摘要
目的:探讨健脾补肾解毒方对骨髓增生异常综合征(MDS)患者骨髓间充质基质细胞(MSC)增殖的影响及作用机制.方法:收集骨髓样本共12例,其中MDS组患者样本与对照组人群样本各6例.Ficoll淋巴细胞分离液提取单个核细胞,通过多次换液提取MSC,分为对照组、MDS组、含药血清干预组及空白血清干预组.含药血清干预组采用健脾补肾解毒方含药血清干预MDS患者MSC,空白血清组采用相同浓度空白血清干预MDS患者MSC.比较对照组与MDS组P1代MSC细胞形态、占接种时骨髓单个核细胞的比例及比较两组细胞最大传代数.检测4组P3～P5代MSC的增殖能力,qRT-PCT检测锌指蛋白A20、核因子-κB(NF-κB)抑制蛋白α(IκB-α)mRNA水平,Western Blot检测锌指蛋白A20、IκB-α、NF-κB蛋白表达.液相芯片检测上清液中各炎症细胞因子的水平.结果:对照组与MDS组MSC形态上基本一致,均呈梭形,且都贴壁生长.但MDS组MSC最大传代数、P1代细胞占提取时单个核细胞比例显著低于对照组(P＜0.05).与对照组比较,MDS组P3～P5代MSC增殖能力显著降低(P＜0.01),锌指蛋白A20、p-NF-κB p65、p-IκB α蛋白表达显著升高(P＜0.01),上清液中白细胞介素(IL)-6、IL-8、肿瘤坏死因子-α(TNF-α)、IL-1β及转化生长因子-β1(TGF-β1)水平显著增加(P＜0.01).与MDS组比较,含药血清组MSC增殖能力显著改善(P＜0.05),锌指蛋白A20、p-NF-κB p65及p-IκB α蛋白表达显著降低(P＜0.01),上清液中IL-1β、TNF-α及TGF-β1水平显著降低(P＜0.01),IL-6水平显著升高(P＜0.01).结论:健脾补肾解毒方可明显改善MDS患者MSC体外增殖能力,其机制可能与降低NF-κB的表达、抑制炎症因子水平,从而减轻对MSC的炎症刺激,间接降低锌指蛋白A20水平有关.
Abstract
Objective:To explore the effect and mechanism of Jianpi Bushen Jiedu Formula on the proliferation of mesenchymal stroma cell(MSC)in myelodysplastic syndrome(MDS)patients.Methods:A total of 12 bone marrow samples were collected,including 6 samples from MDS patients and 6 samples from the control population.Mononuclear cells were extracted from Ficoll lymphocyte separation medium,and MSC were extracted by changing the medium several times.MSC were divided into control group,MDS group,drug-containing serum intervention group and blank serum group.In the drug-containing serum intervention group,Jianpi Bushen Jiedu Formula drug-containing serum was used to intervene MSC of MDS patients,and in the blank serum group,the same concentration of blank serum was used to intervene MSC of MDS patients.The morphology of P1 MSC in the control group and the MDS group were compared,the proportion of MSC in the bone marrow mononuclear cells at the time of inoculation,and the maximum passage number of cells in the two groups were compared.The proliferation ability of P3-P5 MSC in the four groups was detected,the mRNA levels of A20 and IκB-α were detected by qRT-PCT,the protein expressions of A20,IκB-α and NF-κB were detected by Western Blot,and the levels of inflammatory cytokines in the supernatant were detected by liquid chip.Results:The morphology of MSC in the control group and the MDS group was basically the same,both were fusiform and grew adherently.However,the maximum passage number of MSC in the MDS group and the proportion of P1 generation cells in the mononuclear cells at the time of extraction were significantly lower than those in the control group(P＜0.05).Compared with the control group,the proliferation ability of P3-P5 MSC in the MDS group was significantly reduced(P＜0.05),the protein expressions of A20,p-NF-κB p65,and p-IκBα were significantly increased(P＜0.01),and the levels of 1L-6,IL-8,TNF-α,IL-1β and TGF-β1 in the supernatant increased significantly(P＜0.01)in supernants.Compared with the MDS group,the proliferation ability of MSC in the drug-containing serum intervention group was significantly improved(P＜0.05),the protein expressions of A20,p-NF-κB p65 and p-IκB α decreased significantly(P＜0.01),and the levels of IL-1β,TNF-α and TGF-β1 in the supernatant decreased significantly(P＜0.01),the level of IL-6 in the supernatant significantly increased(P＜0.01).Conclusion:Jianpi Bushen Jiedu Formula can significantly improve the proliferation ability of MSC in MDS patients in vitro,and its mechanism may be related to reducing the expression of NF-κB and inhibiting the levels of inflammatory factors,thereby reducing the inflammatory stimulation of MSC and indirectly reducing the expression of A20.
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