| Background:Parkinson’s disease (PD) is generally considered as a neurodegenerative disorder commonly characterized by bradykinesia, resting tremor, rigidity and postural instability, whose pathological changes are dopamine neurons apoptosis and nigrostriatal pathways damage. With the acceleration of the aging of the population, the incidence of people over65years is over1%and it show an increasing trend, causing a heavy burden to families and society and threatening the health of the elderly. Currently, there are about500,000PD patients in the United States, and increasing50,000cases per year.China has gradually entered the aging society and there are about2million PD patients, with increasing of nearly200,000PD patients each year. AS the patient’s condition worsens, they could not take good care of themselves, and they finally often died of various complications often. At present, either medication or surgery can only temporarily improve symptoms but can not prevent progressive disease development. With the rapid development of the regeneration and tissue engineering medicine, the use of stem cell transplantation to replace apoptosis of dopaminergic neurons in the treatment of PD is one kind of new strategy. Stem cell transplantation as a new strategy for the treatment of central nervous system diseases to be widely studied. ADSCs, since Zuk found in2001, as a promising seed cell whose biological characteristics are very similar to bone marrow-derived mesenchymal stem cells. Because of its source readily available, less invasive, rapid proliferation and other advantages, ADSCs is considered as a more better seed cell. How to mark the transplanted stem cells and in vivo tracking is the research focus and difficulty in recent years, benefit from molecular imaging rapid development, its provide the possibility. The magnetic resonance imaging (MRI) is a universal application of imaging technology. Space and time resolution of conventional MR imaging can not display the transplanted cells,with the new MRI contrast enhancer can be repeated non-invasive tracking of transplanted stem cells, ultrasmall superparamagnetic iron oxide (ultrasmall superparamagnetic particles of iron oxide, USPIO) mark is an optimal MR track method. The literature about super paramagnetic iron oxide (super-paramagnetic iron oxide, SPIO) research coverage is more, and there are rare reports about ADSCs mark and USPIO track. How to improve the labeling efficiency while reducing markers of cell toxicity is a prerequisite.In this study, USPIO is used to mark adipose-derived stromal cells of rat.In order to explore the labeling efficiency of different concentration USPIO and its effects on ADSCs in vitro, CCK-8and Alamar blue are respectively employed to test the cell vitality, seeking the ideal labeling concentration.Objective:To isolate, passage, and identify the adipose-derived stromal cells of rat in order to carry out the study further.Method: 1. Primary culture, purification, passage of adipose-derived stromal cells.Primary culture of ADSCs:One of SD rat (weight about100g) was used in the experiment. After weighing, chloral hydrate was injected into intraperitoneal (lml/100g),then the rat was put a prone position after anesthesia, fixed limbs in a flat plate, and shave off the back and belly hair. In the super clean bench, iodine tincture and alcohol were used to disinfected back in turn and then operating under strict sterile conditions, layer separation Organization, to minimize bleeding and red blood cell contamination, remove the perirenal adipose tissue, select the part containing less blood vessels to place in Petri dishes and quickly transferred to the cell room, adipose tissue were repeatedly washed with sterile0.01mmol/L phosphate buffered saline (PBS), and try to remove soft tissue and small blood vessels, then placed in the penicillin bottle, ophthalmic scissors were used to cut it into pieces. Then straw crushing of adipose tissue transferred to a disposable centrifuge tube(15ml), adding0.075%collagenase Ⅰ at37℃to digest for30min～40min. Neutralized with DMEM/F12containing10%FBS, after that filterd by100μm nylon mesh, centrifuged at1200g for10min, the cells were re-suspended by DMEM/F12-10%FBS,5×105cells/ml were plated in25cm2-type culture flask, then incubated at37℃.2. Flow cytometry was used to detect the expression of surface molecules (CD29, CD90,CD44and CD45) on ADSCs of generation passage3.Results:1. Incubated at37℃for12hrs, ADSCs had uniform cobblestone shape,which were adherent growth. After the ADSCs were cultured for1weeks in vitro, ADSCs had cytoplasmic processes, mainly spindle cells, abundant cytoplasm, large nuclei, fine nuclear chromatin, prominent nucleoli, showing that the cells were clone-like growth. After passage, ADSCs were fibroblast-like cell morphology under an inverted microscope. The cells were arranged parallel to the growth or vortex-like growth, it is difficult to distinguish with bone marrow-derived MSC.2. Flow cytometric test results show ADSCs is more homogeneous undifferentiated stem cells.ADSCs expressed surface antigens, including CD4491.05%; CD2996.56%; CD9092.76%;CD45negative expression.Conclusion:The methods of culturing could obtained high purity of the ADSCs, which could be used to the follow experiments.Objective:In order to evaluate the impact of different concentrations of USPIO-PLL complexs on ADSCs. CCK-8and Alamar blue were respectively used to test cell viability, seeking the optimal label concentrations.Method:The experiment were divided into eight groups (negative control group;12.5μg/ml;25μg/ml;50μg/ml;100μg/ml;150μg/ml;200μg/ml; blank control group). USPIO-PLL complexes is made of USPIO and positive charge transfer agent PLL. ADSCs were labeled with different concentrations of USPIO-PLL complexes, at37℃in5%CO2incubator. For the statistical analysis,10μl of CCK-8and Alamar blue reagent were separately added to4wells of different group, incubated for1h, OD value were continuously detected by microplate reader for7days. Labeling efficiency and cellular uptake were analyzed by Prussian blue staining.Data were described by "mean±standard deviation (x±SD)", and analyzed by SPSS (Version13.0). The results of One-Way AN OVA were considered as significant difference as soon as P<0.05.Results:By treating with different concentrations of USPIO-PLL complexes, difference between control group and other groups were significant (F=3.049, P <0.019) in the first days, the differences between the other groups was not statistically significant (F=0.358, P>0.05), indicating except the blank control, the other groups is balanced and consistent. OD values of each group gradually changed after three days, OD value of200μg/ml group compared with the other groups were significant different(F=9.604,P<0.05), showing that cell proliferation were inhibited. After seven days, cell proliferation of150μg/ml group also appeared inhibition, cell proliferation of200μg/ml group was evidently inhibited. Compared with other groups, the difference was statistically significant (F=24.683, P<0.05). The CCK-8and Alamar blue tests consistently indicated that USPIO-PLL complexes of different concentrations(12.5μg/ml～100μg/ml) had no significantly different influence on cell proliferation. Labeling efficiency and cellular uptake were analyzed by Prussian blue staining, approximately95%ADSCs were labeled when the concentration of USPIO above50μg/ml. When USPIO-PLL complexes concentration above100μg/ml, a large number of blue particles can be seen in the ADSCs cytoplasm. In addition the iron uptake rate was about100%, showing a dose dependent relationship.Conclusion:ADSCs could be effectively labeled with USPIO-PLL complexes, approximately95%ADSCs were labeled when the concentration of USPIO above50μg/ml. The CCK-8and Alamar blue tests showed that USPIO-PLL complexes of different concentrations(12.5μg/ml～100μg/ml) had no significantly different influence on cell proliferation. These data demonstrate that ADSCs from the adipose tissue can be effectively labeled with USPIO with minimal effects on cell proliferation and viability. Labeled ADSCs with50μg/ml～100μg/ml USPIO is the optimal choice for labeling ADSCs. |
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