| Corneal disease is one of the most important blinding eye diseases in the world today.Currently,corneal disease blindness caused by decompensation of the corneal endothelium at home and abroad is still a relatively traditional treatment using various types of corneal transplantation,but the donor The world's lack of resources has never been solved.Therefore,finding a cell or method that can replace corneal endothelial cells has become the focus of research on corneal endothelial diseases.In this experiment,10multi-directional differentiation potentials of bone marrow mesenchymal stem cells were used to select 10 rhesus monkeys.BMSCs(Bone marrow mesenchymal stem cells)and rhesus monkey corneal endothelial cells(CECs:Corneal)were taken after general anesthesia.(Isolation,culture and identification of endothelial cells).Rhesus monkey BMSCs were divided into 6 experimental groups according to different induction methods.Normally cultured rhesus monkey BMSCs and CECs were normal control groups,and rhesus monkeys were used in 6 experimental groups.BMSCs were induced to differentiate under different inducing conditions,and the morphological changes of cells after differentiation were observed and the expression changes of cytokines and marker proteins were detected,and the differentiated cells and normal cultured rhesus monkey BMSCs were induced.The differences were compared to observe which of the induction methods induced differentiation of rhesus monkey BMSCs closer to corneal endothelial cells.The experimental results showed that:when rhesus monkey BMSCs under different inducing conditions(in a certain concentration range increase in the medium Aqueous humor concentration,direct contact co-culture of rhesus monkey BMSCs with rhesus monkey CECs,indirect co-culture of rhesus monkey BMSCs with CECs,rhesus monkey B The MSCs were contacted with the rhesus monkey(CEC-CM)and co-cultured.All of them had the potential to differentiate into corneal endothelium-like cells.The tendency of rhesus monkey BMSCs to differentiate into keratocyte-like cells after co-culture with autologous crystal homogenate.Insignificantly,rhesus monkey BMSCs were added with iris tissue extracts to cultivate the orientation-free differentiation trend and potential of corneal endothelium-like cells,and iris tissue significantly inhibited the growth and proliferation of rhesus monkey BMSCs.The feasibility and influencing factors of differentiation of rhesus monkey BMSCs into CECs were analyzed.It lays an important theoretical foundation for the next application of autologous BMSCs to differentiate and treat human corneal endothelium decompensated diseases.At the same time,this experiment succeeded in the isolation,cultivation and cryopreservation of rhesus monkey CEC,which provided successful experience for the culture,identification and cryopreservation of rhesus monkey CECs.Objective(s):Using the multi-directional differentiation potential of rhesus monkey bone marrow mesenchymal stem cells(BMSCs),Rhesus monkey BMSCs and corneal endothelial cells(CECs)were co-cultured in vitro and cultured in different formulations to induce Ganga.Monkey BMSCs differentiate into corneal endothelium-like cells and perform cytomorphological analysis,ultrastructural analysis,detection of specific surface molecules and protein expression,differentiation of specific cytokines during differentiation of rhesus monkey BMSCs and cultured CECs.The detection of Rhesus monkey BMSCs induced differentiation into corneal endothelium-like cells was possible.The induction results under different inducing conditions were compared at the same time.The factors influencing differentiation were explored and the optimal conditions for inducing differentiation of BMSCs were explored.BMSCs induce differentiation to treat human corneal endothelium decompensated diseases to lay a certain theoretical basis.Methods:In this experiment,10 Rhesus monkeys were selected and provided by the Primate Research Center of Kunming Institute of Medical Biology,Chinese Academy of Medical Sciences,both male and female,aged 16-28 months and weighing 4-6.5 kg.After general anesthesia,autologous BMSCs and CECs were isolated and cultured,and the surface markers of cells were detected by flow cytometry and osteogenic adipogenic induction was performed.[1]Assay confirmed that the cells isolated and cultured in this experiment were multidirectional.Bone marrow mesenchymal stem cells(BMSCs)with differentiating potential through immunohistochemical detection of neuron-specific enolase(NSE)and isolation and extraction of rhesus monkey corneal endothelial cells Identification of cell surface specific proteins by immunofluorescence staining[2]proved that the cells isolated and isolated in this experiment were rhesus monkey corneal endothelial cells.BMSCs were divided into 6 experimental groups according to different induction methods,without any induction of normal culture.The BMSCs were used as their own control and a single rhesus monkey corneal endothelial cell culture as a target control group,given different inducing conditions,induced differentiation of BMSCs,morphological observation of the differentiated BMSCs cells by phase contrast microscope,through the Detection of cell surface specific protein expression after induction of differentiation of BMSCs,induction of differentiated BMSCs and normal cells Explore the differences between the raised BMSCs and feasibility analysis and impact factor is close to the corneal endothelial cells,BMSCs were differentiated cells into cells of study CECs.Results:Rhesus monkey corneal endothelial cells can achieve the isolation and culture of cells in vitro,and the cell morphology of rhesus monkey CECs is very typical;rhesus monkey corneal endothelial cells are still strong after several passages(six passages)culture and cryopreservation and resuscitation.The ability of dividing proliferation and expression of corneal endothelial cell specific proteins showed that the cultured corneal endothelial cells were positive for neuron-specific enolase(NSE)expression and the cell ion pump Na+-K+-ATPase protein.Positive expression and positive expression of tight junction protein ZO-1 protein confirmed that the cells isolated and isolated in this experiment were rhesus monkey corneal endothelial cells.In 15%and 25%of the BMSCs cultured in two different concentrations of aqueous humor,the cell shape has a tendency to change in the direction of the polygon,the arrangement becomes loose.and the NSE staining of the two grouds of cells can be seen in the cells.Particles.ZO-1 fluorescence staining showed that the nuclei were stained with blue by DAPI,and no obvious green fluorescence was found on the cell membrane and cytoplasm.The staining results were weakly positive.Na+-K+-ATP fluorescence staining showed that the nucleus was stained with DAPI.Blue,cell membrane and cytoplasm showed obvious red fluorescence staining and positive staining.The indirect contact co-culture of BMSCs with CECs was divided into two groups:indirect co-culture with two groups of cells without aqueous humor and 15%aqueous humor in the culture medium.In the no-aquatic group,long spindle-shaped BMSC cells were seen adherently growing and NSE staining was performed with aqueous humor.Coarctation of cells can be seen in large brown particles;15%aqueous humor group BMSCs/CECs indirectly co-cultured cells become loose,cell shape becomes wider,the cell shape has a tendency to change the polygon,line NSE staining containing room Coarctation of cells in the water coculture showed that the coarse brown particles were significantly more than those without aqueous humor.After indirect co-culture of BMSC and CEC without aqueous humor,the nucleus of ZO-1 staining was stained by DAPI as a blue cell membrane and the cytoplasm was slightly stained green,with weak staining,and Na-K-after indirect co-culture of BMSC and CEC.The ATP-fluorescent nuclei were dyed blue by DAPI,red stained on the cell membrane and.cytoplasm and stained positive.BMSCs and lens homogenates were contactless co-cultured for 14 days.Cells showed a tendency of widening and a directionally oriented polygonal direction.NSE-stained cells were not stained with brown particles.BMSCs were directly contacted with lens homogenate and co-cultured for 14 days.The cells became broad,loosely arranged,and the shape had a tendency to differentiate towards the polygonal direction,but the cell number was lower than that of non-contact co-cultured cells.NSE-stained brown granules in the cells did not stain significantly.Compared with the CEC group,the brown granules in the cells stained significantly.cut back.Immunofluorescence staining of ZO-1 showed that the nucleus was stained with DAPI fuel in blue,no obvious green fluorescence was observed in the cell membrane and cytoplasm,ZO-1 fluorescence staining was negative,and Na+-K+-ATPase immunofluorescence staining was performed.:The nucleus was stained with DAPI fuel in blue,cell membrane and cytoplasm showed obvious red granular fluorescent staining,and Na+-K+-ATPase immunofluorescence staining was positive.Non-contact co-culture of BMSC iris tissue:A small number of spindle-shaped BMSCs adhered to the wall and the cell density significantly decreased.There was no obvious tendency to differentiate in the polygonal direction.NSE-stained cells did not stain significantly,NSE staining was negative,and BMSC iris tissue directly contacted Incubation with an inverted phase contrast microscope for 14 days showed no cell growth and only iris pigment granules.Only BMSC cell debris and iris granules were stained with NSE.Because the growth of BMSC was significantly inhibited after co-culture with MSCs and irises were directly inhibited,non-contact co-culture of BMSCs with the supernatant of the iris resulted in ZO-1 staining of nuclei stained blue by DAPI but no staining was evident in the cell membrane and cytoplasm.After the non-contact co-culture of BMSC with the supernatant of the iris,the Na+-K+-ATP fluorescent nuclei were dyed blue by DAPI,no staining was found on the cell membrane and cytoplasm,and the fluorescence staining was negati-ve.When rhesus monkey BMSCs were contacted in corneal endothelial cell conditioned medium(CEC-CM),the shape of the cells became wider and the shape of the loosened cells changed to the shape of the polygonal shape.The cells were visualized by NSE immunohistochemical staining.Coarse brown granules were positive for NSE immunohistochemical staining.Immunofluorescence staining of ZO-1 protein tightly connected to cells showed that the nuclei were dyed blue by DAPI,slightly stained green fluorescence in cell membrane and cytoplasm,weakly positive by immunofluorescence staining of ZO-1 protein,rhesus monkey BMSC and autologous CEC.Immunofluorescence staining of Na+-K+-ATPase protein after co-culture with CM showed that the nuclei were stained with blue by DAPI,and red fluorescence was observed on the cell membrane and cytoplasm.Immunofluorescence staining of Na+-K+-ATPase protein was positive..Conclusion(s):Rhesus corneal endothelial cells can be successfully isolated and cultured in vitro.The improved membrane-assisted protease digestion method is a reproducible and reliable method for extracting CECs and can significantly improve the culture of rhesus monkey CEC cells.The success rate of cryopreservation and resuscitation provided a successful experimental experience for the culture,identification,and cryopreservation of rhesus monkey CECs.Increasing the concentration of aqueous humor in the culture medium in a certain concentration range favors the differentiation of BMSC cells toward corneal endothelium-like cells;Rhesus monkey BMSC cells exhibit corneal endothelium-like cells after co-culture of rhesus monkey BMSCs and CECs.Differentiation trends and potentials;BMSCs have the potential to differentiate into corneal endothelium-like cells after indirect co-culture of BMSCs and CECs;BMSCs are co-cultured with crystal homogenate and contacted with(CEC-CM).Corneal endothelium-like cells differentiated in direction and potential;rhesus monkey BMSCs were incubated with iris extract;after rhesus monkey BMSCs were co-cultured with iris tissue extracts,not only rhesus monkey BMSCs did not differentiate toward corneal endothelium-like cells.Trends and potentials,and the proliferation of rhesus monkey BMSCs was significantly inhibited. |
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