| Since the eighties of the twentieth century, particularly after the establishment ofhuman embryonic stem cells in1998, we pay more and more attention to applying stemcells in the treatment of refractory diseases, such as central nervous system (CNS) disease,cardiovascular disease and diabetes. In stem cell transplantation therapy areas, A lot ofdoctors have tried to apply embryonic stem cells and adult stem cells (such ashematopoietic stem cells, neural stem cells, bone marrow mesenchymal stem cells, etc.) fortreatment some related diseases, and have gotten some achievement, But embryonic stemcells and adult stem cells applied in clinic also will face many problems, such as security,immune rejection, limited cell sources, ethics, etc., these problems seriously hinder theclinical application of stem cells.In recent years, it was discovered that human amnion also possess such stem cellswhich have multiple differentiational potential. Moreover, some scholars have reportedthat the amniotic stem cells have stronger capability of proliferation than that of bonemarrow. The discovery and study of amniotic stem cells provided a feasible solution forall of the question mentioned above in the current clinical application of stem cells.Amniotic stem cells origins from amnion, which is also called fetal membranes, it is theearly embryo product which is closely linked to the fetus, and it is also a importantorganization in material exchange between matrix and fetus. Amniotic stem cells mainlylay in the cortex and the basal layer of amnion. Amniotic stem cells can express a numberof different tags of early stem cells, such as: OCT-4, alkaline phosphatase, neuron-specificglial fibrillary associated protein (GFAP),microtubule associated protein2(MAP2), neuralstem cell-specific markers (nestin), liver parenchyma cell protein, α-fetoprotein and so on. These show that the amnion was formed in early embryonic development of the inner cellmass, probably remaining immature differentiated stem cells,which retain differentiationalpotential.At the same time human amniotic stem cells also has some of the features:1. theirown lack of major histocompatibility complex I, II antigens such as HLA-A,-B, C and-DRantigens and β2immunoglobulin does not produce immunity Immunogenicity after beingtransplanted, In addition, human amnion cells exist HLA-E, G antigen, which haveimmunosuppressive activity;2. A wide range of sources of human amniotic cells, noethical or moral issue;3. Non-tumorigenic. Meanwhile human amniotic cells is a suitablecell vehicle mediating gene therapy.In addition, a lot of research work proves that the human amniotic stem cells havepotential to treat diseases: Zhao et al found that human amniotic stem cells candifferentiate into cardiomyocyte-like cells after transplanting into the heart; Sakuragawa etal reported that amniotic epithelial cells can effectively inhibit the dopaminergic neuronsdegeneration in PD rats and improve disease behavior in rats significantly; Hideori Okawatransplanted amniotic epithelial cells into rat hippocampus cerebral ischemia model andfound that human amniotic epithelial cells survived in the same way of CA pyramidal cell,suggesting that amniotic cells have potential to transform into neural-like cells, and cantreat ischemic brain damage; Sankar found that amniotic epithelial cells could partiallyrestore neurofunction after SCI; Wei et al reported that diabetic mice can restore normalblood glucose levels after transforming amniotic epithelial cells. In vivo amniotic epithelialcells has the potential to differentiate into pancreatic islet β cells, suggesting that amnioticepithelial cells have the potential of treatment of diabetes and so on.People also use human amniotic cells as cell vector to treat some genetic diseases,such as storage disease mucopolysaccharidosis VII, familial hyperlipidemia steroidsgenetic liver disease et al and have achieved some good results.In China, Shanghai Cell Institute has established and perfected the amniotic stem cellsGM platforms,and carried out a number of animal studies of diseases, including the treatment of neurodegenerative diseases represented by Parkinson's disease and ofischemic brain injury,the study of nerve regeneration mainly including SCI, the treatmentof nervous deafness based on ear hair cell regeneration and the treatment of diabetes. andhas achieved initial results. Now they have completed the preclinical test of the amnioticstem cells and transgenic amniotic stem cell therapy of cerebral ischemic diseases,showing that the amniotic stem cells and transgenic amniotic stem cells have a goodtherapeutic effect for cerebral ischemic disease.These human amniotic cells studies based on animal experiment have displayed theirextensive perspective for clinical application. if combining amniotic cells with tissueengineering technology, a variety of biological products will be developed to meet theclinical demand for various diseases.This study will discuss such as the transformation of amniotic cells to nerve cells,nerve repair and transplantation immunology as a preparation of clinical application ofamniotic cells to repair neurological damage.The first part separation and culture of human amniotic epithelial cellsand human amniotic mesenchymal cell and their comparison ofbiological characteristicsObjective: To compare cell culture methods of human amniotic epithelial cells (AECs)and human amniotic mesenchymal cells (AMSCs) In vitro and the biologicalcharacteristics.Method: Isolation of human amniotic cells with enzyme digestion, utilizing differentadhesive power of two types of cells residing in different level of amniotic membrane andamniotic membrane substrate.separating into different cultured cells and observing theirmorphology by phase contrast microscopy, Comparing surface marker by flow cytometry.At the same time culturing and inducing differentiation and cloning experiments wereperformed. Activated T cells stimulated by PHA were cultured with AECs and AMSCs toobserve the interaction between ampho. Results:(1) the two sources of amniotic cells display totally different characteristics inadherent growth and appearance of morphology. All of them highly expressed CD29,CD44, CD73, CD90, CD105, CD106, CD166, but did not express CD34, CD45, HLA-DR.(2) AMSCs rather than AECs can be cultivated in cloning way in vitro, the two cells can beinduced differentiation into neurons in vitro.(3) the two sources of amniotic cells caninhibit T cells activation and proliferation significantly in vitro.Conclusion: Human AECs and AMSCs which are ideal seed cells have similar biologicalcharacteristics, phenotype, differentiation potential and their negative role in immuneregulation in vitro.The second part Induced differentiation of amniotic cells in vitro andconstruction of complex with silk fibroin scaffolds for repairingcompound spinal cord injury in ratsObjective: To investigate the methods of amniotic cells induction and differentiation invitro, and the growth characteristics of amniotic cells on scaffolds.To explore theperspective of the seed cells transplantation and/or tissue engineering technology in thefield of nerve repair in the future.Methods: Using the methods of enzyme digestion to deal with human amniotictissue,and respectively separating and mass culturing, cell morphology was observed byinverted phase contrast microscope,flow cytometry was used to compare surface markersexpression. identification of amniotic epithelial cells and amniotic mesenchymal cells, anddifferentiation experiments were performed under a certain inducer. focusing on thedirection to become oligodendria. To build amniotic cells and silk fibroin scaffoldcomplex to find if the biological scaffold is conducive to the growth and reproduction ofamniotic cells;Amniotic cells transplantation in groups of animal experiments, Tounderstand the positive role of cell transplantation for tissue engineering in terms ofneurorestoration from multiple angles including animal behavior, pathology,immunocytochemistry, electron microscopy et al. Results: Two types of cells derived from The amniotic membrane were tested adherent growth, but their shape is different, amnioticepithelial cells showed cobblestone-like appearance, with clear boundary, strongrefraction, while the amniotic mesenchymal stem cells showed a fibroblast-likemorphology, expression of it's surface markers is the difference with amniotic epithelialcell, The latter express CK19, while the former express CD49d. Amniotic epithelial cellsthat highly express CD29and CD44suggest that they possess mesenchymal stem cellphenotype. Amniotic cells were induced to differentiate to form oligodendrocytes-like cells,and majority of them expressed galactose cerebroside, some of them expressed MBP. Invitro observing under scanning electron microscopy, three-dimensional porous structure ofsilk fibroin scaffold complex improved cell adhesion and proliferation thereby promotedcells growth. Animal experiments have confirmed the silk fibroin scaffold has favorabletissue compatibility, Glial scar formation in stent complex group animals significantlyreduced, access to the best of nerve repair.Conclus``ion: The amniotic-derived stem cellbs have multilineage differentiation potential,and its lack of major histocompatibility complex I, II antigens such as HLA-A,-B, Cand-DR antigens and β2immunoglobulin does not produce immunogenicity aftertransplantation, Meanwhile human amniotic cells exist HLA-E, G antigen, which haveimmunosuppressive activity.So they can secrete immunosuppressive factors and preventinflammatory reaction after transplantation, Amniotic cells can be regarded as immuneprivilege. This experiment also confirmed that, amniotic cells can differentiate intooligodendrocytes under appropriate condition. Animal experimental results show that thetransformed cells did not show more advantages in nerve repair, and cells transplantationcombined with tissue engineering strategy was conducive to axonal regeneration andmyelin formation.The third part Detection of negative synergism costimulatory PD-L1expression in human amnion cells and its regulation function adv theactivation of microglia in the central nervous systemObjective: To investigate the negative synergism costimulatory molecule PD-L1 expression in human amnion cells and its activation of negative regulation for microglia invitro..Methods: Collecting amniotic epithelial cells about10days in primary culture and amnionmesenchymal cells amplified three generations in vitro, Flow cytometry andimmunofluorescence were adopted to analyze the expression of costimulatory molecules inthese kinds of cells, and adopting method of3H-TdR incorporation to assay the effect ofproliferation of activated microglia stimulated by LPS when amniotic cells were addedin.and the effect of PD-L1monoclonal antibody partially block the amniotic cellsinhibition of microglial proliferation;Meanwhile flow cytometry was used to analysiseffect of amniotic cells on cell cycle of microglial cells under the action oflipopolysaccharide, ELISA was used to assay cytokine level and changes after blockingPD-L1role.Results:(1) Human amniotic epithelial cells and amniotic mesenchymal cells did notexpress positive costimulatory molecules such asCD80/CD83/CD86.but they all highlyexpressed PD-L1..(2) amniotic cells can inhibit the proliferation of microglia, and keep themicroglia which was stimulated by lipopolysaccharide to remain in the G0/G1cell cyclephase, and regulate TNF-α, NO secretion, suggesting that the negative immune regulationmediated by amniotic cells partly was caused by high expression of molecule PD-L1.Conclusions: Human amniotic cells can inhibit microglia activation and proliferation invitro, its PD-L1molecule expression mediate important immune regulation. |
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