前列腺癌PC-3细胞条件培养液对人骨髓间充质干细胞增殖和成骨分化的影响

目的:探讨前列腺癌PC-3细胞条件培养液(PC-3-CM)对人骨髓间充质干细胞(hBMSCs)增殖和成骨分化的影响。方法:从健康成人骨髓分离、培养、扩增hBMSCs。第3代(P3)hBMSCs用于该实验的研究。PC-3细胞培养至对数生长期时换液,继续培养24 h后收集培养上清液即PC-3-CM。hBMSCs分别在常规培养液(对照组)和含50%PC-3-CM的培养液(实验组)中培养,采用WST-8法检测PC-3-CM对hBMSCs增殖活性的影响。根据培养液的不同,将hBMSCs分为4组:常规培养液组(Ⅰ组,对照组),含50%PC-3-CM培养液组(Ⅱ组),成骨诱导剂组(Ⅲ组)和含50%PC-3-CM的成骨诱导剂组(Ⅳ组),通过碱性磷酸酶(ALP)染色、活性检测和Von Kossa钙染色、钙沉积定量来观察PC-3-CM对hBMSCs成骨分化的影响。结果:培养第1、3、5和7天,WST-8法检测显示,实验组细胞吸光度(A)值分别为0.437 0±0.028 5、0.798 0±0.021 3、1.909 0±0.061 2和2.302 3±0.061 0,对照组分别为0.406 0±0.022 3、0.664 3±0.007 5、1.372 7±0.017 6和1.794 7±0.011 5。两组间除第1天的A值无统计学差异(P>0.05)外,其余3 d实验组的A值均高于对照组,差异有统计学意义(P<0.01)。4组细胞ALP染色阳性率和染色强度依次增高,ALP活性分别为0.292 5±0.033 0、1.297 5±0.023 6、2.125 0±0.073 3和3.795 0±0.026 9,依次升高(P<0.01)。4组细胞钙结节数目依次增多,染色强度依次增强,钙沉积含量分别为0.039 0±0.006 4、0.435 0±0.049 2、0.977 5±0.035 2和1.271 3±0.035 2,依次升高(P<0.01)。结论:PC-3-CM可促进人hBMSCs增殖和成骨分化。

Prostate cancer cell line PC-3 conditionedmedium promotes proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells

Objective：To investigate the effects of prostate cancer cell line PC-3 conditioned medium（PC-3-CM） on the proliferation and osteogenic differentiation of human bone marrow human basalis mesenchymal stem cells（hBMSCs）.Methods：hBMSCs were isolated and culture-expanded by density gradient centrifugation from normal volunteers.PC-3 cells were cultured till the time of logarithmic growth and then transferred to a fresh medium,which,after 24 hours of incubation,was collected as PC-3-CM.Passage 3 hBMSCs were cultured in the fresh medium alone（the control group） or that with 50% PC-3-CM（the experimental group）,and the effect of PC-3-CM on the proliferation activity of the hBMSCs was detected by WST-8 assay.Based on the types of medium used,the hBMSCs were divided into Groups Ⅰ（control）,Ⅱ（50% PC-3-CM）,Ⅲ（osteoblast inducer） and Ⅳ（osteoblast inducer containing 50% PC-3 CM）.The effects of PC-3-CM on the osteoblastic differentiation of the hBMSCs were determined by ALP staining,ALP activity detection,Von Kossa staining,and calcium quantitation.Results：At 1,3,5 and 7 days of incubation,the absorbance values of the cells in the experimental group were 0.437 0±0.028 5,0.798 0±0.021 3,1.909 0±0.061 2 and 2.302 3±0.061 0,and those in the control group were 0.406 0±0.022 3,0.664 3±0.007 5,1.372 7±0.017 6 and 1.794 7±0.011 5,respectively,with significant differences between the two groups（P0.01） except on day 1（P0.05）.The positive rate and intensity of ALP staining were gradually increased in the four groups,with the ALP activities of 0.29±0.03,1.30±0.03,2.13±0.08,and 3.80±0.03,respectively（P0.01）,and so was the intensity of Von Kossa staining,with the calcium depositions of 0.04±0.01,0.44±0.05,0.98±0.03,and 1.27±0.04,respectively（P0.01）.Conclusion：PC-3-CM can promote the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells.