CN112206313A

CN112206313A - Application of LAMA3 or THBS1 in preparation of medicine for promoting liver regeneration

Info

Publication number: CN112206313A
Application number: CN202010963747.2A
Authority: CN
Inventors: 古维立; 李桃生; 张帅; 黄子圣
Original assignee: Guangzhou First Peoples Hospital
Current assignee: Guangzhou First Peoples Hospital
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2021-01-12
Anticipated expiration: 2040-09-14
Also published as: CN112206313B

Abstract

The invention relates to the field of medicines, and in particular relates to application of LAMA3 or THBS1 in preparation of a medicine for promoting liver regeneration. Aiming at the insufficient understanding of factors for promoting liver regeneration such as extracellular matrix, cell factors and chemotactic factors in the existing liver regeneration microenvironment, the invention provides the experimental basis that LAMA3 and secretory factor THBS1 in the extracellular matrix are used as the cell factors for treating liver dysfunction and promoting liver regeneration, and the LAMA3 and the THBS1 are used jointly for the first time, and the two factors have certain synergistic effect on the growth of liver cells and liver stem cells.

Description

Application of LAMA3 or THBS1 in preparation of medicine for promoting liver regeneration

Technical Field

The invention relates to the field of medicines, and in particular relates to application of LAMA3 or THBS1 in preparation of a medicine for promoting liver regeneration.

Background

As is known to all, normal liver tissues have strong self-regeneration capacity, and even though 2/3 normal liver tissues are removed by surgery, the residual liver can still be regenerated and restored to the state before the surgical removal, so that the live liver transplantation surgery for treating end-stage liver diseases can be popularized and applied clinically. However, in various pathological conditions, the self-regeneration repair ability of the liver is greatly reduced or completely lost, and the liver function is difficult to recover by itself but continuously worsens and progresses, so that a plurality of end-stage liver disease patients with liver failure and the like appear clinically. Although liver transplantation may be effective in treating end-stage liver disease, including acute liver failure, chronic liver failure, and metabolic liver disease, liver donors are in serious shortage, and liver transplantation surgery itself is vastly invasive and at many risks. This has prompted attempts to promote liver regeneration by transplantation of hepatocytes or immature stem cells derived from various tissues in order to cure liver diseases.

The liver is one of the most regenerative organs in our body. However, the regenerative capacity of the liver is largely impaired in pathological conditions such as viral infection and cholestasis. Some past studies have attempted to understand the molecular/cellular mechanisms of liver regeneration. While both "seeds" (resident tissue cells, including mature hepatocytes and immature hepatic stem cells) and "soil" (local tissue microenvironment) are considered critical, only a few studies have focused on the role of the local tissue microenvironment in regeneration following liver injury.

The tissue microenvironment is composed of complex factors such as extracellular matrix (ECM)/adhesion molecules and cytokines/chemokines. ECM has long been considered to be an inert substrate for cell growth. The dynamic structure of the ECM is composed of a variety of proteins and other macromolecules that provide a scaffold for biological functions of tissue cells (stem cells), including proliferation, migration, and differentiation. ECM has also been shown to play a key role in maintaining hepatic stem cells. Adhesion molecules are a subset of cell surface proteins that bind to the surrounding extracellular matrix and neighboring cells to maintain the structure and function of the cells in various tissues/organs. Although extracellular matrix and adhesion molecules play an important role in maintaining hepatic homeostasis, there is a need to identify ECM and adhesion molecules that play a critical role in liver regeneration.

Laminin (lamin) is an important structural component of ECM, facilitating cell attachment. They are a large family of heterotrimeric basement membrane adhesion proteins, with approximately 16 different isoforms. Each consisting of alpha, beta and gamma chains. Laminins affect the behavior of the associated cell, such as cell adhesion, migration, differentiation, activity, and phenotypic stability. Laminins have been shown to induce the expression of differentiation markers such as tyrosine aminotransferase, tryptophan-2, 3-dioxygenase, and cytochrome P450 in rat hepatocytes.

Thrombospondin (Thrombospondin) belongs to the secreted multifunctional protein family. Thrombospondin-1 (THBS1) is the first member of the family, a 450kda homotrimeric glycoprotein, with multiple functional domains, a stromal cell glycoprotein, secreted by a variety of cell types. By binding to extracellular proteins and/or cell surface receptors, THBS1 regulates a variety of cellular functions, plays an important role in a variety of biological processes, including angiogenesis, apoptosis, potential TGF- β activation and immunomodulation, and is involved in the regulation of many organ functions. THBS1 may play a role in a variety of chronic liver diseases, including mainly non-alcoholic fatty liver disease, liver fibrosis and hepatocellular carcinoma.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of LAMA3 or THBS1 in preparing a medicine for promoting liver regeneration, which has great significance in promoting liver regeneration and treating liver dysfunction.

In order to achieve the purpose, the invention adopts the technical scheme that: provides an application of LAMA3 protein or THBS1 protein in preparing a medicine for promoting liver regeneration.

Laminin alpha 3(LAMA3) gene and laminin coded by the gene participate in proliferation and survival of liver cells, and promote liver regeneration; thrombospondin-1 (THBS1) is involved in the maintenance of the proportion and the dryness of liver stem cells and promotes liver regeneration.

As a preferred embodiment of the use according to the invention, the LAMA3 protein has the NCBI Gene ID: 3909.

as a preferred embodiment of the use of the present invention, the protein THBS1 has NCBI Gene ID: 7057.

the invention provides an application of LAMA3 protein in preparing a medicine for promoting liver regeneration by combined administration with THBS1 protein.

Aiming at the insufficient understanding of factors for promoting liver regeneration such as extracellular matrix, cell factors and chemotactic factors in the existing liver regeneration microenvironment, the invention provides the experimental basis that LAMA3 and secretory factor THBS1 in the extracellular matrix are used as the cell factors for treating liver dysfunction and promoting liver regeneration, the LAMA3 and the THBS1 are combined for the first time, and the two factors have certain synergistic effect on the growth of liver cells and liver stem cells.

The invention also provides a pharmaceutical composition, which comprises at least one of LAMA3 protein and THBS1 protein.

As a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The invention has the beneficial effects that:

(1) the invention finds that the LAMA3 protein in the extracellular matrix has a supporting function on the survival, proliferation and function of the liver cells; experiments prove that the LAMA3 obviously reduces the proportion of the Claudin-3 positive hepatic stem cells, and the effect of LAMA3 on inducing the differentiation of the hepatic stem cells is confirmed by combining the effect of promoting the proliferation of the hepatic cells.

(2) The invention discovers that the secreted protein THBS1 reduces the apoptosis of the newborn liver cells, maintains the proportion of the liver stem cells which are positive to Claudin-3 in the newborn liver cells, and plays a role in the maintenance of the liver stem cells.

(3) The invention discloses that the combined use of LAMA3 and THBS1 can obviously improve the proliferation rate of liver cells for the first time, and in-vitro evaluation, the two factors have a certain synergistic effect on the growth of liver cells and liver stem cells. Has great significance for promoting liver regeneration and treating liver dysfunction.

Drawings

FIG. 1: the results of the effects of LAMA3 and THBS1 on hepatocyte and hepatic stem cell morphology, cell viability are shown.

FIG. 2: results plot of the effect of LAMA3 and THBS1 on albumin expression.

FIG. 3: results of the effect of LAMA3 and THBS1 on the expression of Claudin-3.

FIG. 4: results plot of the effect of LAMA3 and THBS1 on the expression of proliferation index Ki-67.

FIG. 5: and (3) detecting the apoptosis of the hepatic cells of the newborn rat.

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

Observation and counting of positive cells: positive cells were observed and counted using a confocal laser scanning microscope (FV10i-LIV, OLYPUS) and digital images were collected using FV10-ASW software (OLYPUS) with a 60-fold magnification. From the stained slides of each experiment, 15 images were randomly selected for quantitative counting.

Example 1 specific expression of LAMA3 and THBS1

(1) Animal(s) production

Adult male C57BL/6 mice (8-10 weeks) and neonatal mice (within 36 hours after birth).

(2) Liver partial resection and bile duct ligation

Adult mice received 70% liver resection (PH) and half of them were randomly selected for bile duct ligation (PH + BDL). Sham surgery (laparotomy) was used as a negative control for health. 4 mice were sacrificed each group on days 1, 3, 7, 14 post treatment. Total RNA was extracted from liver tissue for PCR chip analysis.

(3) PCR chip of focusing path

By the PCR chip analysis of the focused pathway, ECM and adhesion molecules that may be involved in liver regeneration were sought. The method comprises the following specific steps: total RNA was extracted from liver tissue using the RNase Mini Kit (Qiagen). RNA concentration was determined using a NanoDrop model 2000 spectrophotometer (Thermo Fisher Science), and after mixing 4 RNA samples (0.25. mu.g each), cDNA was reverse transcribed using the RT2 First Strand Kit (Qiagen). Mouse ECM and adhesion molecule PCR chips, containing 84 genes. All data were normalized to housekeeping gene expression and based on cycleThe threshold (Ct) quantifies the expression level of each gene. Based on the different dynamic changes of gene expression between different groups, two factors LAMA3(NCBI website:https：// www.ncbi.nlm.nih.gov/gene/3909(ii) a NCBI Gene ID: 3909) and THBS1(NCBI website:https：// www.ncbi.nlm.nih.gov/gene/7057；NCBI Gene ID：7057)。

example 2 isolation and Primary culture of neonatal mouse hepatocytes

Newborn mice (within 36 hours after birth) are euthanized by cervical dislocation. The liver tissue was removed under sterile conditions and immediately transferred to a buffer (containing Ca)²⁺And Mg²⁺Hanks balanced salt solution, 0.1M EGTA, 1M HEPES). Liver tissue was cut into small pieces, collected in tubes, and centrifuged at 1000rpm for 2 min. Dispersed cells were obtained by collagenase digestion and subsequent filtration through a 40 μm pore size. Freshly isolated neonatal hepatocytes, including mature hepatocytes and immature hepatic stem cells, were suspended in DMEM medium containing 10% fetal bovine serum, 100U/ml penicillin G and 10 μ G/ml streptomycin. Cells were seeded in 6cm petri dishes or 8-well chamber culture slides (Lab-Tek, Thermo Science Nunc, 1.5X 10⁴Perwell), 37 ℃ 5% CO₂Culturing in an incubator. The medium was changed every 3 days.

To evaluate the effect of LAMA3 and THBS1 on hepatocyte growth, hepatocytes were cultured in culture dishes containing the following media, respectively:

control group: no LAMA3 pre-coating, no THBS1 added to the medium;

LAM group: 5 μ g/ml LAMA3 pre-coated, no THBS1 added to the medium;

③ TSP group: no LAMA3 pre-coating, adding 0.5 μ g/ml THBS1 to the culture medium;

LAM + TSP group: 5. mu.g/ml LAMA3 was pre-coated and 0.5. mu.g/ml THBS1 was added to the culture.

Wherein the LAMA3 pre-coating step comprises the following steps:

(1) before use, the stock solution of LAMA3 protein is slowly thawed at 2-8 deg.C.

(2) The amount of LAMA3 protein required for the experiment was calculated and 5ug/mL was used for the initial generation of precoating concentrations. Once the cells are adapted, the coating concentration can be reduced as appropriate empirically for different cell lines (down to 1 ug/mL).

(3) With 1X DPBS (Ca)²⁺/Mg²⁺) The protein stock solution was diluted and the coating solution was added to the petri dish. Ca should be selected²⁺And Mg²⁺The divalent cations of DPBS have an important role in the structure and function of proteins.

(4) Ensuring that the coating solution covers the entire surface of the dish. Surfaces that are not covered with sufficient solution are unable to support cell growth. The culture vessel was sealed with a membrane to prevent evaporation and contamination and incubated overnight at 2-8 ℃.

Example 3 Effect of LAMA3 and THBS1 on cell viability

The total number of viable hepatocytes was measured, and all cells were harvested from the culture dish in example 2 with 0.25% trypsin 7 days after primary culture. The total number of viable cells was counted using a cell counting device (Nucleo Counter, Chemotetec A/S, Denmark).

Results as shown in fig. 1A, all cells, including hepatocytes and hepatic stem cells, were collected from the neonatal liver, and hepatocytes in different media exhibited different shapes and sizes after 7 days of culture: more spindle cells were observed in LAMA 3-coated dishes, and many cells were binuclear and exhibited a fibroblast-like morphology; in contrast, some cells cultured with the addition of THBS1 were elongated.

As shown in fig. 1B, the total number of hepatocytes was significantly increased in the LAM group and LAM + TSP group (P < 0.001) compared to the control group, indicating that LAMA3 significantly increased the number of neonatal hepatocytes; and the total number of the liver cells of the TSP group has no obvious change (P is 0.998).

Example 4 Effect of LAMA3 and THBS1 on albumin expression

Albumin is the most abundant protein synthesized by functional hepatocytes, and all cells were harvested from the dishes in example 2 with 0.25% trypsin 7 days after primary culture, and albumin expression in the cells was examined using immunohistochemical staining.

As a result, as shown in FIG. 2, albumin was widely expressed in each group. The percentage of albumin-positive cells was significantly higher in the LAM, TSP and LAM + TSP groups than in the control group (P < 0.001).

Example 5 Effect of LAMA3 and THBS1 on Claudin-3 expression

Claudin-3 is one of the markers for the identification of hepatic stem/progenitor cells, a small fraction of immature precursor cells, which form hepatocytes and cholangiocytes in the liver. To test Claudin-3 positive hepatic stem cells and their proliferation, all cells were harvested from the plates in example 2 with 0.25% trypsin 7 days after primary culture and double immunostaining for Claudin-3 and Ki-67 expression in hepatocytes was performed.

The results are shown in FIG. 3A, with partially viable neonatal hepatocytes Claudin-3 positive and a few Ki-67 positive. As shown in FIG. 3B, the quantitative data showed that the percentage of claudin-3 positive cells in the LAM group and the LAM + TSP group was significantly lower than that in the control group (P < 0.001). In addition, in the LAM group, the double positive cells Claudin-3⁺/Ki-67⁺(i.e.both protein molecules stain positively in the cell, indicating that both protein molecules are now expressed in this cell.) the percentage is also significantly lower than in the control (P < 0.05). Therefore, LAMA3 can obviously reduce the proportion of Claudin-3 positive hepatic stem cells and induce the differentiation of the hepatic stem cells.

Example 6 Effect of LAMA3 and THBS1 on hepatocyte proliferation Activity

Ki-67 is a nuclear protein bilayer with molecular weight of 345-395kDa, and plays a crucial role in maintaining cell proliferation. Ki-67 is present in all cells in the cell cycle other than G0, beginning in the middle G1 phase, and gradually increasing from S to G2 phase, reaching a peak in M phase, and rapidly degrading in M-neither phase. The Ki-67 marker index (LI), the ratio of Ki-67 staining in tissues, is indicative of cell growth information.

After 7 days of primary culture all cells were harvested from the plates in example 2 using 0.25% trypsin and tested for Ki-67 expression by immunohistochemical methods, indicating that the cells expressed the Ki-67 protein, indicating high cell proliferation activity.

As shown in FIG. 4, Ki-67 expression was higher in all of the LAM, TSP and LAM + TSP groups, but only the LAM + TSP group was statistically significant (P < 0.05).

Example 7 THBS1 significantly reduces neonatal rat hepatocyte apoptosis

All cells were harvested from the culture dish in example 2 with 0.25% trypsin 7 days after primary culture and cell proliferation was estimated by immunostaining. Cells were fixed with 4% Paraformaldehyde (PFA) for 15 min. Membrane permeabilization was performed with 0.1% triton x100 for 10 min. After blocking with 5% bovine serum albumin for 1 hour, the cells were incubated with a rat anti-Ki-67 monoclonal antibody (EBioscience) overnight at 4 ℃ and then treated with Alexa

Conjugated goat anti-rat IgG-H&L polyclonal Secondary antibody (Invertogen) was incubated. Apoptotic cells were incubated with FITC-Annexin V.

As a result, as shown in fig. 5, the percentage of apoptotic cells was significantly decreased in the TSP group and the LAM + TSP group (P < 0.05), and the percentage of apoptotic cells was not significantly changed in the LAM group (P ═ 0.511) as compared with the control group.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

Application of LAMA3 protein or THBS1 protein in preparing medicine for promoting liver regeneration.
2. The use according to claim 1, wherein the LAMA3 protein has the NCBI Gene ID: 3909.
3. the use according to claim 1, wherein the THBS1 protein has the NCBI Gene ID: 7057.
use of LAMA3 protein in the manufacture of a medicament for use in combination with THBS1 protein in the promotion of liver regeneration.
5. A pharmaceutical composition comprising at least one of LAMA3 protein and THBS1 protein.
6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable carrier.