| 1,OverviewParkinson's disease (PD) is a kind of the central nervous system degenerative diseases, extrapyramidal dopaminergic (Dopamine DA) neurons pathological damagewere the main characteristics。the reaserch indicated that when the number of neurons in the substantia nigra DA the decreased in 70%, PD clinical symptoms began to behave, As for the middle and late PD, the number of neurons in the substantia nigra DA reduced more than 90%。Statistics showed that natural infection rate of PD was 0.1% to 0.2%, it increased to 1.4% in people over the age of 55, and it was 3.4% in people over the age of 75。Effective treatment of PD patients not only helped to alleviate suffering, and could greatly reduce the burden on families and society, However, the current conventional treatment (including medical and surgical methods)was limited to control symptoms, and could not slow down, stop the progress of PD pathologys, Statistics showed that 90% of the human brain dopamine (dopamine DA) concentrated in the brain, especially in the substantia nigra-Striatum, when DA decreased more than 70%, the clinical manifestations began to appear for the PD patients, the tyrosine hydroxylase TH is the rate-limiting enzyme, it play an important role in generation of DA in the brain, reserch showed that brain TH decreased obviously in PD patients, And adding in the TH could improve the content of the brain DA, And could ameliorate symptoms of PD., Current studies have shown that stem cells can be induced to differentiate into dopaminergic neurons.。Therefore, stem sell transplantation therapy is an ideal solution for treatment of Parkinson's disease. Stem cells are defined that it could self-replicate in vivo and in vitro, and could continue to maintain the asymmetricly dividing, Including embryonic stem cells, bone marrow derived stem cells, umbilical cord mesenchymal stem cells and so on。Stem cell transplantation therapy in PD experiments, All kinds of stem cells had received good treatment, But the source of embryonic stem cells was limited, and the operation was complex, and it was limited by Ethics。Bone marrow stem cells are an ideal stem cell, But their tumorigenicity and immune exclusion limited its further development。Wharton's jelly umbilical cord mesenchymal stem cells was a new stem cell, it have special advantages。The umbilical cord is a cable which connected between the embryo and placenta-like structure,it was covered by the membranes,and contained mucinous connective tissue, connective tissue contains the blocking yolk sac and allantois, there are umbilical artery and umbilical vein. One end of cord blood is connected to embryonic vascular tube,the other end is connected with the placenta blood vessels., the blood vessel was wrapped mucin-like organization which is known as the umbilical cord glial, It is rich in hyaluronic acid which forms a fibroblast gel structure of water around. Umbilical cord is rich in hematopoietic, mesenchymal, neural and endothelial and other stem/ progenitor cells,,they come from 4 different positions,for example:1,Wharton' sjelly,2. Around umbilical cord blood pipe,3,Cord Blood,4,Under the umbilical vein endothelial。Wharton'sjelly, Wharton'sjelly mesenchymal stem cells derives from umbilical cord Wharton'sjelly organization., Umbilical cord mesenchymal stem cells is a stem cell seed which have Similar biological characteristics with mesenchymal stem cells, its biological characteristics is Similar with mesenchymal stem cells, Under certain conditions, they can be induced to differentiate into liver cells, bone cells and neuronal cells, ete。they are isolated.simply and have No tumorigenicity。In this study, umbilical cord tissue were collected under sterile environment, then dealed with collagenase。purified stem cells by adherent culture, and observed the cell morphology and biological characteristics, then the surface antigens were detected by flow cytometry cell, the stem cell were induced differentiation into dopaminergic neurons by two-step induction under certain programs,and the expression of nestin, nse and TH were detected by immunofluorescence, also the NSE and TH protein expression were identified by weston blot.2,ObjectivesWe were willing to obtain a new seed stem cells:umbilical cord mesenchymal stem cells in this study,then identified its biological characteristics and exploreed the differentiation ability into dopaminergic neurons, so this experiment inclided two parts.1) we planed to the extract umbilical cord mesenchymal stem cells,then purify and identify their biological properties.2) we planed to explored how the umbilical cord mesenchymal stem cells induce into dopamine neurons i, then specific protein expression were deteced by immunofluorescence and weston blot。3,Methods and results1) Human umbilical cords were collected in Hanks' balanced salt solution (PBS) at 4C. After disinfection in 75% ethanol for 30 seconds, the umbilical cord vessels were cleared off while still in PBSS. The mesenchymal tissue (in Wharton's jelly) was then diced into cubes of approximately 1 mm3 and centrifuged at 250g for 5 minutes. After removal of the supernatant fraction,the precipitate (mesenchymal tissue) was washed with DMEM/F12 and centrifuged at 250g for 5 minutes. After aspirationof the supernatant fraction, the precipitate (mesenchymaltissue) was treated with collagenase at 37℃for 18 hours,washed, and further digested with 2.0% trypsin at 37℃for 20 minutes. Fetal bovine serum (FBS) was then added to the mesenchymal tissue to neutralize theexcess trypsin. The dissociated mesenchymal cells were furtherdispersed by treatment with 10% FBS-DMEM/F12 and countedunder the microscope and with the aid of a hemocytometer. Themesenchymal cells were then used directly for cultures.2) Analysis of Umbilical cord MSCs surface markers by flow cytometry:The P3 cells were cultured at the density of 1×106个/ mL, adding fluorescein PE labeled anti-human CD19, CD29, CD31, CD34, CD45, CD73, CD44, CD90, CD105, CD166, HLA-DR, also sat the same type of negative control and blank control, then detected the fluorescence intensity by flow cytometry. 3) the growth curve of the MSCs by CCK8:P2 cells were seeded in 96 well plates at density of 1.5×104个/mL, adding 10% FBS, after 24 h 10μL CCK8 was added, 1 h post-cultured absorbance (OD) values was read at enzyme Measuring standard at 570 nm, keeping testing 7 d.4) Cell cycle:Taking P3 cells, after trypsin digestion, fixed with 4℃70% cold ethanol, then transparent membrane 24 h, incubated for 30 min with 10μg/mL of RnaseA at 37℃, adding 50μg/mL PI Dark, incubation pyridine in4℃at 5 min, detecting the fluorescence intensity by flow cytometry and calculated in the G1, S, G2, M phases of the cell ratio.5) Differentiation:two-step induction method:(1) P3 MSCs were inoculated at the density 5×104个/ mL in 25T flask, adding pre-induction medium (DMEM/F12) with 20 ng/mLBFGF,20 ng/mL EGF, and 2μL N2, after 5d expression of Nestin were detected by immunofluorescence, then antibody was anti-Nestin antibody in rabbit and fluorescent labeled goat anti-rabbit antibody; (2) after 6 day Pre-induction medium was removed, the P1 generation of neural stem cells were seeded in the density of 40000/cm2 in 24-well plates paved Laminin, adding induction medium DMEM/F12, containning 100 ng/mL FGF8,200 ng/mL SHH and 2μLN2, In control Group without FGF8 and SHH, a half medium was changed every 3 days, then incubated in 37℃,5% CO2. after 3,6,9 d the induction was terminated,observed under inverted microsco pe.6) Immunofluorescence:Taking the terminal induced cell, immersed 40 g/L paraformaldehyde 20 min, washed 5 min×3 times with 0.01 mol/L PBS, Reversing Osmosls Membrane 15 min adding 1.5 g/L Triton-100, washed 5 min×3 times with 0.01 mol/L PBS, closed 30 min in 1% BSA, washed 5 min×3 times with 0.01 mol/ L PBS, adding rabbit anti-TH antibody,4℃overnight incubation, washed 5 min×3 times with 0.01 mol/L PBS, adding fluorescent labeled goat anti-rabbit secondary antibody, on 37℃in chamber incubating 1 h, washed 5 min×3 times with 0.01 mol /L PBS, adding DAPI stained, Washed 5 min×3 times with 0.01 mol/L PBS, closed with glycerol, watched on fluorescence microscopy. The same to Nestin, NSE as above,7) Western blotting:after lysising cell, going on the SDS-PAGE electrophoresis, after SDS-PAGE, the SDS-PAGE electrophoresis were transfered to PVDF membrane (45 V,2 h), the PVDF membrane was washed with Tris buffer containing 9 g/ LNaCl, closed 2 h inTBST buffer added with 50 g/L skim milk powder at room temperature, washed 10 min×3 times with TBST buffer, Adding rabbit anti-TH antibody (1:1000), overnight incubation at 4℃,TBST wash, adding 5 mL diluted goat anti-rabbit secondary antibody (1:5000, containing 25g/L nonfat dry milk), at 37℃reacting 1 h, then washed 10 min x 3 times with TBST, adding ECL chemiluminescence reagents, after completely soaked, putted in a film, covered with plastic wrap, placed in X-ray cassette, placed in the darkroom. The same to NSE,8) Statistics:Data as mean (X)±standard deviation (s) said, using SPSS10.0 software for data analysis, using two factor factorial analysis.4% SummaryIn this reaserch the umbilical cord stem cells was successfully extracted, and their biological characterist was identified, cell surface molecules CD29, CD44, CD73 (SH2), CD90, CD105 (SH3), CD 166 were positive, but hematopoietic stem cell surface markers CD34, CD45 and endothelial cell surface molecule CD31 are negative, B cell surface markers CD 19 was negative, it indicates that they are very similar to bone marrow MSCs, while cell surface molecules HLA-DR were negative; the same time it was successfully induced into dopaminergic neuron in neural induction medium in, the expression of opaminergic neurons specific proteins TH and neuronal proteins NSE were detected. In short the mesenchymal stem cells in umbilical cord were successfully isolated and induced dopamine neurons. In the experiment... |
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