| Objective: With the increasing aging of the population,the number of bone defects caused by trauma,degeneration,tumor,deformity,infection and other reasons has increased sharply.At present,bone tissue engineering is the most effective method to treat bone defects,but it is an important problem for seed cells to maintain or even enhance their dry characteristics in the process of proliferation in vitro.At present,the most ideal seed cells are bone marrow mesenchymal stem cells(BMSC),which have strong self-renewal ability,can differentiate into multi-lineage adult cells and low immunogenicity.The Tet(The ten-even translation)family is a group of DNA demethylases that can regulate a variety of epigenetic reactions.Tet protein can convert 5-methylcytosine(5-m C)into 5-hydroxymethylcytosine(5-hm C)and its oxidation derivatives,thus promoting DNA demethylation and affecting gene transcription.It has been reported in relevant literature that Tet2 can negatively regulate BMSC stem expression,but its molecular mechanism is still unclear.Methods: Rat bone marrow mesenchymal stem cells were first extracted,and 10 healthy male rats aged 4 weeks,weighing 300-400 g,were selected from SD rats.After anesthesia,the middle segment of the femur was taken,the bone marrow was washed after full disinfection,and the bone marrow mesenchymal stem cells were retained after three subcultures.Then verify the relationship between mi R-29b-3p and TET2,verify the corresponding relationship between mi R-29b-3p and TET2 gene with double luciferase test,preliminarily verify its corresponding relationship,and then further verify with western blot test and real-time fluorescence quantitative(q RCR)test to observe and compare the changes of TET2 expression in the transfected mi R-29b-3p mimics and mi R-29b-3p inhibitor groups.Secondly,verify the relationship between TET2 and E-cadherin protein,verify the regulatory effect of TET2 on E-cadherin by western blot and q PCR test,compare and observe the expression changes of E-cadherin after transfection of TET2 plasmid and TET2 si RNA,and further verify that TET2 protein is used for E-cadherin by playing the role of deacetylation,add the deacetylation inhibitor MS275 to the wild cell culture medium,and add the same dose of culture medium to the control group,Then Western blot and q PCR tests were used to verify the expression of E-cadherin,and the corresponding relationship was further verified by IP test.TET2 was fished with pan-acetylated antibody,and the changes of TET2 expression in the control group and the dosing group were observed and compared.Then verify the effect of E-cadherin on cell dryness,use western blot and q PCR to verify the regulatory effect of E-cadherin on cell dryness,and compare and observe the expression changes of cell dryness markers Oct4,Sox2,Nanog after transfection of Ecadherin plasmid and si RNA.Then the whole cell pathway was verified from the beginning.Western blot and q PCR experiments were used to further verify the regulation of mi R-29b-3p on the expression of downstream E-cadherin and cell stem markers,and the expression of E-cadherin and cell stem markers after transfection of mimics mi R 29b-3p and inhibitor mi R 29b-3p were compared.Then further verify the effect of mi R-29b-3p,TET2 and E-cadherin on cell stem from the aspect of cell phenotype,verify the ability of cell stem differentiation with cell cloning experiment and induced cell osteogenesis and lipogenic staining experiment,and verify the effect of mi R-29b-3p/TET2 and E-cadherin on cell stem from the aspect of cell phenotype.Results: 1.Rat bone marrow mesenchymal stem cells were first extracted,and then the purity of the extracted cells was verified by CD90 fluorescence.The purity was more than 90%.2.To verify the relationship between mi R-29b-3p and TET2,the results of dual-fluciferase test showed the corresponding relationship between mi R-29b-3p and TET2 gene,and then further verified by western blot and q RCR experiments.When mi R-29b-3p mimics was transfected,the amount of TET2 protein imprinting and RNA expression decreased,and when mi R-29b-3p inhibitor was transfected,the amount of TET2 protein imprinting and RNA expression increased,which proved that mi R-29b-3p and TET2 were negatively correlated.3.To verify the relationship between TET2 and E-cadherin protein,when TET2 plasmid is overexpressed,the amount of protein imprinting and RNA expression decreases;when TET2 si RNA is overexpressed,the amount of protein imprinting and RNA expression increases,which proves that TET2 is negatively correlated with the expression of E-cadherin.It was further verified that TET2 protein was used for E-cadherin through deacetylation.The results showed that the expression of E-cadherin was significantly increased after adding deacetylation inhibitor.It was further verified by IP experiment that after adding MS275,the expression of TET2 was increased by fishing with panacetylation antibody.It further proved that the expression of TET2 was decreased by using deacetylation for Ecadherin.4.To verify the effect of E-cadherin on cell dryness,Western blot and q PCR experiments were used to verify the regulatory effect of E-cadherin on cell dryness.When E-cadherin plasmid was overexpressed,the expression of cell dryness markers Oct4,Sox2,and Nanog increased.When E-cadherin si RNA was transfected,the expression of cell dryness markers Oct4,Sox2,and Nanog decreased,which proved that E-cadherin had a positive regulatory effect on cell dryness.Then verify the complete cell pathway from scratch,and further verify the regulation of the expression of downstream E-cadherin and cell stem markers by mi R-29b-3p with western blot and q RCR experiment.When mi R-29b-3p mimics is transfected,the expression of Ecadherin and cell stem markers Oct4,Sox2,and Nanog are decreased.When mi R-29b-3p inhibitor is transfected,the expression of E-cadherin and cell stem markers Oct4, Sox2,and Nanog are increased,It is proved that mi R-29b-3p has a positive regulatory effect on E-cadherin and cell stem.5.Further verify the effect of mi R-29b-3p,TET2 and E-cadherin on cell dryness from the aspect of cell phenotype,and verify the ability of cell dryness differentiation by cell cloning experiment and induced cell osteogenesis and lipogenesis staining experiment.When the transfected mimics over-expressed mi R-29b-3p,si RNA knocks down TET2,and the plasmid over-expressed E-cadherin,it was found that the cell cloning ability was enhanced,and the staining after induced culture showed that the ability of osteogenesis and lipogenesis was enhanced;When the transfection inhibitor low expressed mi R-29b-3p,the plasmid over-expressed TET2,and the si RNA low expressed E-cadherin,the staining showed that the ability of osteogenesis and lipogenesis decreased after induction and culture.The effect of mi R-29b-3p/TET2 and E-cadherin on cell stem function was fully proved from the aspect of cell phenotype.Conclusion: From multiple levels of cells,it is confirmed that mi R-29b-3p can inhibit the expression of TET2 by targeting,relieve the inhibition of the expression of Ecadherin by deacetylation of TET2,thus promote the expression of E-cadherin,and finally make the expression of stem markers up-regulated,and increase the stem expression of bone marrow mesenchymal stem cells. |
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