Differentiation Of Bone Marrow Stromal Cells Induced By Striatal Extracts Of PD Rats

Idiopathic Parkinson's disease (PD) is a progressive neurodegenerative disorder which is primarily characterized by degeneration of the dopaminergic neurons(DA neurons) of the nigrostriatal pathway. Although levodopa is still considered as the gold standard of antiparkinsonian drug therapy, chronic levodopa treatment is associated with the development of adverse events in the majority of patient, such as motor fluctuations, dyskinesias, and neuropsychiatric problems. Nowdays cell transplantation has attracted great attentions, which could be a potential therapeutics. The most difficult problem is how to obtain enough DA neurons in vitro. Although embryonic stem cells (ESCs) transplantation once obtained some good effects, it could not be taken into widely utility because of the limitation of ethics and resource. Bone Marrow Stromal Cells (BMSCs) are tissue-specific stem cells that are capable of self-renewal and can differentiate into cells of different tissues, exhibiting the capability to overcome the barriers of germ layer. Under some certain conditions, BMSCs may differentiate into neurons, express specific neural markers. Easily got without any serious trauma, and greatly expanded in vitro, the use of BMSCs can eliminate the hazards of immunologic rejection in transplantation and avoid the constraint of ethics. It may constitute an abundant and accessible cellular reservoir for the treatment of a variety of neurological diseases. And the study of BMSCs differentiating into neurons and even into DA neurons has become hot in PD research field.The mechanism of BMSCs differentiating into neurons is still uncertain. Scientists have considered that inherent genetic mechanism and limitation of germ line and cells fates are all plastic. Multi-potency of cells can be induced by environment. And micro-environment of neural tissues or cells can promote the capacity of BMSCs differentiating into neurons. It has been proved in vivo that the behavior of rats or mouse could be improved after BMScs being transplanted into the striatal. The potential mechanism was that BMSCs had the function of excreting neurothophic factors, or that BMSCs transplanted into striatals could differentiate into neurons depending on the micro-environment. And also plenty of research has suggested that in vitro striatal extracts could promote the suvival of DA neurons, enhance differetiation of cells, and help to form synapse. Throughout evolution the mammalian brain has inherited extensive mechanisms to protect itself against environmental insults. A Parkinsonian brain maintains largely normal motor expression despite the loss of 70-80% of its DA neurons, which suggested a possible mechanism of self-protection. So we assume that striatal extracts could promote the differentiation of BMSCs into neurons, even into dopaminnergic neurons. And we furtherly postulate that there may exist some difference on the induction between normal striatal extracts and PD striatal extracts.In this study, we tried to induce BMSCs to differentiate into neurons or even dopaminergic neurons, taking the advantages of promoting differentiation and protecting function of stiatal extracts. And we also compare the possible difference function between normal and PD striatal extracts.Our research is focused on: 1. Culture and observation of juvenile rat bone marrow stromal cells in vitro. 2. Differentiation of bone marrow stromal cells induced by striatal extracts of PD rats.Part One: Culture and observation of juvenile rat bone marrow stromal cells in vitroObjective: To explore the biological characteristics of BMSCs by observing its ultrastructure and detecting its neural markers, for further research on differentiation of BMSCs in vitro.Methods: Bone marrow cells(BM)samples were obtained aseptically from healthy juvenile rat's femur and cells of the third passage were then selected by plastic adhesion. Phase contrast microscope, HE(hematoxylinand eosin)staining, and electron microscope were applied to observe morphocytological characteristic. Cells were finally determined by immunocytochemical stain with Nestin, neuron special enolase(NSE), and glial fibrillary acidic protein(GFAP), and positive cells were statistically counted.Results: Under electron microscope, plenty of microvilli were seen on the surface of the third passage of BMSCs. A great of cells had integrant membrane and abundant organelles in the cytoplasma, and the nuclei were large and chromatin were normally distribution. Gap junction was also found between cells. Immunohistochemical stain indicated that GFAP positive cells were 15.05±3.92% of the total cells, while neither Nestin nor NSE positive cells were found.Conclusion: Plastic adhesion culture may be a stable and reliable system for BMSCs culture in vitro. BMSCs of the third passage proliferated rapidly and the degree of their differentiation was low. A few of them expressed GFAP naturally.Part Two: The differentiation of bone marrow stromal cells induced by striatal extracts of PD rats.Objective: To explore the possibility of BMSCs differentiating into neurons induced by striatal extracts of rats.Methods: PD rats models were established by stereotaxic injections of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB). Bilateral striatals were dissected out and normal striatal extracts (N-SEs) and PD striatal extracts ( PD-SEs) were prepared respectively. Having been isolated and expanded in vitro, the third passage BMSCs were induced by striatal extracts. Cells were observed under phase contrast microscope. The differentiated cells were detected by immunocytochemical stain with Nestin, neuron special enolase(NSE), glial fibrillary acidic protein(GFAP), and tyroxine hydroxylase (TH).Results: Under contrast phase microscope, parts of cells changed morphologically. Some cells had enations. Immunohistochemical stain indicated that GFAP positive cells were significantly increased. In the 10% normal striatal extracts groups: the percentage of NSE positive cells was 3.25±2.37%,of GFAP was 48.44±29.47%. In the 60% normal striatal extracts groups the percentage of NSE was 20±8.62%,of GFAP was 54.60±33.14%. There was no significant difference among groups of different concentration. In the 10% PD striatal extracts groups: the percentage of NSE positive cells was 12.23±5.57%,of GFAP was 55.56±30.23%. In 60% normal PD extracts groups: the percentage of NSE positive cells was 11.92±4.01%,of GFAP was 55.56±30.23%. There was also no significant difference among groups of different concentration. However, no TH positive cells existed in our experiment. Conclusion: Striatal extracts of PD rats could lead to morphological change in BMSCs, and some cells had enations. Compared with the third passage BMSCs, a certain number of cells expressed neural special proteins after being induced, including much more higher percentages of GFAP, and NSE, Nestin, but not TH.Overall Conclusions:1. Plastic adhesion culture may be a stable and reliable system for BMSCs culture in vitro. The third passage BMSCs proliferated rapidly and the degree of their differentiation was low. A few of them expressed GFAP naturally.2. Striatal extracts of PD rats could lead to morphological change in BMSCs, and some cells had enations. After induced, a certain number of cells expressed neural special proteins, including GFAP, NSE, Nestin, but not TH.