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I. Overview of Parkinson's disease (Parkinson's Disease PD) is an extrapyramidal dopaminergic (Dopamine DA) to neuron damage to the main pathological manifestations of central nervous system degenerative diseases. The study found that PD clinical symptoms began to show when the substantia nigra DA neurons reduce the number of 70% for late stage PD, DA quantity decreased by more than 90%. Abroad, the statistics show that the PD natural incidence rate of 0.1% -0.2%, which in people over the age of 55 the increase in incidence of 1.4% in people over 75 years old was 3.4%. China has entered the aging society, an effective treatment of PD will not only help alleviate the patient sufferings, and can greatly reduce the burden on families and society. However, the current conventional treatment (including medical and surgical methods) is limited to the control of symptoms, and can not slow down, to stop the progress of PD pathology. Has made it clear that the human brain dopamine (dopamine DA) content of about 90% concentrated in brain, especially in the substantia nigra - corpus striatum, when here DA decreased more than 70%, the patient will begin to appear PD clinical performance, while the DA in generating metabolic processes in the brain, brain tyrosine hydroxylase (tyrosine hydroxylase TH) play their important role, is the rate-limiting enzyme of DA generated. The study shows: PD patients with intracerebral TH decrease is more noticeable, but added midbrain TH can improve brain DA content, and can improve PD symptoms, studies have shown that stem cells can be induced to differentiate into dopaminergic neurons, therefore, to find a suitable stem cells induced to differentiate into neurons Dogan parallel cell transplantation therapy has become the ideal solution for the treatment of Parkinson's disease. Stem cells are a class of in vivo and in vitro self-replicating and can continue to maintain the asymmetric division characteristic of cell populations, including embryonic stem cells, bone marrow-derived stem cells and umbilical cord mesenchymal stem cells, etc.. Various stem cell stem cell transplant treatment of PD's experiment received good treatment, but embryonic stem cells by sources less, operation complexity and ethics and other restrictions, bone marrow-derived stem cells is one kind of ideal stem cells, but its cause tumor and immune exclusionary limit its further development, mesenchymal stem cells in umbilical cord Wharton's jelly stem cells as a new display its special advantages. The umbilical cord is attached to the cord-like structure between the the embryonic umbilical and placental outside amnion, containing mucinous differentiation connective tissue, connective tissue within. Yolk sac and allantois atresia, umbilical artery and umbilical vein. One end of the umbilical cord is connected to the other end of the placental villous vessels continued even embryonic vascular. Around vessels are mucin-like organization of the parcel, the latter is called the umbilical cord glial, or the Waldorf pass gum (Wharto'n sjelly WJ), it is rich in hyaluronic acid, the formation of fibroblasts around the hydrogel structure. Umbilical cord is rich in hematopoietic, mesenchymal, neural and endothelial various stem / progenitor cells, including mesenchymal stem cell-derived four points: (1) derived from Wharton'sjelly; ② derived from the umbilical cord around; (3) derived from umbilical cord blood ; ④ derived from the umbilical vein endothelial. Wharton'sjelly mesenchymal stem cells are derived from the the umbilical cord Wharton'sjelly Organization. Umbilical cord between the charge-quality stem cells as a stem cell seed biological characteristics similar to bone marrow mesenchymal stem cells, without immune rejection sex, certain induction conditions can differentiate into liver cells into bone cells and neural elements cells and other, non-neoplastic cells drawn easily isolated and cultured simple operation. Umbilical cord mesenchymal stem cells extracted by collecting sterile umbilical cord tissue, sterile environment, the use of collagenase treatment, choose a more purified stem cells by adherent culture, observed biological morphology and biological characteristics of the cells using flow cytometry cytometry cell surface antigen, induction program, two steps induced cells differentiate into dopaminergic neurons using immunofluorescence on technical appraisal of nestin, nse and expression of TH, while weston blot identification NSE and TH protein induces cell Expression. The purpose of this study was the aim of this study is to obtain a new seed stem cells: umbilical cord mesenchymal stem cells and their biological characteristics, to explore their differentiation into dopaminergic neurons capacity, this experiment mainly of two parts. The special protein expression first part explore the umbilical cord mesenchymal stem cells extracted, purified, and their biological characteristics; the second part of the umbilical cord mesenchymal stem cells to differentiate into the possibility of multi dopaminergic neurons, and detected by immunofluorescence and weston blot . Three main methods and results of the test: 1. Isolation and culture of umbilical cord MSCs: under sterile conditions in term pregnancy health of the fetus's umbilical cord 10 cm with 0.01 mol / L PBS soak, wash residual blood in the blood vessels, Clear umbilical cord within the blood vessels, torn umbilical surface of the amnion derived mesenchymal tissue, cut into pieces to a size of 1 mm3 tissue blocks, rinse DMEM/F12 medium by centrifugation at 250 g for 5 min, the supernatant was removed, add 20g / L type Ⅳ collagenase, 37 ° C incubator digestion for 18 h, centrifuged at 250 g for 5 min, the supernatant was removed, adding an equal volume of 20 g / L trypsin digestion for 20 min, dropwise FBS terminate digestion, the straw wind and percussion to the fragmented organization, 250 g centrifugation 5 min, the supernatant was removed, adding fresh DMEM/F12 medium Resuspend cell count at 37 ° C, 5% CO2 incubator culture. 2. Umbilical cord MSCs surface markers by flow cytometry analysis: take the of P3 generation of cells, density of 1 × 106 cells / mL, join the luciferase PE-labeled anti-human CD19, CD29, CD31, CD34, CD45, CD73, CD44, CD90 , CD105, CD166, HLA-DR, at the same time set up the same type of negative control and blank control, flow cytometry fluorescence intensity. 3.CCK8 law draw umbilical cord MSCs growth curve: Take P2 cells, density of 1.5 × 104 / mL, were seeded in 96-well plates, add 10? S, after 24 h of culture, 10 μL of CCK8 cultured for 1 h rear microtiter was measured at 570 nm absorbance (OD) value, continuous detection of 7 d. 4, Determination of the cell cycle: of P3 generation of cells trypsinized, fixed in 70% cold ethanol 4 ° C, transmembrane 24 h the incubated with 10μg/mL RnaseA 37 ℃ for 30 min., Join 50μg/mL propidium iodide 4 ℃ Incubate 5min, the fluorescence intensity of flow cytometry and calculate the proportion of cells in G1, S, G2, M phases. Induced differentiation: induced by two-step method: (1) P3 generation umbilical cord MSCs to a seeded at the density of 5 × 104 / mL2 25T, flasks, adding the pre-induction medium DMEM/F12, 20 ng / mL BFGF, 20 ng / mL EGF and 2μLN2, neural stem cells induced 5d immunofluorescence detection neurospheres Nestin expression, one secondary antibodies were rabbit anti-Nestin antibody and fluorescent-labeled goat anti-rabbit antibody; (2) 6d, remove the pre-induction. liquid P1 generation of neural stem cells to induce liquid DMEM/F12 added to 40000/cm2 density seeded in 24-well plates paved with laminin, and 100 ng / mL FGF8, 200 ng / mL SHH and 2 gLN2, the control group does not FGF8 and SHH add every three days and a half the amount of the medium was changed at 37 ° C, 5? 2 incubator culture 3,6,9 d after termination of induction observed under an inverted microscope. Expression of immunofluorescence staining Nestin, TH, NSE: Take the termination of the induced cells, 40g / L paraformaldehyde for 20 min, 0.01 mol / L PBS rinse 5 min × 3 times by adding 1.5 g / L Triton-100 transmembrane 15 min, 0.01 mol / L PBS rinse 5 min × 3 times 1% BSA closed 30 min, 0.01 mol / L PBS rinse 5 min × 3 times, adding rabbit anti-TH antibody at 4 ° C overnight incubation, 0.01 mol / 5 min × 3 L PBS rinse added fluorescently labeled goat anti-rabbit secondary antibody and incubate at 37 ° C humidity chamber for 1 h, 0.01 mol / L PBS rinse 5 minx3 times, join the DAPI-stained, 0.01 mol / L PBS rinse 5 minx3 times, glycerin closed fluorescence microscopy. Immunofluorescence staining Nestin, NSE as above, the primary antibody were rabbit anti-expression of Nestin, rabbit anti-NSE antibodies, secondary antibodies for fluorescence-labeled goat anti-rabbit antibody. 7. Western blotting of NSE expression of Th after induction cells: each group will terminate induced cell lysis, SDS-PAGE electrophoresis, the SDS-PAGE gel after electrophoresis under cooling transfer PVDF membranes (45 V , 2 h), after the transfer of the PVDF membrane containing 9 g / LNaCl Tris buffer flush, closed joined containing 50g / L skimmed milk powder in TBST buffer at room temperature for 2 h, TBST buffer wash 10 minx3 of times, by adding rabbit anti- TH antibody (1:1000), incubated overnight, washing TBST, add 5 mL of diluted sheep anti-rabbit secondary antibody (1:5000, containing 25g / L skim milk powder) reaction at 37 ° C for 1 h, and then washing with TBST 10 ECL chemiluminescence reagents minx3 times, completely soaked, put in a film, cover with plastic wrap and put it into the X-ray cassette placed in the darkroom. Western blotting of NSE as above, a secondary antibodies were rabbit anti-NSE antibody and goat anti-rabbit secondary antibody. Statistics: data mean (X) ± standard deviation (s) and SPSS10.0 software was used for data analysis, using two-factor factorial analysis. Full Summary successfully extracted umbilical cord mesenchymal stem cells in this experiment, its biological characteristics were identified by flow cytometry showed that cell surface molecules CD29, CD44, CD73 (SH2), CD90, CD105 (SH3), the CD 166 were positive, and hematopoietic stem cell surface markers CD34, CD45, and endothelial cell surface molecule CD31 are negative, B cell surface markers CD19 negative result is very similar to that with bone marrow MSCs, cell surface molecules HLA-DR-negative; cultured in neural induction solution at the same time, successfully induced dopaminergic neurons, the expression of the dopaminergic neuron specific protein TH neurons protein NSE in. In short, the success of the experiment isolated umbilical cord mesenchymal stem cells and neurons induced dopamine tender.
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