The Protective Effect Of Lycopene On The Osteogenic Differentiation Of Mouse Adipose-derived Mesenchymal Stem Cell Under Oxidative Stress

Objective:1.Successfully isolate and culture adipose-derived mesenchymal stem cells(ADSC)from mouse inguinal fat;successfully complete the passage,cryopreservation and resuscitation of ADSC;conduct dryness identification of ADSC.2.Explore the effects of different concentrations of H2O2 on the proliferation of ADSC,and screen out the appropriate concentration of H2O2 to construct an oxidative stress model for ADSC.3.Explore the effect of oxidative stress on the osteogenic differentiation of ADSC,and explore the protective effect of lycopene on the osteogenic differentiation of ADSC under oxidative stress.Method:1.According to the type I collagenase digestion method,separate and extract ADSC from the inguinal fat of mice and culture in vitro;use trypsin digestion method for cell passage;use gradient cryopreservation method for liquid nitrogen storage method for cell cryopreservation;use separately Flow cytometry was used to identify the surface markers of ADSC,and the differentiation experiment was used to identify the multidirectional differentiation ability of ADSC.2.Use different concentrations of H2O2 to intervene in ADSC,observe the growth status of cells under a microscope,use CCK-8 and TUNEL experiments to detect the effects of different concentrations of H2O2 on the proliferation and apoptosis of ADSC,use ROS staining technology to observe different concentrations of H2O2 Effect on the level of ROS produced by ADSC.3.Use lycopene to interfere with the oxidative stress model of ADSC induced by H2O2,use CCK-8,TUNEL experiment,and ROS staining technique to detect the effect of lycopene on the proliferation ability of ADSC under oxidative stress,use ALP staining and activity detection experiment,Alizarin red staining and calcium quantitative detection experiments,PCR and Western blot experiments to detect the effect of lycopene on the osteogenic ability of ADSC under oxidative stress.Results:1.The expression rate of the positive surface markers CD29 and CD90 of the cultured ADSC mesenchymal stem cells are above 90%,and the expression rate of the negative surface marker CD45 is below 5%;orange-red lipids can be seen by oil red O staining in the induction of adipogenic differentiation Drop;Induced osteogenic differentiation and stained with Alizarin Red,brick red calcium nodules can be seen.2.CCK-8,TUNEL experiment,ROS staining technology detection found that H2O2 can induce mouse ADSC to produce ROS and cause oxidative stress.A small amount of ROS can promote the cell proliferation of mouse ADSC,and excessive ROS will inhibit mouse ADSC.The cell proliferation,and at the same time induce cell apoptosis.3.CCK-8,TUNEL experiment,and ROS staining technology test found that the order of the proliferation rate of ADSC in each group is blank control group=lycopene group>co-treatment group>oxidative stress group,and the order of apoptosis rate and ROS level is Oxidative stress group> co-treatment group> blank control group> lycopene group;through ALP staining and activity detection experiment,alizarin red staining and calcium quantitative detection experiment,it was found that the ADSC activity of each group was blank control group=lycopene Vegetarian group> co-treatment group> oxidative stress group,the calcium content of ADSC in each group is lycopene group> blank control group>co-treatment group> oxidative stress group;the COL1 of ADSC in each group is detected by PCR and Western blot experiments,OCN and RUNX2 gene and protein expression.It was found that the expression levels of bone formation-related important genes and proteins in the lycopene group were significantly higher than those in the blank control group,and the gene and protein expression levels in the oxidative stress group were significantly lower than those in the blank control group.In the co-treatment group,due to the intervention of lycopene,the expression levels of various genes and proteins were significantly improved compared with the oxidative stress group.Conclusion:4.The ADSC extracted from adipose tissue has the ability of multidirectional differentiation such as bone formation,and the purity of stem cells is high.5.Low concentration of ROS promotes ADSC proliferation,high concentration of ROS inhibits ADSC proliferation and accelerates apoptosis.6.Oxidative stress inhibits the osteogenic differentiation of ADSC,and lycopene resists oxidative stress to induce apoptosis of ADSC.7.Lycopene promotes the osteogenesis of ADSC while resisting the oxidative stress-induced osteogenesis disorder of ADSC.