软坚清脉颗粒促进骨髓间充质干细胞迁移的实验研究-《西部中医药》

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Title:: Experimental Study on the Promotion of Bone Marrow Mesenchymal Stem Cell Migration by Ruanjian Qingmai Granules

作者:: 宗媛¹, 张永康², 曹烨民^1,2, 马斌³; 1.上海中医药大学，上海 201203
2.上海中医药大学附属上海市中西医结合医院，上海 200082
3.上海交通大学MED-X研究院，上海 202402

Author(s):: ZONG Yuan¹, ZHANG Yongkang², CAO Yemin^1,2, MA Bin³; 1.Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2.Shanghai TCM-integrated Hospital, Shanghai University of TCM, Shanghai 200082, China
3.Med-X Research Institute of Shanghai Jiao Tong University, Shanghai 202402, China

关键词:: 动脉硬化闭塞症; 软坚清脉颗粒; 骨髓间充质干细胞

Keywords:: atherosclerosis obliterans; granules; bone marrow mesenchymal stem cells

分类号:: R543.1+2

DOI:: 10.12174/j.issn.2096-9600.2024.04.01

文献标志码:: A

摘要:: 目的探讨软坚清脉颗粒含药血清对糖氧剥夺条件下促进骨髓间充质干细胞的增殖及其向血管内皮细胞趋化的影响，探讨软坚清脉颗粒治疗动脉硬化闭塞症的可能作用机制。方法制备软坚清脉颗粒含药血清采用血清药理学方法。分离骨髓间充质干细胞（Bone marrow mesenchymal stem cells，BMSCs）后将其分为正常组，模型组，空白血清组，中药低、中、高剂量组共六组。细胞增殖毒性检测（CCK-8）法检测不同浓度软坚清脉颗粒含药血清对糖氧剥夺下BMSCs细胞培养6、12、18、24 h时增殖的影响；实时荧光定量聚合酶链式反应（qRT-PCR）和蛋白免疫印迹法（Western blot）检测不同浓度软坚清脉颗粒含药血清对缺糖氧剥夺条件下BMSCs细胞CCND1、MYC、ERK1/2 mRNA和蛋白的表达情况。Transwell实验检测BMSCs细胞的迁移能力，将BMSCs与人脐静脉血管内皮细胞（Human umbilical vein endothelial cells，HUVECs）在糖氧剥夺条件下分别加入制备的空白血清组老鼠血清与中药中剂量组含药血清，处理24 h后检测细胞迁移情况。Real-time PCR法检测同样条件处理下HUVECs细胞表面趋化因子PDGFB、CXCL12、CCL21mRNA及BMSC细胞表面趋化因子受体PDGFRB、CXCR4、CCR7mRNA表达情况。结果成功分离BMSCs细胞且纯度为95%。1）CCK-8结果示：与正常组相比较，模型组BMSCs细胞在6，12 h时细胞增殖能力上升（P＜0.01）；与正常组相比较，模型组BMSCs在24 h时细胞增殖存活率降低（P＜0.01）；与空白血清组相比较，软坚清脉颗粒含药血清各剂量组在24 h时不同程度的提高缺氧缺血BMSCs细胞增殖能力（P＜0.01）。2）qRT-PCR与Western blot结果显示：与正常组相比较，模型组细胞CCND1、MYC、ERK1/2mRNA和蛋白相对表达量降低（P＜0.01）；与空白血清组相比较，软坚清脉颗粒含药血清各剂量组均可升高CCND1、MYC、ERK1/2 mRNA和蛋白相对表达量（P＜0.01）。3）Transwell结果显示：与对照组相比，共同加药组能促进BMSCs细胞向HUVECs细胞迁移（P＜0.01）。4）Real-time PCR结果显示：与空白血清组相比较，中药中剂量组可升高HUVECs细胞表面趋化因子PDGFRB、CCL21的表达（P＜0.01）；与空白血清组相比较，中药中剂量组可以升高BMSCs表面趋化因子受体PDGFRB、CXCR4的表达（P＜0.01）。结论软坚清脉颗粒含药血清可能通过ERK通路促进BMSCs细胞增殖，同时通过促进HUVEC细胞趋化因子的表达及BMSCs细胞趋化因子受体的表达来实现归巢。

Abstract:: ObjectiveTo investigate the effects of the serum containing Ruanjian Qingmai granules on the proliferation of bone marrow mesenchymal stem cells (BMSCs) and their chemotaxis towards vascular endothelial cells under the condition of glucose-oxygen deprivation, and to explore the possible mechanism of the granules in the treatment of atherosclerosis obliterans (ASO). MethodsSerum pharmacology was used to prepare the serum containing Ruanjian Qingmai granules. BMSCs were isolated and divided into six groups: normal group, model group, blank serum group, and low, medium and high dose groups of Chinese medicine. Cell proliferation toxicity assay (CCK-8) method was used to detect the effects of different concentrations of the serum containing the granules on the proliferation of BMSCs cells cultured under glyco-oxygen deprivation at 6, 12, 18, and 24 h. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to detect the effects of different concentrations of the drug-containing serum on the expression of CCND1, MYC, ERK1/2 mRNAs and proteins in BMSCs cultured under the condition of glycogen-oxygen deprivation. Transwell assay was performed to detect the migratory ability of BMSCs. BMSCs and human umbilical vein endothelial cells (HUVECs) were added to mouse serum of the blank serum group and the serum of medium-dose group of traditional Chinese medicine under the condition of glucose-oxygen deprivation, and the cell migration was detected after 24 h of treatment. Real-time PCR method was used to detect the expressions of chemokines PDGFB, CXCL12, CCL21mRNA on the cell surface of HUVECs and chemokine receptors PDGFRB, CXCR4, CCR7mRNA on the cell surface of BMSCs under the same conditions. ResultsBMSCs cells were successfully isolated with 95% purity. 1) CCK-8 results showed: Compared with the normal group, the cell proliferation ability of BMSCs in the model group increased significantly at 6 and 12 h (P<0.01); compared with the normal group, the cell proliferation survival rate of BMSCs in the model group decreased evidently at 24 h (P<0.01); compared with the blank serum group, each dosage group of the granules-containing serum improved the cell proliferation ability of hypoxic-ischemic BMSCs to varying degrees at 24 h (P<0.01). 2) The results of qRT-PCR and Western blot showed that: Compared with the normal group, the relative expressions of CCND1, MYC, ERK1/2 mRNA and protein in cells of the model group were noticeably reduced (P<0.01); compared with the blank serum group, each dose group of the granules-containing serum could increase the relative expressions of CCND1, MYC, ERK1/2 mRNA and protein (P<0.01). 3) Transwell results displayed: Compared with the control group, the co-addition group could significantly promote the migration of BMSCs cells to HUVECs cells (P<0.01). 4) Real-time PCR results presented: Compared with the blank serum group, the Chinese medicine medium-dose group could elevate the expressions of chemokines PDGFB and CCL21 on the surface of HUVECs (P<0.01); compared with the blank serum group, the Chinese medicine medium-dose group could raise the expression of chemokine receptors PDGFRB and CXCR4 on the surface of BMSCs (P<0.01). ConclusionThe serum containing Ruanjian Qingmai granules may promote the proliferation of BMSCs cells through ERK pathway, and at the same time achieve homing by promoting the expression of chemokines in HUVECs and the expression of chemokine receptors in BMSCs cells.

《西部中医药》[ISSN:2096-9600/CN:62-1204/R]

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