Inducer for differentiation of multi-generation 3T3-L1 cell line and method for differentiating multi-generation 3T3-L1 cell line into white fat cells

Abstract

The invention relates to an inducer for differentiation of a multi-generation 3T3-L1 cell line and a method for differentiating the multi-generation 3T3-L1 cell line into white fat cells. According to the inducer for differentiation of the multi-algebraic 3T3-L1 cell line, disclosed by the invention, 5 [mu] M of Troglitazone (trelaglitazone) is further added into a fat-forming cocktail inducer which takes insulin (Insulin), dexamethasone (DEX, Dexametasone) and 3-isobutyl-1-methylxanthine (IBMX, Isobutylxanthine) as main components, so that the differentiation of the multi-algebraic 3T3-L1 cell line can be inhibited, and the differentiation of the multi-algebraic 3T3-L1 cell line can be inhibited. The 3T3-L1 cell line can still be differentiated into mature white adipocytes instead of beige adipocytes after passage for more than 10 times, and the efficiency of inducing and differentiating the 3T3-L1 cell line into the white adipocytes is improved to 70-80%. Compared with a conventional 3T3-L1 cell line induced differentiation method, the induced differentiation method disclosed by the invention has the advantages that mature white adipocytes instead of beige adipocytes can be obtained; according to the induced differentiation method, the induced differentiation rate can reach 70%-80% without prolonging the number of days; according to the method, the time cost is greatly saved, and the consumption of consumables is greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of adipocyte induction and differentiation, and specifically relates to an inducer for the differentiation of multi-generation 3T3-L1 cell lines, and a method for differentiating multi-generation 3T3-L1 cell lines into white adipocytes. Background Art

[0002] The 3T3-L1 cell is a mouse embryonic fibroblast cell line that can not only be stably passaged but also has the ability to differentiate well into mature adipocytes. It is an internationally recognized cell model for studying fat metabolism and has great scientific research value for studying the role of adipocytes in obesity and its related metabolic diseases.

[0003] The 3T3-L1 preadipocytes were originally derived from Swiss mouse embryonic tissues. Because of their potential to be induced to differentiate from fibroblasts into mature adipocytes, this cell line has been widely used to advance the understanding of the basic cellular mechanisms related to obesity, diabetes, and related diseases.

[0004] The induction and differentiation of conventional 3T3-L1 cell lines usually uses an adipogenic cocktail inducer including insulin, dexamethasone (DEX), and 3-isobutyl-1-methylxanthine (IBMX) as the main components. Through 48 hours of contact inhibition, the 3T3-L1 cells stop proliferating. Under the stimulation of the mixed hormones, the expression program of adipogenic genes is initiated during and after 2-3 rounds of division called mitotic clonal expansion (MCE), and then leads to terminal differentiation.

[0005] There are many factors affecting the differentiation efficiency of 3T3-L1 cells, such as passage number, cell line source, culture dish, etc. The induction and differentiation rate of multi-generation mouse preadipocytes 3T3-L1 is low.

[0006] To improve the differentiation efficiency of multi-generation 3T3-L1 cells, a screening method is disclosed in patent document CN116396927A. After the 3T3-L1 cells are about 1 hour post-confluent and adhere to the culture dish, the fresh complete medium is replaced to remove the aged cells. This method can extend the number of generations of 3T3-L1 cells that can be successfully induced to differentiate and also has a relatively high differentiation success rate. However, the inducer used in this induction differentiation method contains rosiglitazone. Patent documents CN107858326A and CN116396927A both disclose 3T3-L1 induction differentiators containing rosiglitazone. This induction method can indeed increase the differentiation rate of the 3T3-L1 cell line. However, after introducing rosiglitazone, the 3T3-L1 cell line differentiates into beige adipocytes instead of white adipocytes. Beige adipocytes are different from white adipocytes and have a thermogenic function, which is not an ideal product. Summary of the Invention

[0007] One object of the present invention is to provide an inducer capable of efficiently inducing multi-generation 3T3-L1 cells to differentiate into white adipocytes.

[0008] Another object of the present invention is to provide a method capable of efficiently inducing multi-generation 3T3-L1 cells to differentiate into white adipocytes.

[0009] To achieve the above objects of the present invention, the following technical solutions are adopted in the present invention:

[0010] The present invention provides an inducer for the differentiation of multi-generation 3T3-L1 cell lines, which includes Insulin, Dexamethasone (DEX), Isobutylmethylxanthine (IBMX), and Troglitazone.

[0011] Among them, the inducer includes: 0.5 mM Isobutylmethylxanthine, 10 μM Dexamethasone, 1.744 mM Insulin, and 5 μM Troglitazone.

[0012] The present invention provides a method for inducing the differentiation of multi-generation 3T3-L1 cell lines into white adipocytes, and the method includes the following steps:

[0013] 1) Inoculate 3T3-L1 cells into a culture dish, use fresh complete medium, and culture them under the conditions of 37 °C, 5% CO2, and saturated humidity. When the 3T3-L1 cells grow to confluence and reach contact inhibition, start the induction of differentiation, and mark the number of days at each stage during the induction of differentiation;

[0014] The complete medium contains high-glucose DMEM with 10% FBS (fetal bovine serum) and 1% double antibody;

[0015] 2) Discard the complete medium, slowly add the No. 1 medium along the side wall of the culture dish using a micropipette, and record the current day as day0;

[0016] The No. 1 medium is a complete medium containing 0.5 mM Isobutylmethylxanthine + 10 μM Dexamethasone + 1.744 mM Insulin + 5 μM Troglitazone;

[0017] 3) Two days after adding the No. 1 medium, discard the No. 1 medium, slowly add the No. 2 medium along the side wall of the culture dish using a micropipette, and record the current day as day2;

[0018] The No. 2 medium is a complete medium containing 1.744 mM Insulin + 5 μM Troglitazone;

[0019] 4) Two days after adding the No. 2 medium, discard the No. 2 medium, and then slowly add the fresh complete medium along the side wall of the culture dish using a micropipette, and record the current day as day4; After that, replace the fresh complete medium every two days until day8;

[0020] 5) After day8, stop the induction of differentiation.

[0021] In step 1), when the 3T3-L1 cells grow to confluence and reach contact inhibition, it means growing for another 24 h after the cells reach confluence.

[0022] During the induction of differentiation, the present invention adopts the method of changing the entire liquid.

[0023] Since the added drugs have a certain stimulation to 3T3-L1 cells and the 3T3-L1 cells are relatively fragile, the additives need to be gently added along the side wall of the culture dish.

[0024] During the induction of differentiation, observe the differentiation state of the cells every day, and observe the size and number of lipid droplets under a microscope.

[0025] Before induction of differentiation, it may further include the routine culture of 3T3-L1 cells, including the following steps:

[0026] I-1) Resuscitate 3T3-L1 cells: Place 3T3-L1 cells in complete medium and culture them at 37°C, 5% CO2, and saturated humidity conditions;

[0027] The complete medium contains high-glucose DMEM with 10% FBS (fetal bovine serum) and 1% double antibody;

[0028] I-2) After culturing for two days, wash with PBS buffer and replace with fresh complete medium;

[0029] I-3) When the confluence of 3T3-L1 cells is detected to reach more than 80%, perform trypsin digestion and subculture;

[0030] I-4) The subculture ratio is 1:3 to 1:4, and make the 3T3-L1 cells evenly distributed.

[0031] During routine culture, according to the state of 3T3-L1 cells, replace with fresh complete medium one or two days after subculture.

[0032] Compared with the prior art, the advantages of the present invention are as follows:

[0033] The present invention provides a novel inducer for the differentiation of multi-generation 3T3-L1 cell lines. By further adding 5 μM Troglitazone to the adipogenic cocktail inducer including Insulin, Dexamethasone (DEX), and Isobutylmethylxanthine (IBMX) as the main components, it can still differentiate into mature white adipocytes rather than beige adipocytes after more than 10 passages, and improve the efficiency of inducing the differentiation of 3T3-L1 cell lines into white adipocytes to 70%-80%.

[0034] Troglitazone has a thiazolidinedione structure and belongs to thiazolidinedione analogs. The characteristics of thiazolidinedione drugs are that they can significantly enhance the sensitivity of body tissues to insulin, improve the function of pancreatic islet β cells, achieve long-term control of blood glucose, and thus reduce the risk of diabetes complications.

[0035] Compared with the conventional method for inducing differentiation of 3T3-L1 cell line, the method for inducing differentiation of the present invention can obtain mature white adipocytes instead of beige adipocytes; the method for inducing differentiation of the present invention does not require an extended number of days and can achieve an induction differentiation rate of 70%-80%; the method of the present invention greatly saves time costs and the use of consumables. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the process flow chart of Example 1 of the present invention;

[0037] Figure 2 is the result chart of the test example of the present invention;

[0038] Among them,

[0039] Figure 2(a) and Figure 2(b) are the macroscopic view and the results under the phase contrast microscope after Oil Red O staining, respectively;

[0040] Figure 2(c) is the absorbance value measured after extraction with isopropanol;

[0041] Figure 2(d) is the mRNA analysis result of the related indexes of adipocyte differentiation and maturation;

[0042] Figure 2(e) is the mRNA analysis result of the indexes related to adipocyte thermogenesis;

[0043] Figure 2(f) and Figure 2(g) are the protein expression differences and statistical results of UCP1 in four groups of cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] The technical solutions of the present invention will be described in detail below with reference to the drawings and embodiments.

[0045] Comparative Example 1

[0046] 1) Resuscitate 3T3-L1 cells: Place 3T3-L1 cells in a complete medium of high-glucose DMEM containing 10% FBS and 1% double antibody, and culture them under the conditions of 37°C, 5% CO2, and saturated humidity;

[0047] 2) After culturing for two days, wash with PBS buffer and replace with fresh complete medium;

[0048] 3) When the confluence of 3T3-L1 cells is detected to reach 80%-100%, perform trypsin digestion and subculture;

[0049] 4) Subculture ratio 1:3 - 1:4, and make 3T3-L1 cells evenly distributed;

[0050] 5) According to the cell state, choose to replace the fresh complete medium one day or two days after subculture as appropriate;

[0051] 6) According to the experimental needs, 3T3-L1 cells were inoculated into a six-well plate as a culture vessel, and fresh complete medium was used to culture at 37°C, 5% CO2, and saturated humidity. When the 3T3-L1 cells were fully grown and grown for another 24 hours to achieve contact inhibition, induction of differentiation was started, and the days of each stage were marked during the induction of differentiation;

[0052] 7) Discard the old complete medium, and slowly add the prepared 1A medium (complete medium containing 0.5 mM Isobutylmethylxanthine (3-isobutyl-1-methylxanthine) + 10 μM Dexamethasone (dexamethasone) + 1.744 mM Insulin) along the side wall of the culture dish using a micropipette, and record the day as day 0;

[0053] 8) Two days after adding medium 1A, discard the old medium 1A and slowly add the prepared medium 2A (including complete medium with 1.744 mM insulin) along the side wall of the culture dish using a micropipette (the operation must be gentle, otherwise it is easy for cells to curl or float up in pieces, etc.), and record that day as day 2;

[0054] 9) Two days after adding medium 2A, discard the old medium 2A and slowly add fresh complete medium along the side wall of the culture dish using a micropipette (be gentle during the operation, otherwise it is easy for cells to curl or float up in pieces). Record that day as day 4. After that, replace the complete medium with fresh one every two days until day 8.

[0055] 10) After day 8, stop inducing differentiation.

[0056] Comparative Example 2

[0057] Conventional culture and differentiation induction were carried out in the same manner as in Comparative Example 1, except that:

[0058] In step 7), medium 1B (complete medium containing 0.5 mM Isobutylmethylxanthine + 10 μM Dexamethasone + 1.744 mM Insulin + 60 μM Indomethacin) is used instead of medium 1A; and

[0059] In step 8), medium 2B (complete medium containing 1.744 mM Insulin + 60 μM Indomethacin) was used instead of medium 2A.

[0060] Comparative Example 3

[0061] Conventional culture and induced differentiation were carried out in the same manner as in Comparative Example 1, except that:

[0062] In step 7), medium 1C (complete medium containing 0.5 mM Isobutylmethylxanthine + 10 μM Dexamethasone + 1.744 mM Insulin + 1 μM Rosiglitazone) was used instead of medium 1A;

[0063] In step 8), medium 2C (complete medium containing 1.744 mM Insulin + 1 μM Rosiglitazone) was used instead of medium 2A; and

[0064] In step 9), complete medium containing 1 μM Rosiglitazone was used instead of the complete medium.

[0065] Example 1

[0066] Conventional culture and induced differentiation were carried out in the same manner as in Comparative Example 1, except that:

[0067] In step 7), medium 1 (complete medium containing 0.5 mM Isobutylmethylxanthine + 10 μM Dexamethasone + 1.744 mM Insulin + 5 μM Troglitazone) was used instead of medium 1A; and

[0068] In step 8), medium 2 (complete medium containing 1.744 mM Insulin + 5 μM Troglitazone) was used instead of medium 2A.

[0069] Three replicates were set for Example 1 and Comparative Examples 1 to 3 above. During the process of induced differentiation, the cell state was observed every day and the differentiation situation was recorded. It was found that: In Example 1 and Comparative Examples 2 and 3, the phenotype of adipocytes could be observed on the 4th day of the start of induction, lipid droplets appeared, lipid droplets generally appeared on the 6th day, and the differentiation was successful. However, in Comparative Example 1 during the process of induced differentiation, only a few lipid droplets appeared on the 8th day.

[0070] Thus, compared with Comparative Example 1, by introducing troglitazone (Example 1), indomethacin (Comparative Example 2), or rosiglitazone (Comparative Example 3), a higher cell induced differentiation rate can be obtained.

[0071] Test Example

[0072] On the 8th day of induced differentiation, Oil Red O staining was performed on the cells in Comparative Examples 1 to 3 and Example 1, and the absorbance was detected by isopropanol extraction to compare the degree of cell differentiation.

[0073] The specific method of Oil Red staining is as follows: The cells were rinsed three times with PBS buffer for 5 minutes each time, and 4% paraformaldehyde was added and fixed at room temperature for 1 hour, paying attention to avoiding light. After fixation, the cells were rinsed three times with PBS buffer for 5 minutes each time. The Oil Red O stock solution was mixed with water at a ratio of 3:2 to prepare the Oil Red O working solution. After mixing the mixed solution, it was filtered through a 0.22 μm filter, and then the cells were stained for 2 hours and incubated at room temperature in the dark. Discard the Oil Red O working solution, and the cells were washed three times with PBS buffer for 5 minutes each time until no obvious red granular substances were seen in the solution. The cells were placed under an inverted phase contrast microscope for observation and photography.

[0074] After that, isopropanol (500 μl of isopropanol was added per 10 cm 2 culture dish bottom area) was added to the cells after Oil Red O staining to extract the Oil Red O dye in the cells, and the liquid in the dish was carefully pipetted to ensure thorough mixing of the solution. 100 μl of the extract of each group of cells was added to a 96-well plate (3 replicates were set for each group), and 100 μl of isopropanol was pipetted into the 96-well plate as a blank control. The OD value was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 525 nm (or a nearby wavelength). The OD value measured for each group minus the OD value of the blank control group is the relative value of the triglyceride content in the cells.

[0075] The staining results and statistical results are shown in Figure 2.

[0076] Figures 2(a) and 2(b) are the results observed by the naked eye and under an inverted phase contrast microscope after Oil Red O staining, respectively. In Figure 2(a), it can be observed with the naked eye that compared with Comparative Example 1, Comparative Examples 2, 3 and Example 1 all have more red lipid droplets. In Figure 2(b), it can be observed under an inverted phase contrast microscope that compared with Comparative Example 1, Comparative Examples 2, 3 and Example 1 all have more red lipid droplets and darker colors. Therefore, it is proved from the morphological angle that Example 1, Comparative Example 2 and Comparative Example 3 all have an improved adipocyte differentiation and maturation rate.

[0077] Figure 2(c) shows the absorbance values measured after extraction with isopropanol. It can be seen that the absorbance value of Comparative Example 1 using a conventional inducer is the lowest, the absorbance value of Comparative Example 2 with indomethacin introduced as an inducer has increased, and the absorbance values of Example 1 with troglitazone introduced as an inducer and Comparative Example 3 with rosiglitazone introduced as an inducer are the highest. This once again proves from the perspective of optical density analysis that compared with Comparative Example 1, Comparative Examples 2, 3 and Example 1 all have a higher differentiation and maturation rate.

[0078] Figure 2(d) shows the mRNA analysis results of related indicators for adipocyte differentiation and maturation. As can be seen from the figure, the expression levels of the genes Adiponectin, C / ebpα (CCAAT / enhancer-binding protein α), Fabp4 (fatty acid-binding protein 4), and Pparγ (peroxisome proliferator-activated receptor γ) for cell differentiation in Comparative Example 2, Comparative Example 3, and Example 1 are all significantly higher than those in Comparative Example 1. Therefore, at the transcriptional level, it is shown that Example 1, Comparative Example 2, and Comparative Example 3 can all effectively increase the ratio of cell differentiation and maturation.

[0079] Figure 2(e) shows the mRNA analysis results of related indicators for adipocyte thermogenesis. As a positive control, the adipocytes in Comparative Example 3 differentiated into mature beige adipocytes, so the expression levels of the thermogenic genes Ucp1 (uncoupling protein 1), Pgc1-α (peroxisome proliferator-activated receptor γ coactivator 1α), Prdm16 (PR / SET domain-containing protein 16), and Cidea (cell death-inducing DFFA-like effector A) are higher. In contrast, the expression levels of the thermogenic genes in Example 1 are comparable to those in Comparative Example 1 and are much lower than those in Comparative Example 3. Therefore, at the transcriptional level, it is shown that the cells in Example 1 differentiated into white adipocytes required by the present invention, while the cells in Comparative Example 2 and Comparative Example 3 differentiated into beige adipocytes.

[0080] Figures 2(f) and 2(g) show the protein expression differences and statistical results of UCP1 in the four groups of cells of Comparative Example 1 to Comparative Example 3 and Example 1. Among them, Comparative Example 3 is the positive control, and its adipocytes differentiated into mature beige adipocytes, so the expression of the thermogenic gene uncoupling protein 1 is higher (α-Tubulin is α-tubulin, used as an internal reference). Compared with Comparative Example 3, the expression of uncoupling protein 1 in Comparative Example 1, Comparative Example 2, and Example 1 is significantly lower than that in Comparative Example 3 and is not significantly upregulated. Therefore, at the protein level, it is again shown that the cells in Example 1 differentiated into white adipocytes required by the present invention, while the cells in Comparative Example 2 and Comparative Example 3 differentiated into beige adipocytes.

[0081] In summary, taking Comparative Example 3 as the positive control, the cells in Comparative Example 3 differentiated into beige adipocytes. By detecting the expression of thermogenic genes and cell differentiation genes at the protein and mRNA levels of each group of cells, it is proved that the inducer of the present invention can efficiently induce multi-generation 3T3-L1 cells to differentiate into white adipocytes. Compared with the conventional method for inducing the differentiation of 3T3-L1 cell lines, the method for inducing differentiation of the present invention can obtain mature white adipocytes instead of beige adipocytes; the method for inducing differentiation of the present invention does not require an extended number of days and can achieve an induction differentiation rate of 70%-80%; the method of the present invention greatly saves time costs and the use of consumables.

Claims

1. An inducer for the differentiation of multi-generation 3T3-L1 cell lines, comprising: Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and troglitazone.

2. The inducer for differentiating multi-generation 3T3-L1 cell lines according to claim 1, wherein the inducer comprises: 0.5 mM 3-isobutyl-1-methylxanthine, 10 μM dexamethasone, 1.744 mM insulin, and 5 μM troglitazone.

3. A method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes, the method comprising the following steps: 1) Inoculate 3T3-L1 cells into a culture dish, use fresh complete medium, and culture under the conditions of 37 °C, 5% CO2, and saturated humidity. When the 3T3-L1 cells grow to confluence and reach contact inhibition, start the induction of differentiation, and mark the number of days at each stage during the induction of differentiation; The complete medium contains high-glucose DMEM with 10% FBS and 1% double antibiotics; 2) Discard the complete medium, add Medium 1 to the culture dish, and record the current day as day0; Medium 1 is a complete medium containing 0.5 mM 3-isobutyl-1-methylxanthine, 10 μM dexamethasone, 1.744 mM insulin, and 5 μM troglitazone; 3) Two days after adding Medium 1, discard Medium 1, add Medium 2 to the culture dish, and record the current day as day2; Medium 2 is a complete medium containing 1.744 mM insulin and 5 μM troglitazone; 4) Two days after adding Medium 2, discard Medium 2, and then add fresh complete medium to the culture dish, and record the current day as day4; After that, replace the fresh complete medium every two days until day8; 5) After day8, stop the induction of differentiation.

4. The method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes according to claim 3, wherein when the 3T3-L1 cells in step 1) grow to confluence and reach contact inhibition, it is 24 hours after the cells grow to confluence.

5. The method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes according to claim 3, wherein the induction of differentiation uses the method of total liquid replacement.

6. The method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes according to claim 3, wherein In step 2), adding Medium 1 to the culture dish is to slowly add Medium 1 along the side wall of the culture dish with a micropipette; In step 3), adding Medium 2 to the culture dish is to slowly add Medium 2 along the side wall of the culture dish with a micropipette; In step 4), adding the complete medium to the culture dish is to slowly add the complete medium along the side wall of the culture dish with a micropipette.

7. The method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes according to claim 3, wherein During the induction of differentiation, observe the differentiation state of the cells every day, and observe the size and number of lipid droplets with a microscope.

8. The method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes according to claim 3, Before the induction of differentiation, it further includes the conventional culture of 3T3-L1 cells.

9. The method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes according to claim 8, wherein the conventional culture includes the following steps: I-1) Resuscitating 3T3-L1 cells: placing the 3T3-L1 cells in a complete medium and culturing them under the conditions of 37 °C, 5% CO2, and saturated humidity; The complete medium contains high-glucose DMEM with 10% FBS and 1% double antibody; I-2) After culturing for two days, washing with PBS buffer and replacing with fresh complete medium; I-3) When the confluence of 3T3-L1 cells is detected to reach more than 80%, performing trypsin digestion and subculture; I-4) The subculture ratio is 1:3 to 1:4, and the 3T3-L1 cells are evenly distributed.

10. The method for inducing the differentiation of a multi-generation 3T3-L1 cell line into white adipocytes according to claim 8, wherein during the conventional culture, according to the state of the 3T3-L1 cells, fresh complete medium is replaced one or two days after subculture.

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