MACS方法分选CD133 /CD44 前列腺癌干细胞的初步鉴定

Initial identification of CD133 /CD44 prostate cancer stem cell through MACS

摘要:

【摘要】 目的：通过磁珠细胞分选（Magnetic bead cell sorting, MACS）方法从人前列腺癌细胞系PC-3中分选CD133 /CD44 干细胞，为进一步功能性研究奠定基础。方法：运用流式细胞仪检测MACS分选前后PC-3细胞膜上CD133和CD44表达情况；观察无血清培养成球情况，免疫荧光表达情况；比较细胞在分选前后的形态学、增殖能力方面的差异；免疫组化和Western blot检测诱导分化前后PAP蛋白情况。结果：流式细胞术检测PC-3细胞CD133和CD44的阳性表达分别是(1.33?0.05)%和(0.87?0.06)%，而MACS分选后分别为(84.82?0.07)%和(99.91?0.03)%；免疫荧光检测CD133 /CD44 细胞培养后继续呈阳性表达；CD133 /CD44 细胞增殖能力高于PC-3细胞(t=11.0, P=0.008)以及高于诱导后的CD133 /CD44 细胞(t=40.1, P=0.001)；CD133 /CD44 细胞经过TGF-β诱导后免疫组化和Western blot检测PAP表达呈阳性，而未诱导的CD133 /CD44 细胞表达呈阴性。结论：MACS从PC-3细胞株中分选的CD133 /CD44 细胞经过初步功能性鉴定具有干细胞的某些特性，可为前列腺癌干细胞的进一步探索充当铺垫。

Abstract:

【Abstract】 Objective: To sort the CD133 /CD44 stem cell through MACS from human prostate cancer cell line PC-3 and lay the foundation for the further functional research. Methods: We detected the expressions of CD133 and CD44 on the membrane of PC-3 cell before and after MACS through Flow Cytometry (FCM); then observed the formation of spheres cultured by serum-free medium (SFM) and make subsequent immunofluorescence (IF) assay; distinguished the differences of the morphology、proliferation capacity of PC-3 cell between before and after MACS; detected the expression of prostatic acid phosphatase (PAP) before and after the differentiation assay through immunohistochemical (IHC) and Western blot. Results: The positive expressions of the CD133 and CD44 of the PC-3 cell were (1.33?0.05)% and (0.87?0.06)% through FCM, while they were (84.82?0.07)% and (99.91?0.03)% respectively after MACS; the positive expressions of CD133 and CD44 remained after SFM cultured by immunofluorescence (IF); the proliferation capacity of CD133 /CD44 cell was higher than PC-3 cell (t=11, P=0.008) and CD133 /CD44 cell after induced (t=40.1, P=0.001); the expression of the PAP of the CD133 /CD44 cell after induced by TGF-β was positive, while it was negative in the uninduced CD133 /CD44 cell by IHC and Western blot. Conclusions: The CD133 /CD44 cell had some characteristics of stem cell after MACS from PC-3 cell line through initial functional identification, which could be matting for further explore of the prostate cancer stem cell.