Induction culture medium and induction method for quiescent hepatic stellate cells

Abstract

The present invention discloses an induction culture medium for quiescent hepatic stellate cells, comprising 10 μmol / L all-trans retinoic acid (ATRA), 30-50 μmol / L sodium oleate, and 0.1-10 μmol / L S-nitroso-N-acetylopectinamine (SNAP). This culture medium can induce activated hepatic stellate cells to revert to quiescent hepatic stellate cells. The present invention also discloses a method for inducing quiescent hepatic stellate cells, which has a short induction time of only 2-3 days, is easy to operate, and has good efficacy. The induced quiescent hepatic stellate cells are useful for subsequent screening of drugs that inhibit hepatic stellate cell activation.

Description

Technical field:

[0002] The invention belongs to cell biology technology, and in particular relates to an induction culture medium and an induction method for quiescent hepatic stellate cells. Background technology:

[0004] Hepatic fibrosis is a scarring and repair response to chronic liver injury and an intermediate step in the progression of various chronic liver diseases to cirrhosis. Its mechanism is persistent hepatocyte injury and / or inflammation, leading to an imbalance in the synthesis and degradation of the extracellular matrix (ECM), rich in type I and type III collagen. Excessive ECM deposition can disrupt normal liver architecture. Hepatic stellate cells (HSCs) are the primary source of ECM during fibrosis and were the first cell subpopulation identified to be associated with fibrosis. HSCs are dispersed throughout the liver, localized in the perisinusoidal spaces between sinusoidal endothelial cells and hepatocytes. Under physiological conditions, HSCs are relatively quiescent and non-proliferative. However, upon liver injury, stimulated by a series of autocrine and paracrine fibrogenesis signals, HSCs shift from a quiescent state to an activated state. Expression of α-smooth muscle actin (α-SMA) increases, accompanied by a gradual disappearance of cytoplasmic lipid droplets. Simultaneously, expression of factors promoting lipogenesis decreases, while expression of receptors involved in fibrogenesis and cell migration increases, leading to the extensive production of collagen and other extracellular matrix.

[0005] The development of liver fibrosis is associated with the activation of quiescent hepatic stellate cells (HSCs) into myofibroblasts that produce type I collagen (activated HSCs). With the cessation of liver damage, HSCs may transition from an activated state to a quiescent state, or activated HSCs may be reduced through mechanisms such as apoptosis, leading to reversal of liver fibrosis. Therefore, targeting HSCs to inhibit HSC proliferation and activation and induce apoptosis has become an important therapeutic strategy for combating liver fibrosis. Currently, the mechanisms by which HSC activation and proliferation induce liver fibrosis and reverse fibrosis remain unclear. Further research is needed on the regulatory mechanisms of HSCs by cytokines, chemokines, and transcription factors to identify the signal transduction pathways involved in HSC activation. This research aims to develop drugs that inhibit HSC activation or promote apoptosis, which hold great promise for preventing, delaying, or even halting the progression of liver fibrosis.

[0006] Screening for inhibitors of HSC activation requires inactive HSCs as research subjects. Primary HSCs undergo spontaneous activation after 2-3 days of culture on plastic plates not coated with a simulated biomatrix. With prolonged culture, HSCs gradually differentiate from lipid-rich quiescent cells into fibroblasts, with SMA secretion reaching its peak at 15 days. It is currently believed that HSCs are activated after 7 days of primary culture or passaged HSCs.

[0007] The deactivation process of hepatic stellate cells includes apoptosis, cellular senescence, immune clearance, and phenotypic reversal. Apoptosis, senescence, and immune clearance primarily reduce the number of activated HSCs, leading to decreased cell viability. Reversion of activated HSCs to a quiescent state is a relatively effective approach. Studies have reported that culturing primary HSCs on basement membrane matrix-coated culture plates can maintain HSCs in a quiescent state, and that drugs that promote adipocyte differentiation can restore activated HSCs to a quiescent state. The liver is a key site of glucose metabolism. Studies have found that decreased glycogen synthesis in the liver is accompanied by significant increases in various indicators of liver fibrosis. Rosiglitazone and insulin promote glycogen synthesis and inhibit gluconeogenesis. Patent document CN 112779310 A discloses methods for restoring HSCs to a quiescent state using a combination of sodium oleate, vitamins A, and rosiglitazone, or a combination of sodium oleate, vitamins A, and insulin. However, restoration is ineffective, as excessive sodium oleate concentrations and enlarged lipid droplets can lead to cell rupture.

[0008] Nitric oxide (NO) is a biological mediator ubiquitous in various vertebrate cells and a key regulator of intercellular signaling. Nitric oxide synthase (NOS) is widely distributed in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. It can be activated by lipopolysaccharide (LPS), endotoxins, and various cytokines, producing large amounts of endogenous cytotoxic NO, which is involved in the pathogenesis of various liver diseases. NO exhibits both cytotoxic and cytoprotective biological activities. NO plays a protective role in the development of liver fibrosis. High concentrations of NO can inhibit the activation of hepatic stellate cells, reduce collagen synthesis, and alleviate extracellular matrix deposition, thereby alleviating liver fibrosis. SNAP, a nitrosothiol derivative, acts as a NO donor, releasing NO. SNAP is a potent vasodilator in vitro, inhibiting mitosis and proliferation of vascular smooth muscle cells and acting as a stable inhibitor of platelet aggregation. In addition to its vasodilatory properties, NO donors may also exert direct anti-fibrotic properties. Summary of the invention:

[0010] In order to solve the current problem of spontaneous activation of hepatic stellate cells, the present invention provides an induction culture medium and induction method for quiescent hepatic stellate cells. The purpose of the present invention is achieved through the following technical solutions:

[0011] The invention discloses an induction culture medium for quiescent hepatic stellate cells, comprising all-trans retinoic acid, sodium oleate and S-nitroso-N-acetylpenicillamine.

[0012] Preferably, in the above-mentioned induction medium, the concentrations of all-trans retinoic acid (ATRA) are 10 μmol / L, sodium oleate is 30-50 μmol / L, and S-nitroso-N-acetylpenicillamine (SNAP) is 0.1-1 μmol / L.

[0013] Preferably, in the above-mentioned induction medium, the concentrations of all-trans retinoic acid (ATRA) are 10 μmol / L, sodium oleate is 30 μmol / L, and S-nitroso-N-acetylpenicillamine (SNAP) is 1 μmol / L.

[0014] Preferably, the above culture medium is 1640 or DMEM or DMEM / F12.

[0015] Preferably, the above culture medium further contains 10% serum, 100 U / mL penicillin and 100 μg / mL streptomycin;

[0016] Preferably, the serum is fetal bovine serum, calf serum or horse serum.

[0017] The present invention also provides a method for inducing quiescent hepatic stellate cells, comprising the following steps:

[0018] Step 1: Inoculate activated hepatic stellate cells into a culture plate or dish with an initial cell density of 20-30%;

[0019] Step 2: After the cells adhere to the wall, the induction medium is replaced with the induction medium according to claim 1 or 2, and the medium is changed every 24 hours for 2-3 days;

[0020] Step 3: The cells were tested for Oil Red O and hepatic stellate cell marker protein.

[0021] Technical effects:

[0022] 1. The induction culture medium for quiescent hepatic stellate cells provided by the present invention can induce activated hepatic stellate cells to revert to quiescent hepatic stellate cells, with a significant increase in Oil Red O, a decrease in α-SMA, and an increase in GFAP in the cells.

[0023] 2. The induction method of quiescent hepatic stellate cells provided by the present invention has a short induction time of only 1-2 days.

[0024] The method is easy to operate and has good effects. The induced quiescent hepatic stellate cells are helpful for the subsequent screening of drugs that inhibit the activation of hepatic stellate cells.

[0025] Attached drawings:

[0026] Figure 1Oil Red O staining of LX-2 cells (A. Control group; B. 10 μmol / L ATRA + 50 μmol / L sodium oleate + 0.1 μmol / L SNAP; C. 10 μmol / L ATRA + 40 μmol / L sodium oleate + 0.33 μmol / L SNAP; D. 10 μmol / L ATRA + 30 μmol / L sodium oleate + 1 μmol / L SNAP; E. 250 nmol / L Insulin + 10 μmol / L Vitamin A + 50 μmol / L sodium oleate + 2% FBS; F. 1 μmol / L Rosiglitazone + 10 μmol / L Vitamin A + 50 μmol / L sodium oleate + 2% FBS) 200×;

[0027] Figure 2 Oil Red O staining of HSC-T6 cells (A. Control group; B. 10μmol / L ATRA + 50μmol / L sodium oleate + 0.1μmol / L SNAP; C. 10μmol / L ATRA + 40μmol / L sodium oleate + 0.33μmol / L SNAP; D. 10μmol / L ATRA + 30μmol / L sodium oleate + 1μmol / L SNAP; E. 250nmol / L Insulin + 10μmol / L Vitamin A + 50μmol / L sodium oleate + 2% FBS; F. 1μmol / L Rosiglitazone + 10μmol / L Vitamin A + 50μmol / L sodium oleate + 2% FBS) 200×;

[0028] Figure 3 It is the OD value at 492 nm wavelength on a microplate reader after elution with Oil Red O;

[0029] Figure 4 is the immunofluorescence staining of LX-2 cells;

[0030] Figure 5 Immunofluorescence staining of HSC-T6 cells;

[0031] Figure 6 Western blot images of LX-2 and HSC-T6 cells;

[0032] Figure 7 Electron micrograph of HSC-T6 cells (black arrow: endoplasmic reticulum, white arrow: lipid droplet; black hollow arrow: mitochondria). Specific implementation method:

[0034] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments to the present invention based on the above invention content.

[0035] Passaged hepatic stellate cells (LX-2) or HSC-T6 were seeded into 24-well plates at an initial density of 30%. After overnight adherence, LX-2 cells were replaced with 1640 medium supplemented with 10 μmol / L all-trans retinoic acid (ATRA), 30-50 μmol / L sodium oleate, 0.1-1 μmol / L S-nitroso-N-acetylpenicillamine (S-N-acetylpenicillamine), 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. The medium was changed every 24 hours for 48 hours. HSC-T6 cells were replaced with DMEM supplemented with 10 μmol / L ATRA, 30-50 μmol / L sodium oleate, 0.1-1 μmol / L S-nitroso-N-acetylpenicillamine (S-N-acetylpenicillamine), 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Positive experimental groups were treated with culture medium containing 250 nmol / L insulin, 10 μmol / L vitamin A, 2% FBS, 1% anti-antibody, and 50 μmol / L sodium oleate-BSA, or culture medium containing 1 μmol / L rosiglitazone, 10 μM vitamin A, 2% FBS, 1% anti-antibody, and 50 μmol / L sodium oleate-BSA. The medium was changed every 24 hours for 48 hours.

[0036] Oil Red O staining to observe lipid droplets in cells: aspirate the culture medium, add 500μl PBS to each well, wash 1-2 times, add 4% paraformaldehyde to fix for 20-30 minutes; aspirate the fixative, add 500μl PBS to each well, wash 3 times. Aspirate the PBS in the well plate, add 500μl 60% isopropanol to wash for 15-20 seconds to remove water; add 200μl Oil Red O staining solution and incubate at room temperature for 10-15 minutes; aspirate the Oil Red O staining solution, add 500μl 60% isopropanol to wash for 15-20 seconds to remove excess staining solution; wash with PBS for more than 3 times until the liquid is clear, and take pictures under a microscope. The results are as follows Figure 1 and Figure 2As shown, in the control group, HSC-T6 cells showed minimal Oil Red staining and smaller lipid droplets, and LX-2 cells showed almost no Oil Red O staining. In hepatic stellate cells, lipid droplet size and Oil Red staining increased significantly in three different induction groups (B. 10μmol / L ATRA + 50μmol / L sodium oleate + 0.1μmol / L SNAP; C. 10μmol / L ATRA + 40μmol / L sodium oleate + 0.33μmol / L SNAP; D. 10μmol / L ATRA + 30μmol / L sodium oleate + 1μmol / L SNAP). Group D exhibited the best results and was selected for subsequent experiments. In the positive control groups (Groups E and F), lipid droplet size increased, but the number of lipid droplets and the degree of Oil Red O staining were less pronounced than in the three induction groups. Add 100 μl of 100% ethanol to elute the Oil Red O dye in the cells, take 50 μl and put it into a 96-well plate, and measure the OD value at a wavelength of 492 nm using a microplate reader. Figure 3 As shown, the OD492 value of the Oil Red O eluate in the induced group was significantly higher than that in the control group (p<0.05). Among the three induced groups, the OD492 values ​​of group D were higher than those of groups B and C (p<0.05).

[0037] Immunofluorescence detection of hepatic stellate cell surface marker proteins α-smooth muscle actin (α-SMA) and glial fibrillary acidic protein (GFAP): Hepatic stellate cells LX-2 or HSC-T6 were seeded into confocal culture dishes and induced as above. The culture medium was discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde; washed 3 times with PBS, treated with 0.5% Triton for 20 min; washed 3 times with PBS; blocked with 5% goat serum for 1 h, and washed 3 times with PBS. Mouse-derived anti-α-SMA and mouse-derived anti-GFAP primary antibodies were added respectively, incubated overnight at 4°C, and washed 3 times with PBS. FITC-labeled fluorescent secondary antibodies were incubated at room temperature in the dark for 1 h, washed 3 times with PBS, and DAPI dye was added. The cells were incubated at room temperature in the dark for 5 min, washed with PBS, and observed and photographed under a laser confocal microscope. The results are shown in Figure 2. Figure 4 As shown, red fluorescence shows the expression of α-SMA, and green fluorescence shows the expression of GFAP. Both LX-2 and HSC-T6 cells highly express α-SMA, and the red fluorescence in LX-2 is more significant, while the expression level of GFAP is lower. After 48 hours of induction, the red fluorescence expression of α-SMA in both hepatic stellate cells was significantly reduced, while the green fluorescence expression of GFAP was significantly enhanced ( Figure 4 , Figure 5 ).

[0038] Western blot detection of hepatic stellate cell surface marker proteins: Hepatic stellate cells LX-2 or HSC-T6 were seeded into 6mm culture dishes and induced as above. °Wash cells three times with pre-chilled PBS at C, add 1 mL of PBS, scrape off the cells with a cell scraper, transfer to a 1.5 mL EP tube, centrifuge at 13,000 rpm for 5 min, discard the supernatant, add an appropriate amount of protein lysis buffer, and lyse the cells on ice, shaking vigorously on a shaker every 10 min for a total of 30 min. Centrifuge at 13,000 rpm for 5 min, and remove the supernatant. Determine protein concentration by BCA assay, run on 12% SD S PAGE at 100 V, transfer to a PVDF membrane at 100 V, block with 5% skim milk at room temperature for 1 h, and add mouse anti-α-SMA and mouse anti-GFAP primary antibodies for 4 min. ° C overnight, and the corresponding secondary antibody was incubated at room temperature for 1 hour. ECL luminescence was developed, and the gray value of the Western blot band was analyzed using ImageJ software. The results are shown in Figure 2. Figure 6 As shown, LX-2 or HSC-T6 cells highly expressed α-SAM, while the basal expression level of GFAP was low. After 48 hours of induction, the expression of α-SMA in both hepatic stellate cells was significantly decreased, while the expression level of GFAP was significantly increased (p<0.05).

[0039] Electron microscopy of HSC-T6 cells Figure 7 As shown, in the control group, the ER cisternae were enlarged, dark in color, rich in protein, and exhibited vigorous protein synthesis. Mitochondrial ridges were visible and structurally normal, with occasional lipid droplets. However, after induction with the 10μmol / L ATRA + 30μmol / L sodium oleate + 1μmol / L SNAP regimen, ER expansion was significantly reduced, mitochondria condensed, ridge spaces widened, dorsal processes increased in density and darkened in color, and intracellular lipid droplets increased in number and size, as well as glycogen. This suggests that after induction, protein synthesis activity in hepatic stellate cells decreased and lipid droplet storage increased.

[0040] This induction culture medium acts on activated hepatic stellate cells for 48 hours, which can significantly increase the storage of lipid droplets in hepatic stellate cells, reduce the expression of the activation marker α-SMA, restore the expression of the quiescent marker GFAP, and induce activated hepatic stellate cells to return to the quiescent phase.

[0041] This induction method is not only used for LX-2 and HSC-T6 hepatic stellate cell lines, but also has the same induction effect on primary hepatic stellate cells and other hepatic stellate cell lines cultured in vitro for more than 7 days.

Claims

1. A quiescent hepatic stellate cell induction culture medium, comprising 1640 medium, DMEM, or DMEM / F12 medium, containing 10% serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, wherein: It also contains 10 μmol / L all-trans retinoic acid, 30-50 μmol / L sodium oleate, and 0.1-1 μmol / L S-nitroso-N-acetylpenicillamine.

2. The induction medium according to claim 1, wherein: Contains 10 μmol / L all-trans retinoic acid, 30 μmol / L sodium oleate, and 1 μmol / L S-nitroso-N-acetylpenicillamine.

3. The induction medium according to claim 2, wherein: The serum is fetal bovine serum, calf serum or horse serum.

4. A method for inducing quiescent hepatic stellate cells, comprising the following steps: Step 1: Inoculate activated hepatic stellate cells into a culture plate or dish with an initial cell density of 20-30%; Step 2: After the cells adhere to the wall, the culture medium is replaced with the induction medium according to any one of claims 1 to 3, and the cells are induced for 2 to 3 days; Step 3: The cells were tested for Oil Red O and hepatic stellate cell marker protein.

Images