Construction method of endothelial progenitor cell model for pulmonary arterial hypertension research

Abstract

The invention discloses a construction method of an endothelial progenitor cell model for pulmonary arterial hypertension research, which comprises the following steps: (1) enriching and separating mononuclear cells centrally existing in CD34 cells in peripheral blood of a pulmonary arterial hypertension patient by adopting Histopaqui-10771, then culturing and resuspending the mononuclear cells in an EGM-2MV culture medium containing growth factors, then spreading in a culture plate coated with fibronectin for culturing; (2) 2 days after planking, sucking away non-adherent cells; and cleaning the adherent cells, adding the EGM-2MV culture medium containing the growth factors again for continuous culture, changing the culture medium and adding fibronectin at regular time during culture, inducing directional differentiation of the endothelial progenitor cells, and culturing for 2 weeks to start to appear the endothelial cells. The injury state of the endothelial cells in the disease is represented by the endothelial cells differentiated from the endothelial progenitor cells of the pulmonary hypertension patient, the research on the pulmonary hypertension cells and molecular mechanisms is facilitated, and the method is used for screening pulmonary hypertension treatment drugs and exploring potential pulmonary hypertension treatment targets.

Description

Technical Field

[0001] The invention relates to a method for constructing a cell model, and in particular to a method for constructing an endothelial progenitor cell model for studying pulmonary hypertension. Background Art

[0002] Pulmonary Hypertension (PH) is a life-threatening progressive disease characterized by pulmonary endothelial cell dysfunction and vascular remodeling. Pulmonary artery pressure and pulmonary vascular resistance gradually increase, eventually developing into right ventricular hypertrophy, heart failure, and even death. With the in-depth understanding of the etiology and pathogenesis of PH, the current clinical treatment methods for PH include oxygen therapy, vasodilators, targeted drugs, and lung transplantation. These methods can improve the quality of life of patients and increase the survival rate to a certain extent, but they cannot reverse the disease process and cause huge clinical and economic burdens. The results of the REVEAL registration study showed that the 5-year survival rate of PH patients was only 57%. Therefore, it is necessary to deepen the research on the mechanism of pulmonary hypertension.

[0003] The pathogenesis of pulmonary hypertension is very complex, and further exploration of its pathogenesis and treatment strategies is urgently needed. The occurrence and development of pulmonary hypertension is closely related to pulmonary vascular remodeling, involving environmental factors (hypoxia, oxidative stress, inflammation, etc.), genetic factors (gene mutations), and epigenetic factors (DNA methylation, histone acetylation, and non-coding RNA regulation, etc.). Among them, pulmonary artery endothelial cells play an important role in maintaining the normal structure and function of blood vessels. Endothelial cells are involved in the synthesis and secretion of a variety of growth factors and vasoactive substances, such as endothelin, nitric oxide, serotonin, thromboxane, and prostacyclin, etc., which can directly or indirectly affect the growth and contraction of pulmonary artery smooth muscle cells. Therefore, further exploration of the mechanism of action of endothelial cells in the occurrence and development of pulmonary hypertension can provide a scientific basis for the clinical treatment of pulmonary arterial hypertension.

[0004] At present, animal models are often used to study pulmonary hypertension, mainly including simple chronic hypoxia, monocrotaline (MCT) injection, chronic hypoxia combined with SU5416, surgical shunt, etc. Experimental animals can be selected from pigs, cattle, sheep, rats, mice, etc. Animal models are important media for experimental research. The construction of the above-mentioned animal models of pulmonary hypertension has laid the foundation for the current understanding of pulmonary hypertension. However, due to the differences between the genomes of experimental animals and humans, the differences in modeling methods, and the complexity and heterogeneity of pulmonary hypertension itself, the current animal models of pulmonary hypertension still cannot fully simulate the characteristics of human disease. For example, although the MCT model has the advantages of good repeatability and simple operation, it can quickly cause vascular endothelial cell dysfunction, but it cannot simulate severe angiogenic pulmonary hypertension well; for example, the modeling conditions of the simple chronic hypoxia model are easy to set, but different species have different tolerance to chronic hypoxia, and hypoxia induction is reversible, so the simulation of severe pulmonary hypertension is very limited, and even only causes mild pulmonary vascular endothelial damage.

[0005] With the development of cell biology, the application of new technologies and culture reagents has made it possible to use human primary cells as research objects. Compared with animal models and cell lines, human primary cells retain more biological characteristics of original tissues in the human body and can better establish cell disease models. However, it is very difficult to obtain lung tissue from patients with pulmonary hypertension for primary cell culture. Only a small number of patients with end-stage pulmonary hypertension can obtain primary pulmonary vascular endothelial cells during lung transplantation. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing an endothelial progenitor cell model for studying pulmonary hypertension.

[0007] The method for constructing an endothelial progenitor cell model for pulmonary hypertension research includes the following steps:

[0008] (1) Using Histopaque-10771 to enrich and separate mononuclear cells concentrated in CD34 cells from peripheral blood of patients with pulmonary hypertension, the mononuclear cells were then cultured and resuspended in EGM-2MV medium containing growth factors, and then plated on a culture plate coated with fibronectin for culture;

[0009] (2) After 2 days of plating, the non-adherent cells were removed; the adherent cells were washed, and the EGM-2MV medium containing growth factors was added again to continue culturing. During the culture period, the medium was changed regularly and fibronectin was added to induce the directional differentiation of endothelial progenitor cells. Endothelial cells began to appear after 2 weeks of culture.

[0010] Endothelial progenitor cells (EPC), as precursor cells of endothelial cells, play an important role in maintaining the dynamic repair and angiogenesis of blood vessels. Endothelial progenitor cells are derived from bone marrow, circulate in peripheral blood, and have good proliferation potential, and endothelial progenitor cells are closely related to the onset of pulmonary hypertension. Therefore, peripheral blood of patients with pulmonary hypertension can be collected, endothelial progenitor cells can be enriched, primary cells can be cultured in vitro, endothelial progenitor cells can be induced to differentiate into endothelial cells, and a pulmonary hypertension endothelial cell model can be constructed. In the present invention, step (1) uses Histopaque-10771 reagent to enrich endothelial progenitor cells, and mononuclear cells concentrated in CD34 cells can be efficiently obtained.

[0011] During the culture process, the inventors found that endothelial progenitor cells are more likely to adhere to the wall at an early stage, so the non-adherent cells can be aspirated 2 days after the cells are plated to increase the specificity of the obtained endothelial progenitor cells and improve the purity of the endothelial progenitor cells.

[0012] The EGM-2MV culture medium containing growth factors of the present invention contains the following components: 25 mL of 10% fetal bovine serum, 0.2 mL of hydrocortisone, 2 mL of recombinant human basic fibroblast growth factor, 0.5 mL of vascular growth factor, 0.5 mL of regenerated human R3 insulin-like growth factor-1, 0.5 mL of ascorbic acid, 0.5 mL of human epidermal growth factor, and 0.5 mL of GA-1000.

[0013] In step (1), the Histopaque-10771 reagent is mixed with peripheral blood from patients with pulmonary hypertension and then centrifuged at 400×g for 30 minutes at room temperature, and the braking and acceleration of the centrifuge are ensured to be at the lowest setting to avoid severe braking and acceleration affecting layer separation.

[0014] In step (1), the concentration of fibronectin coated on the culture plate is 0.1 mg / mL. Fibronectin, as an extracellular matrix, simulates the in vivo environment as much as possible and can promote the adhesion and colonization of endothelial progenitor cells.

[0015] In step (1), the cell density of the culture plate was 2×10 6 / hole.

[0016] In step (2), the adherent cells are washed with phosphate buffered saline (PBS) or an appropriate amount of cell culture medium.

[0017] In step (2), during the culture period, the cell medium is replaced every 3 days.

[0018] The present invention also includes an expansion culture step, namely step (3): endothelial cells differentiated from endothelial progenitor cells begin to appear, showing a typical cobblestone-like structure, and the cells are digested with trypsin containing 0.25% EDTA, the cell density is adjusted, and the cells are plated in a cell culture dish coated with fibronectin for expansion culture.

[0019] The culture conditions in the present invention are 5% CO2 concentration and 37°C.

[0020] Compared with the prior art, the present invention collects peripheral blood from patients with pulmonary hypertension, enriches endothelial progenitor cells and induces differentiation to construct a primary endothelial cell model that can be used for the study of the mechanism of pulmonary hypertension. Its advantages are as follows:

[0021] 1. Compared with culturing primary endothelial cells by obtaining human lung tissue, the threshold for obtaining peripheral blood is lower and more convenient, and only a small amount of peripheral blood is needed for primary cell culture and induced differentiation;

[0022] 2. Compared with traditional animal models, the human primary cells used in the present invention can retain more biological characteristics of the original tissues in vivo, and the constructed cell model can more realistically reflect the endothelial injury state in vascular remodeling of pulmonary hypertension;

[0023] 3. The present invention differentiates endothelial progenitor cells into endothelial cells. The results show that endothelial cells differentiated from endothelial progenitor cells of patients with pulmonary arterial hypertension can well characterize the state of endothelial cell damage in the disease. Therefore, the constructed pulmonary arterial hypertension endothelial cell model is helpful to study the cellular and molecular mechanisms of pulmonary arterial hypertension, and can be used to screen drugs for the treatment of pulmonary arterial hypertension and explore potential therapeutic targets for pulmonary arterial hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the EPC isolation and differentiation induction experimental results of an embodiment of the present invention.

[0025] Figure 2 The expression of endothelial lineage marker genes in the embodiments of the present invention at different culture times in vitro.

[0026] Figure 3 This is a schematic diagram of immunofluorescence of EPCs from patients with pulmonary hypertension after induced differentiation according to an embodiment of the present invention. DETAILED DESCRIPTION

[0027] Example 1 Acquisition and culture of endothelial progenitor cells from peripheral blood of patients with pulmonary hypertension

[0028] Acquisition and processing of clinical samples: After passing ethical review, obtaining the patient's right to know and signing the consent form, 20 mL of peripheral venous blood was collected from the patient, placed on ice for stable transportation, and sent to the cell laboratory for immediate processing of fresh blood. To ensure the activity of endothelial progenitor cells in the blood, the blood was kept at 4°C for no more than 12 hours. The control group was healthy people whose gender and age matched those of the patients with pulmonary hypertension.

[0029] (1) First add 3 mL of Histopaque-10771 to a 15 mL centrifuge tube, and then carefully add 3 mL of peripheral blood to the Histopaque-10771 reagent. Centrifuge at 400 × g for 30 minutes at room temperature. Make sure the centrifuge's brake and acceleration are at the lowest setting, as severe brake and acceleration may affect layer separation.

[0030] (2) After centrifugation, the centrifuge tube can be divided into a plasma layer, a mononuclear cell layer, a separation fluid layer, and a red blood cell layer. Carefully discard the plasma with a pipette, collect the mononuclear cells, and add 10 mL of phosphate buffered saline (PBS) or an appropriate amount of cell culture medium to wash the cells. Gently pipette to mix. Centrifuge at 250 × g for 10 minutes, discard the supernatant, and repeat the washing process.

[0031] (3) Add EGM-2MV culture medium containing 10% fetal bovine serum to resuspend the mononuclear cell pellet.

[0032] The EGM-2MV culture medium containing 10% fetal bovine serum contains 25 mL of fetal bovine serum, 0.2 mL of hydrocortisone, 2 mL of recombinant human basic fibroblast growth factor, 0.5 mL of vascular growth factor, 0.5 mL of regenerated human R3 insulin-like growth factor-1, 0.5 mL of ascorbic acid, 0.5 mL of human epidermal growth factor, and 0.5 mL of GA-1000.

[0033] (4) The cell solution was spread on a six-well cell culture plate and the cell density was adjusted to 2×10 6 / well, cultured in a 5% CO2 concentration, 37°C incubator. The six-well cell culture plate was coated with 0.1 mg / mL fibronectin in advance.

[0034] (5) Using the differential cell adhesion characteristics, 2 days after cell plating, remove the non-adherent cells in the culture plate, wash with 2 mL PBS, and then add 2.5 mL of EGM-2MV culture medium containing 10% fetal bovine serum.

[0035] (6) The cell culture medium was changed every 3 days, and endothelial progenitor cells were induced to differentiate into endothelial cells by adding cell growth factors and extracellular matrix (0.1 mg / mL fibronectin). The use of the extracellular matrix is ​​to simulate the in vivo environment as much as possible, which can promote the adhesion and colonization of endothelial progenitor cells. After 2 weeks of culture, endothelial cells differentiated from endothelial progenitor cells began to appear, showing a typical paving stone-like structure.

[0036] (7) Digest the cells with trypsin containing 0.25% EDTA and adjust the cell density to 1×10 6 / mL, and plated into a cell culture dish coated with fibronectin for expansion culture.

[0037] like Figure 2 As shown in the figure, with the increase of in vitro culture time, the proportion of cells expressing endothelial lineage genes gradually increased, among which the expression changes of VEGFR2 and CD133 were the most significant. At the beginning of culture, a considerable proportion of cells expressed CD34 and CD31, and the expression of CD34 and CD31 gradually increased over time.

[0038] Example 2 Identification of Endothelial Cells Differentiated from Endothelial Progenitor Cells in Patients with Pulmonary Hypertension

[0039] Endothelial cells and vascular endothelial cells (HUVEC) differentiated from endothelial progenitor cells of patients with pulmonary hypertension were digested with trypsin containing 0.25% EDTA, and then plated into 24-well cell culture plates coated with fibronectin. When the cell growth confluence reached 90%, the cell culture medium was discarded, and after washing with PBS three times, 4% paraformaldehyde was added to each well to fix the cells for 20 minutes, and the cells were washed again with PBS for 3 times, each time for 5 minutes, and blocked with a blocking solution containing 3% BSA at room temperature for 1 hour. Anti-CD31, VEGFR2, TIE2, ACVRL1, and vWF antibodies were added to each well separately, and the primary antibodies were incubated at 4°C overnight. The next day, the antibodies in each well were discarded, PBS was added for washing three times, and the secondary antibodies coupled with fluorescein were added, and incubated at room temperature for 2 hours in the dark. After 2 hours, the secondary antibodies were discarded, DAPI was added to stain the cell nuclei, and after washing with PBS three times, the cells were photographed using a fluorescence microscope. The results are shown as follows Figure 3 The results show that the endothelial cells differentiated from endothelial progenitor cells of patients with pulmonary hypertension in the present invention have the same morphology as vascular endothelial cells (HUVEC), but have the endothelial cell damage state in pulmonary hypertension disease.

Claims

1. A method for constructing an endothelial progenitor cell model for pulmonary hypertension research, characterized in that: The following steps are involved: (1) Using Histopaque-10771 to enrich and separate mononuclear cells concentrated in CD34 cells from peripheral blood of patients with pulmonary hypertension, the mononuclear cells were then cultured and resuspended in EGM-2MV medium containing growth factors, and then plated on a culture plate coated with fibronectin for culture; (2) After 2 days of plating, the non-adherent cells were removed; the adherent cells were washed, and the EGM-2MV medium containing growth factors was added again to continue culturing. During the culture period, the medium was changed regularly and fibronectin was added to induce the directional differentiation of endothelial progenitor cells. Endothelial cells began to appear after 2 weeks of culture.

2. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: The EGM-2MV culture medium containing growth factors contains the following components: 25 mL of 10% fetal bovine serum, 0.2 mL of hydrocortisone, 2 mL of recombinant human basic fibroblast growth factor, 0.5 mL of vascular growth factor, 0.5 mL of regenerated human R3 insulin-like growth factor-1, 0.5 mL of ascorbic acid, 0.5 mL of human epidermal growth factor, and 0.5 mL of GA-1000.

3. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: In the step (1), the Histopaque-10771 reagent is mixed with the peripheral blood of patients with pulmonary hypertension and then centrifuged at 400×g for 30 minutes at room temperature, and the braking and acceleration of the centrifuge are ensured to be at the lowest setting to avoid severe braking and acceleration affecting layer separation.

4. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: In the step (1), the concentration of fibronectin coated on the culture plate is 0.1 mg / mL.

5. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: In step (1), the cell density of the culture plate is 2×10 6 / hole.

6. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: In the step (2), the adherent cells are washed with phosphate buffer or cell culture medium.

7. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: In the step (2), during the culture period, the cell medium is replaced every 3 days.

8. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: The method also includes an expansion culture step, namely step (3): endothelial cells differentiated from endothelial progenitor cells begin to appear, showing a typical cobblestone-like structure, and the cells are digested with trypsin containing 0.25% EDTA, the cell density is adjusted, and the cells are plated in a cell culture dish coated with fibronectin for expansion culture.

9. The method for constructing an endothelial progenitor cell model for pulmonary hypertension research according to claim 1, characterized in that: The culture conditions in the present invention are 5% CO2 concentration and 37°C.

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