The invention discloses a
tree shrew SMSCs
separation method,
tree shrew immortalized SMSCs, a
tree shrew immortalized SMSCs construction method and application, and relates to the technical field of tree shrew immortalized SMSCs construction.The tree shrew immortalized SMSCs construction method includes the steps that mixed
enzyme digestion is adopted to obtain adult primary tree shrew SMSCs, repeated differential adherent purification is utilized, and the tree shrew immortalized SMSCs are obtained; the SMSCs
growth curve is observed through a CCK8 proliferation test, the
cell morphology is observed through HE
staining, the
cell cycle and
apoptosis conditions are detected through
flow cytometry, and related
marker gene and
protein expression is identified through
immunofluorescence and RT-PCR.
Myogenic differentiation is induced by adopting a serum evacuation method, and the
myogenic differentiation condition is detected by immunofluorescent
staining. The adipogenic differentiation is induced by adopting a adipogenic
inducer (DMEM / F12 + 0.5 mmol / L IBMX + 10 [mu] g / mL INS + 1 [mu] mol / L DEX + 1 [mu] mol / L
rosiglitazone + 10% FBS + 10% HS + 1%
double antibody), and the formation condition of lipid droplets after the adipogenic differentiation is identified by adopting oil fruit O
dyeing. The separated and purified P3 generation cells are transfected by SV40 overexpression
lentivirus, the
cell viability after
transfection and immortalization is detected through CCK8, symbolic
protein expression is identified through
immunofluorescence, SV40
gene expression is detected through RT-PCR, and cell
senescence is detected.