ABSTRACT
BACKGROUND:In the past, the culture and differentiation of bone marrow mesenchymal stem celsin vitrowere mostly reported in the adult or animal rather than in children. OBJECTIVE: To explore the ability of bone marrow mesenchymal stem cels from children differentiating into neural stem cels and nerve cels. METHODS: Bone marrow mesenchymal stem cels from children were isolated and cultured, and passage 12 cels were cultured in the pre-induction medium (DMEM culture medium containing 10% fetal bovine serum and 1 mmol/L β-mercapto ethanol) and induction medium (DMEM containing 2% dimethyl sulfoxide and 150 μmol/L butylated hydroxyanisole). Expression of nestin and β-tublin III was detected using immunocytochemistry method at 30 minutes and 7 days after induction, while RT-PCR was used to detect nestin mRNA expression at 0, 5.5, 6 days after induction. RESULTS AND CONCLUSION: After combined induction, the cels shrank from round shape to tapered, polygonal or oval shape, and cel processes extended gradualy and became filament-like shape. Interconnected cels formed a network at 6 days after combined induction. The expression of nestin antigen was positive at 30 minutes after induction, while the expression of β-tublin was positive at 7 days. RT-PCR findings showed that positive expression of nestin mRNA was detected at 5.5 hours of induction, and then disappeared at 6 days. These findings show that the combined use of dimethyl sulfoxide and butylated hydroxyanisole can induce bone marrow mesenchymal stem cels from children to differentiate into neural stem cels and nerve cels in vitro.
ABSTRACT
Objective To explore whether gap junction disturbances are involved in the pathogenesis of levodopa-induced dyskinesia ( LID ). Methods The hemi-parkinsonian ( PD ) rat was treated intraperitoneally with L-dopa methylester (20 mg/kg) and benserazid (10 mg/kg) for 21 days and abnormal involuntary movement was evaluated to establish LID rat model. The experimental animals were divided into three groups: LID group, PD group and normal control group, respectively. The behavior responses of intraperitoneal injection of different doses of carbenoxolon and intracerebroventricular injection of quinine were observed to estimate the effects of gap junctional blockade on the abnormal involuntary movement ( AIM ) in the rat model of LID. Double immunofluorescence labeling was used to analyze the expression of connexin 36 ( Cx36 ) in enkephalin positive medium spiny neurons and parvalbumin ( PV ) positive interneurons in the striatum. Western blottings was used to observe the expression of Cx36 in the striatum and moter cortex. Results Behavioral characteristics indicated that high dose of carbenoxolone ( >60 mg/kg) intraperitoneal injection and intracerebroventricular injection of quinine ( 0.5, 1.0, 2.0 μmol/L, > 2.5 μmol/L ) could decrease the AIM score of LID rats. Western blotting indicated that expression of Cx36 in lesioned striatum and motor cortex of LID rat model was 219.56% ±18.12% and 226.03% ±16.33%, respectively, which induced a significant upregulation in comparison with the normal control group (104.05% ±3.82%, t=15.389, P<0.01;105.27% ±2.82%,t=8.074, P<0.01) and untreated PD group (119.31% ±8.92%, t=13.356, P<0.01; 138.20% ±17.88%, t=5.872, P<0.01). Double immunofluorescence labeling staining revealed that Cx36 expression was increased in Enk-positive striatum neurons in LID model ( 57.59% ±5.36%) compared with that in normal control group (32.67% ±4.22%) and PD group (37.24% ±0.86%, F=78.060, P<0.01). The expression of Cx36 in PV-positive interneurons was also elevated in LID group (68.49% ±11.60%) in comparison with normal control group ( 40.43% ± 2.30%) and PD group ( 31.92% ± 5.68%, F = 39.567, P < 0.01 ).Conclusions The Cx36 expression is generally increased in lesioned striatum and motor cortex of LID rat model. In the striatum, the up-regulation of Cx36 is specifically observed in Enk-positive striatum neurons and in PV-positive interneurons. The dyskinesia behavior of LID rats can be significantly reduced by treatment with gap junction blockade. All these results suggest that gap junction dysfunction may play an important role in the pathogenesis of LID.