CN116789798A

CN116789798A - Protein composition, preparation method and application thereof

Info

Publication number: CN116789798A
Application number: CN202210264243.0A
Authority: CN
Inventors: 周徐; 俞卫锋; 唐丹
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2023-09-22

Abstract

The present invention provides protein compositions comprising amphiregulin and fibroblast growth factor to inhibit liver fibrosis. The invention also provides a preparation method of the protein composition, which comprises the steps of performing transformation and amplification culture on primary human hepatocytes by using a small molecule reprogramming culture medium to obtain liver precursor-like cells, culturing the liver precursor-like cells by using a basal culture medium, and collecting in vitro culture supernatant containing the amphiregulin and the fibroblast growth factor. The preparation method has no exogenous gene introduction, reduces the risk of protein molecular structure and space conformation change of the protein composition caused by gene change, and ensures the in vivo application safety. The invention also provides an in vitro application of the protein composition and hepatic stellate cells in co-culture and an application of the protein composition in preparation of anti-hepatic fibrosis drugs, which proves that the protein composition has the effect of inducing hepatic stellate cells to apoptosis.

Description

Protein composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a protein composition, a preparation method and application thereof.

Background

Hepatic stellate cells (Hepatic stellate cells, HSCs) are located within the dise gap, in close proximity to the liver sinusoidal endothelial cells and hepatocytes. Hepatic stellate cells in normal liver are in resting state; when the liver is damaged by inflammation or mechanical stimulation, hepatic stellate cells are activated and transformed into myofibroblast-like cells (MFCs). Continuous activation of hepatic stellate cells is a key link in the development and development of hepatic fibrosis, and can exacerbate hepatic injury.

Accordingly, there is a need to develop novel protein compositions to solve the above problems in the prior art.

Disclosure of Invention

The invention aims to provide a protein composition, a preparation method and application thereof, so as to play a role in inhibiting hepatic fibrosis.

To achieve the above object, the protein composition of the present invention includes amphiregulin and fibroblast growth factor to exert an inhibitory effect on liver fibrosis.

Preferably, the fibroblast growth factor is FGF19.

The preparation method of the protein composition comprises the following steps: performing transformation amplification culture on the primary human liver cells by using a small molecule reprogramming culture medium to obtain liver precursor-like cells, culturing the liver precursor-like cells by using a basal culture medium, and collecting an in vitro culture supernatant containing the amphiregulin and the fibroblast growth factor.

Preferably, the small molecule reprogramming media comprises the composition of basal media, N2 additives, B27 additives, epidermal growth factor, hepatocyte growth factor, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors, sphingosine monophosphate, and indoleacetic acid.

Further preferably, the content of the epidermal growth factor is 10-30 nanograms per milliliter, the content of the hepatocyte growth factor is 10-30 nanograms per milliliter, the content of the ROCK kinase inhibitor is 5-15 micromoles per liter, the content of the Wnt signal pathway agonist is 1-4 micromoles per liter, the content of the TGF-beta signal inhibitor is 0.1-2 micromoles per liter, the content of the sphingosine monophosphate is 0.1-2 micromoles per liter, the content of the indoleacetic acid is 4-6 micromoles per liter, and the volume percentage of the N2 additive and the B27 additive is not more than 1%.

Preferably, the step of transforming expansion culture of human primary hepatocytes using a small molecule reprogramming medium comprises: human primary hepatocytes were grown at 1X 10 ⁴ -3×10 ⁴ Culturing with the small molecule reprogramming medium at inoculation density of 0.5X10 after confluence rate of not less than 90% ⁴ -1×10 ⁴ The inoculation density of each square centimeter is used for 3-4 times of continuous subculture until the fusion rate reaches 40% -50%.

Further preferably, the step of culturing the liver precursor-like cells using a basal medium comprises: after the continuous subculture is finished, continuous culture is performed for not less than 24 hours using the basal medium.

Further preferably, the step of collecting the in vitro culture supernatant comprising said amphiregulin and said fibroblast growth factor comprises: after the continuous culture is finished, the collected culture supernatant is centrifuged for not less than 10 minutes under the centrifugal force of not less than 300g to remove cell debris to obtain a purified culture supernatant, and then the purified culture supernatant is concentrated by adopting an ultrafiltration centrifuge tube for 20-25 times to obtain the in vitro culture supernatant.

The in vitro application of the protein composition of the invention comprises co-culturing the protein composition with hepatic stellate cells to promote apoptosis of the hepatic stellate cells.

The application of the protein composition in preparation of anti-liver fibrosis medicines comprises the use of the protein composition for interfering with thioacetamide-induced mouse liver fibrosis models.

Drawings

FIG. 1 is a graph showing comparison of expression levels of genes involved in collagen secretion in LX-2 cells in a co-culture group, an LX-2 culture group and an LX-2 activation culture group;

FIG. 2 is a graph showing comparison of protein expression of LX-2 cells in co-culture, LX-2 culture and LX-2 activation culture;

FIG. 3 is a comparative schematic diagram showing apoptosis of LX-2 cells in a co-culture group and an LX-2 activation culture group after 48 hours of co-culture;

FIG. 4 is a comparative schematic diagram of LX-2 apoptosis in rhFGF19 Ab and rhAREG Ab-mediated co-culture, rhFGF19 and rhAREG-mediated co-culture, LX-2 activation culture and co-culture;

FIG. 5 is a photograph of microscopic morphology of tissue sections obtained by HE staining, marsen trichromatic staining, sirius staining of liver tissue of a normal control group, a sham operation group and a protein composition treatment group;

FIG. 6 is a graph comparing the number of α -SMA positive cells in a normal control group, a sham operated group, and a protein composition treated group.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the word "comprising" and the like means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof without precluding other elements or items.

The embodiment of the invention provides that the protein composition of the invention comprises amphiregulin and fibroblast growth factor to inhibit liver fibrosis.

In some embodiments, the fibroblast growth factor is FGF19.

The preparation method of the protein composition provided by the embodiment of the invention comprises the following steps: performing transformation amplification culture on the primary human liver cells by using a small molecule reprogramming culture medium to obtain liver precursor-like cells, culturing the liver precursor-like cells by using a basal culture medium, and collecting an in vitro culture supernatant containing the amphiregulin and the fibroblast growth factor.

In the examples of the present invention, cell culture was performed in a cell incubator at 37℃and a carbon dioxide concentration of 5%, unless otherwise specified. The culture medium used for cell culture and various reagents used for treating cells, such as buffer, are sterilized and filtered through a 0.22 μm filter to remove impurities before use.

The data relating to statistical analysis in the various embodiments of the present invention were repeated at least 3 times per set of experiments, and the experimental result data were statistically analyzed using GraphPad Prism 8.0 software. Comparison between two sets of data statistical differences were calculated using a two-tailed unpaired t-test, and comparison of differences between multiple sets of data was calculated using ANOVA analysis of variance. p <0.05 is considered to be statistically different in the drawings of the specification: * Represents P <0.05; * Represents P <0.005; * Represents P <0.001; * Representing P <0.0001.

The following is a detailed description of specific examples:

example 1

The present example provides a method for preparing a protein composition, comprising the following steps:

human primary hepatocytes were processed at a rate of 2X 10 ⁴ The cells were inoculated into a petri dish (from Corning) at a density of one square centimeter, cultured with a transformation proliferation medium for 8 to 10 days, and then grown to 90% confluency, and passaged. Cell count 1X 10 ⁴ The inoculation density of each square centimeter is continuously passaged, after the inoculation density is passaged to P3-P4 generation secondary cells with the fusion rate of 40-50%, the transformation proliferation culture medium is changed into a basic culture medium DMEM (from Gibco) without adding any serum and cytokine components, and continuous culture is carried out for 24 hours. After the continuous culture, the culture supernatant is centrifuged by using a centrifugal force of 300 Xg for 10 minutesTreatment to remove cell debris gives a purified culture supernatant. And concentrating the purified culture supernatant 20-25 times by using an ultrafiltration centrifuge tube (Millipore, UFC901096,10 KD) to obtain a protein composition, sterilizing the protein composition by using a 0.22um filter, and storing the protein composition in a low-temperature refrigerator (-80 ℃) for later use.

Specifically, the main components of the transformation proliferation culture are as follows: DMEM/F12 medium (Gibco), supplemental mix additives, and small molecule and growth factor additives, wherein N2 additive (1%), B27 additive (1%) is the supplemental mix additive; the growth factor additive is epidermal growth factor EGF (20 ng/mL) and hepatocyte growth factor HGF (20 ng/mL); the small molecule additives are ROCK kinase inhibitor Y27632 (10. Mu. Mol/L), wnt signal path agonist CHIR99021 (3. Mu. Mol/L), TGF-beta signal inhibitor A83-01 (1. Mu. Mol/L), sphingosine monophosphate S1P (1. Mu. Mol/L) and indoleacetic acid LPA (5. Mu. Mol/L).

The BCA protein quantification kit (from shanghai bi yun biotechnology limited) was used to detect total protein content in different batches of protein compositions. The BAC quantification step was performed according to the kit instructions. The results show that the total protein content of the liver precursor cell secretor groups of three different batches is 1.908 mg/ml, 2.177 mg/ml and 1.787 mg/ml respectively, and the protein composition content of each batch is close, so that the batch-to-batch stability is high.

Example 2

This example examined the pro-apoptotic effect of the protein composition of example 1 on LX-2 by co-culturing it with the human immortalized hepatic stellate cell line LX-2. LX-2 of this example was purchased from Procell.

Specifically, the hepatic stellate cell line LX-2 was expressed as 1X 10 ⁴ The density of each square centimeter was plated in Matrigel (BD) coated 6-well plate dishes (Corning). The hepatic stellate cell culture medium was DMEM (source culture), and 10% fetal bovine serum (BD) and 1% antibiotics (source culture) were added. Matrigel plating was performed at a concentration of 100ug/ml. The hepatic stellate cell line LX-2 will be in a relatively resting state in a matrigel coated culture dish.

Further, the ready-to-use protein composition was removed from the freezer (-80 ℃) and thawed overnight at 4℃and formulated into 2mL of a medium solution containing 10% protein composition, followed by the addition of TGF- β1 (2.5 ng/mL) to give a protein composition suspension. When hepatic stellate cells grow to 50% fusion rate, the prepared protein composition suspension is added as a co-culture group to perform co-culture for not less than 48 hours, and the volume content of the protein composition in the co-culture system is 1%. Among them, when LX-2 is treated with TGF-. Beta.1 cytokine, hepatic stellate cells are activated, and the secretion of collagen and the increase of fibrosis-related genes are caused.

In this example, the hepatic stellate cell line LX-2 plated on Matrigel (BD) coated 6-well plate dishes (Corning) was used as LX-2 culture medium, and 2.5ng/ml TGF- β1 culture medium was added to the LX-2 culture medium as LX-2 activation medium for simultaneous co-culture.

After 48 hours of co-culture, LX-2 cell RNA was extracted from the co-culture, LX-2 culture and LX-2 activation culture, respectively, using an RNA extraction kit (purchased from Promega). Quantitative fluorescent PCR (Real-Time PCR) was performed on a PCR instrument (available from Roche) using SYBR GreenPCR kit (available from Norvezan Corp.). The verification genes comprise collagen secretion related genes COL3A1, COL1A1 and fibrosis related genes a-SMA, vimentin, desmin, TGF-beta 1 and Pdgfb.

FIG. 1 is a graph showing comparison of expression levels of genes involved in collagen secretion in LX-2 cells in a co-culture group, an LX-2 culture group and an LX-2 activation culture group. Referring to FIG. 1, LX-2 is stimulated and activated by TGF-beta 1, COL3A1 and COL1A1 are elevated, and the liver precursor cells secrete the protein composition to treat and inhibit LX-2 related gene activation caused by TGF-beta 1 stimulation.

After 72 hours of co-culture, protein content of LX-2 cells in the co-culture group, the LX-2 culture group and the LX-2 activation culture group is detected by using a BCA protein quantitative kit. The protein was diluted with 1X SDS solution to a final concentration of 1mg/ml protein solution according to the detection concentration. After denaturation, western blot immunoblotting is performed to detect the expression of a-SMA, vimentin, desmin protein.

FIG. 2 is a graph showing comparison of protein expression of LX-2 cells in co-culture, LX-2 culture and LX-2 activation culture. Referring to FIG. 2, LX-2 was co-cultured with the protein composition, and the expression level of the a-SMA, vimentin, desmin protein was significantly reduced, indicating that the protein composition inhibited LX-2 fibrosis-related protein expression.

After co-culturing for 72 hours, the LX-2 cells of the co-cultured group and the LX-2 activated cultured group are digested by pancreatin to obtain single cell suspension, and the single cell suspension is re-suspended to be 1 multiplied by 10 after being washed twice by PBS ⁶ The concentration of/ml was followed by the procedure of Annexin V-FITC apoptosis assay kit (Beyotime). Specifically, 100ul of cell suspension is sucked, supernatant is centrifugally taken, 5 mu LAnnexinV-FITC dye solution and 10 mu L of Propidium Iodide (PI) dye solution are added, a pipette gun is used for mixing cells and reagents, incubation is carried out for 20min at room temperature and in a dark place, and after incubation is finished, the detection operation of the machine is carried out. Flow cytometry was performed using a FACS Calibur flow cytometer (BD) and flow jo software was used for flow result analysis.

FIG. 3 is a comparative schematic diagram showing apoptosis of LX-2 cells in the co-culture group and the LX-2 activation culture group after 48 hours of co-culture. As shown in FIG. 3, compared with the apoptosis rate of 7.96% of the LX-2 activation culture group, the apoptosis rate of cells reaches 34% after 48 hours of co-culture of the protein composition and LX-2, and hepatic stellate cell apoptosis can be obviously induced.

Example 3

This example uses tandem mass spectrometry tag (TMT) analysis of proteomics to the protein composition of example 1, and protein-protein interaction (PPI) analysis constructs to find that Leukemia Inhibitory Factor (LIF), endothelin 1 (EDN 1), colony stimulating factor 1 (CSF 1), amphiregulin (AREG), fibroblast growth factor 19 (FGF 19) interact directly or indirectly with intermediate molecules of the JAK-STAT pathway in the protein composition. Whereas the JAK/STAT pathway has important regulatory roles in the development of liver fibrosis.

To further explore what factors inhibited hepatic stellate activation and even led to hepatic stellate apoptosis by participating in JAK/STAT pathway modulation, this example added at least one of FGF19 neutralizing antibodies FGF19 Ab and AREG neutralizing antibody AREG Ab to each of the culture systems of example 2 and used rhFGF19 and rhAREG in place of the protein composition. Wherein, the concentration of the rhFGF19 and the rhFGF19 in the co-culture system is 100 nanograms/milliliter, and the concentration of the neutralizing antibody FGF19 Ab of the FGF19 and the neutralizing antibody AREGAb of the AREG in the co-culture system is 10 micrograms/milliliter.

After 48 hours of co-culture, flow analyses were performed on each group of cell aggregates, according to the comparison graph of the number percent apoptotic cells for each group shown in fig. 4. Referring to FIG. 4, the combined use of rhFGF19 and rhAREG induces apoptosis of LX-2 to some extent in the presence of TGF- β1 as compared to the co-culture system of TGF- β1 and LX-2; while the combined use of FGF19 Ab and aregmab reduced the pro-LX-2 apoptosis, suggesting that the combined use of rhFGF19 and rhAREG helps induce STAT 1-mediated HSC apoptosis.

Example 4

This example provides the use of a protein composition in the preparation of an anti-liver fibrosis drug.

First, in order to induce liver fibrosis, thioacetamide (TAA) is used to induce liver fibrosis. Liver fibrosis model was induced by Thioacetamide (TAA) in 5-6 week old female mice (C57 BL/C), which were diluted in physiological saline and injected intraperitoneally at 200mg/kg dose for 3 weeks 1 and 7 weeks total. Animals were in three groups, normal control group, sham operation group (PBS injection group), protein composition treatment group, and animal numbers were: 8, 8.

The concentrated secreted protein was quantified by BCA protein quantification kit. The protein was diluted to 2mg/ml with PBS. 7 weeks after TAA injection, the normal control group was untreated, the sham operated group was given 250ul of PBS solution by spleen injection, and the protein composition treated group was given 500ug of protein by spleen injection. After 7 days of injection of secretory proteome, the liver of the mouse is soaked in formalin solution for fixation, and then embedded and sliced for immunomorphology detection, namely HE staining detection, marsen trichromatic detection and sirius detection, and comprehensive analysis is carried out on the liver fibrosis degree of the mouse.

As shown in fig. 5, after 7 weeks of TAA drug induction, the liver surface of the PBS-injected mice was uneven, had a rough texture, and the immunomorphology showed extensive collagen presence, and the normal liver tissue interval was defined by the interconnections between fibers. After twice protein secretion treatment, the liver texture of the mice in the treatment group is closer to that of the mice in the normal group, and the immune morphology shows that the fibrous tissue is obviously reduced in the liver and the fibrous tissue presents a more slender structure, which suggests that the protein group treatment obviously improves the liver fibrosis degree of the mice induced by TAA.

As shown in fig. 6, after 7 weeks of TAA drug induction, the number of activated hepatic stellate cells (α -SMA positive cells) in the livers of rats in PBS injection group was significantly increased, while the number of activated hepatic stellate cells (α -SMA positive cells) in the treatment group was significantly decreased compared to the sham operation group, which indicates that proteome treatment can inhibit hepatic stellate cell activation in vivo.

Claims

1. A protein composition comprising amphiregulin and a fibroblast growth factor.

2. The protein composition of claim 1, wherein the fibroblast growth factor is FGF19.

3. The method for producing a protein composition according to claim 1, wherein human primary hepatocytes are subjected to a transformation expansion culture using a small molecule reprogramming medium to obtain liver precursor-like cells, the liver precursor-like cells are cultured using a basal medium, and then an in vitro culture supernatant containing the amphiregulin and the fibroblast growth factor is collected.

4. The method of claim 3, wherein the small molecule reprogramming media comprises the basal medium, N2 additive, B27 additive, epidermal growth factor, hepatocyte growth factor, ROCK kinase inhibitor, wnt signaling pathway agonist, TGF- β signaling inhibitor, sphingosine monophosphate, and indoleacetic acid.

5. The method according to claim 4, wherein the content of the epidermal growth factor is 10-30 ng/ml, the content of the hepatocyte growth factor is 10-30 ng/ml, the content of the ROCK kinase inhibitor is 5-15 μmol/l, the content of the Wnt signaling pathway agonist is 1-4 μmol/l, the content of the TGF- β signaling inhibitor is 0.1-2 μmol/l, the content of the sphingosine monophosphate is 0.1-2 μmol/l, the content of the indoleacetic acid is 4-6 μmol/l, and the volume percentages of the N2 additive and the B27 additive do not exceed 1%.

6. The method of preparing a protein composition according to claim 3, wherein the step of performing transformation expansion culture on the human primary hepatocytes using the small molecule reprogramming media comprises:

human primary hepatocytes were grown at 1X 10 ⁴ -3×10 ⁴ Culturing with the small molecule reprogramming medium at inoculation density of 0.5X10 after confluence rate of not less than 90% ⁴ -1×10 ⁴ The inoculation density of each square centimeter is used for 3-4 times of continuous subculture until the fusion rate reaches 40% -50%.

7. The method of preparing a protein composition according to claim 6, wherein the step of culturing the liver precursor-like cells using a basal medium comprises:

after the continuous subculture is finished, continuous culture is performed for not less than 24 hours using the basal medium.

8. The method of preparing a protein composition of claim 7, wherein the step of collecting an in vitro culture supernatant comprising said amphiregulin and said fibroblast growth factor comprises:

after the continuous culture is finished, the collected culture supernatant is centrifuged for not less than 10 minutes under the centrifugal force of not less than 300g to remove cell debris to obtain a purified culture supernatant, and then the purified culture supernatant is concentrated by adopting an ultrafiltration centrifuge tube for 20-25 times to obtain the in vitro culture supernatant.

9. The in vitro use of a protein composition according to claim 1, comprising co-culturing said protein composition with hepatic stellate cells to promote apoptosis of said hepatic stellate cells.

10. The use of a protein composition according to claim 1 for the preparation of an anti-liver fibrosis drug comprising the intervention of a thioacetamide-induced mouse liver fibrosis model using said protein composition.