磁珠细胞分选CD133+/CD44+前列腺癌干细胞的初步鉴定

Initial identification of CD133+/CD44+ prostate cancer stem cell through magnetic bead cell sorting

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（flow cytometry，FCM）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（immunofluorescence，IF）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（immunohistochemistry，IHC）和Western blot检测诱导分化前后前列腺酸性磷酸酶（prostatic acid phos－phatase，PAP）蛋白情况。结果：FCM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PAP分别为（84.82±0.07）%和（99.91±0.03）%；IF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+细胞经过转化生长因子-β诱导后IHC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:

Objective：To sort the CD133+/CD44+ stem cell through magnetic bead cell sorting（MACS） from human prostate cancer cell line PC-3 and to lay the foundation for the further functional research. Methods：Expressions of CD133 and CD44 on the membrane of PC-3 cell before and after MACS were detected through flow cytometry（FCM）. Formation of spheres cultured by serum-free medi－um（SFM） was observed and subsequent immunofluorescence（IF） assay was made. Differences in the morphology and proliferation capacity of PC-3 cell before and after MACS were distinguished. Expressions of prostatic acid phosphatase（PAP） before and after the differentiation assay were detected by immunohistochemistry（IHC） and Western blot. Results：Positive expressions of CD133 and CD44 of PC-3 cell were （1.33±0.05）% and （0.87±0.06）% through FCM，while those after MACS were （84.82±0.07）% and （99.91±0.03）% respectively. Positive expressions of CD133 and CD44 remained after SFM detected by IF. Proliferation capacity was higher in CD133+/CD44+ cell than in PC-3 cell（t=11，P=0.008） and CD133+/CD44+ cell after inducement（t=40.1，P=0.001）. Ex－pressions of PAP of CD133+/CD44+ cell were positive after being induced by transforming growth factor-β and were negative in the uninduced CD133+/CD44+ cell based on IHC and Western blot. Conclusions：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.