Construction method and application of wuzhishan pig mitral valve in vitro model

Abstract

The application belongs to the field of modern agricultural technology, and particularly relates to a construction method and application of a Wuzhishan pig bicuspid valve in-vitro model. The application first constructs a three-dimensional in-vitro culture model of Wuzhishan pig heart bicuspid valve interstitial cells, which can maintain a low activation level of MVIC with the culture days, and tends to be mature after 14 days of culture. The method solves the problem that MVIC is easily activated in two-dimensional in-vitro culture, and a culture medium that can maintain a low activation level of MVIC in-vitro is screened out, and the three-dimensional in-vitro model is constructed by using the culture medium, which can be used as an in-vitro model of the static state of MVIC, and provides a platform for the mechanism research of MVIC activation. The model of some construction methods of the application can reduce the activation of Wuzhishan pig bicuspid valve interstitial cells, which can not only be used as an animal medical model to study the pathogenesis, but also provide help for the preservation of Wuzhishan pig heart cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of modern agricultural technology, and particularly relates to a construction method and application of a Wuzhishan pig mitral valve in vitro model. BACKGROUND

[0002] Wuzhishan pig is a characteristic pig breed in Hainan Province, China, and is the rarest pig breed with the smallest body size and the lightest weight. The adult pig weighs no more than 35 kg, has a gentle temperament, a low metabolic rate, strong adaptability, and is resistant to coarse feeding. In addition, the Wuzhishan pig has excellent meat quality and adaptability, stable genetics, and is easy to raise and manage. [1] The Wuzhishan pig can also be used as a male parent to breed new varieties with high-quality meat through crossbreeding. At the same time, due to its closed population and stable genetic traits, and the similarity in anatomy and physiology to humans, the Wuzhishan pig is often used as a medical model pig to study the pathogenesis of cardiovascular diseases. [2] The mitral valve is an important anatomical structure between the left atrium and the left ventricle of the heart. Mitral valve disease has a high incidence in mammals, and is commonly seen in humans, dogs, and pigs. The incidence of mild valve changes in piglets under 2 months of age is about 26%; the incidence in pigs of 6 months of age at slaughter is nearly 30%; and the incidence in pigs of 3-4 years of age is about 90%. [3] Streptococcus suis type II is a zoonosis that can cause endocarditis and acute death, and is highly prevalent in pigs of slaughter age. The endocarditis caused by it is mainly located in the valve, and the mitral valve lesion is dominant (59%). [4] When mitral valve disease occurs, the activation of mitral valve interstitial cells (MVICs) plays a key role. The use of MVICs to construct an in vitro model can provide a platform for the study of various mitral valve diseases and help the development of the livestock and veterinary industry. However, the current culture of MVICs in two-dimensional and three-dimensional systems spontaneously transforms the cells into an activated state, thereby increasing the difficulty of studying the activation of MVICs and the specific pathogenesis of mitral valve disease. Therefore, it is necessary to develop a method for constructing a mitral valve in vitro model that can maintain a low activation level of MVICs in vitro.

[0003] [1]. XU Wenjuan, et al. Comparison of carcass traits and meat quality between Wuzhishan and Landrace pigs. Chinese Journal of Animal Husbandry, 2013. 40(05): pp. 93-96.

[0004] [2]. TIAN Yulong and ZHONG Hongshan. Application of Wuzhishan miniature pig in modeling of cardiovascular system diseases. Journal of Interventional Radiology, 2016. 25(04): pp. 363-366.

[0005] [3]. Gagna C, Meier D, Ru G, Pospischil A, Guarda F. Pathology of mitral valve in regularly slaughtered pigs: an abattoir survey on the occurrence of myxoid degeneration (endocardiosis), fibrosis and valvulitis. Zentralbl Veterinarmed A. 1998 Sep;45(6-7):383-95.

[0006] [4]. Jensen HE, Gyllensten J, Hofman C, Leifsson PS, Agerholm JS, Boye M, Aalbæk B. Histologic and bacteriologic findings in valvular endocarditis of slaughter-age pigs. J Vet Diagn Invest. 2010 Nov;22(6):921-7. SUMMARY

[0007] The first aspect of the present application aims to provide a method for constructing a Wuzhishan pig mitral valve in vitro model.

[0008] The second aspect of the present application aims to provide a Wuzhishan pig mitral valve interstitial cell culture medium.

[0009] The third aspect of the present application aims to provide the use of the culture medium of the second aspect of the present application in reducing the activation level of Wuzhishan pig mitral valve interstitial cells cultured in vitro.

[0010] The fourth aspect of the present application aims to provide the use of the culture medium of the second aspect of the present application in preparing a Wuzhishan pig cardiovascular disease cell model.

[0011] In order to achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows:

[0012] The first aspect of the present application provides a method for constructing a Wuzhishan pig mitral valve in vitro model, comprising the following steps: culturing Wuzhishan pig mitral valve interstitial cells using an activation inhibiting culture medium.

[0013] In some embodiments of the present application, the activation inhibiting culture medium comprises a basic culture medium, serum.

[0014] In some embodiments of the present application, the basal medium is MCDB131 medium.

[0015] In some embodiments of the present application, the MCDB131 medium comprises 0.5-2 wt% L-Glu.

[0016] In some embodiments of the present application, the serum comprises fetal bovine serum.

[0017] In some embodiments of the present application, the serum comprises fetal bovine serum.

[0018] In some embodiments of the present application, the inhibitory activation medium comprises growth factors; the growth factors comprise EGF, FGFb.

[0019] In some embodiments of the present application, the concentration of EGF is 1-10 ng / mL.

[0020] In some embodiments of the present application, the concentration of FGFb is 0.1-1 ng / mL.

[0021] In some embodiments of the present application, the inhibitory activation medium comprises Y27632.

[0022] In some embodiments of the present application, the concentration of Y27632 is 1-20 μM.

[0023] In some embodiments of the present application, the Y27632 is a ROCK-I and ROCK-II inhibitor, with the molecular formula of C 14 H 21 N3O, and CAS number of 146986-50-7.

[0024] In some embodiments of the present application, the inhibitory activation medium comprises antibiotics.

[0025] In some embodiments of the present application, the heart valve disease cell model comprises a mitral valve disease model.

[0026] In some embodiments of the present application, the Wuzhishan pig mitral valve interstitial cells are isolated from the anterior leaflet of the Wuzhishan pig mitral valve, and the isolation method comprises the following steps:

[0027] The surface endothelial cells of the anterior leaflet of the Wuzhishan pig mitral valve are removed, the anterior leaflet of the Wuzhishan pig mitral valve is digested using collagenase, and the digestion solution is inoculated in a culture medium.

[0028] In a second aspect of the present application, a Wuzhishan pig mitral valve interstitial cell culture medium is provided, comprising MCDB131 basic medium, 0.5-2 wt% L-Glu, 1-12 v / v% serum, 1-10 ng / mL EGF, 0.1-1 ng / mL FGFb, and 1-20 μM Y27632.

[0029] In some embodiments of the present application, the Wuzhishan pig mitral valve interstitial cell culture medium comprises an antibiotic.

[0030] In a third aspect of the present application, the Wuzhishan pig mitral valve interstitial cell culture medium according to the second aspect of the present application is used to reduce the activation level of in-vitro cultured Wuzhishan pig mitral valve interstitial cells.

[0031] In some embodiments of the present application, the activation level is manifested as a decrease in the expression levels of ACTA2, Collagen I, Collagen III, and alpha-SMA.

[0032] In a fourth aspect of the present application, the Wuzhishan pig mitral valve interstitial cell culture medium according to the second aspect of the present application is used to prepare a Wuzhishan pig heart valve disease cell model.

[0033] In some embodiments of the present application, the heart valve disease model comprises one of a mitral valve disease model, preferably a myxomatous mitral valve disease model.

[0034] The present application has the following beneficial effects:

[0035] The present application first constructs an in-vitro three-dimensional culture model of Wuzhishan pig heart mitral valve interstitial cells, a characteristic pig breed in Hainan Province, which can significantly reduce the activation level of MVICs with the culture time, and tends to mature after 14 days of culture. The present application solves the problem that MVICs are easily activated when cultured in two dimensions in-vitro, and a culture medium that can maintain a low activation level of MVICs in-vitro is obtained through screening, and the three-dimensional model is constructed using this culture medium, which can be used as an in-vitro model of the resting state of MVICs, providing a platform for the mechanism research of MVIC activation. The model that can reduce the activation of Wuzhishan pig mitral valve interstitial cells according to certain construction methods of the present application can not only be used as an animal medical model to study the pathogenesis of various mitral valve diseases, but also can provide help for the preservation of Wuzhishan pig heart cells. BRIEF DESCRIPTION OF DRAWINGS

[0036] The present application will be further described below in conjunction with the drawings and examples, in which:

[0037] Figure 1 Results of the effects of different culture media on the expression of alpha-SMA protein of Wuzhishan pig heart mitral valve interstitial cells under two-dimensional culture.

[0038] Figure 2 Results of expression of ACTA2, Collagen I, and Collagen III of the Wuzhishan pig heart mitral valve interstitial cells under the influence of different culture media in two-dimensional culture.

[0039] Figure 3 Results of expression of α-SMA and Collagen I protein of the Wuzhishan pig heart mitral valve interstitial cells in three-dimensional culture in the Advanced F-12 culture system.

[0040] Figure 4 Results of H&E / Masson staining of the Wuzhishan pig heart mitral valve interstitial cells in three-dimensional culture in the MCDB131 culture system.

[0041] Figure 5 Results of expression of α-SMA and Collagen I protein of the Wuzhishan pig heart mitral valve interstitial cells in three-dimensional culture in the MCDB131 culture system.

[0042] Figure 6 Comparison of results of expression of α-SMA and Collagen I protein of the Wuzhishan pig heart mitral valve interstitial cells in three-dimensional culture in the MCDB131 and Advanced F-12 culture systems. DETAILED DESCRIPTION

[0043] The concept and technical effects of the present application will be described below in combination with examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0044] The reagent consumables of the present application are shown in Table 1:

[0045] Table 1

[0046]

[0047] Example 1: Screening of two-dimensional culture conditions of Wuzhishan pig mitral valve interstitial cells

[0048] 1. Isolation of Wuzhishan pig mitral valve interstitial cells

[0049] Wuzhishan pig anterior leaflet of mitral valve was digested by enzyme digestion method using 300 U / mL collagenase I. The surface endothelial cells were scraped off with a sterile swab 10 min before digestion, and then the mitral valve was placed in 3 mL collagenase for 10 h. After digestion, the mixture was mixed by blowing and then passed through a cell strainer to remove undigested residual tissue. After centrifugation at 1000 g for 5 min, the cells were resuspended in complete medium containing 10 v / v% FBS prepared with MCDB131 and inoculated in a T75 culture flask.

[0050] 2. Two-dimensional adherent culture of Wuzhishan pig interstitial cells using different culture media

[0051] Culture medium groups:

[0052] 1) Advanced F-12 + 10 v / v% FBS + 1 v / v% P / S + 1 wt% L-Glu;

[0053] 2) MCDB131 + 1% v / v P / S + 1 wt% L-Glu + 2 v / v% FBS + 5 ng / mL EGF + 0.25 ng / mL FGFb;

[0054] 3) MCDB131 + 1% v / v P / S + 1 wt% L-Glu + 2 v / v% FBS + 10 μM Y27632 + 5 ng / mL EGF + 0.25 ng / mL FGFb;

[0055] 4) MCDB131 + 1% v / v P / S + 1 wt% L-Glu + 10 v / v% FBS + 5 ng / mL EGF + 0.25 ng / mL FGFb;

[0056] 5) MCDB131 + 1% v / v P / S + 1 wt% L-Glu + 10 v / v% FBS + 10 μM Y27632 + 5 ng / mL EGF + 0.25 ng / mL FGFb.

[0057] The culture medium was replaced every 48 h, and the cells were subcultured when they reached 80% confluence. P1, P3, and P5 cells were collected for MVIC activation level detection.

[0058] MVIC activation level detection included Western Blot detection of α-SMA protein expression, qPCR detection of ACTA2, Collagen I, and Collagen III.

[0059] The relevant detection primer sequence information is as follows:

[0060]

[0061] 3. Cell passage and preservation

[0062] When the confluence of the cells reached 80%, the cells were digested with TrypLE Express enzyme, centrifuged at 1500 rpm for 5 min, and the supernatant was removed. The cells were resuspended in a cryopreservation solution (50 v / v% MCDB131 complete medium + 40 v / v% FBS + 10 v / v% DMSO) to a density of 3.5*10 6 MVIC / mL. 1 mL of the cell suspension was added to each cryopreservation tube, and the gradient cooling method (4°C for 1 h, -20°C for 2 h, and -80°C for 1 day, then transferred to liquid nitrogen for long-term storage) was used for cryopreservation. When the cells needed to be recovered, the cells were quickly thawed at 37°C after being taken out of the liquid nitrogen tank, 4 mL of complete medium was used to resuspend the thawed cell suspension, centrifuged at 1500 rpm for 5 min, the supernatant was removed, resuspended with complete medium, and inoculated in a T25 culture flask, and the medium was changed every 48 h. The confluence of the recovered cells generally reached 80% on d3.

[0063] 4. Experimental results

[0064] The results of α-SMA protein expression are shown in Figure 1 Compared with Advanced F-12, the four complete media prepared based on MCDB131 as the basic medium can reduce the activation level of MVIC. Among the combinations of MCDB131 as the basic medium, the 10 v / v% FBS concentration has a more obvious effect on the α-SMA protein expression of MVIC, and the addition of Y27632 can further reduce the α-SMA protein expression.

[0065] The results of ACTA2, Collagen I, and Collagen III detection are shown in Figure 2 According to the continuous comparison of P1, P3, and P5, it can be known that the 10 v / v% FBS group can better maintain the low activation level of MVIC. Therefore, it is determined that MCDB131 + 10 v / v% FBS + 1 v / v% P / S + 1 wt% L-Glu + 5 ng / mL EGF + 0.25 ng / mL FGFb can be used as a better medium for maintaining the low activation level of MVIC in vitro.

[0066] Example 2 Influence of Advanced F-12 culture system on the activation state of MVIC in three-dimensional culture

[0067] 1. Isolated culture: Five-finger mountain pig bicuspid anterior leaflet was digested by enzyme digestion method using 300 U / mL collagenase I. The anterior leaflet was digested for 10 min, and the surface endothelial cells were scraped off with a sterile swab. Then the bicuspid was placed in 3 mL of collagenase for 10 h of digestion. After digestion, the mixture was mixed by blowing and centrifuged at 1000 g for 5 min. The undigested residual tissue was removed using a cell sieve, and resuspended with complete medium (MCDB131 containing 10 v / v% FBS). After adhesion, the two-dimensional cultured MVICs were obtained.

[0068] 2. In vitro three-dimensional model construction: The two-dimensional cultured VICs were digested using TrypLE Express enzyme, and after centrifugation at 1500 rpm for 5 min, the supernatant was removed and resuspended to 3.75*10 5 4 mL. 400 μL of cell suspension was combined with 200 μL of 3 mg / mL Cultrex rat collagen I (R&D Systems) at 4°C and 12 μL of filtered 1 M NaOH. 500 μL of the solution was quickly transferred to each well of a 24-well culture dish, and incubated at 37°C for 1 h for coagulation. The surrounding of the gel was separated from the wall of the plate using a 10 μL gun head to form MVIC-COL, and then 1 mL of complete medium (Advanced F-12 + 10 v / v% FBS + 1 v / v% P / S + 1 wt% L-Glu) was added to each well, and the medium was changed every 24 h for 14 days of culture.

[0069] 3. In vitro 3D model identification: The 1D, 3D, 5D, 7D, 14D cultured MVIC-COL was collected, paraffin sectioned, and subjected to H&E staining and Masson staining to observe the tissue composition and cell distribution. Then the protein sample was collected to detect the expression level of α-SMA to detect the activation level.

[0070] 4. Experimental results

[0071] The expression results of MVIC activation markers Collagen I and α-SMA are shown in Table 1. Figure 3

[0072] The results of Masson staining and H&E staining showed that the collagen fibers and extracellular matrix content increased with the increase of culture days. The original type I rat tail collagen was gradually degraded and new collagen was generated. The expression of MVIC activation markers Collagen I and α-SMA was detected, and the results showed that the MVIC activation level gradually decreased with the increase of culture days.

[0073] Example 32 Effect of MCDB131 culture system on the activation state of MVIC in three-dimensional culture

[0074] ​1. Isolated culture: Five-finger mountain pig bicuspid anterior leaflet was digested by enzyme digestion method using 300 U / mL collagenase I. The anterior leaflet was digested for 10 min, and the surface endothelial cells were scraped off with a sterile swab. Then the bicuspid was placed in 3 mL of collagenase for 10 h of digestion. After digestion, the mixture was mixed by blowing and centrifuged at 1000 g for 5 min. The undigested residual tissue was removed using a cell sieve, and resuspended with complete medium (MCDB131 containing 10 v / v% FBS). After adhesion, the two-dimensional cultured MVICs were obtained.

[0075] 2. In vitro three-dimensional model construction: The two-dimensional cultured VICs were digested using TrypLE Express enzyme, and after centrifugation at 1500 rpm for 5 min, the supernatant was removed and resuspended to 3.75*10 5 4 mL. 400 μL of cell suspension was combined with 200 μL of 3 mg / mL Cultrex rat collagen I (R&D Systems) at 4°C and 12 μL of filtered 1 M NaOH. 500 μL of the solution was quickly transferred to each well of a 24-well culture dish, and incubated at 37°C for 1 h for coagulation. The surrounding of the gel was separated from the wall of the plate using a 10 μL gun head to form MVIC-COL, and then 1 mL of complete medium (MCDB131 + 10 v / v% FBS + 1 v / v% P / S + 1 wt% L-Glu + 5 ng / mL EGF + 0.25 ng / mL FGFb) was added to each well, and the medium was changed every 24 h for 14 days of culture.

[0076] 3. In vitro 3D model identification: MVIC-COL cultured for 1D, 3D, 5D, 7D, 14D was collected, paraffin sections were prepared for H&E staining and Masson staining to observe the tissue composition and cell distribution. Then protein samples were collected to detect the expression level of α-SMA to detect the activation level.

[0077] 4. Experimental results

[0078] The results of H&E / Masson staining to detect the cell distribution and collagen arrangement of MVIC-COL are shown in Figure 4 The results of MVIC activation markers Collagen I and α-SMA expression are shown in Figure 5 With the increase of culture days, MVIC-COL gradually produced new extracellular matrix. The expression of MVIC activation markers Collagen I and α-SMA was detected, and the results showed that under the MCDB131 culture system, the activation level of MVIC decreased to a low level after D1.

[0079] In addition, Figure 6The culture effects of MCDB131 and Advanced F-12 culture systems are compared, and MCDB131 is significantly stronger than Advanced F-12 in reducing the activation ability of MVIC.

[0080] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1.A method for constructing a Wuzhishan pig mitral valve in vitro model, comprising the following steps: culturing Wuzhishan pig mitral valve interstitial cells using an activation-inhibiting medium; the activation-inhibiting medium comprises a basal medium and serum; the serum is fetal bovine serum; the basal medium is MCDB131 medium; the MCDB131 medium comprises 1 wt% L-Glu; the volume fraction of the serum is 10%; the activation-inhibiting medium comprises growth factors; the growth factors comprise EGF and bFGF; the concentration of the EGF is 5 ng / mL; the concentration of the bFGF is 0.25 ng / mL; the activation-inhibiting medium comprises Y27632; the concentration of the Y27632 is 10 μM. 2.The method according to claim 1, characterized in that: the activation-inhibiting medium comprises antibiotics. 3.Use of the method according to claim 1 or 2 in the preparation of a Wuzhishan pig mitral valve in vitro model.

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