Establishment Of Sheep Embryonic Muscle-derived Stem Cells Line And Effects On Its Proliferation From Acanthopanax Injection

Objective The isolating, culturing, biological characteristics and induced differentiation potential of muscle-derived stem cells(MDSCs) were investigatived, and the proliferation effect of Acanthopanax senticosus injection on MDSCs was explored.Methods ①The combined enzyme digestion and differential adherence preplate were used to isolate MDSCs; ②Cell were cultured in the whole medium containing serum and embryo extract, and the cell morphology was observed, then, different passaged muscle-derived stem cell-specific surface molecular markers Sca-1, CD34, CD144, Desmin and CD45 were detected using RT-PCR and immunofluorescence; ③To analysis proliferative capacity of the cells, the cryopreservation and recovery efficiency and growth curves of different passaged cells were measured; ④To view chromosomal abnormalities and clonogenic ability of different passaged cells, the karyotype analysis and detection of clonogenic ability were made; ⑤MDSCs were induced into three germ layer cells(adipocytes, osteoblasts, chondrocytes, muscle cells, liver-like cells secreting islet-like cells and neuron-like cells) and the different germ layer cells were identifed using various methods; ⑥The proliferation ability of MDSCs was tested by different concentrations of Acanthopanax senticosus injection, the concentration was screened by the MTT assay, the cell proliferation was analyzed by flow cytometry, and the proliferation genes c-erbB2, ki-67 and PCNA were identified by RT-PCR. Drugs, paraffin and frozen sections analysis were used to analyze and test animal muscle injury model.Results ①Differential adherence had been used for five times to get the purified musclederived stem cells; ②The cell morphology was mostly fusiform and round in vitro, musclederived stem cells surface markers Sca-1, CD34, CD144, Desmin were positively, CD45 negatively expressed through RT-PCR and immunofluorescence; ③The cell frozen survival rate was more than 90%, and the PDT of different passaged cells were 28.62 h, 35.85 h, 34.39 h, and 43.34 h according to the growth curves; ④Different passaged cells had no chromosomal abnormalities, indicating that the cells had genetic stability in vitro culture. Different passaged cells had the ability to form a clone group, which indicated that the cells had a strong cell proliferation ability; ⑤Under the appropriate induce conditions, muscle-derived stem cells could differentiate into mesoderm cells, such as adipocytes, osteoblasts, chondrocytes and myoblasts; endoderm cells, such as liver-like cells and pancreatic secretion-like cells; and the ectoderm cell, such as neuron-like cells; ⑥The best Acanthopanax senticosus injection concentration was 2 μg·mL-1 though MTT detection, the proliferation gene c-erbB2, ki-67 and PCNA expression were positively expressed, and the paraffin and frozen section results had showed that muscle-derived stem cells had the ability of repairing muscle damage.Conclusion The experiment proved that the sheep embryo muscle-derived stem cells have rich resources to access and they could be cultued and separated in vitro. The low, medium and high passages MDSCs still have a strong ability of self-renewal and proliferation differentiation after repeated subculture, Acanthopanax injection could facilitate the cell proliferation, so they have great prospects in the medical organization, and provide a reliable experimental evidence on the clinical application and the combined application between Chinese herbal medicine and stem cells.