Therapeutic Effect Of Bone Marrow Mesenchymal Stem Cells Transplantation On Manganism Rats

?Objective?1.To explore the most suitable conditions for the model of manganism rats and use the discriminant analysis method finding out the general formula to screen out manganism rats as treatment objects;2.Bone marrow mesenchymal stem cells?BMSCs?were transplanted into the manganism rats and were observed about their growth and differentiation.3.To observe the therapeutic effect of bone marrow mesenchymal stem cell transplantation on manganism rats by detecting the changes of dopamine content and the related components of Lewy bodies.;?Methods?1.Twenty-four healthy male SD rats were randomly divided into control group,low dose group?15mg/kg?,middle dose group?25mg/kg?and high dosegroup?50mg/kg?,and tested for behavioral scores,muscle tension change,balance beam test,water maze test four behavior tests every other month.All rats were kept treating with intraperitoneal injection of MnCl₂·4H₂O until the obvious behavioral changes arise.Then detect its brain striatum?Tyrosine Hydroxylase,TH?,explore the most suitable modeling conditions?dose and time?,divide into the manganism group and non-manganism group by 30% of the mean level of the control group.Establish a discriminant analysis function through the four behavioral indicators combined to in order to judge whether the rats were manganism rats.2.Thirty SD rats were treated with the most suitable modeling conditions according to the above method.Every rat was tested and recorded the muscle tension change,balance beam tes,etc.and judged according to the obtained discriminant formula.All manganism rats were selected to be randomly divided into Bone marrow stromal cell?BMSCs?transplantation group blank control group and PBS control group.BMSCs were injected into the BMSCs group by brain stereotaxis.PBS control group was injected with PBS.And the blank control group did not do any treatment.A month later every rat was tested the neurobehavioralchanges.The concentration of dopamine?DA?in the striatum were measured,the ultrastructural changes were observed by electron microscopy,the growth,migration and differentiation of BMSCs were observed by immunofluorescence and the main components of Lewy body detected.?Results?1.The result of model conditions:There was a death in the high-dose group.The rest Twenty-three rats were detected behavior changes by behavioral test,muscle tension change,balance beam tes and water maze test to evaluate the poisoning effect.The behavioral changes of rats in the control group were negative for three months.Low-dose group behavior had changed,but compared with the control group,the difference was statistically significant.The behavioral changes occurring of the high dose and middle dose groups was in shorter time.According to the expression of TH,the success rate ofhigh dose composition power was100%,and the medium dose was 66.67%.We can get the conclusion synthetically that 25mg/kgBW concentration of MnCl₂·4H₂O for three months is the best modeling conditions.2.Discriminant analysis results:The mean of the TH expression in the control group was taken as normal level.The TH's expression lower than 30% of the normal level was identified as manganism rats.Thus,all rats were re-divided into manganism rats group and non-manganism rats,the difference of TH content between the two groups was statistically significant?P <0.01?,manganism group was lower than non-manganism group.The scores of the two groups were statistically significant?P<0.01?,and the behavioral scores of the rats in the manganism group were higher than those in the non-manganism group.Fisher discriminant function:Manganism group: Y=-31.830+8.867x1-4.995x2+9.862x3+0.952x4+8.877x5Non-manganism group:Y=-22.831+2.792x1-3.318x2+7.043x3+0.874x4+8.241x5,where x1-x5 respectively mean the results of the muscle tension score,the balance beam score,the behavioral score,the water maze escape latency,and the water maze platform search.All results were independently taken into the two functions above.Which function value is greater,then the rat belongs to which group.The coincidence rate was 95.7%?22/23?,and the falsepositive rate was 4.3%?1/23?.The correct rate of the discriminant function for nonmanganism group and the manganism group was 92.3%?12/13?and 100%?10/10?.3.Effects of BMSCs transplantation:19 rats were manganism in 30 poisoned rats and the success rate was 63.3%.These manganism rats grew well after transplantation,no infection or other side effects appearing,The rat head skin completely healed about one week.There were no significant differences in behavior score,muscle tension score,the balance beam score between the three groups before and after treatment.There was no significant difference in dopamine content between the control group and the control group.The content of dopamine in the BMSCs group was higher than that before treatment.There were TH positive cells in both groups,but the positive rate of TH in BMSCs group was significantly higher than that in PBS control group,which indicated that DA neurons existed in both groups and DA neurons in BMSCs treated group were more than PBS control group.BMSCs treatment group can see the accumulation of H-Nuc expression,a few began to spread,and with cerebrospinal fluid into the ventricles,indicating that exogenous BMSCs in the rat brain can survive at least one month,and can migrate to other areas.BMSCs treated with TH and H-Nuc double-labeled positive cells,PBS control group without double positive cells.It is suggested that human BMSCs can be successfully differentiated into DA neurons after implantation of SD rat brain.4.Louis body composition observation:In TEM,it can be seen that there are more vacuoles around the neurons in the striatum of the rats before transplantation,nucleus pool dilatation,nuclear condensation,chromatin aggregation,glial cell chromatin aggregation.It suggested that the damage and apoptosis of striatum neurons in rats with manganese poisoning were serious.Neurons can also be seen within a number of protein aggregation and part of the protein aggregates and lysosomal fusion,myelin sheath visible nerve abnormalities.After treatment,the nucleus of the striatum neurons in the BMSCs group was round or oval,with complete cell structure.High mitochondria,endoplasmic reticulum,and other organelles are clear and clear,mitochondrial structure is complete and clear,the body of the body of the protein aggregation less,myelin nerve fibers without aggregation,mitochondrial morphologynormal.PBS control group before treatment difference is not obvious.Western blot showed that the expression of ?-syn was increased compared with the control group and the content of NF-L was increased after BMSCs transplantation.?Conclusions?1.25mg/ kg BW of MnCl₂·4H₂O exposure for three months can be established a better model of manganism.The formula can be used to determine whether rat was the manganism rat through the behavioral tests combination.2.BMSCs transplantation was less invasive,and the transplanted stem cells grew well.3.The content of DA increased,the aggregation of Lewy bodies decreased,and the damage of DA neurons was improved obviously after BMSCs transplantation.