| Cell hypoxia is a common cell state in cardio-cerebrovascular diseases and cancers.The cellular hypoxia response is regulated by multiple factors and multiple pathways.The tolerance of different cells to hypoxia was significantly different.Exploring cell-associated regulatory mechanisms under hypoxia will instruct people to reduce the damage caused by hypoxia on cells through the regulation of related mechanisms,thereby inhibiting cell apoptosis and promoting cell regeneration and repair.Human dental pulp cells(h DPCs)are separated from the pulp tissue.Because the pulp tissue is surrounded by hard dentin,the oxygen reaching the pulp tissue needs to pass through the blood vessels in the narrow root canal.The lack of an effective collateral circulation results in a much lower oxygen concentration in the pulp tissue than in the normal environment.This shows that human dental pulp cells adapt to hypoxic conditions and have a certain tolerance to hypoxia.One of the organelles that is particularly sensitive to cellular hypoxia is mitochondria,the main site for cellular respiration and energy supply.Hypoxia can lead to adaptive changes in the morphological structure of the mitochondria,or even pathological changes.Mi R-210,as a hypoxia-specific mi RNA,there are twoversions of mi R-210,namely,mi R-210-3p and mi R210-5p.It will protect cells from hypoxia and inhibit mitochondrial respiration and apoptosis.Mi R-210 has been widely studied in hypoxic cardiovascular and cerebrovascular diseases and tumors.However,the regulation of mi R-210 and related studies in human dental pulp cells after hypoxia have been rarely reported.Objective:To explore the changes in mitochondrial morphology and function of human dental pulp cells under hypoxia,observe the ultrastructural changes of mitochondria after hypoxia,and further explore the role of mi R-210 in the regulation of human dental pulp cells in the condition of hypoxia.Methods:human dental pulp cells were cultured in vitro by tissue explant collagenase method The inoculated fifth generation cells were placed in the anaerobic tank(5%CO2,95%N2)and 5% CO2 in a constant temperature incubator for 6h,12 h,18h and 24 h,the mitochondrial membrane potential was detected by flow cytometry using Rhodamine123 staining;The inoculated fifth generation cells were placed in the anaerobic tank(5%CO2,95%N2)for 24 h and 5% CO2 in a constant temperature incubator for 6h,12 h,18h and 24 h,they were fixed,stained,sectioned,and the ultrastructural changes of mitochondria were observed by transmission electron microscopy;The inoculated fifth generation cells were placed in the anaerobic tank(5%CO2,95%N2)and 5% CO2 in a constant temperature incubator for 6h,12 h,18h and 24 h,using real-time PCR to detect the expression of mi R-210-3p.Results:Human dental pulp cells were successfully obtained by tissue explant collagenase method.After culturing in a constant temperature incubator for 3 to 5 days,it was observed under the microscope that the cells around the tissue blocks swam out,and it could also be seen that the individual cells or cell populations digested by the enzyme adhered to the wall.The cells are spindle-shaped,with protrusions that resemble the morphology of fibroblasts,with clear and central nuclei,rich cytoplasm and abundant cytoplasm.After passage,cells can adhere to the wall within 24 hours,and the cells grow well after adherence.The value-added rate is faster,and it can be passed once in an average of 3 to 5 days.After the passage,the cells were long spindle-shaped,with protrusions,and the cells were arranged radially or whirlpool-shaped.The flow cytometry was used to detect the mitochondrial membrane potential of the cells.The results showed that the mitochondrial membrane potential of normoxic cells increased slightly with time,and the mitochondrial membrane potential of hypoxia was significantly decreased(P<0.01),and the mitochondrial membrane potential in the hypoxia group was significantly lower than that in the normoxia group(P<0.01).The results of transmission electron microscopy showed that the cytomembrane structure of the normoxic group was intact,the organelles in the cytoplasm were abundant,the nuclear membrane was complete,the size and quantity of mitochondria were normal.The mitochondrial membrane structure is complete,the ridges are clear,and the ridges are arranged perpendicular to the long axis of the mitochondria.A clear bilayer membrane structure was observed in the nuclear membrane;In the hypoxia 6h group,the membrane structure was intact,the number of mitochondria was increased compared with the normoxic group,the mitochondrial structure was intact,and the hypertrophic mitochondria were seen;in the hypoxia group of 12 h,vacuole-like changes occur in the cytoplasm of cells,and various morphological changes occur in the mitochondria: mitochondrial swelling,deformation,mitochondrial rupture,mitochondrial cristae disintegration,vacuolar degeneration,longitudinal mitochondrial(parallel to the mitochondrial long axis),and myelin-like structures etc..In the hypoxia group of 18 h,cytoplasm vacuoles increased,and many pathological changes were also observed.The mitochondrial rupture was increased more than 12h;in the hypoxia group of 24 h,A large number of vacuoles appeared in the cytoplasm,chromatin margins and nuclear membrane rupture.4.The expression of mi R-210-3p in hypoxic group increased significantly after 6 h,but decreased after 24 h.There was no obvious change in the expression of mi R-210-3p in normoxic group.The expression levels were significantly increased and there was a statistical significance(P<0.01).Conclusions:1.With prolonged hypoxia,the mitochondrial membrane potential of human dental pulp cells was decreased,cell injury was aggravated,and the expression of mi R-210-3p was up-regulated,which was positively correlated with the degree of cell injury.2.mi R-210-3p is closely related to the hypoxia of human dental pulp cells and may be involved in the process of cell apoptosis. |
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