| PartⅠTHE ISOLATION,CULTURE AND IDENTIFICATION OF HUMAN UMBILICAL CORD MES ENCHYMAL STEM CELLSObjective:Human umbilical cord mesenchymal stem cells(HUC-MSCs)was isolated,cultured and identified in vitro to prepare for HUC-MSCs treatment of Neutrophilic allergic rhinitis model in mice.Methods:1.To isolated and cultured HUC-MSCs.Under sterile condition,use D-Hanks buffer to wash the normal full-term healthy baby umbilical cord tissue repeatly in order to remove residual blood,artery,vein and adventitial tissue.The clean umbilical cord was cutted to the1mm~3 size of tissue block and by trypsin digestion.The collected cells were precipitated evenly in the culture bottle.The complete culture medium was added to the cell culture bottle.The culture bottle was turned in turn every4h,the medium was added every 24h,and changed half of the culture medium every 3d.When the cells were adhered to 80%of culture bottle,which were digested with trypsin,then cultured to three or four generation according to the proportion of 2:1.2.Identification of HUC-MSCs.The expression of cell surface markers including CD34,CD45,HLA-DR,CD73,CD90 and CD105 was detected by flow cytometry.At the same time,osteogenic,adipogenic and chondrogenic conditions were used to induce the differentiation of the third generation of cells.The osteogenesis,adipogenesis and chondrogenic differentiation ability of cells were observed after 7 days.After the osteogenesis was induced by 20d,alizarin red staining was used to identify the osteogenic potential of HUC-MSCs.After adipogenic induced 15d,oil red O staining was used to identify the ability of fat differentiation.Alcian blue staining was identified chondrogenic differentiation ability after the formation of cartilage tissue.Results:1.Under inverted microscope,the cells showed fibroblast growth like whirlpool.After continuous passage for 10 times,the cells became more homogeneous fibroblasts,and there was no obvious change in cell morphology.2.Flow cytometry results showed that the expression rates of CD34,CD45,CD73,CD90,CD105 and HLA-DR on HUC-MSCs cell surface were 0.12,0.17,99.93,99.83,99.77,0.09,respectively,which were consistent with the characteristics of HUC-MSCs cell surface markers.Osteogenic induction showed that there was a floating white calcium nodule above the cell induced by 10d,and the white calcium nodules were stained with rust red by 20d.Lipid induced results showed that lipid droplets were formed in cytoplasm by 5d and oil red O staining showed red dye in 15d.Chondrogenic differentiation showed that the cells became dense clumps by 25d.Cells could be stained dark blue by alcian blue staining.Conclusion:The method of separating and cultivating HUC-MSCs with adherent culture method is stable and reliable.HUC-MSCs can be obtained with high purity and multi differentiation ability.Part Ⅱ ESTABLISH MICE MODEL OF NEUTROPHILS ALLERGIC RHINITIS AND SFFECT OF HUC-MSCS BY REGULATE EXPRESSION OF GLUCOCORTICOID RECEPTOR ALPHAObjective: To establish mice model of neutrophils allergic rhinitis induced by ovalbumin(OVA)combined with lipopolysaccharide(LPS).Observe the effect of HUC-MSCs on AR model about behavior,cytokines and glucocorticoid receptor alpha in nasal mucosa epithelial cells.To verify that Neu AR model of mice can lead to glucocorticoid resistance t ALPHAhrough inhibit GRa nuclear transcription.HUC-MSCs plays a therapeutic role in Neu AR model by enhancing the nuclear transcription of GRa.Methods: 1.Establishment of Neu AR mice model.The BALB/c female mice were basic sensitized with intraperitoneal injection ovalbumin(OVA)on 1d,7d and 14 d after desensitization feeding of ovalbumin during 4 weeks.Use OVA and LPS to intranasal once a day during 22d-28 d of challenge phase.Observation of 30 min nose and runny nose,sneezing and nasal drops after the end of intranasally.The control group was replaced by PBS solution and pure OVA.2.Treatment of Neu AR model by HUC-MSCs.Mice were divided into three groups:AR+HUC-MSCs+Dex group(stem cell therapy group),AR+Dex group(dexamethasone treatment group)and OVA+Dex group(control group).After the end of the basic sensitization,the stem cell therapy group and the dexamethasone group were given HUC-MSCs suspension(the number of cells 1×10~6)and Dex(1mg/ml)intraperitoneal injection,respectively.Observe the behavioral changes of mice in each group.After the intervention,the mice were killed and the nasal cavity lavage fluid and nasal tissue were collected to observe the pathological changes of the nasal mucosa,the cell classification count,the cytokine(IL-4,IL-17,IFN-γ)and the cytoplasm of the nasal mucosa and GRa in cytoplasm and nucleus of nasal mucosa epithelial cells.Results: Compared with pure OVA mode,OVA combined with LPS model can induce runny nose,nose and sneezing.The pathological findings of nasal mucosa showed obvious infiltration of Eosinophil and Neu.The number of Eos and Neu in NLF was significantly increased.The level of IL-4 and IL-17 was up-regulated,and the level of IFN-γ decreased in NLF.The level of serum Ig E is elevated.Using dexamethasone to intervene the Neu AR model,which showed GRa could not transport from cytoplasm to nucleus.After HUC-MSCs treatment of Neu AR model,the score of behavior improved significantly,the levels of Eos,Neu,Ig E,IL-4 and IL-17 decreased,and the level of nuclear GRa increased.Conclusion: OVA combined with LPS continuous nasal dripping can set up Neu AR mice model successfully which cause the insensitivity to glucocorticoid.HUC-MSCs can improve Neu AR with glucocorticoid resistance by enhance GRa nuclear translocation.Part Ⅲ EFFECT OF HUC-MSCS ON GLUCOCORTICOID RECEPTOR EXPRESSION IN HUMAN NASAL EPITHELIAL CELLS ANS ITS MECHANISMObjective: To study the changes of GRa and glucocorticoid receptor beta(Glucocorticoid receptor beta,GRβ)in human nasal epithelial cells(HNEPCs)induced by OVA combined with LPS.Methods: 1.According to the intervention methods,HNECs group was divided groups as follows: OVA group,OVA + LPS group,OVA+LPS+Dex group,OVA+LPS+Dex+HUC-MSCs group.The expression of GRa and GRβ in each group was detected by immunofluorescence,m RNA and western blot.The supernatant was collected for the detection of IL-4 and IL-17.2.HNECs was divided two groups according to added the different cytokines: IL-4,IL-17.The expression sites of GRa and GRβ were detected by immunofluorescence.The m RNA and protein of GRa and GR β were detected in each group.3.HUC-MSCs and HNECs were co cultured with Transwell.According to the addition of intervention factors,different groups were grouped as follows: OVA+LPS group,OVA+LPS+Dex group and OVA+LPS+ HUC-MSCs+Dex group.The expression of GRa and GRβ was detected by immunofluorescence,RT-PCR and western blot.Results: HNECs were stimulated by OVA combined with LPS,which the m RNA and protein levels of GRa decreased,m RNA and protein levels of GRβ increased.IL-4 and IL-17 secreted by HNECs was increased.After co culture with HUC-MSCs,the level of m RNA and protein of GRβ in HNECs was decreased through the intervention of IL-17.Conclusion: The imbalance between GRa and GRβ of HNECs induced by OVA combined with LPS.IL-17 is an important cytokine which leads to GRβ increased in HNECs.HUC-MSCs can reduce the GRβ expression of HNECs by antagonistic effect of paracrine action to IL-17. |
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