Experimental Study On Therapeutic Effect Of CDNF Gene Modified Bone Mesenchymal Stem Cells Transplantation In PD Model Rats

As a new conservative factor, Cerebral dopamiane neurotrophic factor (CDNF) has specific protection and restoration function for dopaminergic neuron. The major pathological change of Parkinson’s disease is the decrease of dopaminergic neuron. As a kind of pluripotent stem cells, bone mesenchymal stem cells can migrate and differentiate into neuron-like cells in the brain after transplanting into the rat model of Parkinson’s disease. Bone Mesenchymal Stem Cells is useful for Parkinson’s disease from some certain degree. This paper discusses whether the development of PD is associated with the change of CDNF quantity in brain, Then we attempt to transfect CDNF gene to BMSCs successfully to study the combined treatment effect of CDNF and BMSCs in PD model rats.Objective:To investigate the relationship between the Cerebral dopamiane neurotrophic factor (CDNF) and Parkinson’s disease (PD) and the treatment of CDNF modified bone marrow mesenchymal stem cells (BMSCs) for PD. We conduct a preliminary experiment to study the expression of CDNF in the PD model rats and the treatment of CDNF modified BMSCs for PD rat model, to provide experimental basis for the clinical use of CDNF gene in Parkinson’s disease.Methods:(1) We use the stereotaxic instrument to prepare the PD rat model, the PD symptom was induced by 6-OHDA. Rat rotating behavior was induced by morphine though intraperitoneal injection after three weeks, we detect the expression of TH with Western blot (WB) and immune histochemical to screen success models. The expression of CDNF in the nigra-striatum of Parkinson’s disease rat model was detected by immunohistochemistry, Western blot and Q-PCR.(2) Construction of eukaryotic expression plasmid vector. Eukaryotic expression plasmid pCDNA3.1 (+)- CDNF was recombined to BMSCs by the Lipo2000, then detected by RT-PCR and immunocytochemistry. Using β-mercaptoethanol induce BMSCs to differentiate into neuron-like cells directionally in vitro to explore the differentiation potential and CDNF protein expression of CDNF-BMSCs after differentiation.(3) Successful PD model rats were divided into three groups:Control Group, BMSCs Group and CDNF-BMSCs group. According to the experimental design. Respectively, PBS (10ul), BMSCs (lOul,1 × 104/ul cell), CDNF-BMSCs (10ul,1 × 104/ul cell) were injected in the striatum of rats of the control group, BMSCs group and CDNF-BMSCs group. Rotation behavior was induced by the morphine before,2 weeks,5 weeks,8 weeks after the injection treatment. And in the above time points, detect the expression of CDNF and TH in rat striatum by immunohistochemical and WB respectively.Results:(1) The immunohistochemical results show that the CDNF and TH positive cells were decreased significantly in the substantia nigra of model rat; WB results show that the expression of CDNF and TH in the model group were different from the control group significantly:model group significantly lower than the control group (p< 0.01, n= 10). Q-PCR analysis result showes that the expression of CDNF mRNA has no significant difference between normal group and model group (p> 0.05, n= 5).(2) The study tested in vitro shows that we transfect CDNF gene to BMSCs successfully. After the treatment of β-mercaptoethanol, CDNF gene modified BMSCs can also differentiate into neuron-like cells and express CDNF protein.(3) The PD rats rotational behavior improved significantly in CDNF-BMSCs Group to BMSCs Group two weeks later after cell transplantation therapy (P< 0.05, n=10).5 weeks after the treatment of BMSCs and CDNF-BMSCs,the PD rats behavior of CDNF-BMSCs Group and BMSCs Group significantly differents from Control Group(P< 0.01, n=10); WB results show that the TH protein is significantly different from BMSCs-CDNF group to BMSCs group 2 weeks after transplantation (P<0.05,n=5); But the result of WB shows that the relative expression of CDNF protein between the two groups is 0.191 ±0.006 (BMSCs group) 、 0.305±0.016 (CDNF-BMSCs group) two weeks later after transplanttion (P<0.01, n=5). TH, CDNF protein expression in substantia nigra were dected by Immunohistochemistry, the number of TH positive and CDNF positive cells in the brain increases gradually with time (n= 5) both in BMSCs group and CDNF-BMSCs group.Conclusion:The expression of CDNF is associated with the development of Parkinson’s disease, Eukaryotic expression plasmid pCDNA3.1(+)-CDNF was constructed successfully, which might decrease apomorphine-induced rotations, increase TH in substantia nigra and afford a new therapeutic schedule in clinic.