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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。
Objective: To investigate the effects of PC-3 cell conditioned medium (PC-3-CM) on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: hBMSCs were isolated, cultured and expanded from healthy adult bone marrow. Third generation (P3) hBMSCs were used in the study. PC-3 cells were cultured until the logarithmic growth phase was changed, and the culture supernatant, PC-3-CM, was harvested 24 hours later. hBMSCs were cultured in normal medium (control group) and 50% PC-3-CM medium (experimental group) respectively. The effect of PC-3-CM on proliferation of hBMSCs was detected by WST-8 method. The hBMSCs were divided into 4 groups according to the different culture broth: group Ⅰ (control group), medium group Ⅱ (group Ⅱ), group Ⅱ (group Ⅲ) And osteogenic agent group containing 50% PC-3-CM (group Ⅳ). The effects of PC-3-CM on hBMSCs were observed by alkaline phosphatase (ALP) staining, activity assay and Von Kossa calcium staining, Osteogenic differentiation. Results: On the 1st, 3rd, 5th and 7th day of culture, the WST-8 assay showed that the absorbance (A) values of the experimental group were 0.437 ± 0.028 5, 0.798 ± 0.021 3, 1.909 ± 0.061 2 and 2.302 3 ± 0.061 0 in the control group and 0.406 ± 0.022 3,0.664 3 ± 0.007 5, 1.372 ± 0.017 6 and 1.794 7 ± 0.011 5 in the control group, respectively. There was no significant difference in the A value between the two groups except the first day (P> 0.05). The A values of the other three days in the experimental group were significantly higher than those in the control group (P <0.01). The ALP staining positive rate and staining intensity of 4 groups of cells increased in turn, ALP activity were 0.292 5 ± 0.033 0,1.297 5 ± 0.023 6,2.125 0 ± 0.073 3 and 3.795 0 ± 0.026 9, respectively (P <0.01). The numbers of calcium nodules in four groups increased in turn, the staining intensity increased in turn, and the contents of calcium deposits were 0.039 0 ± 0.006 4, 0.435 ± 0.049 2, 0.977 5 ± 0.035 2 and 1.271 3 ± 0.035 2, respectively (P < 0.01). Conclusion: PC-3-CM can promote the proliferation and osteogenic differentiation of human hBMSCs.