEAS 2B细胞发生恶性转化的过程中可能涉及了p16基因的失活。
Objective To investigate the mutations of p16 and p15 genes in BEAS CP and BEAS TE cells induced by cyclophosphamide (CP) and cetipiperazine (TEPA) in immortalized human bronchial epithelial cells (BEAS 2B). Methods Polymerase chain reaction (PCR), polymerase chain reaction single strand conformation polymorphism (PCR SSCP) and DNA sequence analysis were used. Results The aberrant bands of exon 1 and exon 2 of p15 gene were found in BEAS CP cells. Exon 1 and exon 2a of p16 gene in BEAS TE cells showed abnormal banding and mobility hysteresis. Abnormal bands were found between two single strands of exon 1 of p15 gene. DNA sequence analysis revealed that BEAS CP cells had a C insertion at position 182 of exon 1 of p15 gene and a G insertion at position 206, and C → T (TCC → TTC) at exon 2 ) Conversion (Ser → Phe) and codon 32 with T → A (AAT → AAA) transversion (Asn → Lys). The codon 12 of exon 2a of p16 gene of BEAS TE cells has G → A (CGC → CAC) transition (Arg → His). Conclusion The p15 gene plays an important role in the CP-induced malignant transformation of BEAS 2B. Inactivation of p16 gene may be involved in the TEPA-induced malignant transformation of BEAS 2B cells.