Biological Function Research Of Dedifferentiated Human Umbilical Cord Mesenchymal Stem Cells

Objective: To study the basic biological functions and anti-oxidative stress survival ability and mechanism of dedifferentiation of human umbilical cord mesenchymal stem cells,in order to further broaden people's understanding of the biological properties of human umbilical cord mesenchymal stem cells,and provide reference for the clinical development and utilization of this type of mesenchymal stem cells,it has theoretical and practical significance.Method: The mesenchymal stem cells were isolated and cultured from the umbilical cord of the newborn fetus by the tissue block method,the human umbilical cord mesenchymal stem cells(h MSCs)were subjected to transient adipogenic induction culture in a medium of adipogenic differentiation-inducing complete medium,and the induction conditions were removed,and the initial stem cell culture medium was changed to become De-h MSCs,and observed the morphology of the obtained cells under an inverted microscope;In order to identify and evaluate the basic biological functions of De-h MSCs,the experiments were divided into two groups: control group(h MSCs)and experimental group(De-h MSCs).By comparing the biological functions of the two groups of cells,in order to find the biological functional advantages of De-h MSCs.The specific contents include: using CCK-8 to detect the proliferative capacity of the cells,and detecting the immunophenotype of the cslls by flow cytometry,and detecting the cells to adipogenic and osteoblasts by oil red O and alizarin red staining,the differentiation ability of the cells was evaluated by RT-PCR,and the in vitro migration ability of the cells was detected by transwell;In order to further study the anti-oxidative stress survival ability and mechanism of dedifferentiation of human umbilical cord mesenchymal stem cells,this experiment the concentration of t-butyl hydroperoxide(t-BHP)was determined by CCK-8 method to establish a cell injury model,flow cytometry was used to measure the cycle distribution and apoptosis of the two groups of cells,the signal molecules related to apoptosis and autophagy were detected by Western blot,and the autophagy of the two groups was observed by laser confocal microscopy.Result: Human umbilical cord mesenchymal stem cells can be isolated from human umbilical cord tissue within 2-3 weeks by tissue method,the method has high success rate and stable separation effect,the obtained cells are easy to be expanded in vitro,and have stable morphological characteristics of mesenchymal stem cells after multiple passages.After h MSCs enter into adipogenic differentiation,the differentiation induction medium is removed,and the initial stem cell culture is carried out to become De-h MSCs,the cells have the same shape as h MSCs,and have a long spindle shape with uniform morphology;and the basic biological functions of the experimental group De-h MSCs were compared with the control group,this cell had the same proliferation and migration ability in vitro as the control group,but there was no significant difference between the two groups.Both surface markers CD90,CD73,and CD105 associated with stromal cells were highly expressed,and surface markers CD34 and human leukocyte common antigen CD45 were not expressed.Both have the ability to differentiate into adipogenic and osteoblasts,and the efficiency of adipogenic differentiation of De-h MSCs is significantly higher than that of control h MSCs(p<0.05);We also found that after treatment of 300?M t-BHP in both groups,the cell cycle of both groups was blocked,but the experimental group is more tolerant to cell cycle arrest caused by t-BHP,at the same time,the survival rate of the cells was also affected,and the cell survival rate of the control group was significantly lower than that of the experimental group(p<0.05).Western blot was used to detect the changes of key molecules in the signal transduction pathways such as apoptosis and autophagy in the control group and the experimental group before and after t-BHP treatment,the expression of anti-apoptotic protein molecules in the experimental group was significantly higher than that in the control group(p<0.05),and the expression of autophagy molecule LC-3B-II in the control group was significantly higher than that in the experimental group(p<0.05),by laser confocal observation,it was found that both groups of cells showed different degrees of autophagy,but the degree of autophagy in the control group was significantly higher than that in the experimental group,it is indicated that the change of autophagy activity caused by dedifferentiation is also involved in the tolerance of De-h MSCs to t-BHP.Conclusion: De-h MSCs and h MSCs have the same morphological characteristics,and both have strong proliferation and migration ability in vitro,uniform expression of stromal cell-associated surface markers CD90?CD73?CD105,does not express hematopoietic stem cells surface marker CD34 and human leukocyte common antigen CD45,both have the ability to differentiate into adipogenic and osteoblasts,and the efficiency of adipogenic re-differentiation is significantly higher than that of h MSCs.The above results: on the one hand,it is confirmed that our primary isolated cells are MSCs;on the one hand,it indicates that dedifferentiation does not affect the proliferation,immunophenotype,differentiation and migration ability of h MSCs in vitro,and has stronger adipogenic re-differentiation ability after dedifferentiation;De-h MSCs are significantly superior to h MSCs in their ability to resist apoptosis and alter autophagy activity,this ability is to make up for the low survival rate of h MSCs after transplantation,and it is expected to become a seed cell with more advantages in clinical regenerative therapy,and provide theoretical basis and experimental reference for the clinical optimization application of De-h MSCs.