The Differentiation Potential Of Ovine Amnion Epithelial Cells And Its Application In Treatment Of Bone Injury Model

The amnion epithelial cells(AECs) is a type of cells possessed with stem cell characteristics and derive from superseded placenta amnion. AECs are not only have multi-directional differentiation potentiality, but possess immune exemption potency that immunological rejection and tumorigenesis are not generated after post transplant because of terminal transferas telomere defected, and AECs are gained readily and not cause ethics dispute, which make it become one hot spot of cell researches. To apply AECs curing some serious disease is the final objective of clinical researchers. Furthermore, successful clinical application and treat are base on safe and reliable animal experiment. At present, human amnion epithelial cells are studied chiefly and adopted to treat liver disease, pulmonary tuberculosis, heart disease, diabetes, nervous system disease and so on. However, the research on ovine amnion epithelial cells is not report in domestic and scarced in internationally. Therefore, isolating, culture and assessing stem cell characteristics, differentiation in vivo and direction location induction and differentiation in vitro of ovine amnion epithelial cells are proceed system research in this study.1. Isolating and culture of ovine amnion epithelial cellsThe isolating and digestion methods of ovine amnion epithelial cells in accordance with the means of digestion human amnion epithelial cells were explored. And pancreatic enzyme surrogate TrypLE was adopted lastly to digest ovine amnion and isolate primary cell that are obtained with the features of high vigor and purity coefficient. The culture solution contained with15%fetal bovine serum was applied and maintained excellently the favourable growth tendency of ovine amnion epithelial cells.The optimum density and scope of cultural density of cells inoculation were detected through observation and comparision in different density gradient culture experiment. It was found, via observing cell growth cycle in optimum density scope, that the incubation period of ovine amnion epithelial cells was1to2days, and exponential growth phase was3to5days, afterward maintaining invariably.The freezing and storing scheme of cells was definited by freezing and storing experiments, which DMSO was conducted as freezing and storing protective additive and FBS and DMSO were mingled according to certain ratio(FBS:DMSO=9:1) as freezing and storing solution.Moreover, to adopt the simple and quick freezing and storing ways that removed cells from-80℃to liquid nitrogen were efficiently preserve ovine amnion epithelial cells.2. Assessment the stem cell characteristics of ovine amnion epithelial cellsCell immunofluorescence and RT-PCR were performed to detect respectively the expression of embryonic stem cell of ovine AECs marker proteins Oct-4, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81and totipotency related genes OCT-4, SOX-2, Nanog, Rex-1and so on. The observed Oct-4, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81immunoreactivity of ovine AECs were predominant. RT-PCR experiments confirmed that the genes of Oct-4, Sox-2, Rex-1were expressed and Nanog was not expressed. The results indicated that the ovine AECs have the multi-directional differentiation potentiality of stem cell properties.3. Directional induction and differentiation of ovine amnion epithelial cells in vitroTo check the multi-directional differentiation potentiality of ovine amnion epithelial cells in vitro, ovine AECs were induced to directional differentiate into nervous systemcells, osteoblast and adipocyte utilizing some additional factors. Cell immunofluorescence and RT-PCR technologies, alkaline phosphatase activities detection, alizarin bordeaux stain, oil red O dyeing were performed respectively to accredit induced and differentiated cells. The results showed:①the specific marker β-Ⅲ-tubulin in cellula nervosa and GFAP in nerve cement cells were observed positive immunofluorescence signals after28day in induced nerves direction; the genes of nestin, β-Ⅲ-tubulin and MAP-2were expressed.②alkaline phosphatase activities in bone formation induction and differentiation were elevate slightly after28day and begin to higher obviously than control group after35days. Moreover, the activities were to continue increase as cultural time prolong and the differences between induce and control group were evident; calcify nodus were visible in alizarin monosulfonate stain.③it could be observed lipid droplet formation by oil red O dyeing in cellula adiposa induction and differentiation on42d; alkaline phosphatase activities in foundation culture group were reach to maximum and higher conspicuously than each induced group. From above research to the morphous characteristics of differentiated cells, we may safely draw the suppose that ovine amnion epithelial cells have already induced and differentiated into positive pro-mature neuron cells expressed β-Ⅲ-tubulin, positive mature neuron cells expressed MAP-2, positive nerve cement cells expressed GFAP, osteoblast with calcify nodus and cellula adiposa with lipid droplet.4. study of therapy efficacy applied ovine amnion epithelial cells in rabbit radius defected models Injecting directly ovine amnion epithelial cells into ovine vivo to cure achilles tendon injury had reported and therapeutic efficacy was extremely excellent. To test the possibility of acquired ovine amnion epithelial cells differentiation in rabbit vivo and similar therapeutic efficacy with reported above, the13-centimetre long radius defected models were made artificially in30rabbits, divided into high dose group, low dose group and control group randomly. To carry out cellular transplant by intravenous injection according to setting dose after dealing with ovine aminion epithelial cells. Took the models for X-ray in2weeks,4weeks and8weeks before the ovine aminion epithelial cells transplantations, after operation and injection cells respectively and meanwhile observed histomorphology. The resultes hint:defect tissues were repaired in the high dose group at8weeks, observed from the X-ray and histological section of new bone, and the quality and quantities of trabecular bones in high dose group were better and higher than in the low dose group and it was the same situation between the low dose group and the control group. Accordingly, it was presume that ovine amnion epithelial cells in rabbit radius defected models in vivo were differentiated into bone and accelerated repaired speed and quality of bone coloboma.And the study indicated that it is feasible to apply ovine amnion epithelial cells to heal small bone defect.In this study successfully gain ovine amnion epithelial cells possessed with stem cell characteristics are have the multi-directional differentiation potency in vivo and in vitro. And the results in this research supply with the theory knowledges of AECs in stem cell characteristics and differentiation in vivo and in vitro, and establish rationale and provide practice base for clinical exploratory development.