Research On The DA And AADC Expression By BMSCs Transplantation In PD Rats

As a precursor of norepinephrine (NE), Dopamine (DA) is a key transmitter in the central nervous system which plays a selective role in inhibitory neurons. If dopamine is insufficiently synthesized, excitatory cholinergic neurons will be excessive, so as to cause the Parkinson’s disease (PD). Current clinical treatments of PD emphasize on restoring the DA level in brain in order to control the symptoms of PD. As a result, the focus of PD research is to study the DA content as well as the factors influencing DA synthesis. This study examines the dopamine levels, as well as romatic amino acid decarboxylase (AADC) gene and protein expression in the PD rats’ corpus striatum. In order to explore the mechanism of exogenous cell transplantation in the treatment of PD, and to provide the experimental evidence for the clinical gene therapy of PD, this study analyzes the dynamic changes of DA and AADC content in PD model rats’ corpus striatum before and after the transplantation of bone marrow mesenchymal stem cells (BMSCs). Part 1 Research on the DA level by BMSCs transplantation in the corpus striatum of PD ratsThe unilateral stereotaxic intra-striatal single point injection of 6-hydroxydopamine (6-OH) was adopted to establish PD model rats. The rats were induced to rotate by injecting apomorphine (APO) after operation, in order to screen the successful PD rat model. Bone marrow mesenchymal stem cells (BMSCs) from normal rats’ femur were cultured and purified to the third generation, and then in situ transplanted into the PD model rats’ damaged corpus striatum. The UV test was be selected to detect concentration gradient of DA standard to determine the standard curve, the brain dopamine in the striatum of high performance liquid chromatographic method was be set up.The treated rats were induced to rotate by injecting APO after operation as well, in order to determine whether cell transplantation would lead to behavioral improvement of PD model rats. Rats were grouped into five:Normal rats (N group), PD model rats after 4 weeks (M4 group), PD model rats after 8 weeks (M8 group), BMSCs transplantation rats after 4 weeks (T4 group) and BMSCs transplantation rats after 8 weeks (T8 group); their striatal homogenate was selected. High performance liquid chromatography (HPLC) was used to detect the dopamine level in each corpus striatum. Finally, how DA changed after BMSCs transplantation was discussed.The number of rotations for the successful PD model rats is 8.47±1.12 r/min. After BMSCs transplantation, the number of rotations gradually decreased to 6.49±0.46 r/min at the 8th week, indicating statistically significant difference(P<0.05). The experiment established HPLC chromatographic conditions successfully:the detection is Waters SymmrtryshieldTM RP18 column, the mobile phase is 0.05 mol/L sodium dihydrogen phosphate,0.02 mol/L citric acid and methanol (74:24:2), the flow rate is 1.0 mL/min, using external standard method for quantitative determination, and the maximum ultraviolet absorption wavelength lambda is 280 nm. Based on the determination of standard, we set dopamine concentrations (X) as the abscissa and dopamine standard chromatographic fengfeng area (Y) as the ordinate, conducted regression analysis, and obtained Y= 16.343+3.0812 X as a result. The correlation coefficient R2= 0.9996, indicating a good correlation. The average recovery rates are 90.4%,91.1%, and 90.4%; RSD are 3.4%,8.1%, and 3.4%; the detection of limit is 0.5 mg/L; and the limit of quantitation is 10.8 mg/L. They all met the requirements of biological sample pretreatment.The sampled test results based on the established HPLC approach showed that the DA content in the corpus striatum of N group rats was 15.56±2.16 ug/g. The DA contents in the corpus striatum of M4 group and M8 group rats were 10.67±1.54 ug/g and 10.50 g±2.32 ug/g respectively; there is no significant difference between M4 and M8 group (P>0.05), and the DA contents in both M4 and M8 group were significantly lower than that in N group (P<0.01). The DA content in the corpus striatum of T4 and T8 group rats were 12.70±2.91 ug/g and 13.71±2.42 ug/g respectively; the DA level in T4 group was significantly higher than that in M4 and M8 group (P<0.05), and the DA level in T8 group was extremely significantly higher than that in M4 and M8 group (P<0.01). The results showed that the DA levels gradually restored when the cell transplantation extended.The research showed that BMSCs transplantation treatment technology improved the behavior of the PD model rats obviously and promoted DA content in the corpus striatum. The research also indicated that after BMSCs transplantation, the BMSCs were probably differentiated into dopaminergic neurons, promoted the functional recovery and increased the secretion of DA, so as to improve the symptoms of PD models.Part 2 Research on the AADC Expression by BMSCs transplantation in the corpus striatum of PD ratsThis study aims to explore the mechanism of content change of DA in BMSCs transplantation in PD treatment by detecting the expression of the speed limit enzyme-the aromatic amino acid decarboxylase (AADC) used in dopamine synthesis. AADC total RNA, miRNA and total protein in normal, PD model and BMSCs transplanted rats’corpus striatum were extracted. The AADC mRNA, miRNA and protein in corpus striatum were detected using fluorescence quantitative PRC and Western Blot method. Moreover, the changes of AADC mRNA, miRNA and protein were analyzed coupling with the level of DA. The study clarified the relationship among the PD rats’behavior after BMSCs transplantation, the dopamine levels in corpus striatum, as well as the speed limit enzyme.The results showed that, there was no significant difference between N group, M4 group, M8 group, T4 group and T8 group in AADC mRNA level (P>0.05).The expression level of miRNA in model rats was significantly higher than normal rats’(P<0.01); and the protein content significantly decreased (P<0.01). After BMSCs transplantation treatment, the expression of AADC miRNA in the damaged side gradually decreased; however, the AADC protein expression increased. In this experiment, miRNA and the protein expression changed in opposite direction.It was possibly suggest that AADC protein expression in the transcription regulation by miRNA. Immunohistochemical results showed that specific staining cells of the model group decreased compared to the normal group, and BMSCs transplantation significantly increased after treatment.In this experiment, the AADC proteins expression in the damaged side of PD model rats’ brain was significantly lower than that in the normal side. AADC protein content increased after BMSCs transplantation treatment, and the corresponding DA content also increased in corpus striatum synchronously. The results indicated that the expression of AADC regulated the synthesis of DA, which effectively improved the content of DA. Also, aromatic amino acid decarboxylase made a huge contribution to PD treatment.