CN115125190B - Culture medium for promoting vascular endothelial cell proliferation - Google Patents
Culture medium for promoting vascular endothelial cell proliferation Download PDFInfo
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- CN115125190B CN115125190B CN202210884159.9A CN202210884159A CN115125190B CN 115125190 B CN115125190 B CN 115125190B CN 202210884159 A CN202210884159 A CN 202210884159A CN 115125190 B CN115125190 B CN 115125190B
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Abstract
The invention discloses a culture medium for promoting vascular endothelial cell proliferation, which contains glaucocalyxin B, wherein glaucocalyxin B can promote vascular endothelial cell proliferation and inhibit intracellular oxidative stress and expression of senescence proteins p16 and p 19.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a culture medium for promoting vascular endothelial cell proliferation, wherein the culture medium contains glaucocalyxin B.
Background
Vascular endothelial cells, commonly referred to as single-layered, flattened epithelium lining the inner surfaces of the heart, blood vessels and lymphatic vessels, form the inner wall of blood vessels. They have the function of engulfing foreign bodies, bacteria, necrotic and senescent tissues, and also participate in the immune activity of the body. The endothelial cells are seen by high magnification observation of capillaries on mesentery, and are smaller than mesothelial cells and are closely arranged. Endothelial cells or vascular endothelium are a thin layer of specialized epithelial cells, consisting of a layer of squamous cells. It forms the inner wall of a blood vessel and is the interface of blood in the lumen of the blood vessel and other vessel walls (single-layer squamous epithelium). Endothelial cells are microvasculature that run along the entire circulatory system, from the heart to a minimum. Endothelial cells on the inner surface of the ventricle are called endocardium. Microvasculature and lymphatic microtubules are composed of a single layer of endothelial cells. Endothelial cells are easy to separate and culture into monolayer cells from large blood vessels, and have great value in researching endothelial cell regeneration, tumor angiogenesis promoting growth factors (tumor angiogenesis factor, TAF) and the like. Thus, experiments require a large amount of vascular endothelial cell proliferation. The research results of primary vascular endothelial cells in mice show that the expression level of senile proteins such as primary endothelial cells P16, P19, P21, P53, rb and the like of old mice is obviously increased compared with that of young and middle aged mice.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a culture medium for promoting vascular endothelial cell proliferation, wherein the culture medium contains glaucocalyxin B.
The invention aims at realizing the following technical scheme:
a culture medium for promoting proliferation of vascular endothelial cells contains glaucocalyxin B.
The culture medium for promoting vascular endothelial cell proliferation takes glaucocalyxin B as the only effective component in the culture medium.
The culture medium for promoting vascular endothelial cell proliferation can promote vascular endothelial cell proliferation by glaucocalyxin B.
The culture medium for promoting vascular endothelial cell proliferation inhibits intracellular oxidative stress and expression of senescence proteins p16 and p 19.
Glaucocalyxin b, CAS No.:80508-81-2, molecular formula: c (C) 22 H 30 O 5 Molecular weight: 374.477 from Chengdoremineramexane Biotechnology Co.
The technical effects are as follows:
the research result of the invention shows that glaucocalyxin B can promote vascular endothelial cell proliferation and inhibit the expression of senescence proteins p16 and p 19.
Drawings
FIG. 1 influence of glaucocalyxin B on vascular endothelial cell proliferation activity;
FIG. 2 influence of glaucocalyxin B on expression levels of vascular endothelial cell senescence proteins p16, p 19.
Detailed Description
The following describes the essential aspects of the present invention in detail with reference to examples, but those skilled in the art will recognize that the scope of the present invention should not be limited to the specific examples.
Example 1: influence of glaucocalyxin B on vascular endothelial cell proliferation
1.1 obtaining materials
(1) After alcohol is used for wiping the ultra-clean table top, the required packaged and sterilized equipment is put in, and ultraviolet rays are irradiated for 30min.
(2) 4C 57BL/6 mice are taken, the eyeballs are subjected to bloodletting and sacrificed, the mice are placed in 75% alcohol for whole body soaking and disinfection for 10s, the skin is cut off along the midline of the abdomen of the mice in an ultra-clean workbench, the thoracic cavity and the abdominal cavity are opened, the thoracic and abdominal aorta is cut off along the spinal column, and the mice are placed in a sterile culture dish containing PBS. The external fat and connective tissue of the blood vessel are peeled off by using fine forceps under a microscope, and the whole blood vessel is sheared by a spring. The vessel was washed 6 times with sterile PBS to wash away residual blood clots and mouse hair, etc.
1.2 digestion and centrifugation to obtain cells
(1) Digestion of endothelial cells: digesting the blood vessel with 0.02% type II collagenase after filtering by a sterilizing filter, sealing the centrifugal tube orifice by a sealing film, carrying out water bath at 37 ℃ for 40min, and shaking for about 30s every 10min. Centrifuging at 1500r/min for 10min. (2) Planting: the blood vessel and the cells are resuspended in 1ml of RPMI1640 medium containing 20% fetal bovine serum, transferred into a gelatin-coated culture flask, and then a small amount of culture solution is added, and the flask bottom is paved just before the blood vessel is fully covered, and the mixture is placed in a 5% CO2 incubator at 37 ℃ for standing for 2 hours, so that endothelial cells are attached to the wall. (3) Liquid replacement: after standing for 2 hours, the vascular tissue and the culture solution are discarded, other cells are prevented from migrating out, and the culture solution is added again. The culture solution was replaced the next day, and then replaced every 2 days. (4) Primary passage: when the density is higher, the cells are passaged again, 1ml of cell digestive juice is added, the cells are placed in a 5% CO2 incubator at 37 ℃ for about 1min, obvious shrinkage and rounding of endothelial cells are visible under an inverted microscope, the cell gap is increased, and at the moment, 4ml of complete culture medium is added to terminate digestion, and the cells are passaged 1:2.
1.3 coculture of glaucocalyxin B with vascular endothelial cells
To explore the effect of glaucocalyxin B on IL-17A-induced vascular endothelial cell viability, appropriate intervention concentrations of IL-17A were selected, we set concentration gradients of 1, 5, 10, 50, 100nM for glaucocalyxin B, and co-intervened with 10 μM IL-17A for 24h after pre-incubation for 2h.
1.4MTT method for detecting cell viability
(1) Cells were seeded into 96-well plates at a density of 8000 cells/well and drug intervention was performed after 2 days of culture.
(2) After removal of the original medium and PBS rinse, 100. Mu.L of complete medium and 10. Mu.L of MTT solution were added to each well and incubation was continued for 4h in the cell incubator.
(3) 150 μl DMSO was added to each well, mixed well and crystal violet was dissolved.
(4) Absorbance was measured at 490nm using an enzyme-labeled instrument.
The results show that the cell viability of vascular endothelial cells gradually increased with increasing glaucocalyxin b intervention concentration, as shown in figure 1.
Example 2: influence of glaucocalyxin B on expression level of vascular endothelial cell senescence proteins p16 and p19 (Western blot detection)
(1) Cell culture and grouping
The vascular endothelial cells obtained in example 1 above were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin diabody. All cells were culturedIncubator at constant temperature of 6-well plate, 37 ℃ (humidity 95%,5% CO) 2 Concentration). Three groups are divided, 10 mu M IL-17A is added into the three groups, and medicines (glaucocalyxin B) with different concentrations are added into the two glaucocalyxin B groups for culturing for 24 hours.
(2) Total protein extraction: cells were digested and single cell suspensions were collected in 1.5mL EP tubes, cell count 1X 10 6 And adding 260 mu L of whole protein extraction mixed solution (extraction reagent ratio Lysis Buffer: phosphatase inhibitor: protease inhibitor: PMSF=1000:5:1:10), lightly blowing and beating uniformly mixed cells by using a gun head, placing the uniformly mixed cells on ice for cracking for 30min, and swirling for 3-5 times during the process to enhance the cracking effect. Centrifuge at 13500rpm and 4℃for 15min. Carefully aspirate the supernatant, place in a new 1.5m LEP tube, measure concentration using BCA protein assay, add an equal volume of 2 x Loading Buffer, mix well, after 10min in a 97 ℃ water bath, store at-20 ℃.
(3) And (3) glue preparation: repeatedly cleaning the glass plate by using double distilled water, and then wiping the glass plate by using filter paper. Glass plates of proper specifications (1.0 mm specification is generally used enough) are placed into a glue making groove, fixed, and then proper water is added for leakage detection. A separation gel (typically 8-15%) is added to suit the molecular weight configuration of the protein of interest. After being prepared according to the proportion of the separation glue, the mixture is slightly and uniformly mixed, the mixture is added into a glass plate (no bubbles can be added into the glue, and the intensity of attention is needed in the mixing process), the upper layer is sealed by double distilled water for 1cm, the phenomenon of glue leakage is observed, the glue leakage causes uneven concentration of the glue, and the glue needs to be reconfigured. After about 30-40min at room temperature, when obvious limit appears between the colloid and the liquid seal layer, the double distilled water of the upper liquid seal layer is poured off, and the filter paper is stretched between the glass plates to suck the residual water (note that the colloid is not touched). Adding 5% concentrated gel, inserting into clean comb of 1.0mm, standing at room temperature for 10-15min until gel is completely coagulated.
(4) Electrophoresis: the gel rack is placed in an electrophoresis tank, 1 Xelectrophoresis buffer is poured into the tank to be filled up, a comb is pulled out slowly upwards (if the gel is deformed, a needle is used for pulling back for resetting, if the gel is deformed too much, reconfiguration is needed), and the sample is added by a micropipette in an equal volume. The positive electrode and the negative electrode of the electrophoresis apparatus are correctly connected, electrophoresis is carried out under the constant voltage condition of 80V until the marker enters the separation gel, and when the separation gel is slightly separated, the voltage is regulated according to the target protein, generally, the electrophoresis is carried out under the constant voltage of 120V for 90min, and the electrophoresis is stopped until the electrophoresis is suitable for gel cutting.
(5) Transferring: and (3) pre-estimating the size of the cut gel before electrophoresis is finished, cutting out PVDF membranes with similar sizes, marking the edges of the membranes in advance, and soaking in methanol for standby. Turning off the power supply, taking out the gel clamping plate, making a sandwich membrane transferring clamp, sequentially placing the sponge, the thick filter paper, the gel, the PVDF, the thick filter paper and the sponge into the membrane transferring clamp, and placing the colloid on the black surface. And placing the film transferring frame into an electrophoresis tank, and covering the electrophoresis tank with ice after the film transferring frame is installed. The constant current 200mA film transferring time is about 2 hours, the film transferring time is related to the size of protein, and the larger the protein is, the longer the film transferring time is needed.
(6) Blocking and antibody incubation: after finishing the film transfer, 10% of skimmed milk powder prepared by 1 XTBST is taken out, the PVDF film is placed in the skimmed milk powder for sealing, and the milk is ensured to permeate the PVDF film for about 2 hours. After the sealing is finished, the recovered milk is stored at 4 ℃ and can be reused for 4-5 times. The antibody was diluted with 1 XTBST in the appropriate ratio (see instructions and personal experience), the diluted primary antibody was added to the antibody incubation cassette, and the PVDF membrane was placed in the incubation cassette and incubated overnight at 4 ℃. Re-heating at room temperature for 30min every other day, recovering primary antibody, and storing to 4deg.C for 2-3 times. PVDF membrane was washed 3 times with 1 XTBE at shaking table shaking for 15min each time. The corresponding secondary antibody diluted by 1 XTBE is added into the incubation box, incubated for 1-2h at room temperature, and the PVDF membrane is washed again for 3 times each for 15min. Preparing beta-actin according to the proportion of 1:10000; p16 and p19 were prepared at a ratio of 1:1000, secondary antibody dilutions were prepared with blocking solution, and the coat Anti-rabit IgG/HRP was prepared at a ratio of 1:10000.
(7) Exposure: the hypersensitive luminescent reagent needs to be prepared at present, and after being uniformly mixed according to the ratio of 1:1, the luminescent liquid is uniformly added on the PVDF film, and the film is not required to be dehydrated in the period. Rapidly placing the sample into a C300 analysis imager, incubating the sample in a darkroom for 10 seconds, and exposing the sample. The exposure time is set automatically, or the exposure can be performed manually according to the experience value. The pictures were subjected to grey scale analysis using ImageJ (Version 1.44 p) software, see fig. 2.
The results of gray quantitative analysis show that glaucocalyxin B inhibits the expression of vascular endothelial cell senescence proteins p16 and p19, and has statistical difference (p < 0.05) compared with a model group.
The research result of the invention shows that glaucocalyxin B can inhibit the expression of senescence proteins p16 and p19, promote the proliferation of vascular endothelial cells, and can be used as a culture medium of vascular endothelial cells.
The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.
Claims (3)
1. The glaucocalyxin B is the only effective component in the preparation of the culture medium for promoting the proliferation of vascular endothelial cells.
2. The use of glaucocalyxin b as the sole active ingredient in the preparation of a medium for promoting vascular endothelial cell proliferation according to claim 1, wherein: glaucocalyxin B is the only effective component and can promote vascular endothelial cell proliferation.
3. The use of glaucocalyxin b as the sole active ingredient in the preparation of a medium for promoting vascular endothelial cell proliferation according to claim 1, wherein: glaucocalyxin B is the only effective component for inhibiting the expression of senescent proteins p16 and p 19.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851272A (en) * | 2009-03-31 | 2010-10-06 | 苏州金昊药业开发有限公司 | Glaucocalyxin B, derivative, preparation method and application thereof |
| CN114369566A (en) * | 2022-01-20 | 2022-04-19 | 中国中医科学院医学实验中心 | Culture solution for promoting proliferation and angiogenesis of vascular endothelial cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851272A (en) * | 2009-03-31 | 2010-10-06 | 苏州金昊药业开发有限公司 | Glaucocalyxin B, derivative, preparation method and application thereof |
| CN114369566A (en) * | 2022-01-20 | 2022-04-19 | 中国中医科学院医学实验中心 | Culture solution for promoting proliferation and angiogenesis of vascular endothelial cells |
Non-Patent Citations (1)
| Title |
|---|
| 蓝萼甲素对血管内皮细胞的保护作用;夏龙等;《中医药导报》;第第20卷卷(第第6期期);第22-24页 * |
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