| Research background Obesity is a type of metabolic syndrome caused by the body’s energy intake exceeding its energy expenditure.Its main manifestations are excessive deposition of white adipose tissue,abnormal proliferation of adipocytes,and imbalance of differentiation.At the same time,because adipose tissue has the functions of regulating body temperature,storing energy,and buffering mechanical shocks,and obesity exacerbates the inflammatory effect of adipose tissue,resulting in increased incidence and mortality of some cardiovascular diseases such as hypertension and thrombosis.In terms of cell biology,pre-adipocytes are a type of cells that have the ability to proliferate and differentiate into mature adipocytes at the same time.This precursor cell undergoes four stages of mitotic cloning,growth arrest,further cloning,and terminal differentiation under the action of different growth factors and hormones.The cells lose their fibroblastic morphology,mature adipocytes characterized by the accumulation of triglycerides in the cytoplasm.This process is called "adipogenic differentiation".This process has been a hot topic in the field of obesity research.In order to understand intuitively the process of lipid accumulation inside cell differentiation,it is necessary to establish a controllable and stable differentiation model of mature adipocytes in vitro.The mouse’s 3T3L1 is derived from Swiss mouse embryos at gestational age of about 18 days.It can differentiate into mature adipocytes under appropriate induction conditions.At the same time,it can also undergo single differentiation after transplanting into mice,which can simulate living adipose tissue,so the 3T3-L1 fibroblast cell line has been one of the most extensive cell lines for related in vitro studies [1,2] The classic 3T3-L1 preadipocyte induction method is to use a fixed dose of dexamethasone,3-isobutyl-1-methylxanthine(IBMX),insulin and cell culture Mixed,and then different induction culture medium to culture cells according to a certain time difference,called the cocktail method [3].This method comes with disadvantages such as the influence of the culture equipment and the number of cell generations,the low conversion rate,and the unstable differentiation process [4].Therefore,the conditions for inducing differentiation of 3T3L1 cells are first explored and optimized,and the establishment of a suitable cell model is crucial for subsequent research.Therefore,based on the classic literature reports,combined with the actual working conditions of the laboratory,a series of explorations on the adipogenic differentiation conditions of the cell line were carried out,and a method with a high differentiation rate was established.Micro RNA has been one of the hot areas of biological research in the past three decades.The role of such regulatory non-coding RNAs in the differentiation,proliferation and survival of adipocytes has been widely revealed,suggesting that it may serve as a new biological target for obesity-related diseases[5].Mi R-222-3p has been implicated as a potential regulator of lipogeneeis.Therefore,using 3T3L1 cells as a model,the selected was transfected into the cells,and the transfection conditions were initially determined.The mechanism was predicted in order to provide basic reference for subsequent research.Objective In this study,in vitro culture of mouse 3T3L1 fibroblasts was used.Based on the classic "cocktail" induction method,a set of highly efficient differentiations suitable for the laboratory was explored by changing the inducing time and the glucose concentration in the culture medium.At the same time,the conditions for specific miR-222-3p transfection into cells were explored and the possible targets genes were predicted.Method 1.3T3L1 cells,including recovery,basic culture,and cryopreservation.2.The classic "cocktail" hormone method induces differentiation of preadipocytes,oil red O staining,and observes the rate of induced differentiation under a microscope.3.To investigate the effect of glucose concentration on the adipogenic differentiation rate of 3T3L1 cells by changing the glucose concentration of basal DMEM medium in the induction solution.4.Investigate the effect of induction time on the rate of adipogenic differentiation of 3T3L1 cells by changing the effect time of the two inducers in the induction process.5.Liposomal transfection of miR-222-3p mimic,inhibitor and corresponding controls into 3T3L1 preadipocytes,and RT-q PCR was used to confirm transfection efficiency.6.Use Target Scan7,miRDB.Pic Tar,miRanda and other prediction software to predict the possible target genes of miR-222-3p on adipocyte differentiation.Result 1.The classic "cocktail" method was used to induce 3T3L1 preadipocytes.After induction,they were stained with oil red O,and lipid droplets were observed under the microscope.2.High-glucose DMEM medium(glucose concentration 4500 mg / L), low-sugar DMEM medium(glucose concentration 1000 mg / L),and RPMI-1640 medium(glucose concentration 2000 mg / L)were used as induction basal medium to induce cells.After the induction process,the staining results showed the rate of positive cells in the low glucose-induced group was significantly higher than that in the two groups(*P <0.05),while the induction rate in the high-glucose group and the 1640 group have similar differentiation efficiency.3.On the basis of the classic "cocktail" induction method,by changing the action time of inducer Ⅱ,the staining results after the inducer showed that the number of positive cells in day 3 group was significantly higher than that in day 2 group and day 4 group(*P <0.05),but there was no significant difference between day2 and day4 groups.The experimental grouping induction rate was significantly higher than the control group.4.Transfect miR-222-3p mimic,inhibitor and the corresponding control with 3T3L1 preadipocytes with liposome transfection reagents.Use the total miRNA extraction kit to perform RT-q PCR to verify the transfection effect.Results The miR-222-3p content was found to be significantly higher in the mimic group and significantly lower in the inhibitor group than in the control group.(*P <0.05)5.The four miRNA target online prediction tools were used to predict the possible targets of miR-222-3p,and the results of each group were compared online using Venny2.1.0 to obtain 12 potential targets.Conclusion 1.The classic "cocktail" method induces 3T3L1 preadipocytes to induce differentiation,and the differentiated cells show lipid droplet aggregation,but the ratio is not high.The control group is undifferentiated cells without lipid droplets in the cytoplasm.2.Changing the glucose concentration of the basal medium in the induction solution found that the low-glucose group had a higher effect of inducing differentiation.3.Change the action time of the induction solution Ⅱ.The results show that the cells induced by the induction solution II for 3 days have a higher rate of induced differentiation than the traditional cocktail method for 2 days.When it is extended,the differentiation rate will no longer increase.4.Different target prediction software predicted multiple identical miR-222-3p targets. |
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