前列腺癌相关成纤维细胞的分离培养与鉴定

The primary culture and characteristics of prostate cancer-associated fibroblasts

目的 分离培养前列腺癌相关成纤维细胞（CAF）及正常成纤维细胞（NPF）,探讨两者生物学差异.方法 用Percoll技术分离培养CAF及NPF细胞,用免疫荧光法检测上皮细胞标志物（Pan-CK）,成纤维细胞标志物波形蛋白（Vimentin）和平滑肌细胞标志物平滑肌肌动蛋白（SMA）;用噻唑蓝（MTT）法检测细胞生长能力;用软琼脂糖实验检测两种细胞诱导良性细胞BPH-1恶变.结果 CAF与NPF细胞形态相似,均高表达Vimentin,不表达Pan-CK,CAF细胞高表达SMA.CAF细胞生长速度快于NPF细胞,第4天吸光度值分别为0.98 ±0.07、0.74±0.04,P＜0.05;第6天吸光度值分别为2.06±0.13、1.59±0.14（P ＜0.05）,且能诱导BPH-1恶性变.结论 CAF细胞高表达SMA,增殖能力强,能促进前列腺良性细胞恶性变.

Objective To culture the prostate cancer-associated fibroblasts （CAF） and normal prostate fibroblasts （NPF）,and compare their biological characteristics.Methods The CAF and NPF cells were isolated by percoll gradient centrifugation.The cell morphology was detected by phase contrast microscope; the Pan-CK （epithelium marker）,Vimentin （fibroblast marker）,and smooth muscle actin （SMA） （smooth muscle marker） was stained by immunofluorescence; the cell growth ability was detected by methyl thiazol tetrazolium （MTT） assay.The non-malignant prostatic BPH-1 cells were co-cultured with CAF or NPF cells,and the malignant transformation was detected by soft agar assay.Results The morphology of the CAF and NPF cells was similar.In the immunofluorescence assay,the Vimentin was expressed at high levels in both cells; the Pan-CK did not express in either cells; the SMA was expressed at a higher level in CAF cells.The CAF cells grew faster than the NPF cells （At 4 d,the A values were 0.98 ± 0.07 vs.0.74 ± 0.04,P ＜ 0.05 ; At 6 d,the A values were 2.06 ± 0.13 vs.1.59 ± 0.14,P ＜0.05）,and could induce the malignant transformation of BPH-1 cells.Conclusion The CAFs cells are more active than NPF cells,and could promote the malignant transformation of prostate non-malignant cells.