前列腺癌CK5^+CK8^+细胞的分选及其分子生物学特性

Sorting and molecular biological characteristics of CK5^+CK8^+ prostate cancer cells

目的在人前列腺癌细胞系中分选CK5^+CK8^+细胞,了解其分子生物学特性。方法流式细胞术在人前列腺癌细胞系PC3和LNCaP中分选CK5^+CK8^+细胞,用实时荧光定量聚合酶链反应(RT-PCR)和蛋白质印迹法方法检测LNCaP细胞分选的CK5^+CK8^+细胞CK5、CK8、雄激素受体(AR)和前列腺特异抗原(PSA)的表达,细胞迁移实验检测CK5^+CK8^+细胞迁移能力,琼脂糖凝胶克隆形成实验检测CK5^+CK8^+细胞成瘤能力。采用t检验进行统计学分析。结果采用流式细胞术可以在PC3中分选出(21.3±4.6)%的CK5^+CK8^+细胞,在LNCaP中分选出(1.2±0.4)%的CK5^+CK8^+细胞。与非CK5^+CK8^+细胞相比,LNCaP中分选的CK5^+CK8^+细胞CK5 mRNA表达差异有统计学意义(t=10.435,P<0.001),CK8 mRNA表达差异无统计学意义(t=1.974,P=0.121),AR和PSA mRNA表达差异有统计学意义(t=4.317,3.232;P=0.016,0.037)。蛋白质印迹法检测得到类似结果。细胞培养7 d后,CK5^+CK8^+细胞和非CK5^+CK8^+细胞均可以在Transwell小室迁移生长,迁移细胞数分别为(60±7)个和(32±5)个,2者比较差异有统计学意义(t=6.031,P=0.004)。细胞培养14 d后,CK5^+CK8^+细胞和非CK5^+CK8^+细胞均可以在软琼脂糖凝胶中克隆性生长,阳性克隆数分别为(71±5)个和(27±3)个,差异有统计学意义(t=13.009,P<0.001)。结论人前列腺癌细胞系LNCaP和PC3都可以分选出CK5^+CK8^+细胞,LNCaP中CK5^+CK8^+细胞AR和PSA均有一定程度表达,迁移和成瘤能力增强。

Objective To explore CK5^+CK8^+ cells from human prostate cancer cell lines,and to investigate their molecular biological characteristics.Methods Flow cytometry was used to sort CK5^+CK8^+ cells from human prostate cancer cell lines PC3 and LNCaP.CK5^+CK8^+ cells sorted from LNCaP cells were detected by quantitative RT-PCR and Western blotting for the expression of androgen receptor(AR)and prostate specific antigen(PSA).Cell migration assay were used to measure the CK5^+CK8^+ cell migration ability,and agarose gel formation study was used to detect CK5^+CK8^+ cell tumorigenicity.Statistical analysis was performed using t test.Results Flow cytometry was successful in sorting out CK5^+CK8^+ cells that accounted for(21.3±4.6)%of PC3 cells and(1.2±0.4)% of LNCaP cells.Compared with non-CK5^+CK8^+ cells,the CK5 mRNA expression(t=10.435,P<0.001),but not the CK8 mRNA expression(t=1.974,P=0.121)in CK5^+CK8^+ cells sorted from LNCaP cells was statistically different.There was no statistically significant difference in AR and PSA mRNA expression between non-CK5^+CK8^+ cells and CK5^+CK8^+ cells(t=4.317 and 3.232;P=0.016 and 0.037).Similar results were obtained by Western blotting.After 7 days of cell culture,both CK5^+CK8^+cells and non-CK5^+CK8^+ cells were able to migrate and grow in the Transwell chamber,where the numbers of migrating cells were(60±7)and(32±5),respectively,with statistically significant difference between the both(t=6.031,P=0.004).After 14 days of cell culture,both CK5^+CK8^+ cells and non-CK5^+CK8^+ cells showed clonal growth in soft agarose gels,where the numbers of positive clones was(71±5)and(27±3),respectively,with statistically significant difference between the both(t=13.009,P<0.001).Conclusion Both human prostate cancer cell lines LNCaP and PC3 can be used for sorting of CK5^+CK8^+ cells.CK5^+CK8^+ cells sorted from LNCaP express a certain level of AR and PSA,and also show enhanced migration and tumorigenicity.