| BackgroundOcular injury is one of the main reasons of blindness, the most common cause of which is chemical burns and thermal burns. The damage to the ocular surface is serious and can result in poor prognosis. If they are not treated in time or accurately, it will cause blindness, even loss of the eye because it is difficult to treat. Chemical burns of the eye are a common ocular trauma that account for a large proportion of corneal disease. Corneal alkali burns are the most serious injury of chemical burns, and alkaline substances can cause subsequent damaging effects such as corneal tissue liquefaction necrosis, then which can lead to the corneal ulceration and perforation, secondary glaucoma and complicated cataract. All of these are severe damage to the anatomy of the eye and visual function.Currently, there is no ideal treatment for severe ocular surface damage. Corneal transplantation is still the primary means of treatment of corneal alkali burn which often induces corneal blindness. However, high rejection rate after corneal transplantation and low long-term graft survival are the thorny issue of clinical practice. Traditional transplants operations include: ① limbal stem cell transplantation have a good effect for some patients, but there are lack of supports related to laboratory studies. Limbal grafts are taken from the healthy eye, which are absence of allograft rejection and can solve the shortage of donor-derived disadvantage. The high survival rate of limbal stem cell transplantation and the partial deletion of stem cells relative to the healthy eye will not affect the normal ocular function make it become one of the increasingly reliable method of the treatments clinically. However, it is still an unresolved issue on the number of limbal tissue taken from the healthy eye which can ensure the surgical needs and cannot affect the ocular surface stability of the healthy eye at the same time. And it is not available for the large area of the injured ocular damage for both eyes.② Limbal allograft transplantation that is able to provide sufficient limbal grafts, but the source is limited, is widely limited in clinical application. The limbus with a rich capillary is without corneal immune privilege status, so after allogeneic transplantation, its rejection cannot be ignored;③ amniotic membrane transplantation for ocular surface disorders is widely applied to clinical, but for corneal severe corneal alkali burn injury patients, its efficacy is still poor due to the transparency of the cornea amniotic inferior even if successful transplant.Corneal alkali burns are the most severe corneal chemical damage. The alkaline substances can rapidly react with the cell lipids because it can dissolve in both water and fat. The compounds formed through the saponification of the alkaline substances can quickly penetrate both the hydrophilic corneal stroma and the lipophilic conjunctiva, corneal epithelium,endothelium, which can cause serious damage to limbal stem cells. The serious lack of limbal stem cells will lead to the persistent inflammation, corneal and conjunctival metaplasia, the corneal scarring. In recent years, there have appeared in limbal stem cell transplantation, amniotic membrane transplantation and cultured limbal stem cell transplantation therapy according to the research on the biological characteristics of limbal stem cells. The combine of stem cells and tissue engineering provides a new way for the treatment of ocular surface diseases. The following is the hope for the treatment of corneal alkali burns stem cells:1. Embryonic stem cells:It is a kind of pluripotent stem cells which derived from early embryonic cells and urogenital ridge can be induced to differentiate into all three embryonic layer cells under certain conditions. He took limbal corneal fibroblasts as substrates and type Ⅳ collagen as planking to induce ESCs differentiate into corneal epithelium cells successfully. Transplanted ESCs which was induced to differentiate into corneal epithelium cells into mouse model with cornea injury, corneal epithelium regeneration was observed in 24 hours.These results suggest that ESCs have the potential to be induced to differentiate into corneal epithelial cells. However, because ESCs involves ethical issues and has the tumorigenic potential risk, thus limiting its further research and application.2. Mesenchymal stem cells:Pluripotent cells with strong ability of proliferation and differentiation, can be directed to differentiate into myocardial cells, epithelial cells, fat cells, nerve cells, osteoblasts and other cells in vitro under different conditions of induction.This kind of cell origin is not clear, the key point is to avoid ethical issues which the embryo stem cell may face, the rejection rate is very low because of autologous tissue as well, thus it has good application prospect.At present, there have been research on in vitro transplantation of human bone marrow mesenchymal stem cells on rabbit model of alkali burn to explore the possibilities of MSCs in treatment of ocular surface damage, good results have been achieved.In recent years, more reports have pointed out that the rat MSCs co-cultured with corneal stromal cells (corneal stromal cells, CSCs) can be successfully induced differentiate into cells with characteristics of corneal stromal cells to be transplanted in alkali-burned mouse model, it has a good curative effect for ocular surface reconstruction. However, due to the complexity of bone marrow cells composition, separation is difficult, very low content of stem cells limits its extensive application in clinic.3. Adipose-derived stem cells:It with MSCs in biological phenotype and function similarly, experiment proves that it had differentiation potential.Studies in vitro found that ADSCs can be differentiation into different tissue cells under different conditions, such as vascular endothelial, muscle, cartilage, nerve, such as fat cells.Because the body fat content is rich, far greater than bone marrow tissue, accessible, it overcomes the drawback of MSCs source shortage, so become more and more popular. But, ADSCs purification problem is still not well solved, at the same time (simultaneously), most studies stay in animal experiments and cell phenotypic differentiation stage, its differentiation into some sort of mature cell phenotype whether still have the corresponding cell function and signaling pathways is still not clear, studied with ADSCs transplantation for the treatment of ocular surface damage were very little at home and abroad.In recent years, research on the pathological mechanism of corneal alkali burn has made considerable progress:corneal alkali burn is a complex pathological process, involving a variety of cells and molecules also plays an important role, except for alkaline substances directly damage participate in the immune systemThe immune inflammatory cells, including lymphocytes, polymorphonuclear leukocytes, langerhans cell, etc.) and immune molecules (such as cytokines, chemotactic factor, adherence factor etc.) influence each other in mediating the pathologic changes of the corneal alkali burn.The present research on stem cell transplantation how to affect is still exist many unsolved problems.We adopted New Zealand white rabbits to isolate ADSCs and CSCs firstly,then ADSCs was induced to co-culture with cells with characteristics of corneal stromal cells in transwell Chambers. Finally, ADSCs were transplanted with amniotic membrane as carrier into rabbit corneal alkali burn model to observe the recovery of corneal morphology and explore its molecular mechanism. After successfully induced, ADSCs may reduce the new blood vessels and corneal opacity effectively by lowering the expression of vascular endothelial growth factor and the expression of related immune inflammation factor so that improve the therapeutic effect.In order to get the following results and conclusions:this research will be studied in following four aspects:PartOne:Isolation and idendification of the rabbit adipose mesenchymal stem cells (ADSCs)ObjectiveTo establish effective system for ADSCs to be isolated,cultured and amplified in vitro, and confirmed that multi-directional differentiation potential of stem cells do exist in the adipose tissue.MethodsRabbit inguinal fat tissue was digested by trypsin, cultured in DMEM medium with 10% fetal bovine serum and amplificated by enzyme digestion. Special phenotype of ADSCs is distinguished through observing the cell morphology and detected by flow cytometry instrument.ResultsAdipose mesenchymal stem cells which come from fat tissue grow into fiber shuttle samples morphology, and stick a wall into a single arrangement. Flow cytometry identification:CD34 (-), CD45 (-), CD105 (+), CD29 (+), CD44 (+), positive rate was 90.23%,88.56% and 90.23% respectively.ConclusionSuccess in isolatationADSCs from the adipose tissue which were with characteristics of stem cell proliferation such as specific surface molecules of cells (CD29+, CD44+, CD34-, CD45-) that are expected to become the source of seed cells of the tissue engineering.PartTwo:ADSCs co-cultivation with CSCs were induced to differentiation into corneal stromal cell in vitro.ObjectiveTo co-cultivate ADSCs with CSCs which were induced to differentiation into corneal stromal cell so that it can lay the foundation for further study of effect of ADSCs transplantation of corneal alkali burn and its molecular mechanism.MethodsADSCs and CSCs were cultivated In lower level of transwell culture system (aperture 0.45 microns),1 week after induction, keratin-pan fluorescence staining and RT-PCR were used to appraisal expression of corneal stromal cells markers(keratin-pan fluorescence staining).ResultsThe results of of keratin-pan mRNA RT-PCR showed that expression of corneal stromal cells’markers (keratin-pan) was positive.ConclusionADSCs can be differentiation into corneal stromal cells under certain conditions in vitro.PartThree:Observation of clinical effect of adipose mesenchymal stem cell transplantation for rabbit corneal alkali burn.ObjectiveTo observe whetherrabbit adipose mesenchymal stem cell transplantation can improve therapeutic effect in treating of corneal alkali burn.MethodsThe New Zealand white rabbits (60 eyes) with corneal alkali burn models were randomly divided into four groups:A group will be ADSCs which co-cultured with corneal stromal cells (corneal stromal cells, CSCs) with amniotic membrane as the carrier for transplantation; group B is directly ADSCs with amniotic membrane for transplantation, group C is amniotic membrane transplantation; D group receive no treatment.Observing the degree of corneal opacity, formation of neovascularization after one week, two weeks, one month after transplantation.ResultsAfter transplantation, the degree of corneal opacity score and neovascularization area after one week, two weeks, one month, pairwise comparisons between the four groups were statistically significant (P<0.05), moreover, A group of the degree of corneal opacity score and neovascularization area in three time points were significantly smaller than the other three groups.ConclusionRabbit adipose-derived mesenchymal stem cell transplantation was effectivefor repairing theocular surface alkali damage. ADSCs transplantation treatment can obviously reduce the new blood vessels formation after alkali burn, improve state of corneal opacity and transparency, and promote the restoration of tissue injury after corneal alkali burn.Part Four:Observation effect of Adipose mesenchymal stem cell transplantation on the concentration of related factors after treatment of the corneal alkali burn.ObjectiveTo detectthe concentration of related factors after adipose mesenchymal stem cell transplantation in treatment of the corneal alkali burn.MethodsNew Zealand rabbit of four groups were killed one month after transplantation, aqueous humor and corneal tissue samples were taken, the content of VEGF and corneal tissue inflammation related factor IL-10, CD-45, IFN-γ of posterior chamber water were determined using ELISA method after transplantation, and then changes of factor concentration in four groups were observed.ResultsOne month after transplantation, concentration of VEGF, CD45, IL-10, IFN-γ in four groups by ELISA assay, pairwise comparisons were statistically significant (P <0.01), concentration of CD45, IFN-γ were significantly lower in group A than in the other three groups, concentration of IL-10 in group A was significantly higher than the other three groups; and VEGF concentration pairwise comparisons between the four groups, except C, D group (P=0.525), others were statistically significant, and A group was significantly lower than the other three groups ((P<0.01).ConclusionThe mechanism of ADSCs transplantation for treatment of alkali burn may be that ADSCs could be differentiation into a kind of cell with the characteristics of corneal cells under certain condition, together with which could reduce the immune inflammatory reaction and inhibit the formation of new blood vessels. |
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