CN104726393A

CN104726393A - Preparation method of TNF-alpha-induced human umbilical vein endothelial cell HUVEC inflammatory reaction model

Info

Publication number: CN104726393A
Application number: CN201310718727.9A
Authority: CN
Inventors: 黄午阳; 王兴娜; 李春阳; 闫征; 王帆; 王健
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2013-12-24
Filing date: 2013-12-24
Publication date: 2015-06-24

Abstract

The invention relates to a preparation method of the TNF-alpha-induced human umbilical vein endothelial cell HUVEC inflammatory reaction model and belongs to the field of biological technology. The preparation method comprises the following steps: (1) cultivation of HUVEC cells, namely cultivating the HUVEC cells in an M199 complete culture medium at 37 DEG C in an incubator with 5% of CO2; (2) preparation of HUVEC samples, namely digesting the cells by 15ml of 1mg/ml I-type collagenase for 15-20 min when the cells grow to a 80%-90% monolayer, so as to prepare a cell suspension, and then adjusting the cell concentration to 1*10<6> /ml; (3) synchronization of HUVEC cells, namely culturing 2ml of the cell suspension in a 6-well plate and then performing starvation culture for 24 hours by a serum-free culture medium when the cells grow to 90% of the culture medium, so that all the cells are synchronized; and (4) induction of HUVEC cell inflammatory reaction by TNF-alpha, namely adding the TNF-alpha with a final concentration of 10ng/ml into the HUVEC cells to stimulate the HUVEC cells for 6 hours, taking the cell supernatant and assaying the cell inflammatory cytokines MCP-1, ICAM-1 and VCAM-1 by an ELISA method, wherein the protein content is all increased. The HUVEC inflammatory reaction model built according to the invention is simple and easy to operate and can be used in mechanism studies of the inflammatory reactions as well as in in-vitro cell experiments of anti-inflammatory drugs.

Description

The preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model that TNF-α induces

One, technical field

The present invention is the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model that a kind of TNF-α induces, and belongs to biological technical field.

Two, background technology

Human umbilical vein endothelial cells HUVEC is separated from normal human's umbilical cord the vein endothelial cell obtained, and can express the inflammation factor and the multiple key enzyme relevant to vasoconstriction or diastole, is the cell model system of widespread use in physiology, pharmacological research.

TNF-α is important pro-inflammatory cytokine, plays an important role in cardiovascular disorder.TNF-α is the glycoprotein that a kind of mononuclear macrophage primarily of activation produces, and is a kind of important pro-inflammatory cytokine, plays an important role in the generation and evolution of cardiovascular disorder.TNF-α can cause Dysfunction of vascular endothlial cells, then cytokine profiles is produced as intercellular adhesionmolecule1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), monocyte chemotactic factor (MCP-1) etc., thus cause white corpuscle and monocyte emigration to shield to blood vessel, cause vascular inflammation to react.Normal endothelial cell expresses MCP-1 hardly, ICAM-1 and VCAM-1 albumen, or expression amount is little.After TNF-α stimulates, the expression of endotheliocyte MCP-1, ICAM-1 and VCAM-1 albumen obviously increases, and the concentration of the change of expressing quantity and TNF-α and stimulation time have substantial connection.Entered us and test discovery when TNF-α concentration is 10ng/ml and stimulation 6h, three's expression amount all reaches peak value.

TNF-α is utilized to induce Human umbilical vein endothelial cells HUVEC to produce inflammatory response cell model; may be used for the mechanism of further investigated vascular endothelial cell inflammation damage; prevent and regulate too drastic inflammatory reaction; protection inner skin cell function, has positive meaning to prevention and therapy vascular inflammation disease.

Three, summary of the invention

Technical problem the present invention mainly provides a kind of TNF-α to induce the method preparing Human umbilical vein endothelial cells HUVEC inflammatory cell reaction model.

Technical scheme the present invention is the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model that a kind of TNF-α induces, and comprising:

1) HUVEC cell cultures: HUVEC cell M199 perfect medium (containing phenol red, 20% newborn calf serum, 2mmol/L L-glutaminate, 100U/ml penicillin, 100U/ml Streptomycin sulphate, 100 μ g/ml vascular endothelial growth factor-heparin) is placed in 37 DEG C, 5%CO ₂incubator in cultivate.

2) HUVEC sample preparation: treat that cell covers with to 80%-90% individual layer, be prepared into cell suspension with the type i collagen enzymic digestion 15-20min of the 1mg/ml of 15ml, get 10ul and count on blood counting chamber, adjustment cell concn is 1*10 ⁶/ ml.

3) HUVEC cell synchronization: get 2ml cell suspension to 6 orifice plate and cultivate, when cell cover with to 90% time, the substratum changing serum-free carries out hunger and cultivates 24h, makes all cells synchronization.

4) TNF-α induces the reaction of HUVEC cellular inflammation: add the TNF-α that final concentration is 10ng/ml, stimulates HUVEC cell 6h, and get cell conditioned medium liquid ELISA method and measure cellular inflammation factor M CP-1, ICAM-1, VCAM-1, protein content all raises.

Beneficial effect the present invention is intended to set up a kind of simple inflammatory reaction In vitro cell model.The inflammation factor of the Human umbilical vein endothelial cells HUVEC that present method selects TNF-α to induce, significantly improves the protein expression content of HUVEC cell MCP-1, ICAM-1, VCAM-1.This inflammatory response cell model, may be used for the mechanism of further investigated vascular endothelial cell inflammation damage, prevents and regulate too drastic inflammatory reaction, and protection inner skin cell function, has positive meaning to prevention and therapy vascular inflammation disease.

Four, embodiment

Below in conjunction with specific embodiment, the invention will be further described:

Embodiment 1

With M199 perfect medium at 37 DEG C, 5%CO ₂incubator in cultivate HUVEC cell to cell and cover with 80%-90% individual layer, be prepared into cell suspension with the type i collagen enzymic digestion 15-20min of the 1mg/ml of 15ml, get 10ul and count on blood counting chamber, adjustment cell concn is 1*10 ⁶/ ml.Get 2ml cell suspension to 6 orifice plate to cultivate, when cell cover with to 90% time, the substratum changing serum-free carries out hunger and cultivates 24h, makes all cells synchronization.Add the TNF-α that final concentration is 10ng/ml, stimulate HUVEC cell 6h, get cell conditioned medium liquid ELISA method and measure OD value under microplate reader 450nm, detect cellular inflammation factor M CP-1, ICAM-1, VCAM-1 albumen, result shows that MCP-1 concentration rises to 0.608 ± 0.018 μ g/L (P < 0.001) from 0.130 ± 0.007 μ g/L, ICAM-1 concentration rises to 0.669 ± 0.033 μ g/L (P < 0.01) from 0.133 ± 0.002 μ g/L, VCAM-1 concentration rises to 0.538 ± 0.019 μ g/L (P < 0.01) from 0.147 μ g/L ± 0.011 μ g/L, the protein content of three all raises.

Claims

The preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model that 1.TNF-α induces, includes following steps:

1) 37 DEG C are placed in, 5%CO with M199 perfect medium (1 containing phenol red, 20% newborn calf serum, 2mmol/L L-glutaminate, 100U/ml penicillin, 100U/ml Streptomycin sulphate, 100 μ g/ml vascular endothelial growth factor-heparin) ₂incubator in cultivate HUVEC cell

2) HUVEC cell covers with to 80%-90% individual layer, and be prepared into cell suspension with the type i collagen enzymic digestion 15-20min of the 1mg/ml of 15ml, get 10ul and count on blood counting chamber, adjustment cell concn is 1*10 ⁶/ ml.

3) get 2ml cell suspension to 6 orifice plate to cultivate, when cell cover with to 90% time, the substratum changing serum-free carries out hunger and cultivates 24h, makes all HUVEC cell synchronizations.

4) add the TNF-α that final concentration is 10ng/ml, stimulate 6h, get cell conditioned medium, according to ELISA method, get cell conditioned medium liquid and under microplate reader 450nm, measure OD value see cellular inflammation factor M CP-1, ICAM-1, VCAM-1, discovery protein content all raises.
2. the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model of TNF-α induction according to claim 1, it is characterized in that: HUVEC cell M199 perfect medium (containing phenol red, 20% newborn calf serum, 2mmol/L L-glutaminate, 100U/ml penicillin, 100U/ml Streptomycin sulphate, 100 μ g/ml vascular endothelial growth factor-heparin) in 37 DEG C, 5%CO ₂cultivate, cover with to 80%-90% individual layer.
3. the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model of TNF-α induction according to claim 1, is characterized in that: the collagenase I type (1mg/ml) adding 15ml is prepared into cell suspension 37 DEG C of digestion.
4. the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model of TNF-α induction according to claim 1, is characterized in that: cell suspension adjustment concentration is 1*10 ⁶/ ml.
5. the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model of TNF-α induction according to claim 1, it is characterized in that: 2ml cell suspension to 6 orifice plate is cultivated, when cell cover with to 90% time change serum-free substratum carry out hunger cultivation 24h.
6. the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model of TNF-α induction according to claim 1, is characterized in that: add the TNF-α that final concentration is 10ng/ml, stimulates HUVEC cell 6h.
7. the preparation method of the Human umbilical vein endothelial cells HUVEC inflammatory response model of TNF-α induction according to claim 1, it is characterized in that: get cell conditioned medium liquid EELISA method, OD value is measured under microplate reader 450nm, detect cellular inflammation factor M CP-1, ICAM-1, VCAM-1, protein content all raises.