ABSTRACT
Objective To screen the active components of Bushen Huoxue Decoction ( BSHXD) involved in promoting the proliferation of bone marrow mesenchymal stem cells ( MSCs). Methods BSHXD and its subdivisions were extracted with petroleum ether, ethyl acetate, water-free ethanol and water respectively. MSCs were isolated and cultured by the bone marrow adherent method. At the third passage, MSCs were identified by the specific surface markers with immunofluorescence, and their osteogenic and adipogenic differentiation were tested by alizarin red staining and oil red “O” staining. After treated with the extracts of BSHXD and its subdivisions at gradient concentrations for 24 hours, cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay for the screening of active components and optimal concentration. MTT assay was used to describe the growth curve of MSCs treated with the most effective components, and cell cycle was analyzed by flow cytometry. Results Compared with the blank control group, the extracts of BSHXD and its subdivisions could protect MSCs from death to various degrees. Of all the extracts, the ethyl acetate extract of Bushen Division ( BSD) , ethyl acetate extract of BSHXD, ethyl acetate extract of Huoxue Division ( HXD) had the strongest effect, and the effect was dose-dependent, 100 μg/mL being the optimal active concentration while having no any cytotoxic reaction. The results of MTT assay revealed that BSD extracts promoted the proliferation of MSCs significantly and was the most effective component, and then came BSHXD. The results of flow cytometry indicated that BSD extract had the most strongest effect on increasing the amount of MSCs at proliferative phase, and then came BSHXD. Conclusion BSD ethyl acetate extract is the active component of BSHXD for promoting the proliferation of MSCs, showing an effect on increasing the proportion of MSCs at proliferative phase.
ABSTRACT
【Objective】To observe the in-vitro effect of panax notoginseng saponins(PNS) on bone marrow mesenchymal stem cells(MSCs) proliferation and on inducing MSCs to differentiate into cardiomyogenic cells.【Methods】MSCs were isolated from adult SD rats,and then were cultured and cloned by density gradient method and adhesive cultivation.The cell surface markers of MSCs were detected with flow cytometer.The effect of PNS on MSCs proliferation was observed with methyl thiazolyl tetrazolium(MTT) assay.The 8th generation of continuous cultured MSCs was used to observe the in-vitro differentiation of cardiomyogenic cells.The cardiomyogenic cells were identified under phase contrast microscope by immunohistochemical method and reverse transcription-polymerase chain reaction(RT-PCR) analysis.【Results】CD44 expression of MSCs was positive while that of CD34 expression of bone marrow hematopoietic stem cells was negative,indicating the success of the MSCs culture.After induction with PNS,the increase of MSCs was obvious as compared with the blank control group,and there existed histological changes of MSCs after in-vitro induction.The expression of specific proteins of Desmin and cardiac troponin I(cTnI) in MSCs was positive,and the results of RT-PCR analysis showed that the expression of myosin heavy chain(MHC) was increased.【Conclusion】PNS can increase the in-vitro proliferation of MSCs and induce MSCs to differentiate into cardiomyogenic cells in vitro,and this will supply experimental evidence of cell transplantation for the treatment of myocardial infarction.