| ObjectiveSpinal cord injury (SCI) is a common severe disease of orthopedics department. The etiology of SCI is trauma including traffic incidence, high fall, heavy crash and fall down. SCI can cause hematomyel ia, dropsy, necrosis, capsular space, gummosis and so on, which can lead to neuron deprivation, demyelination of neurite. Clinical symptoms mainly manifest funct ion disorder of movement, sense, reflectance, and musculus sphincter below injury plan. Astrocyte cell, locate in central nervous system, can change microenvironment post-SCI, prevent secondary lesion as well as form guidance passage of regenerating axonal. Astrocyte cell can also secrete massive neurotrophic factor and cytokine which might promote and induce reparative regeneration of nerve tissue. It is helpful for promoting functional recovery of limbs. YaoTongDing Injection (YTDI) is composed by Chinese herbs which can promote qi to activate blood. In this experiment, YTDI was made into containing drugs serum which was used to cultivate astrocyte cell. Cell proliferation was observed in this experiment. All these data can provide evidence for investigating the mechanism of treating spinal injury with Chinese herbs and help researchers choose proper dose for YTDI.MethodsIn the cytotoxicity experiment, mice astrocyte cell of serial subcultivation was digested by pancreatic enzyme, and then inoculated onto culture plate. The cell was randomly divided into experimental group and control group. Containing drug serum of different density was added into each group. The cell form was observed under microscope after the cell was cultivated for 24 hours. Drug toxicity of cell was analyzed with MTT colorimetric method. The drug density of cell multiplication experiment was determined according to analysis result.According to the drug density determined by toxicity experiment, astrocyte cell of serial subcultivation was randomly divided into experimental group and control group in cell multiplication experiment. The cell proliferation change of each group was observed respectively after 24 hours, 48hours and 72 hours in vitro, and experiment result was recorded after MTT analysis.Flow cytometry was used to estimate cell proliferation too. Inoculating astrocyte cell in the petri dish. Containing drug serum of different density was added into each group. The cell percentage of every phase was determined through flow cytometry after the cell was cultivated in vitro for 72 hours. The proliferation index and the percentage of every phase can reflect proliferation activity of astrocyte cell.Numerical data are expressed as the mean ± standard deviation. Numeration data and rate comparison were T-test, Probabilities (P) <0.05 were considered significant.ResultsIn the cytotoxicity experiment, the cell of high density (100mg/ml) group was massively disaggregated and died. The cell of middle dose (50mg/ml and 25mg/ml) group obviously contractedIt indicated that containing drugs serum of YTDI at high dose have obvious toxic effect. The ID50 of cell growth was about lOOmg/ml resulted from MTT test. Containing drugs serum of YTDI at low dose can promote cell multiplication. No significant difference was presented as the density of containing drug serum was less than 12mg/ml (P<0.05). As a result, the density of containing drugs serum was 50mg/ml, 25mg/ml, 12mg/ml, 6mg/ml respectively.In cell multiplication experiment, Containing drugs serum of YTDI at low dose (6mg/ml) can promote cell multiplication, compared with control group, significant difference was present (P<0. 05)especially after 72 hours. Containing drugs serum of YTDI at high dose (50mg/ml, 25mg/ml, 12mg/ml) can inhabit cell multiplication, compared with control group, significant difference was also present (P<0. 05).The percentage of cell at each phage in flow cytometry and Pr I optimum indicated that Containing drugs serum of YTDI at low dose (6mg/ml) can promote cell multiplication. The cell percentage at DNA synthesis period (including DNA synthesis period, DNA post-synthetic phase, M-period) and PrI optim... |
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