| The mesenchymal stem cells possess the ability to self-sustain,renew,and differentiate.Mesenchymal stem cells can differentiate into multiple cellular types and have various potential medical benefits.Argali is classified in second-class catalog of state animal protection project.It is very difficult to obtain fresh tissue samples under the strict requirement of national environmental protection.For the purpose to carry out the research of hand-made clone on Argali without damage to the donor,the umbilical cord mesenchymal stem cells from umbilical cord of a newborn Argali was collected,isolated and cultured,and the differentiation ability of the cells were detected in vitro.The details are as below:The umbilical cord attached to the placenta was cut off after the delivery and expulsion of the placenta by the female Argali,and Wharton’s jelly was obtained under aseptic condition.The cells were cultured by the method of tissue block culture.After the cells were cultured stably,the P5,P1 0 and P15 cells were selected for a proliferation test,and cell doubling time was measured.Osteogenic differentiation was performed by adding 10 mmol/L β-glycerophosphate,0.05 mmol/L ascorbic acid and 0.1 μmol/L dexamethasone in DMEM/F12 medium to the P8 cells.Chondrogenic differentiation was performed by adding 10 ng/mL TGF-P3,0.1 μmol/L dexamethasone,1%ITS(100×),0.3 mmol/L ascorbic acid,and adipogenic differentiation was performed by adding 60 μmol/L indomethacin,5 μg/mL insulin,1 μmol/L dexamethasone,0.5 mmol/L IBMX.On the 7th,14th and 21st day after induction of differentiation,the cells of each group were stained with tissue-specific staining to determine the degree of specific differentiation,and the total RNA was extracted from each group,and the differentiation-specific genes were detected by fluorescence quantitative analysis for evaluating the changes in genes expression.The main results were as follows:(1)The mesenchymal stem cell isolated from Argali umbilical cord was attached to the wall in the form of fibril and formed many stable clones.Being cultured to 15 passages,the cells morphology was kept properly.The cellular growth curve was a "S" type,and the doubling time was on average of 33.5 h.(2)Cells in osteoblast-induced differentiated group grew in clusters and formed clonal protuberances with large network-like rough surface processes in vitro.The clone-like protuberances showed typical orange-red calcium deposits with alizarin red S staining.With the increase of differentiation time,the shape and color of the calcium deposits increased.Real-Time PCR results showed that with the extension of inducting time,the relative expression of BGLAP,OPN and RUNX2 genes in the osteogenic differentiation group increased extremely significantly.(3)When which were induced with chondrogenic differentiation media in vitro to the 5th day,the cells showed cartilage matrix-like structure and increased cartilage matrix on the 21st day.After staining with alcian blue solution,the cartilage matrix appeared blue.The results of real-time PCR showed that the expression of LUM gene in the chondrogenic differentiation group increased significantly,while the expression of BGN and SOX9 genes increased extremely significantly.(4)On the 3rd day after adipogenesis,the cells showed short rods and small fat droplets,and on the 21st day after adipogenesis,most of cells were adhered to the wall in a network while a little of cells shed off.The fat globules in cells sample from the 7th and 14th day induced groups appeared dark red with oil red O staining.Real-Time PCR results showed that the relative expression of LPL and PPARy genes in the adipogenic differentiation group increased extremely significantly,while the relative expression of SCD gene decreased significantly.These results indicated that the umbilical cord mesenchymal stem cell from the Argali could be isolated and cultured by tissue block culture method,and the stem cells could be differentiated into osteoblast,chondroblast and adipocyte.These results showed a new way for collection and utilization of wild animal mesenchymal stem cell successfully and provided a reference for the rational utilization of wild animal resources. |
PDF Full Text Request