Regeneration of skeletal muscles is crucial to the therapeutic effect on skeletal muscle dysfunction, such as skeletal muscle injury, sarcopenia, and muscle atrophy. Lots of research indicate that
Chinese acupuncture treatment can promote regeneration of skeletal muscles and help repair muscle function. C2C12 cells, the mouse myoblastic cell line, have been widely applied as the model of muscle
regeneration in vitro. This study is meant to detect the effects of acupuncture serum (collected from subjects who received acupuncture treatment) on proliferation and differentiation of C2C12
myoblasts, and then investigate the possible mechanism of acupuncture inducing regeneration of skeletal muscle.
(1)Preparation of serums: 4-month-old male Sprague-Dawley rats were randomly assigned into two groups: acupuncture group (immobilized and then acupunctured), and control group (only immobilized for
the same time as acupuncture group). Rats of acupuncture group underwent needling at Housanli (ST 36) and Huantiao (GB 30) in both hind limbs. After 2 weeks, blood was collected from abdominal aorta
and acupuncture serum and control serum were separately isolated for preparation; (2)Assessment of proliferation of C2C12: Cells were divided into four groups and were cultured in proliferation
medium, consisting of Dulbecco's modified Eagle's medium (DMEM) separately supplemented with 10% fetal bovine serum(FBS) (group A), 10% acupuncture serum (group B), 10% control serum (group C) and 10%
mixed serum (composed of 5% acupuncture serum and 5% FBS) (group D). The cell proliferation was determined by MTT assay and cell growth curve, which was formed by counting the number of cells every
day under an inverted microscope; (3)Assessment of differentiation of C2C12: Morphological observation determined the appropriate concentration of acupuncture serum to induce differentiation of C2C12,
and then cells were assigned into three groups, cultured in differentiated medium respectively containing 2% acupuncture serum (group AS), 2% control serum (group CS), and 2% horse serum (group HS).
Fluorescent immunocytochemistry were applied to detect the expression of myosin heavy chain (MHC) as a specific marker of myoblast differentiation, and the morphological changes of C2C12 were observed
with phase contrast microscopy.
(1)The results of MTT assay and cell growth curve indicate that group B has a significantly stronger proliferative response than group A and C, and group C has a significantly stronger proliferative
response than group A; (2)Acupuncture serum can promote differentiation of C2C12 more significantly at the concentration of 2% than other concentration; (3)The level of MHC in group AS is
significantly higher than group CS and lower than group HS, as is the same with the level of fusion of cells in morphological observing.
Acupuncture serum at the concentration of 10% and 2% can respectively promote proliferation and differentiation of C2C12 in vitro; Compared with FBS, serum of rats can accelerate proliferation of
C2C12 at a markedly higher rate, which suggests serum of rodents may be more suitable to maintain C2C12.