Construction And Application Of Visualization Model For Bone Cell-culture Of Bone Micro-environment

Background:The components of bone cells in the bone microenvironment are mainly composed of osteoblasts,osteoclasts and osteocytes,of which osteoblasts and osteoclasts protect the integrity of the skeletal system through bone remodeling;it is also used by scholars to study cell models commonly associated with bone diseases such as osteoporosis,osteoarthritis,and periodontal diseases.Existing models of osteoblast co-cultivation have been studied in the effects of osteogenesis on osteogenesis and differentiation,the interaction between osteogenesis and osteogenesis,as well as in osteopathy pharmacology research and application in medical biomaterials.The research on the proportion of co-cultivation is relatively small,so a new co-cultivation model is constructed in this paper,and a new cell line is constructed using the transfection method and a co-cultivation system is established.Objective:In this experiment,the osteoblast precursor cells and osteoclast precursor cells were carried with different fluorescent reporter genes by transfection method.Through co-cultivation at different ratios,a new visual co-culture model was finally constructed.The model is as follows The one-step microfluidic platform provides a reliable co-cultivation system,and also guarantees the cultivation technology.Methods:(1)MC3T3E1 was divided into experimental group plus induction solution under induction of osteogenic induction solution,and normal culture solution of control group,then ALP and alizarin red staining was done after 7 days of induction,and Q-PCR fluorescence quantitative analysis was measured of ALP m RNA;RAW264.7 in Bone induction fluid was divided into experimental group plus induction fluid,and control group was cultured normally,TRAP staining was performed for 5 days after induction,and TRAP m RNA was quantitatively analyzed by Q-PCR;(2)MC3T3E1 with fluorescent target gene was established by virus transfection.The fluorescence intensity analysis was performed after 5 days of culture in the experimental group and the control group with normal culture medium;MC3T3E1cell line was in the experimental group and normal culture with induction medium The liquid control group was analyzed by fluorescence microscopy after 7 days of culture;(3)Q-PCR quantitative fluorescence analysis was performed on MC3T3E1 and RAW264.7 induced after transfection,respectively,to detect the expression of target genes COL?m RNA and CTSKm RAN;(4)Establish a co-culture system of MC3T3E1 and RAW264.7 after induction with a fluorescent target gene,and culture them at a ratio of 1: 1,1: 2,1: 3,1: 4,2: 1,and 3: 1,respectively1,3,5,7 days,and then perform fluorescence intensity analysis;(5)Establish a co-culture system of MC3T3E1 and RAW264.7 after induction with a fluorescent target gene,and culture for 1,3,and 5 days at 1: 1,1: 2,and 2: 1,respectively,and then perform Q-PCR to quantify fluorescence The analysis of OPG /RANK / COL? / CTSK gene expression was performed.Results:(1)MC3T3E1 was induced by the induction solution,and the experimental group and the control group were stained with ALP and Alizarin Red,respectively.The staining results of the osteoblast precursor cells in the experimental group were more obvious than those in the control group.Trap staining analysis was performed under induction,and more osteoclasts were formed in the experimental and control groups;(2)Quantitative analysis of ALP was performed by Q-PCR on MC3T3E1 after induction,and the gene expression of ALP m RNA in the experimental group washigher than that of the control group;RAW264.7 after induction was used for quantitative analysis of TARP by Q-PCR.The gene expression of TRAP m RNA was more obvious than that of the control group;(3)MC3T3E1 virus-transfected with the target gene was induced by the induction solution.The experimental group began to increase in fluorescence intensity on the fifth day of induction,while the control group had almost no significant change.RAW264.7 induced by the induction solution,the fluorescence intensity of the experimental group began to increase on the 3rd day,and there was no significant change in the control group;(4)MC3T3E1 and osteoclast precursor cells were induced after transfection,and Q-PCR fluorescence quantitative analysis was performed in the experimental group and the control group.The expression of CTSK m RAN in the experimental group was higher in osteoblast precursor cells,and in RAW264.7 The expression of COL?m RNA in the experimental group was higher than that in the control group;(5)Co-cultured osteoblasts and RAW264.7 at a ratio of 1: 1,1: 2,1: 3,1: 4,2: 1and 3: 1,the fluorescence intensity analysis showed that 1: 2 was more Ideal co-cultivation ratio;(6)Co-cultured osteoblasts and RAW264.7 at a ratio of 1: 1,1: 2 and 2: 1,the OPG / RANK m RNA first increased and then decreased with the increase of time,COL? / CTSK m RNA With the increase of time,it also rises first and then decreases.Conclusions:Through the establishment of cell lines and the construction of co-cultivation methods,it is finally concluded that the ratio of 1:2 in different ratios of the co-cultivation system is the ratio;and the ratio of gene expression and co-culture of OPG / RANK / COL? / CTSK in the co-cultivation ratio The proportion of the corresponding cells in the medium and the culture time increased first and then decreased.