| 【Objective】To observe the DA neurons were differentiated from hBMSCs transplanted into the striatum of manganese-poisoned rat and related Proteins were expressed【Methods】1.Select 70 male Sprague-Dawley（SD）male Sprague-Dawley rats and intraperitoneally inject MnCl2·4H2O 0.2mg/100g into the rat model of manganese poisoning.According to rat muscle tension,balance beam,behavior,water maze escape latency,water maze platform search times,etc.,a comprehensive model of manganese poisoning was established[1],and 40manganese poisoning models were selected.The manganese poisoning model was randomly divided into experimental group（B group）and control group（P group）,each group of 20.Each group of hBMSCs rats given in group B brain striatum transplanted in the same way P group was only given PBS transplantation.After the observation Period of rats was divided into 1,2,3,and 4 months,according to the observation period,group B rats were divided into B1,B2,B3,B4 four subgroups,group P rats were divided into P1,P2,P3,P4 subgroup,5 rats in each group.At the end of the observation period,the rats were observed for various observations the manganese poisoning model was randomly divided into exPerimental group（B group）and control group（P group）,each group had 20 rats.In group B,hBMSCs were given and brain striatum was transplanted in rats.In group P,only PBS was given.The observation period of the transplanted rats was divided into 1,2,3,and 4 months.According to the observation period,the rats in the B experiment group were divided into four subgroups:B1 group,B2group,B3 group,and B4 group,and the control group P group.Rats were divided into P1group,P2 group,P3 group,P4 subgroup,5 rats in each group.2.The bone marrow cells were maintained in DMEM（Gibco）maintenance medium and cultured to third generation hBMSCs.The phenotype of hBMSCs was identified by flow cytometry.3.The rat striatum coordinates（AP=-0.24 mm,L=-0.40 mm,V=-0.55 mm）were determined with reference to a stereotaxic map of the brain and the hBMSCs/PBS were transplanted into the striatum of rats with manganese poisoning.4.The muscle tension of each rat in the experimental group and the control group was measured at the end of 1,2,3,and 4 months after transplantation.Two rats from the exPerimental group and the control group were each taken to make frozen sections of brain tissue under an immunofluorescence microscope.NeuN/hNUC,TH/hNUC,VMAT2/hNUC staining was observed.In the remaining three rats in each group,striatum tissues were rapidly isolated after anesthesia.The protein contents of NeuN,TH,and VMAT2 in the brain striatum of each rat were determined by Western Blot,and the striatum of the rats was detected by ELISA.DA content,BDNF and GDNF levels were measured at the end of April and April.【Results】1.Under the electron microscope,the edema around the neurons in the striatum of rats before transplantation can be seen,and the nuclear nucleus is small,pyknosis,chromatin aggregation.Multiple protein aggregations were observed in neurons,and some protein aggregates were fused with lysosomes and myelinated nerve filaments accumulated.After transplantation,the nucleus membrane of striatum of the experimental group showed an oval shape,which was more complete than that before transplantation,and the protein aggregation was less.The aggregation of nerve fibers in the myelin sheath was less,and there was no significant difference in the control group before transplantation.This shows that the hBMSCs Post-transplant lesions that reduce the accumulation of striatum neuronal proteins.2.Changes in muscle tone:Analysis of variance showed that there was no significant difference in the scores of muscle tone increase between the experimental group and the control group before transplantation（F=0.255,P>0.05）,and the score of muscle tension increased significantly in the experimental group after transplantation.Lower than the control group（t=2.568,P<0.05）,the difference was significant 4 months after transplantation（t=2.351,P<0.05）.The muscle tension scores of the control group before and after transplantation were significantly higher（t=4.806,P<0.01）,but there was no significant difference in the score of muscle tension increase before and after transplantation in the control group（t=1.614,P>0.05）.,indicating that striatum stem cell transplantation may slow the Progression of lesions in manganese poisoning rats.3.Differentiation of hBMSCs into dopaminergic neurons（1）NeuN/hNUC double-label fluorescence analysis:hBNCs were specifically labeled with hNUC and NeuN-specific labeled mature neurons were analyzed by fluorescence microscopy.The brain sections of the experimental group showed the presence of NeuN/hNUC double-label positive cells,and no NeuN/hNUC double-label positive cells were seen in the control group.NeuN-labeled positive cells could be observed in the control group,but fewer than the experimental group,indicating that hBMSCs can differentiate into mature neurons.The number of B3 NeuN/hNUC double-labeled cells in the brain sections of the experimental group from the first to the fourth month was higher than that in the control group,and B4 was also decreased.This indicated that most of the neurons differentiated from hBMSCs matured at 3 months and then gradually decreased.（2）TH/hNUC double-label fluorescence analysis:TH is a specific marker of dopaminergic neurons,brain sections of the experimental group showed TH/hNUC double-label positive cells,no TH/hNUC double-label positive in the control group In the cells,TH-positive cells were observed,but less than the experimental group.It shows that hBMSCs can differentiate into dopaminergic neurons.（3）VMAT2/hNUC double-label fluorescence analysis:The expression of type II vesicular monoamine transporter（VMAT2）demonstrated that dopaminergic neurons have differentiated and matured and are capable of secreting dopamine.The brain sections of the experimental group showed the presence of VMAT2/hNUC double-label positive cells,and no VMAT2/hNUC double-label positive cells were observed in the control group.VMAT2-labeled positive cells could be observed,but fewer than the experimental group,indicating that hBMSCs differentiated Dopaminergic neurons secrete dopamine.（4）Determination of NeuN protein content:The experimental group and the control group were statistically analyzed.The NeuN protein content of the experimental group was significantly higher than that of the control group（t=2.743,P<0.01）,indicating that the mature neurons content of the experimental group was higher than that of the control group.There was no significant difference between the experimental group and the control group at each time point（P>0.05）.The content of NeuN protein in the experimental group increased gradually from January to March after transplantation,peaked at the third month,and then gradually decreased.There was no significant difference in the content of NeuN protein in the striatum of the experimental group within 4 months（F=0.074,P>0.05）.There was no significant difference in NeuN protein content in the striatum of the control group（F=0.844,P>0.05）.This shows that most of the neurons differentiated from hBMSCs gradually matured over time.（5）Determination of TH protein content:Statistical analysis was performed between the experimental group and the control group.The results showed that the TH protein content in the experimental group was significantly higher than the control group,with significant differences（t=2.229,P<0.05）,indicating that the experimental group dopaminergic The neurons content was significantly higher than that of the control group.The TH protein in the experimental group was significantly higher than that in the control group at the first month after transplantation（t=5.757,P<0.01）,indicating that the tyrosine hydroxylation in the striatum of the experimental group rats was the first month after transplantation.The enzyme content was significantly higher than that of the control group,and there was no significant difference between the other time points and the control group（P>0.05）.The TH protein content of the experimental group decreased from 1 to 3 months after transplantation,decreased to the minimum at the 3rd month,and then began to increase.There was no significant difference within 4 months（F=0.515,P>0.05）.The control group also had no Significant difference（F=0.095,P>0.05）.These results indicate that hBMSCs can differentiate into dopaminergic neurons and the differentiation process may be regulated by other factors such as neurotrophic factors.（6）Determination of VMAT2 Protein content:The experimental group and the control group were statistically analyzed and the results showed that the VMAT2 protein content in the experimental group was significantly higher than the control group,with significant differences（t=2.102,P<0.05）,indicating that the experimental group was mature The content of dopaminergic neurons was significantly higher than that of the control group.After the first month of transplantation,statistical analysis was performed between the experimental group and the control group.The results showed that the VMAT2 protein in the striatum of the experimental group was significantly higher than that of the control group（t=4.233,P<0.01）.After the first month,the content of mature dopaminergic neurons in the striatum of the experimental group was significantly higher than that of the control group.There was no significant difference between the other time points and the control group（P>0.05）.There was no significant difference in the content of VMAT2 protein in the striatum of the experimental group within 4 months（F=0.512,P>0.05）.The overall trend of changes was similar to that of dopaminergic neurons,indicating that the dopaminergic neurons were further matured.After starting to secrete dopamine.（7）Determination of BDNF protein content:Statistical analysis was performed between the experimental group and the control group.The results showed that the BDNF protein in the striatum of the experimental group was significantly higher than that of the control group at the first month after transplantation（t=6.036,P<0.01）.The striatum BDNF protein content in the experimental group and the control group first decreased and then increased from January to April.This indicated that hBMSCs could secrete BDNF and gradually increase with time.BDNF may regulate the differentiation and growth of dopaminergic neurons in an autocrine manner.（8）Determination of GDNF protein content:Statistical analysis was performed between the experimental group and the control group.The results showed that the GDNF protein content in the experimental group was higher than that in the control group,and there was no significant difference（t=1.626,P>0.05）.The content of GDNF protein in the striatum of experimental and control rats increased gradually from January to April.This indicates that transplanted human bone marrow mesenchymal stem cells may secrete GDNF and increase gradually over time.（9）Determination of DA protein content:Statistical analysis was conducted between the experimental group and the control group.The results showed that the dopamine content in the experimental group was significantly higher than that in the control group,with a significant difference（t=2.109,P<0.05）.The dopamine content in the experimental group decreased from 1 to 4 months after transplantation.Within 4 months,there was no significant difference in the content of dopamine in the striatum of the experimental group（F=1.675,P>0.05）.There was no significant difference in the content of dopamine in the striatum of the control group（F=1.491,P>0.05）.).It is suggested that dopaminergic neurons differentiated from hBMSCs secrete dopamine and increase the dopamine content in the striatum,and their secretion may be regulated by neurotrophic factors.（10）The correlation between DA and TH,VMAT2,NeuN,BDNF and GDNF was analyzed by pearson correlation.The results showed that DA was related to TH（r=0.488,P<0.05）,and TH was related to BDNF（r=0.517,P<0.05）,VMAT2 was associated with NeuN（r=0.726,P<0.05）.There was no correlation between other indicators（P>0.05）.A Partial correlation analysis was performed on the correlation indicators.The results showed that DA was still associated with TH（r=0.583,P<0.05）,and TH was still associated with BDNF（r=0.581,P<0.05）,VMAT2 and NeuN were still correlated（r=0.774,P<0.05）.【Conclusions】In this experiment,it was observed that hBMSCs could slow down the progression of muscle tone in Mn-poisoned rats,and could slow down the trend of protein accumulation in striatum.The hBMSCs can differentiate into dopaminergic neurons after striatum transplantation,and the content of dopamine in the striatum increases.It showed that hBMSCs had a certain therapeutic effect on manganese poisoning rats. |
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