Method for inducing human adipose-derived stem cells to be differentiated into insulin-secreting cells
Through the method of synergistic induction of photobiomodulation technology and inducing factors, the problems of low differentiation efficiency and long cycle of human adipose stem cells are solved, and efficient and accurate differentiation of insulin secretion cells is achieved.
Patent Information
- Application Number
- CN202510435209.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, the differentiation efficiency of human adipose stem cells to insulin secreting cells is low and the induction cycle is long, especially the efficiency of one-step induction method, and the process of step-by-step induction method is complicated.
The method of synergistic induction of photobiomodulation technology and inducing factors is adopted to activate intracellular signaling pathways, improve energy supply, and regulate human adipose stem cell differentiation by adding inducing factors in stages and combining near-infrared light irradiation.
It significantly improves the efficiency of cell differentiation, simplifies the induction process, shortens the induction cycle, and ensures the stability and accuracy of the differentiation process.
Smart Images
Figure CN120249183A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for inducing human adipose stem cells to differentiate into insulin-secreting cells. Background Art
[0002] Diabetes is a chronic metabolic disease. The main cause is that the body of patients fails to secrete enough insulin, resulting in the body's inability to maintain normal glucose metabolism balance, and thus hyperglycemia symptoms occur. The traditional treatment methods for diabetes are mostly exogenous insulin injection. However, this method cannot achieve the physiological blood glucose control effect provided by native islet cells and cannot get rid of drug control. Stem cells have the characteristics of self-renewal, unlimited proliferation and multi-directional differentiation potential, and can be successfully differentiated into insulin-secreting cells with functions similar to islet β cells under in vitro induction conditions. The transplantation of such insulin-secreting cells differentiated from stem cells provides a new solution for the treatment of diabetes.
[0003] Human adipose stem cells are rich in sources, easy to obtain and have low immunogenicity, and are important cell sources for differentiating into insulin-secreting cells. Currently, the methods for inducing human adipose stem cells to differentiate into insulin-secreting cells mainly include one-step induction method and stepwise induction method. Among them, the one-step induction method does not require changing the culture medium and is simple to operate. However, because a variety of induction factors added simultaneously will affect the cell differentiation at different stages, the cell-directed differentiation efficiency is relatively low; the stepwise induction method can avoid the influence of induction factors at different stages on cell differentiation, but it requires changing the culture medium multiple times, the induction process is complex, and the induction period is long. Summary of the Invention
[0004] Aiming at the problems of low induction and differentiation efficiency and long induction period of the existing methods, the present invention provides a method for inducing human adipose stem cells to differentiate into insulin-secreting cells. Compared with the existing methods, the present invention combines photobiomodulation technology, and regulates the differentiation of human adipose stem cells by activating intracellular signal pathways and improving cell energy supply, and this method can significantly improve the cell differentiation efficiency.
[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for inducing human adipose stem cells to differentiate into insulin-secreting cells, the specific content is as follows: Step 1: Add an equal volume of collagenase to human adipose tissue, digest at 37 °C for 1 h, then add serum-containing medium to terminate digestion, centrifuge, discard the supernatant, add serum-containing medium to resuspend again, then passage and culture 3 times at 37 °C, collect the human adipose stem cells after passage and culture, dilute them with PBS buffer solution, and place the diluted human adipose stem cells in a well plate to self-aggregate to obtain human adipose stem cells that self-aggregate into spheres; Step 2: Place the self-aggregated human adipose stem cells obtained in Step 1 into an induction differentiation medium, and induce differentiation at 37°C for 2 days to complete the first-stage induction differentiation and obtain a first induction differentiation system; Step 3: Add retinoic acid and transforming growth factor-β1 to the first induction differentiation system described in Step 2, and irradiate with near-infrared light at 10 minutes per day at 37°C for 2 consecutive days to complete the second-stage induction differentiation and obtain a second induction differentiation system; Step 4: Add taurine, betacellulin, insulin-like growth factor, nicotinamide, and BMP type I receptor kinase inhibitor to the second induction differentiation system described in Step 3, and irradiate with near-infrared light at 10 minutes per day at 37°C for 5 consecutive days to collect insulin-secreting cells.
[0006] Preferably, the collagenase in Step 1 is 1 mg / mL collagenase IV. Selecting collagenase IV with high activity and high catalytic efficiency can fully digest human adipose tissue in a relatively short time, obtain a large number of dispersed human adipose stem cells faster, and reduce the time required for separating adipose stem cells; the action conditions of collagenase IV are mild, which can better maintain the integrity and biological activity of human adipose stem cells and is beneficial to subsequent induction differentiation.
[0007] Preferably, the serum-containing medium in Step 1 is 10% fetal bovine serum αMEM medium. αMEM medium contains various nutrients such as amino acids, vitamins, inorganic salts, and glucose, and fetal bovine serum contains rich bioactive substances such as growth factors, hormones, and transferrin; in the present invention, αMEM medium containing fetal bovine serum is used for culturing human adipose stem cells. On the one hand, it can provide the basic substances required for the growth and metabolism of human adipose stem cells, and on the other hand, the active components such as growth factors in fetal bovine serum can significantly enhance the vitality and metabolic function of human adipose stem cells, which is beneficial to cell proliferation and differentiation. Preferably, the formula of the induction differentiation medium in Step 2 is DMEM / F12 basal medium, 3% fetal bovine serum (FBS), and 100 ng / mL activin A. In the present invention, DMEM / F12 basal medium provides rich nutrients for human adipose stem cells, ensuring the rapid growth and proliferation of cells; 3% fetal bovine serum can not only provide the required growth factors for cells but also avoid some problems caused by too high serum concentration to a certain extent; activin A induces human adipose stem cells to differentiate in the endoderm direction, laying a foundation for subsequent differentiation into insulin-secreting cells.
[0008] Preferably, the final mass concentration of retinoic acid in step three is 1-5 mg / L, and the final mass concentration of transforming growth factor-β1 is 2-20 μg / L. It should be noted that the final mass concentration involved in the present invention refers to the mass contained per unit volume in the final state of the induction and differentiation system of the inducer. The present invention induces human adipose stem cells to form a cell population similar to pancreatic endocrine progenitor cells, preparing for subsequent further differentiation into mature insulin-secreting cells; transforming growth factor-β1 plays a role in inhibiting cell proliferation, appropriately slowing down the division rate of human adipose stem cells, thereby creating favorable conditions for cell differentiation; within the above mass concentration range, retinoic acid and transforming growth factor-β1 can not only effectively play an inducing role, but also have no other effects on cells.
[0009] Preferably, the intensity of the near-infrared light in step three is 10 J / cm 2 , and the wavelength range is 760-1100 nm. The present invention combines the technology of photobiomodulation, and through the synergistic induction of near-infrared light irradiation and inducers, the usage amount of the inducer is significantly reduced; at the same time, since photobiomodulation can rapidly activate the signal transduction in cells and effectively improve the energy supply in cells, there is no need to perform the operation of changing the culture medium during the culture process, greatly simplifying the induction process and significantly improving the cell differentiation efficiency.
[0010] Preferably, in step 4, the final mass concentration of taurine is 0.1-1.0 ug / L, the final mass concentration of betacellulin is 10-20 ug / L, the final mass concentration of insulin-like growth factor is 20-50 ug / L, the final mass concentration of nicotinamide is 0.5-1.0 g / L, and the final mass concentration of BMPI type receptor kinase inhibitor is 0.1-3.0 ug / L. The taurine used in the present invention can maintain the osmotic pressure balance in cells. Within the above mass concentration range, it can create a stable intracellular environment for the differentiation of adipose stem cells into insulin-secreting cells, ensuring the normal progress of biochemical reactions and signal transduction in cells; betacellulin can bind to receptors on the cell surface, activate the Ras-Raf-MEK-ERK signal pathway in cells, and at the same time up-regulate the gene expression related to the differentiation of insulin-secreting cells, promoting the differentiation of cells into insulin-secreting cells; insulin-like growth factor can regulate the gene expression profile in cells and jointly promote the expression of insulin-secreting cell-specific genes with factors such as betacellulin, promoting cell differentiation and functional maturation; nicotinamide participates in the redox reaction in cells, regulates the energy metabolism of cells, and provides sufficient energy for the differentiation of human adipose stem cells into insulin-secreting cells; the BMPI type receptor kinase inhibitor can specifically inhibit the activity of type I receptor kinase in the BMP signal pathway, preventing cells from differentiating into other non-insulin-secreting cells, thereby promoting the differentiation process of human adipose stem cells into insulin-secreting cells; the above-mentioned inducing factors cooperate and regulate each other within an appropriate mass concentration range to jointly regulate the differentiation process of human adipose stem cells into insulin-secreting cells.
[0011] The beneficial effects of the present invention are as follows: (1) In the present invention, through the pre-stage well plate culture, human adipose stem cells are self-aggregated to form cell clusters, and their morphology is highly similar to that of the cell clusters of natural islets. This unique cell cluster structure lays a good foundation for subsequent cell differentiation, greatly improving the stability and efficiency of the cell differentiation process; (2) The present invention innovatively uses the method of synergistic induction of photobiomodulation and inducing factors, significantly reducing the usage amount of inducing factors. At the same time, because photobiomodulation can quickly activate signal transduction in cells and effectively improve the energy supply in cells, there is no need to replace the culture medium during the culture process, greatly simplifying the induction process and significantly improving the cell differentiation efficiency, having outstanding application value in related fields; (3) The present invention adopts the method of adding cell growth factors in stages according to needs to precisely control the cell culture process. This method can effectively avoid the unexpected effects that may be caused by the simultaneous action of multiple inducing factors on cells at different differentiation stages, ensuring that the cell differentiation process is more precise and efficient; (4)The method provided by the present invention can exert a powerful positive driving force on the cell differentiation process at the molecular and cellular levels through a unique technical path and regulatory mechanism, achieving a significant improvement in the degree of differentiation, significantly shortening the induction period, and effectively improving the differentiation efficiency. Description of the Drawings
[0012] Figure 1 It is a detection result diagram of human adipose stem cell surface markers.
[0013] Figure 2 It is a staining result diagram for identifying the differentiation ability of human adipose stem cells.
[0014] Figure 3 It is a detection diagram of the expression of insulin-secreting cell marker genes.
[0015] Figure 4 It is a detection diagram of the insulin secretion amount of insulin-secreting cells. Detailed Embodiments
[0016] The technical solution of the present invention will be further described in detail below through specific examples, but the present invention is not limited thereto.
[0017] Example 1 A method for inducing the differentiation of human adipose stem cells into insulin-secreting cells, the specific steps are as follows: Step 1: Rinse the minced human adipose tissue repeatedly with PBS buffer in a laminar flow hood, then take 10 mL and put it into a 50 mL sterile centrifuge tube. Add collagenase IV at a concentration of 1 mg / mL equal in volume to the human adipose tissue to the centrifuge tube, and incubate and digest in a constant temperature shaker at 37°C for 1 h. Then add αMEM medium containing 10% fetal bovine serum equal in volume to the centrifuge tube to terminate the digestion. Place the centrifuge tube in a centrifuge, set the centrifuge speed to 1000 r / min, and centrifuge for 10 min. Discard the supernatant. Then add αMEM medium containing 10% fetal bovine serum equal in volume to the centrifuge tube, gently pipette and mix well with a pipette tip, resuspend the cells, and place the centrifuge tube in an incubator at 37°C and 5% CO2 for culture. After subculturing 3 times, centrifuge at 1000 r / min for 10 min, collect the obtained human adipose stem cells, dilute them with PBS buffer, adjust the cell density to 1×10 6 cells / mL, and then place them in a sterile 96-well plate to allow them to aggregate by themselves.
[0018] Step 2: Inoculate the human adipose stem cells aggregated into spheres obtained in Step 1 into the induction differentiation medium at a volume ratio of 1%. The medium formula is: DMEM / F12 basal medium, 3% fetal bovine serum (FBS), 100 ng / mL activin A, and induce differentiation at 37°C for 2 days to complete the first-stage induction differentiation and obtain the first induction differentiation system.
[0019] Step 3: Add retinoic acid with a final mass concentration of 3 mg / L and transforming growth factor β1 with a final mass concentration of 11 μg / L to the first induction and differentiation system described in Step 2. Under the condition of 37 °C, place it in an incubator equipped with light-emitting diodes. Use near-infrared light with an intensity of 10 J / cm 2 , a wavelength of 760 nm, to irradiate the cells for 10 min every day, then turn off the diode, and continuously induce and differentiate for 2 days to complete the second-stage induction and differentiation, obtaining the second induction and differentiation system.
[0020] Step 4: Add taurine with a final mass concentration of 0.55 μg / L, betacellulin with a final mass concentration of 15 μg / L, insulin-like growth factor with a final mass concentration of 35 μg / L, nicotinamide with a final mass concentration of 0.75 g / L, and BMP type I receptor kinase inhibitor with a final mass concentration of 1.55 μg / L to the second induction and differentiation system described in Step 3. Under the condition of 37 °C, irradiate the cells with near-infrared light for 10 min every day, then turn off the diode, and continuously induce and differentiate for 5 days, and collect the cells to obtain insulin-secreting cells.
[0021] Comparative Example 1 A method for inducing human adipose stem cells to differentiate into insulin-secreting cells, the specific steps are as follows: Step 1: Rinse the minced human adipose tissue repeatedly with PBS buffer in a laminar flow hood, then take 10 mL and put it into a 50 mL sterile centrifuge tube. Add collagenase IV with a concentration of 1 mg / mL equal to the volume of the human adipose tissue to the centrifuge tube, and oscillate and digest in a constant temperature shaker at 37 °C for 1 h. Then add an equal volume of αMEM medium containing 10% fetal bovine serum to the centrifuge tube to terminate the digestion. Place the centrifuge tube in a centrifuge, set the centrifuge speed to 1000 r / min, centrifuge for 10 min, discard the supernatant, then add an equal volume of αMEM medium containing 10% fetal bovine serum to the centrifuge tube, gently pipette and mix well, resuspend the cells, and place the centrifuge tube in an incubator at 37 °C and 5% CO2 for culture. After passage 3 times, centrifuge at 1000 r / min for 10 min, collect the obtained human adipose stem cells, dilute them with PBS buffer, and adjust the cell density to 1×10 6 cells / mL, and then place them in a sterile 96-well plate to let them self-aggregate.
[0022] Step 2: Inoculate the self-aggregated human adipose stem cells obtained in Step 1 into the induction and differentiation medium at a volume ratio of 1%. The formula of this medium is: DMEM / F12 basal medium, 3% fetal bovine serum (FBS), 100 ng / mL activin A. Induce and differentiate at 37 °C for 2 days to complete the first-stage induction and differentiation, obtaining the first induction and differentiation system.
[0023] Step 3: Add retinoic acid with a final mass concentration of 3 mg / L and transforming growth factor β1 with a final mass concentration of 11 μg / L into the first induction and differentiation system described in Step 2, and continuously induce differentiation for 2 days at 37°C to complete the second-stage induction and differentiation, thereby obtaining the second induction and differentiation system.
[0024] Step 4: Add taurine with a final mass concentration of 0.55 μg / L, betacellulin with a final mass concentration of 15 μg / L, insulin-like growth factor with a final mass concentration of 35 μg / L, nicotinamide with a final mass concentration of 0.75 g / L, and BMP type I receptor kinase inhibitor with a final mass concentration of 1.55 μg / L into the second induction and differentiation system described in Step 3, and continuously induce differentiation for 5 days at 37°C, then collect the cells to obtain insulin-secreting cells.
[0025] Example 2 A method for inducing human adipose stem cells to differentiate into insulin-secreting cells, comprising the following specific steps: Step 1: Rinse the minced human adipose tissue repeatedly with PBS buffer in a laminar flow hood, then take 10 mL and place it into a 50 mL sterile centrifuge tube. Add collagenase type IV with a concentration of 1 mg / mL equal in volume to the human adipose tissue into the centrifuge tube, and digest it in a constant temperature shaker at 37°C for 1 h. Then add αMEM medium containing 10% fetal bovine serum equal in volume to the centrifuge tube to terminate digestion. Place the centrifuge tube in a centrifuge, set the centrifuge speed to 1000 r / min, centrifuge for 10 min, discard the supernatant, add αMEM medium containing 10% fetal bovine serum equal in volume to the centrifuge tube again, gently pipette and mix well, resuspend the cells, and then place the centrifuge tube in an incubator at 37°C and 5% CO2 for culture. After subculturing 3 times, centrifuge at 1000 r / min for 10 min, collect the obtained human adipose stem cells, dilute them with PBS buffer, adjust the cell density to 1×10 6 cells / mL, and then place them in a sterile 96-well plate to allow them to self-aggregate.
[0026] Step 2: Inoculate the self-aggregated human adipose stem cells obtained in Step 1 into the induction and differentiation medium at a volume ratio of 1%, and the medium formula is: DMEM / F12 basal medium, 3% fetal bovine serum (FBS), 100 ng / mL activin A. Induce differentiation at 37°C for 2 days to complete the first-stage induction and differentiation, thereby obtaining the first induction and differentiation system.
[0027] Step 3: Add retinoic acid with a final mass concentration of 1 mg / L and transforming growth factor β1 with a final mass concentration of 2 μg / L into the first induction and differentiation system described in Step 2, and place it in an incubator equipped with light-emitting diodes at 37°C, and use light with an intensity of 10 J / cm 2, After irradiating the cells with near-infrared light with a wavelength of 760 nm for 10 minutes every day, turn off the diode, and continuously induce differentiation for 2 days to complete the second-stage induction of differentiation, obtaining the second induced differentiation system.
[0028] Step 4: Add taurine with a final mass concentration of 0.1 μg / L, betacellulin with a mass concentration of 10 μg / L, insulin-like growth factor with a mass concentration of 20 μg / L, nicotinamide with a mass concentration of 0.5 g / L, and BMP type I receptor kinase inhibitor with a mass concentration of 0.1 μg / L to the second induced differentiation system described in Step 3. Under the condition of 37°C, irradiate the cells with near-infrared light for 10 minutes every day and then turn off the diode, continuously induce differentiation for 5 days, collect the cells, and insulin-secreting cells can be obtained.
[0029] Example 3 A method for inducing human adipose stem cells to differentiate into insulin-secreting cells, the specific steps are as follows: Step 1: Rinse the minced human adipose tissue repeatedly with PBS buffer in a laminar flow hood, then take 10 mL and put it into a 50 mL sterile centrifuge tube. Add collagenase IV with a concentration of 1 mg / mL equal to the volume of the human adipose tissue to the centrifuge tube, oscillate and digest in a 37°C constant temperature shaker for 1 h, then add an equal volume of αMEM medium containing 10% fetal bovine serum to the centrifuge tube to terminate digestion. Place the centrifuge tube in a centrifuge, set the centrifuge speed to 1000 r / min, centrifuge for 10 min, discard the supernatant, add an equal volume of αMEM medium containing 10% fetal bovine serum to the centrifuge tube again, gently pipette and mix well, resuspend the cells, and place the centrifuge tube in a 37°C, 5% CO2 incubator for culture. After subculturing 3 times, centrifuge at 1000 r / min for 10 min, collect the obtained human adipose stem cells, dilute with PBS buffer, adjust the cell density to 1×10 6 cells / mL and place them in a sterile 96-well plate to self-aggregate.
[0030] Step 2: Inoculate the self-aggregated human adipose stem cells obtained in Step 1 into the induction differentiation medium at a volume ratio of 1%, and the medium formula is: DMEM / F12 basal medium, 3% fetal bovine serum (FBS), 100 ng / mL activin A. Induce differentiation at 37°C for 2 days to complete the first-stage induction of differentiation, obtaining the first induced differentiation system.
[0031] Step 3: Add retinoic acid with a final mass concentration of 5 mg / L and transforming growth factor β1 with a mass concentration of 20 μg / L to the first induced differentiation system described in Step 2. Under the condition of 37°C, place it in an incubator equipped with a light-emitting diode, and use an intensity of 10 J / cm 2, after irradiating the cells with near-infrared light with a wavelength of 760 nm for 10 minutes every day, turn off the diode, and continuously induce differentiation for 2 days to complete the second-stage induction of differentiation and obtain the second induced differentiation system.
[0032] Step 4: Add taurine with a final mass concentration of 1.0 μg / L, betacellulin with a concentration of 20 μg / L, insulin-like growth factor with a concentration of 50 μg / L, nicotinamide with a concentration of 1.0 g / L, and BMP type I receptor kinase inhibitor with a concentration of 3.0 μg / L to the second induced differentiation system described in Step 3. Under the condition of 37°C, after irradiating the cells with near-infrared light for 10 minutes every day, turn off the diode, continuously induce differentiation for 5 days, collect the cells, and insulin-secreting cells can be obtained.
[0033] Experimental results: 1. Characterization of human adipose stem cells and detection of their differentiation ability (1) Take 100 μL of human adipose stem cell suspension with a density of 1×10 6 cells / mL and add it to a flow cytometry tube. Except for the blank tube without adding antibodies, add 5 μL of CD90 and CD105 antibodies to the other detection tubes, vortex and mix well, incubate in the dark at 4°C for 20 minutes, add 500 μL of PBS buffer for washing once, centrifuge at 1200 r / min for 5 minutes, discard the supernatant, resuspend the precipitate with PBS buffer, and detect by flow cytometry. Figure 1 The results show that more than 90% of the obtained human adipose stem cells simultaneously express the surface markers CD90 and CD105 of human adipose stem cells, indicating that the obtained human adipose stem cells have a high purity.
[0034] (2) Take 10 μL of the above cell suspension and inoculate it in DMEM high-glucose medium for culture until more than 80% of the cells are confluent. Replace the medium with adipogenic induction medium, change the induction medium after 3 days of culture, repeat the replacement 3 times, and then continue the culture. After lipid droplets appear, rinse with PBS buffer, fix with 10% paraformaldehyde at room temperature for 30 minutes, discard the fixing solution, rinse with PBS buffer 2 times, add Oil Red O staining solution and stain at room temperature for 30 minutes, wash with PBS 2-3 times, and observe lipid droplets under a microscope. The staining results are as Figure 2 shown in -A. Orange-red lipid droplets exist in the cells, indicating that the obtained human adipose stem cells have the ability of adipogenic differentiation.
[0035] (3) Inoculate 10 μL of the above cell suspension into DMEM high-glucose medium for culture until more than 80% of the cells are confluent. Replace the medium with osteogenic induction medium, change the induction medium after 3 days of culture, repeat the replacement 3 times, and then continue the culture until calcified nodules appear. After rinsing with PBS buffer, fix with 10% paraformaldehyde at room temperature for 30 min, add alizarin red staining solution, stain at room temperature for 30 min, then wash the cells with distilled water, and observe the calcified nodules under a microscope. The staining results are as Figure 2 shown in -B. The calcium precipitation area appears red, indicating that the obtained human adipose stem cells have osteogenic differentiation ability.
[0036] 2. Identification of insulin-secreting cells (1) Collect the obtained insulin-secreting cells, use the cells before induced differentiation as a control, extract RNA and reverse transcribe it into cDNA respectively, use GAPDH as an internal control, and detect the gene expression of two markers of mature β cells, nuclear transcription factors NKX6.1 and PDX1, and endocrine protein INS by real-time fluorescence quantitative PCR reaction. Figure 3 The results show that in the cells after induced differentiation, the gene expressions of NKX6.1, PDX1 and INS are significantly higher than those of the cells without induced differentiation, proving that the cell induced differentiation is successful; compared with the comparative example that only uses the induction factor for induction, in the example, photobiomodulation and the induction factor are used for synergistic induction, and the expression levels of the marker genes are significantly increased, that is, the cell differentiation efficiency in the example is higher within the same time.
[0037] (2) Incubate the obtained insulin-secreting cells in KRBH buffer for 1 h as a basal control first, then incubate them in KRBH buffer with glucose concentrations of 2 mmol / L, 10 mmol / L, and 20 mmol / L without water for 1 h in sequence, and finally incubate them in KRBH buffer containing 30 mmol / L potassium chloride for 1 h. Use an enzyme-linked immunosorbent assay kit to detect the insulin production levels in the buffer at each stage. After the stimulation test, collect the insulin-secreting cells and digest them into a single-cell suspension, and calculate the total cell number for normalization. The results of the insulin production levels in the buffer at each stage are as Figure 4 shown. Under glucose stimulation, the obtained insulin-secreting cells can secrete insulin normally, and with the increase of glucose concentration, the insulin secretion amount also increases significantly. After the stimulation ends, the high-concentration KCL solution depolarizes the cell membrane, and the intracellular insulin is completely released; the above results show that the obtained insulin-secreting cells can dynamically regulate the insulin secretion amount according to the glucose content, that is, the cells have mature insulin secretion function.
[0038] The above-described embodiments only represent the selected embodiments of the present invention. All other embodiments obtained without creative efforts fall within the scope of protection of the present invention.
Claims
1. A method for inducing human adipose stem cells to differentiate into insulin-secreting cells, characterized in that, Comprising the following steps: Step 1: Add an equal volume of collagenase to human adipose tissue, digest at 37°C for 1 h, then add serum-containing medium to terminate digestion, centrifuge, discard the supernatant, resuspend with serum-containing medium, and then passage culture 3 times at 37°C. Collect the human adipose stem cells after passage culture, dilute them with PBS buffer, and place the diluted human adipose stem cells in a well plate to self-aggregate to obtain self-aggregated spherical human adipose stem cells; Step 2: Place the self-aggregated spherical human adipose stem cells obtained in Step 1 in an induction differentiation medium, and induce differentiation at 37°C for 2 days to complete the first-stage induction differentiation and obtain the first induction differentiation system; Step 3: Add retinoic acid and transforming growth factor β1 to the first induction differentiation system described in Step 2, irradiate with near-infrared light for 10 minutes per day at 37°C, and continuously induce differentiation for 2 days to complete the second-stage induction differentiation and obtain the second induction differentiation system; Step 4: Add taurine, betacellulin, insulin-like growth factor, nicotinamide, and BMP type I receptor kinase inhibitor to the second induction differentiation system described in Step 3, irradiate with near-infrared light for 10 minutes per day at 37°C, and continuously induce differentiation for 5 days, then insulin-secreting cells can be collected.
2. The method according to claim 1, wherein The collagenase described in Step 1 is 1 mg / mL collagenase IV.
3. The method according to claim 1, characterized in that The serum-containing medium described in Step 1 is 10% fetal bovine serum αMEM medium.
4. The method according to claim 1, wherein The formula of the induction differentiation medium described in Step 2 is DMEM / F12 basal medium, 3% fetal bovine serum (FBS), 100 ng / mL activin A.
5. The method according to claim 1, wherein The final mass concentration of retinoic acid described in Step 3 is 1 - 5 mg / L, and the final mass concentration of transforming growth factor β1 is 2 - 20 μg / L.
6. The method according to claim 1, wherein The near-infrared light intensity described in Step 3 is 10 J / cm 2 , and the wavelength range is 760 - 1100 nm.
7. The method according to claim 1, characterized in that, The final mass concentration of taurine described in Step 4 is 0.1 - 1.0 μg / L, the final mass concentration of betacellulin is 10 - 20 μg / L, the final mass concentration of insulin-like growth factor is 20 - 50 μg / L, the final mass concentration of nicotinamide is 0.5 - 1.0 g / L, and the final mass concentration of BMP type I receptor kinase inhibitor is 0.1 - 3.0 μg / L.