CN111893090B - Atherosclerosis prevention and treatment drug screening cell model, construction and application thereof - Google Patents

Atherosclerosis prevention and treatment drug screening cell model, construction and application thereof Download PDF

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CN111893090B
CN111893090B CN202010825420.9A CN202010825420A CN111893090B CN 111893090 B CN111893090 B CN 111893090B CN 202010825420 A CN202010825420 A CN 202010825420A CN 111893090 B CN111893090 B CN 111893090B
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vsmc
cell
model
cac
cvf
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CN111893090A (en
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孙黔云
李娇
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Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention discloses a construction method of a cell model for screening atherosclerosis prevention and treatment drugs, which comprises the following steps: preparing CVF; diluting CVF with normal saline, uniformly mixing with NHP in equal volume, and obtaining CAC after one period of time in a constant-temperature water bath; cell culture: human vascular smooth muscle cell line was cultured in DMEM high-sugar medium containing 15% FBS at 37deg.CContaining 5% CO 2 Culturing in a cell incubator; diluting VSMC in logarithmic growth phase, inoculating, performing liquid exchange treatment after a period of time, adding CAC, and reacting for a period of time to obtain the cell model; the invention also discloses a cell model obtained by using the construction method; the invention also discloses application of the cell model in screening atherosclerosis prevention and treatment medicines. The invention uses CVF to specifically activate the alternative complement pathway of blood plasma, constructs a VSMC proliferation activation model induced by the activation of the alternative complement pathway, and provides a novel cell model for developing the screening of AS prevention and treatment drugs.

Description

Atherosclerosis prevention and treatment drug screening cell model, construction and application thereof
Technical Field
The invention relates to the technical field of atherosclerosis cell models, in particular to an atherosclerosis control drug screening cell model, and construction and application thereof.
Background
With the development of economy and society and the continuous improvement of human living conditions, the incidence and mortality of cardiovascular diseases are increasing and the trend of younger is presented. Atherosclerosis (AS) is a common high-frequency chronic cardiovascular inflammatory disease and is one of the main causes of cardiovascular and cerebrovascular death. Vascular smooth muscle cells (vascular smooth muscle cells, VSMC) are important components of the vessel wall, and abnormal proliferation and migration of VSMC play a critical role in the pathogenesis of AS lesions. VSMC activation and dysfunction caused by various pathogenic factors are important links of inflammation development, and can induce VSMC to generate inflammatory cytokines to further aggravate inflammatory response, and play a key role in AS occurrence and development. Many factors are involved in VSMC proliferation activation, such as lipopolysaccharide and oxidized low density lipoprotein, and previous studies have focused on VSMC proliferation activation and injury induced by oxidized low density lipoprotein (ox-LDL), and screening models for VSMC proliferation activation inhibition are constructed based on this principle in drug screening at the corresponding cellular level.
The current method for damaging vascular smooth muscle cells by ox-LDL has the following defects:
ox-LDL plays an important role in the pathogenesis of AS, a well-recognized mechanism of which is: ox-LDL causes oxidative stress injury and dysfunction of endothelial cells, thereby triggering inflammation, released inflammatory mediators and infiltration activation of inflammatory cells and injury of VSMC, and in this pathological process, foam cells are formed with macrophages and VSMC, which in turn cause activation, calcification, injury and apoptosis of VSMC, gradually forming atheromatous plaques. Therefore, ox-LDL directly stimulates vascular smooth muscle cell proliferation and activation and AS pathogenesis are greatly different, and AS a screening model, AS pathogenesis cannot be reflected well.
Disclosure of Invention
In order to solve the defects in the related fields, the invention provides an atherosclerosis prevention and treatment drug screening cell model, and construction and application thereof.
The invention relates to an atherosclerosis prevention and treatment drug screening cell model and construction and application thereof, which are realized by the following technical scheme:
a construction method of an atherosclerosis prevention and treatment drug screening cell model comprises the following steps:
step 1, preparing cobra venom factor CVF;
step 2, diluting the prepared cobra venom factor CVF with normal saline, uniformly mixing with NHP in equal volume, and obtaining a complement alternative pathway activation product CAC after one period of time in a constant temperature water bath;
step 3, cell culture: human vascular smooth muscle cell line VSMC was cultured in DMEM high-sugar medium containing 15% FBS at 37deg.C with 5% CO 2 Culturing in a cell incubator;
and 4, diluting the human vascular smooth muscle cells VSMC in the logarithmic growth phase cultured in the step 3, inoculating, performing liquid exchange treatment after a period of time, adding the complement alternative pathway activation product CAC obtained in the step 2, and reacting for a period of time to obtain the cell model for screening the atherosclerosis prevention and treatment drugs.
Further, the concentration of cobra venom factor CVF diluted in step 2 is 6.5X10 4 U·L -1 。
Further, the ratio of the complement alternative pathway activation product CAC to the human vascular smooth muscle cell VSMC diluted in step 4 is 30 to 50 μl/well: 100. Mu.L/well.
Further, the method comprises the steps of,the concentration of the human vascular smooth muscle cell sap diluted in the step 4 is 7.5X10 4 And each mL.
Further, the cobra venom factor is obtained by the following method:
the method comprises the steps of subjecting lyophilized powder of cobra venom to SP Sephadex C-25 cation exchange chromatography, collecting an activity peak, subjecting the obtained product to Sephadex S-200 molecular sieve gel chromatography, collecting the activity peak, subjecting the obtained product to HiTrap Q HP column anion exchange chromatography to obtain a purified preparation product of CVF, wherein the purified preparation product is a band in SDS-PAGE electrophoresis under a non-reducing condition, the band characteristic under the reducing condition accords with the characteristic of CVF, the anticomplement activity unit is 1500U/mg, and the prepared product is packaged after protein quantification by a BCA method and is frozen at the temperature of-80 ℃ for later use.
Further, the reaction time after the addition of the alternative complement pathway activation product from step 2 in step 4 is at least 6 hours.
Further, the time of the constant temperature water bath in the step 2 is 0.5h.
Further, the liquid change treatment is performed using serum-free DMEM medium.
Further, the ratio of the amount of complement alternative pathway activation product to the amount of human vascular smooth muscle cell fluid in step 4 was 40 μl/well: 100. Mu.L/well.
The cell model constructed based on the construction method is a VSMC proliferation activation model.
Use of the above cell model for screening an atherosclerosis-preventing drug by:
after a to-be-detected medicine sample is added into the cell model for culturing for a period of time, cell viability, adhesion molecules ICAM-1 and VCAM-1 are used as screening indexes, E-selectin is used as a detection index, and after detection, the comparison is carried out with a CAC model group without the to-be-detected medicine sample, the difference of data reduction has significance (P is less than 0.05), and then the proliferation of the sample on VSMC, the activation of the VSMC and the inhibition effect of inflammatory reaction can be judged, namely the sample has potential as an atherosclerosis prevention and treatment medicine; otherwise, the medicine has no potential as an atherosclerosis preventing and treating medicine.
Compared with the prior art, the invention has the following beneficial effects:
based on the important role that alternative complement activation has in the pathogenesis of AS, alternative complement activation products directly stimulate vascular smooth muscle cell proliferation activation and inflammatory reactions. Thus, CVF is used to specifically activate the alternative complement pathway, and the alternative complement pathway activation product is used to induce VSMC proliferation activation, thereby forming a VSMC proliferation activation model. This way of obtaining a full component product of the alternative complement pathway by CVF-specific activation of the alternative complement pathway is highly consistent with the way that the body stimulates VSMC under pathophysiological conditions with excessive activation of the alternative complement pathway. Therefore, the invention utilizes CVF to specifically activate the alternative complement pathway of blood plasma, constructs a VSMC proliferation activation model induced by the activation of the alternative complement pathway, and provides a novel cell model for developing the screening of AS prevention and treatment drugs.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
FIG. 1 shows the effect of different amounts of CAC on VSMC proliferationWherein the method comprises the steps of ** Indicating that the experimental data of this group were statistically significantly different (P < 0.01) from that of the control group;
FIG. 2 is an age-related relationship of CAC to promote VSMC proliferationWherein the method comprises the steps of ** Indicating that the experimental data of this group were very significantly statistically different (P < 0.01) compared to the control group; ## indicating that the experimental data of this group were very significantly statistically different (P < 0.01) compared to the 0h model group; FIG. 3 shows the effect of CAC on VSMC expressing adhesion molecules +.>E-selectin; ICAM-1; c (C)VCAM-1; wherein the method comprises the steps of ** Indicating that the experimental data of this group were statistically significantly different (P < 0.01) from that of the control group; # indicating that the experimental data for this group were statistically significantly different (P < 0.05) compared to the 0h model group, ## indicating that the experimental data of this group were statistically significantly different (P < 0.01) compared to the 0h model group;
FIG. 4 shows the effect of CAC on the nuclear transcriptional activity of VSMC NF-. Kappa. B p65 Wherein the method comprises the steps of * Indicating that the experimental data of this group were statistically significantly different (P < 0.05) compared to the control group;
FIG. 5 shows the effect of Western blot detection of CAC on VSMC expression of p-NF- κ B p65, NF- κ B p65 and IKKWherein the method comprises the steps of * Indicating that the experimental data of this group were statistically significantly different (P < 0.05) compared to the control group, ** the experimental data showed statistically significant differences (P < 0.01) between the control group and the control group
FIG. 6 shows the effect of PDTC on VSMC cell viabilityWherein the method comprises the steps of ** Indicating that the experimental data of this group were statistically significantly different (P < 0.01) from that of the control group; # indicating that the experimental data for this group were statistically significantly different (P < 0.05) compared to the model group, ## indicating that the experimental data of this group were statistically significantly different (P < 0.01) from the model group;
FIG. 7 is the effect of PDTC on CAC induced VSMC expression adhesion molecules, wherein A:B:/>C:/> ** indicating that the experimental data of this group were statistically significantly different (P < 0.01) from that of the control group; # indicating that the experimental data for this group were statistically significantly different (P < 0.05) compared to the model group, ## indicating that the experimental data of this group were statistically significantly different (P < 0.01) from the model group;
FIG. 8 shows the effect of PDTC on CAC-induced VSMC NF- κ B p65 transcriptional activity Wherein the method comprises the steps of ** Indicating that the experimental data of this group were statistically significantly different (P < 0.01) from that of the control group; # indicating that the experimental data of this group were statistically significantly different (P < 0.05) compared to the model group;
FIG. 9 shows the effect of PDTC on p-NF- κ B p65, NF- κ B p65 and IKK protein expressionWherein the method comprises the steps of ** Indicating that the experimental data of this group were statistically significantly different (P < 0.01) from that of the control group; # this set of experimental data is shown to be statistically significantly different (P < 0.05) compared to the model set.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which is to be read in light of the specific examples. The working principle not described in detail in the present invention belongs to the prior art and the common general knowledge in the art, and the person skilled in the art should know.
1. Materials and instruments
1.1 reagents
Fetal bovine serum (fetal bovine serum, FBS) (Argentina Natocor-Industrial Biol, inc.); DMEM high sugar medium (Sigma, usa); renilla luciferase reporter plasmid, NF- κbp65 signaling pathway plasmid and Dual-Luciferase reporter assy systemKit (Promega company, usa); plasmid extraction kit (Beijing Tiangen Biochemical technology Co., ltd.), lipo6000TM liposome transfection reagent (Jiangsu Biyun biotechnology institute); plasma standards (normal human plasma, NHP) (Siemens, germany), lot 503264C; inactivating NHP in water bath at 56 deg.C for 0.5 hr to obtain inactivated human blood plasma (inactivated normal human plasma, INHP); human E-selectin, ICAM-1 and VCAM-1 ELISAKis (Bodhisattva, inc.); p-NF- κ B p65, NF- κ B p65 and IKK Rabbit mAb (CST Co., USA); PDTC (Sigma Co., USA).
The human vascular smooth muscle cell line (VSMC) used in the present invention was taught by university of south China Chen Linxi.
1.2 instruments
CLM-170B-8-NF CO 2 Incubator (ESCO company, singapore); continuous wavelength microplate reader Gen5 (Bio Tek Co., U.S.A.); chemiluminescent detector (Promega, usa); fusion-FX western blotting system (Vilber, france); TS2FL inverted phase contrast microscope and imaging system (Nikon Corp.).
2. Example 1
The embodiment provides a method for constructing a cell model for screening atherosclerosis prevention and treatment drugs, which comprises the following steps:
step 1, preparing cobra venom factor: the method comprises the steps of (1) subjecting snake venom freeze-dried powder of cobra (Naja atra) to SP Sephadex C-25 cation exchange chromatography, collecting an activity peak, subjecting the activity peak to Sephacryl S-200 molecular sieve gel chromatography, collecting the activity peak, subjecting the activity peak to HiTrap Q HP column anion exchange chromatography to obtain a purified preparation product of CVF, wherein the purified preparation product is a band in SDS-PAGE electrophoresis under a non-reducing condition, the band characteristic under the reducing condition accords with the characteristic of CVF, the anticomplement activity unit is 1500U/mg, and the preparation product is packaged after protein quantification by a BCA method, and is frozen at the temperature of-80 ℃ for standby;
the methods for separating and purifying CVF and measuring anticomplement activity are specifically disclosed in published documents of the subject group:
(1)Sun QY,Chen G,Guo H,Chen S,Wang WY,Xiong YL.Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor.Toxicon,2003,42(3):257-262.
(2) Sun Qianyun, wang Wanyu, xiong Yuliang. In vitro haemolytic activity studies of cobra venom highly active anticomplement factor. Chinese pharmacological report, 2003,19 (8): 909-913.
Step 2, the cobra venom factor obtained in step 2 is diluted to a concentration of 6.5X10 with Normal Saline (NS) 4 U·L -1 Mixing with NHP in equal volume, and heating in 37deg.C constant temperature water bath for 0.5 hr to obtain alternative complement pathway activation product;
step 3, cell culture: human vascular smooth muscle cell line was cultured in DMEM high-sugar medium containing 15% FBS at 37deg.C with 5% CO 2 Culturing in a cell incubator;
step 4, diluting the human vascular smooth muscle cells in the logarithmic phase cultured in the step 3 to 7.5X10 4 Inoculating the mixture to a 96-well cell culture plate after each mL, performing liquid exchange treatment after 24 hours, adding the complement alternative pathway activation product obtained in the step 2, and reacting for a period of time at 37 ℃ to obtain the cell model for screening the atherosclerosis prevention and treatment medicine.
Further, the ratio of the amount of complement alternative pathway activation product to the amount of human vascular smooth muscle cell sap in step 4 was 30: 100. Mu.L/well.
Further, the liquid change treatment is performed using serum-free DMEM medium.
3. Example 2
The present embodiment provides a method for constructing a cell model for screening an atherosclerosis-preventing drug, which is different from embodiment 1 in that: the ratio of the amount of complement alternative pathway activation product to the amount of human vascular smooth muscle cell sap in step 4 was 40: 100. Mu.L/well. The other steps are the same.
4. Example 3
The present embodiment provides a method for constructing a cell model for screening an atherosclerosis-preventing drug, which is different from embodiment 1 in that: the ratio of the amount of complement alternative pathway activation product to the amount of human vascular smooth muscle cell sap in step 4 was 50: 100. Mu.L/well. The other steps are the same.
5. Example 4
The present example provides a cell model constructed based on the construction method of example 1, which is a VSMC proliferation activation model.
6. Example 5
The present example provides a cell model constructed based on the construction method of example 2, which is a VSMC proliferation activation model.
7. Example 6
The present example provides a cell model constructed based on the construction method of example 3, which is a VSMC proliferation activation model.
8. Example 7
This example provides the use of the cell model of example 5 in screening drugs for the prevention and treatment of atherosclerosis.
9. Experimental part
9.1 Effect of different amounts of CAC on VSMC cell proliferation
DMEM high sugar medium containing 15% FBS for culturing human vascular smooth muscle cell line (VSMC) at 37deg.C with 5% CO 2 Culturing in a cell culture incubator.
Cobra venom factor (cobra venom factor, CVF) is prepared by separating and purifying the subject components of the applicant, and the specific method is as follows: the snake venom freeze-dried powder of cobra (Naja atra) is subjected to SP Sephadex C-25 cation exchange chromatography, activity peaks are collected, then Sephacryl S-200 molecular sieve gel chromatography is carried out, activity peaks are collected, hiTrap Q HP column anion exchange chromatography is carried out, and a purified preparation product of CVF is obtained, wherein the purified preparation product is a band in SDS-PAGE electrophoresis under a non-reducing condition, the band characteristic under the reducing condition accords with the characteristic of CVF, the anticomplement activity unit is 1500U/mg, and the preparation product is packaged after protein quantification by a BCA method, and is frozen at the temperature of-80 ℃ for standby.
The method for separating and purifying CVF and measuring anticomplement activity is specifically disclosed in published documents of the subject group of the applicant: (1) Sun QY, chen G, guo H, chen S, wang WY, xiong YL.Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor. Toxicon,2003,42 (3): 257-262; (2) Sun Qianyun, wang Wanyu, xiong Yuliang. In vitro haemolytic activity studies of cobra venom highly active anticomplement factor. Chinese pharmacological report, 2003,19 (8): 909-913.
CVF (concentration 6.5X10) diluted with Normal Saline (NS) 4 U·L -1 ) Equal volumes were gently mixed with NHP and incubated with INHP instead of NHP for 0.5h in a constant temperature water bath at 37℃to prepare alternative complement pathway activation products (CVF-activated complement, CAC) as controls.
Diluting VSMC in logarithmic growth phase to 7.5X10 4 The cells are inoculated into 96-well cell culture plates after each mL, the volume of the plates is 100 mu L/well, the plates are subjected to liquid exchange treatment after 24 hours, 30 mu L, 40 mu L and 50 mu L/well CAC are respectively added to stimulate VSMC, the MTT method is adopted to detect the cell viability after 24 hours of stimulation at 37 ℃, and meanwhile, an inactivated plasma incubation product control group (control) is arranged.
As a result, as shown in FIG. 1, CAC was able to stimulate VSMC cell proliferation, and at 40. Mu.L of CAC for 24 hours, cell proliferation was maximized, and the difference was significant (P < 0.01) compared with the control group.
9.2 Effect of CAC on VSMC cell viability at different times
Referring to "9.1" test method, after 40. Mu.L CAC was added, MTT method was used to detect cell viability and observe changes in cell morphology by microscopic examination at 0, 6, 12, 24h, respectively, while an inactivated plasma incubation product control (control) was set.
As a result, as shown in FIG. 2, the cell viability increased with the extension of the stimulation time, and after 6 hours, the difference between the model group and the control group was statistically significant (P < 0.01).
9.3 Effect of CAC on VSMC adhesion molecule expression
Cell inoculation was carried out according to the method of "9.1", 100. Mu.L per well was incubated for 24 hours, then subjected to liquid exchange treatment 2 times, and after addition of 40. Mu.L of CAC, 100. Mu.L was supplemented with serum-free DMEM high-sugar medium, and the cell culture supernatants were taken out of 0, 6, 12 and 24 hours, centrifuged at 2000rpm for 10 minutes, and the supernatants were taken out to determine the content of the related adhesion molecules (E-selectin, ICAM-1 and VCAM-1).
The results are shown in FIG. 3, where CAC stimulated VSMC cells to express E-selectin, ICAM-1 and VCAM-1. Wherein ICAM-1 and VCAM-1 are expressed at the highest time of 6h and E-selectin is expressed at the highest time of 12 h.
9.4 Effect of CAC on VSMC NF- κB Signal pathway
9.4.1 plasmid preparation and concentration determination reference (Li Jiao, guo Jing, li Min, sun Qianyun. Intervention of ligustrazine on endothelial cell inflammatory response to alternative complement pathway activation. Chinese pharmacological notification, 2019, 35 (1): 90-95.), are performed by the methods described herein.
9.4.2 double luciferase reporter Gene detection of the Effect of CAC on the nuclear transcriptional Activity of VSMC NF-kappa B p65
VSMC grown in log phase was diluted to 7.5X10 4 Plating at a concentration of 100 mu L per well, inoculating into 96-well black opaque cell culture plate, culturing for 24 hr, performing liquid exchange treatment with serum-free DMEM high sugar culture medium, discarding supernatant, adding 90 mu L of DMEM high sugar culture medium containing serum and no antibiotics and 10 mu L of transfection mixture solution per well, and mixing according to Lipo6000 TM Liposome transfection reagent instructions overnight transfection, after successful transfection, the supernatant was discarded, and after 4h treatment with 40. Mu.LCAC and 60. Mu.L serum-free DMEM high sugar medium, fluorescence intensity detection (Model) was performed using INHP CVF incubation as a control, specific detection methods and calculation formulas refer to the methods described in literature (Li Jiao, guo Jing, li Min, sun Qianyun. Intervention of ligustrazine on endothelial cell inflammatory response to alternative complement pathway activation. Chinese pharmacological report, 2019, 35 (1): 90-95.), which are not described in detail herein.
As shown in FIG. 4, the results show that the CAC acts on VSMC and can induce up-regulation of nuclear transcription activity of NF- κ B P65, and the difference is statistically significant (P < 0.05) compared with the control group.
9.4.3 Detection of VSMC expression of p-NF- κ B p65, NF- κ B p65 and IKK by CAC
VSMC grown in log phase was inoculated at 25cm 2 In disposable cell culture flasks of 5X 10 inoculation density 5 And each mL. Culturing for 24h, changing the liquid when the cells are fully paved by 80% -85%, adding 1200 mu L of CAC into a model group (model), adding 1.8mL of serum-free DMEM culture medium to supplement 3mL, respectively stimulating for 3h and 24h, extracting cytoplasmic proteins according to the operation of the Biyun Tian cytoplasmic protein extraction kit, and taking INHP CVF incubation products as a control. And (3) separating the protein by polyacrylamide gel electrophoresis, wet transfer film, incubating the primary antibody overnight and incubating the secondary antibody for 1h, and performing exposure development detection and quantitative analysis by a chemiluminescent chromogenic imaging system.
As shown in FIG. 5, after VSMC was stimulated by CAC, the expression of P-NF- κ B P65, NF- κ B P65 and IKK was up-regulated, where the difference in IKK protein expression between stimulation 3h and 24h was statistically significant (P < 0.01) compared to the control group, and the difference in P-NF- κ B P65 and NF- κ B P65 expression between stimulation 24h was significant (P < 0.05, P < 0.01) compared to the control group.
9.5 statistical analysis
Software SPSS 19.0 was used to perform one-way analysis of variance on the data, and LSD was used for multiple comparisons between groups.
From the above experimental results, it can be seen that the alternative complement pathway activation product CAC can stimulate VSMC proliferation and activation, and can be used for screening active substances with effect of inhibiting VSMC proliferation and activation by using the alternative complement pathway activation product to stimulate VSMC proliferation and activation to construct a cell model.
9.6 screening experiments of cell model verification
9.6.1 Effect of PDTC on CAC induced VSMC proliferation
Reference is made to the aforementioned "9.1" method (dilution of VSMC in logarithmic growth phase to 7.5X10) 4 Inoculating to 96-well cell culture plate with volume of 100 μL/well, performing liquid exchange treatment after 24 hr, adding 10 μL/well PDTC (final concentration: 1.0X10 respectively) -5 、1.0×10 -6 、1.0×10 -7 mol·L -1 ) After pre-incubation for 2h, 40. Mu.L/well CAC was added to stimulate for 24h, and MTT assay was used to measure cell viability. Experimental setup control group, model group, PDTC group.
As shown in FIG. 6, PDTC with different concentrations can inhibit VSMC proliferation caused by stimulation of complement activator, and the inhibition effect is statistically significant (P < 0.05, P < 0.01) compared with the model group, so that a certain dose-effect relationship is shown.
9.6.2 Effect of PDTC on VSMC adhesion molecule expression
Referring to the foregoing "9.3" experimental procedure, PDTC (final concentration 1.0X10) was performed at the highest time point of each index expression -5 、1.0×10 -6 、1.0×10 -7 mol·L -1 ) Is pre-incubated for 2h. The experiments set up control, model, PDTC.
CAC stimulates VSMC cells to express E-selectin, ICAM-1 and VCAM-1. When pretreated with PDTC, the expression of both E-selectin, ICAM-1 and VCAM-1 were significantly down-regulated, wherein the differences between E-selectin and ICAM-1 in the three concentration PDTC-treated groups compared to the model group were statistically significant (P < 0.01, P < 0.05, FIGS. 7A and 7C); the differences between the VCAM-1 treated with PDTC at the high and medium concentrations compared with the model group were statistically significant (P < 0.01, FIG. 7B), and the concentrations showed a clear dose relationship.
9.6.3 Effect of PDTC on VSMC NF- κ B p65 Nuclear transcription Activity
Cell plating and transfection were performed according to the method "9.4.2" described above, wherein the intervention groups were each added to a final concentration of 1.0X10 -5 、1.0×10 -6 、1.0×10 -7 mol·L -1 PDTC is pretreated for 2 hours, CAC is added into a constant temperature incubator at 37 ℃ for 4 hours to detect fluorescence intensity, and Ra value is calculated according to a relative nuclear transcription activity formula. The experiments set up control, model, PDTC.
As shown in FIG. 8, PDTC was able to significantly down-regulate the increase in nuclear transcription activity of NF-. Kappa. B p65 by CAC stimulation VSMC, at a concentration of 1.0X10 -5 mol·L -1 The differences between the groups and the model groups were all statistically significant (P < 0.05).
9.6.4 Effect of PDTC on CAC induced VSMC expression of p-NF- κ B p65, NF- κ B p65 and IKK
Protein extraction was performed according to the method "9.4.3". Wherein the intervention group is added with 1.0X10 -5 mol·L -1 PDTC is pretreated for 2 hours, the supernatant is discarded as much as possible after liquid exchange, 1.8mL of serum-free DMEM medium is added, 3mL of 1.2mLCAC is added for supplementing, and after 3 hours and 24 hours of stimulation respectively, cytoplasmic proteins are extracted. Protein is separated through polyacrylamide gel electrophoresis according to a Western bolt method, and subjected to wet transfer membrane, primary antibody overnight incubation and secondary antibody incubation for 1h, and then exposure development detection and quantitative analysis are carried out through a chemiluminescent chromogenic imaging system.
As shown in FIG. 9, P-NF-. Kappa.Bp 65, NF-. Kappa.Bp 65 and IKK expression were significantly upregulated after CAC stimulation of VSMC, whereas PDTC had an intervention in its upregulation, wherein at 24h CAC treatment the differences were statistically significant (P < 0.05, P < 0.01) compared to the model group.
9.6.5 statistical analysis
Software SPSS 19.0 was used to perform one-way analysis of variance on the data, and LSD was used for multiple comparisons between groups.
The experimental result shows that PDTC has better intervention effect on the cell model constructed by the invention, and the intervention mechanism is to specifically inhibit the phosphorylation of NF-kappa B p65, thereby effectively inhibiting the activation of NF-kappa B signaling pathway, obviously inhibiting the proliferation activation of VSMC and the expression of adhesion molecules, and the experimental result is consistent with the currently recognized important effect of the NF-kappa B signaling pathway in AS pathogenesis. The experiment is taken as an embodiment of a screening model constructed by the invention, and meanwhile, the effectiveness of the screening model is also verified by adopting the PDTC which is a specific inhibitor of NF- κB signal channel.
The model of the invention is characterized in that CVF is used for specifically activating the alternative complement pathway, and then the alternative complement pathway activation product is used for inducing VSMC proliferation activation, so that a VSMC proliferation activation model is formed. This way of obtaining a full complement component product by CVF-specific activation of the alternative complement pathway is highly consistent with the way of stimulation of VSMC by complement over-activation under pathophysiological conditions in the body. Therefore, the invention utilizes CVF to specifically activate the alternative complement pathway of plasma, constructs a VSMC proliferation activation model induced by the activation of the alternative complement pathway, and provides a proper cell model for developing the screening of AS control drugs.
In the present invention, stimulation of VSMC by alternative complement pathway activators results in activation of cell proliferation, resulting in upregulation of expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin, increased levels of NF- κ B p65 phosphorylation, increased nuclear transcriptional activity of NF- κ B p65, and increased expression of NF- κ B p65 and IKK proteins. As a screening example of the model, the result of the cell model detection shows that the NF- κB specific inhibitor PDTC has a certain inhibition effect on the change of the inflammatory response related index.
NF-. Kappa.B is a protein dimer consisting mainly of two subunits, p50 and p65, and is the point of collection of multiple signal pathways in activated cells, and plays a vital role in the development and progression of inflammation. In the invention, after VSMC is stimulated by CAC, the phosphorylation level of NF-kappa Bp65 is increased, the nuclear transcriptional activity of NF-kappa B p is increased, and the expression of NF-kappa B p and IKK proteins is obviously up-regulated, which indicates that after VSMC is stimulated by CAC, the NF-kappa B signal channel is activated and the transcriptional activity of NF-kappa B p65 responds to the signal up-regulation, thereby regulating and controlling the expression of adhesion molecules, p65 and IKK proteins. Therefore, the up-regulation of the expression of related indexes such as NF- κB signal channel activation and adhesion molecules shows that CAC can induce VSMC proliferation activation and model construction success. The PDTC adopting the NF- κB signal pathway activation specific inhibitor can definitely inhibit proliferation activation and inflammatory reaction of VSMC, which not only shows that the model can be successfully used for screening AS control drugs, but also reveals that the PDTC has a certain intervention effect on the proliferation activation of the VSMC caused by activation of alternative complement pathway, and can be used AS an effective positive drug in screening application of the model.

Claims (4)

1. A method for constructing a cell model for screening an atherosclerosis prevention and treatment drug, which is characterized in that the cell model is a VSMC proliferation activation model induced by complement alternative pathway activation; the method comprises the following steps:
step 1, preparing cobra venom factor CVF;
step 2, diluting the prepared cobra venom factor CVF with normal saline, uniformly mixing with the plasma standard substance in an equal volume, and obtaining a complement alternative pathway activation product CAC after one period of time in a constant temperature water bath;
step 3, cell culture:human vascular smooth muscle cell line VSMC was cultured in DMEM high-sugar medium containing 15% FBS at 37deg.C with 5% CO 2 Culturing in a cell incubator;
step 4, diluting the human vascular smooth muscle cells VSMC in the logarithmic growth phase cultured in the step 3, inoculating, performing liquid exchange treatment after a period of time, adding the complement alternative pathway activation product CAC obtained in the step 2, and reacting for a period of time to obtain the cell model for screening the atherosclerosis prevention and treatment drugs;
the concentration of cobra venom factor CVF diluted in step 2 was 6.5X10 4 U·L-1；
The ratio of the complement alternative pathway activation product CAC to the human vascular smooth muscle cell VSMC diluted in step 4 was 40 μl/well: 100. Mu.L/well;
the concentration of the human vascular smooth muscle cell sap diluted in the step 4 is 7.5X10 4 individual/mL;
the cobra venom factor is obtained by the following method:
the method comprises the steps of subjecting lyophilized powder of cobra venom to SP Sephadex C-25 cation exchange chromatography, collecting an activity peak, subjecting the obtained product to Sephacryl S-200 molecular sieve gel chromatography, collecting the activity peak, subjecting the obtained product to HiTrap Q HP column anion exchange chromatography to obtain a purified preparation product of CVF, wherein the purified preparation product is a band in SDS-PAGE electrophoresis under a non-reducing condition, the band characteristic under the reducing condition accords with the characteristic of CVF, the anticomplement activity unit is 1500U/mg, and the prepared product is packaged after quantitative protein quantification by a BCA method and is frozen at-80 ℃ for later use;
the reaction time after the addition of the alternative complement pathway activation product from step 2 in step 4 is at least 6 hours.
2. The method according to claim 1, wherein the constant temperature water bath at 37℃in step 2 is performed for 0.5h.
3. The method of claim 1, wherein the plating is performed using serum-free DMEM medium.
4. Use of a cell model prepared by the construction method according to claim 3 for screening an atherosclerosis-preventing drug, wherein the drug is screened by:
after a to-be-detected medicine sample is added into the cell model for culturing for a period of time, cell viability, adhesion molecules ICAM-1 and VCAM-1 are used as screening indexes, E-selectin is used as a detection index, and after detection, the comparison is carried out with a CAC model group without the to-be-detected medicine sample, the difference of data reduction has significance (P is less than 0.05), and then the proliferation of the sample on VSMC, the activation of the VSMC and the inhibition effect of inflammatory reaction can be judged, namely the sample has potential as an atherosclerosis prevention and treatment medicine; otherwise, the medicine has no potential as an atherosclerosis preventing and treating medicine.
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