The Research On Bone Mesenchymal Stem Cells As Replacement Cells For Parkinson Disease

Parkinson disease is a common neurological disorder characterized by occurrence in the middle and old-aged, and main lesion of which is in the substantia nigra and striatum. And paralysis agitans happen for decrease of dopamine production. So far, the etiology of PD has been unclear, and the exact therapy unfounded. Cell transplantation is a kind of ideal cure method. The goal of this research is to discuss the cytology mechanism of bone mesenchymal stem cell transplantation in the treatment of PD. Presentation of this experiment will come from the acquisition, identification and directional induction of BMSCs, and cells grafting on treating on PD as follows:Part ⅠThe Acquisition, Identification and Directional Induction in Vitro of BMSCsPurpose:To isolate and culture BMSCs in vitro, and to identify the purity and the feasibility of differentiation to neurons after purification and proliferation. Methods: BMSCs were collected from adult rat by flushing femurs, and inoculated with completed medium constituted of DMEM-LG,10%fetal bovine serum. Cells were cultured through whole marrow method with regular change of fluid and digestion and passage of BMSCs to amplify cells. The vigor of different generations of BMSCs were tested blue colorimetric test and the growth curve was drawed. The purified BMSCs were determined of phenotype and cell cycle through the method of flow cytometry (FCM).5-Bromodeoxyuridine was added to the medium to mix into the DNA of BMSCs, and the BrdU mark effect on BMSCs was detected through the method of immune fluorescence staining. Medium containing low concentration of β-mercaptoethanol(β-ME) was adopted as preinducer, and medium containing dimethylsulfoxide(DMSO) and β-ME was adopted as inducer to induce BMSCs to differentiate. Cells were observed under microscope, and the expression of neurons characteristic marker nestin was detected to exam the ability of BMSCs differentiate into neuron and glial. Result:BMSCs isolated and cultured through the method of whole marrow were attached after0.5h. Cells began to form cell colony after7-10d and covered the bottle bottom gradually. The morphology of the passaged cells was homogeneous and the time of cell fusion was shortened. The vigor of different passages of BMSCs was not different from each other. The test result from FCM indicated that the positive rate of CD29, CD34and CD90are96.8%,2.6%and95.3%, and they conform to the characters of BMSCs. Cells with BrdU mixed can send yellow-green fluorescence under fluoroscope. Cells treated through immunocytochemistry showed us that neuron-like cells are nestin and NSE-positive, but no obvious GFAP-positive cells can be seen. Conclusions:BMSCs can be successfully isolated and cultured through the method of whole marrow. BMSCs of the3rd passage are of high purity, and the phenotype corresponds with the characteristics of BMSCs. BrdU can serve as the marker of BMSCs, and BMSCs have the potential to differentiateinto nerve cells.Part ⅡThe Research of BMSCs Transplantation on the Treatment of PD RatPurpose:To establish successful PD models of rats and measure the survival and differentiation of BMSCs after being transplanted. And to probe the mechanism of BMSCs’ treatment on PD. Methods:The unilateral stereotaxic intra-striatal double point injection of6-OHDA was adopted to establish PD models of rats. The rats were induced to rotate by apomorphine(APO)2weeks after6-OHDA administration. The TH expression of striatum in damaged side was tested by immunohistochemical staining for Pathology test. BrdU-marked BMSCs were transplanted into the striatum in damaged side of successful PD models of rats.2weeks after the experiment, the TH expression and the survival, migration and differentiation of transplanted BMSCs through the method of immunohistochemistry and immuno fluorescence. Result:Inducing by APO,22rats continued to rotate to the healthy side among the30rats with average circle number exceeds7r/min, and TH-positive cells of the striatum and nigra in damaged side were obviously decreased. PD models of rats were successfully established. The average circle number of PD rats transplanted with BMSCs was6.49+0.46r/min4weeks after operation, and a large number of BrdU-positive cells were scattered, with visible nestin, NSE and GFAP-positive cells. TH positive cells were more than before operation. Conclusions:The unilateral stereotaxic intra-striatal double point injection of6-OHDA can establish successful PD models of rats. BMSCs transplanted into striatal can survive, migrate and differentiate. And BMSCs have treatment effect on PD rats, the possible reason of which is that BMSCs functioned in stead of the damaged cells.