Migration Of Embryonic Stem Cells In The Brain Of Parkinson's Disease Mice Induced By Stromal Cell-Derived Factor-1

Parkinson's disease (PD) is a neurodegenerative disorder in the central nervous system which characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and now is also the second common incidence following Alzheimer's Disease(AD) of the neurodegenerative disorder in the central nervous. The etiopathogenisis and mechanism of PD is not known. Up to now, it was identified that there are about of 90% dopamine (DA) localized in midbrain, especially in nigra-striatum. The symptoms of PD will occur when the content of DA in this area decreases more than 70%, which inclede onset of motor features such as freezing, falling, and dementia, etc. And it is reported that there are about 0.1%-0.2% of the incidence rate in the world, compared to 3.4% of the people over 75-year-old. There is a huge burden for the families and the societies resulted from increasing PD patients.However, the current medicinal and surgical treatments are significantly limited within controlling the parts of symptoms for the patients in mid-late, and could not be able to prevent the neurodegenerative progress of PD. With the developed technique of the genetic engineering and the developing research of nerve stem cells (NSCs), it is be possible for NSCs modified with medicative gene to treat PD, which become a cheerful prospect.Stromal cell derived factor-1 (SDF-1α) is a new chemotactic factor belong to the family ofα(CXC), and is derived from stromal cells. SDF-1αplays a chemotactic and adjustic role, which is impoetant in neurodevelopment and brain injury. CXCR4 is the only ligand for SDF-1αand is a marker for several kinds of stem cells including neural stem cells. High expression of SDF-1α/CXCR4 is observed early in embryonic development, and expression decreases progressively after birth; the expression of SDF-1αand its ligand are low in adults. A relation between SDF-1α/CXCR4 expression and nervous system development has been reported. In addition, the expression of SDF-1αis high in the liver, kidney, and heart after hypoxia or radiation injury. Studies have found that SDF-1αCXCR4 are involved in chemotaxis and mobilization of neural stem cells and play a critical role in the repair and reconstruction of the injured brain. Therefor, SDF-1αwould be play a important role in inducing the migration of stem cells outside into the specific sites after injuried.The seed cells are always the focus in the research of PD treatment by transplantation. Fetal cells cross the placenta and persist in maternal blood for rather long time after delivery. In the solid tissue, fetal cells were found to differentiate into cells with the tissue specific marker, and fetal cells can differentiate into astrocytes, oligodentrocytes and neurons in maternal hippocampus area and excitotoxic chemical NMDA lesion will increase the number of migrated fetal cells significantly. Gestation-derived stem cells(GdSCs) are a group of stem cells derived from embryos, fetus and subsidiary organs of fetal, including embryo-derived stem cells(EdSCs),fetal-derived stem cells(FdSCs) and fetal appendage-derived stem cells(FAdSCs) involved in umbilical cord blood mesenchymal stem cells(UCBMSCs),umbilical cord mesenchymal stem cells(UCMSCs) and placenta mesenchymal stem cells(PMSCs), etc. It is already certified that EdSCs could differentiate into DAegic neurons, and excret DA. Therefore, it is well credited that it is the most effective method on treating PD by FdSCs-transplantion. Netherless, it is the ethics and the source that limit the usage of FdSCs-transplantion.GdSCs are the most potential source for stem cells. Howerver, the methods of segregating GdSCs, in present, are completely destructive. Thus, if only we could induce the stem cells in different phase from embryo and fetus into the required positions by a nature way of some factors, the above problems seems to be solved. And the migration path could be named by "vertical transplatation". The natural induced transplatation would not hurt the baby, because every cells in embryo and fetus have huge capability of reproducement. Therefore, the induced factor is important for the vertical transplatation. And we speculate that SDF-la would be the induced factor as the most possible. For this reason, we would abserve the migrated cells in the special places in which there were over-expressed SDF-la in the brain of PD models, and evaluate the treatment on PD, which provide experimental evidence for clinical therapeuty. There are three parts in this paper.Part I Establishment and evaluation of PD mice injected by MPTP In this part, we establisehed PD female mice of C57BL/6j by low-dose interval of several subcutaneous-injections. The behaviors of mice injected MPTP were observed every time, and also after model-made were continue at 9:00 every Monday for one hour. Time spent in pole-test of mice were recorded at 0,4th,7th,13th,16thday after injection in order to examine the coordination of motor ability; And the Daengic neurons by TH-immunoreactivity in SNc and the DA levels by HPLC in SN of mice in two groups were detected at 10th,16th and 30th day after the beginning. The results showed that, the abnormal behaviors, such as tremor, hunchback, erect tail, piloerection, straddled hindlimbs, bradykinesia, and gait instability, were observed in MPTP group. There were 3 times longer spent on pole-test in MPTP group than that in control group. And the loss of DA content and TH positive cells in MPTP group were more than 80% of that in the control group, which was similar to clinic PD in mid-late. Therefore, we concluded that it was a good method to establish PD mice models by low-dose interval of several subcutaneous-injections, which provided a prerequsite for the further research, because of low mortality, high successful rate and the similar changes of behaviors and loss of DA and TH-neurons.Part II To construct and detect of plasmids pDsRFP-SDF-la, explore the methods of transfection by Lipofectin(LP2000), and detect the coexpression of target gene after transfection. The SDF-la cDNA was amplificated by RT-PCR from total mRNA of mouse tissues, and subcloned into pDsRFP-C2 vector to obtain new plasmid pDsRFP-SDF-la. Both of SDF-la gene and RFP gene were proved in the plasmid of pDsRFP-SDF-1α, which was identificated by restriction enzyme analysis of Xhol and EcoRI and target fragment sequenced. And then, the plasmid of pDsRFP-SDF-1αwas transfected into SMMC7721 cells assisted by LP2000. And the transfection efficiency was 41.2% fter 3 days. SMMC7721 cells transfected expressed RFP together with SDF-1αidentified by immunocytochemistry, and the percent of gomphosis was about 71%. The co-expression of RFP and SDF-la provided a method to detect intuitively the existence of SDF-la, which was convenient to locate and detect the exogenous factor in the further study.Part III To detection of exogenous cells migrated in the brain of PD female mice, and explore the mechanism of migration. The successful models were the PD female mice of SDF-la-overexpressed, which were established by steroatactic-injection of pDsRed2-N1-SDF-la into the right caudate nucleus(CPU)of PD mice, in whose filial generation there were more than one expressed with GFP after mated with GFP-engineered male mice. And the changes of behaviors, DA levels, location of GFP-cells, ratio of GFP(+) in blood and content of SDF-la in different tissues were detected. The results showed that:the behaviors of two groups both improved, and successful models expressed more apperently than unsuccessful ones in two groups. There was a significant different between successful models and unsuccessful ones in two groups, and DA levels in successful models of SDF-la group was the highest. However, there were mo significant different between the unsuccessful models of two groups. The ratio of GFP(+) cells in blood of successful models in SDF-la group was 7 times higher than that in sham-operated group, but there were no GFP(+) cells detected in unsuccessful model of two groups. Under the fluorescent light microscope, a great qualtity of RFP zonally distributed in the brain of SDF-la group were oberserved. And in the tissus of successful models in SDF-1αgroups, there were GFP(+) cells expressed on the vessel wall and around the sinusoid in liver and spleen. However, the most exciting found was that there were both of GFP(+) and RFP(+) cells in the same position. According to results of Western blots, the content of SDF-1αexpressed the highest in the location of transplatation, next in the around of transplanted position, and the lowest in blood. The results demonstrated that SDF-1αinduced the migration of exogenous cells from fetus by the gradient.In summary, the fetus cells indeed migrated into the location of SDF-la over-expressed in the brain, and PD mice improved in behaviors and DA content. According to the results above, We concluded that SDF-la induced the fetus cells by content increased progressively, while blood-chanel was one of the migration path. The target of this experiment was to testify the induced effect of SDF-1αby vertical transplatation, and provide potential therapetic usage of FadSCs.