CN102346128A - Method for testing surface friction performance of living vascular endothelial cell - Google Patents
Method for testing surface friction performance of living vascular endothelial cell Download PDFInfo
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- CN102346128A CN102346128A CN2011101915517A CN201110191551A CN102346128A CN 102346128 A CN102346128 A CN 102346128A CN 2011101915517 A CN2011101915517 A CN 2011101915517A CN 201110191551 A CN201110191551 A CN 201110191551A CN 102346128 A CN102346128 A CN 102346128A
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Abstract
The invention relates to a method for testing the surface friction performance of a living vascular endothelial cell, which is characterized in that a soft material living cell prosthetic vessel model is formed by polymer hydrogel with a similar structure and function to a physiological vessel and a monolayer vascular endothelial cell membrane, the method is used for directly, quantificationally and accurately testing the surface friction performance of the human body vascular endothelial cell which is cultured in vitro or is alive for a long time in vitro, and the adhesive force of the cell on the surface of a hydrogel support is strengthened, so that the cell is not easily removed from the surface of the hydrogel in a test process, and the friction of the living cell can be conveniently and accurately tested for a long time; and due to the polymer hydrogel used in the method, the vascular endothelial cell is at a sandwich state between the softhydrogel cell cultivating support and a softhydrogel friction substrate, and stress borne by the cell can be effectively reduced through the viscoelastic polymer hydrogel, so that the cell survival state is protected, the defect that the cell is easy to be removed and damaged is overcome, and the method provided by the invention has the advantage of accurately evaluating the friction performance of the living cell.
Description
Technical field
The invention belongs to cell biological mechanics determination techniques field, be specifically related to a kind of method of measuring vascular endothelial cell mantle friction performance alive.
Background technology
The minimally invasive medical technology is one of 21 century medical domain medical skill with fastest developing speed, and its reason is numerous needs of patients treatment.It is the medical skill that adopts a series of mis instruments, material and modernized digital diagnosis and treat equipment to carry out the Clinics and Practices operation that Wicresoft gets involved medical treatment, and it has the advantage of " timely, Wicresoft, painless, comfortable ", particularly outstanding aspect the treatment angiocardiopathy.Along with the arrival in aging population epoch and the raising of quality of life, Wicresoft's interventional medicine engineering has demonstrated unprecedented application prospect.Statistics shows, China patient that need accept operation on vessels of heart has reached about 4,000,000 people now, and annual speed with 20% increases.Be about 5,000,000,000 yuan in China in 2005 with the Wicresoft relevant market sales revenue of interventional medical device material, estimate that market demand might reach 300～40,000,000,000 yuan in 2015.So at present all in the world medicine equipments are made the commercial city and have been targeted China, with the medicine equipment potential market of China as the maximum treating cardiovascular disease in the whole world.
Yet, be a long-term existence and open question still by the vascular diseases of catheter/vascular endothelial cell friction Induced.In operative process; Length is that the catheter of 80-150cm is usually from shallow table leg arteries or vein approach; Through tortuous blood vessel therapeutic device (support etc.) is transported to lesions (like heart or brain); In this process; Catheter contacts with the monolayer endothelial cell film on blood vessel surface inevitably and produces friction; The too high endothelial cell that will cause that rubs is impaired; And then bring out complication such as operative hemorrhage, thrombosis and postoperative reangiostenosis, finally bring out vascular diseases such as atherosclerotic.So catheter/human vas endothelial cell frictional behaviour is significant to the interventional therapy success or not of angiocardiopathy Wicresoft.But at present catheter/human vas endothelial cell frictional behaviour and mechanism are still indeterminate, get involved the medical utmost point with the Wicresoft that develops rapidly and do not match, and its main cause is to measure at present the following shortcoming of the method for catheter frictional behaviour:
1) there is limitation in catheter material/material friction research pair cell; This type research accounts for the overwhelming majority, mainly with methods such as little tribometer, rotary flow graph and multi-functional little frictional testing machine estimate between the tube material, frictional behaviour between the substrates such as material and glass, macromolecule hydrogel.But, be difficult to conclude whether institute's research material has the function that reduces the vascular endothelial cell friction equally owing to be not research object with the vascular endothelial cell.
2) catheter material/animal blood vessels inner wall surface friction research is difficult to expand to human body, and relevant report is still rare, has report to study tube material/animal blood vessels inwall frictional behaviour with pin-on-disc friction wear testing machine, reciprocating type friction testing appearance.Yet, be that the major limitation property of research object need to be a large amount of fresh blood vessel samples with the animal blood vessels, and it is very limited to obtain the chance of the fresh blood vessel sample of a large amount of human bodies, so this method is difficult to expand to human vas inwall Study on Friction Properties.
3) in addition aspect the test of in vitro culture vascular endothelial cell frictional behaviour, the W.G.Sawyer of Univ Florida USA etc. with little frictional testing machine preliminary assessment the ox main artery vascular endothelial cell/glass frictional behaviour of cultivating at glass surface.Though macroscopical friction behavior on vascular endothelial cell surface has been reacted in this research to a certain extent, also has following point: 1. research object problem, do not estimate human body vascular endothelial cell frictional behaviour, the result is difficult to use for reference human body; 2. System Design problem; Cell is in " sandwich " state between " hard " glass cultivation support and " hard " glass rubbed substrate in the test; Therefore; Stress is concentrated in " softness " cell surface; Cell is stripped from damage very soon in the test, causes being difficult to accurately estimating the cell frictional behaviour.Another is in the human vas endothelial cell surface of macromolecule hydrogel surface cultivation and the frictional behaviour between the glass plate with the rotational rheometer test; Because friction force is bigger; Increase with the test duration; Cell is peeled off damage by glass substrate gradually, and information accurately can not be rubbed after the 200s.In addition, above-mentioned two kinds of methods all do not have account temperature and CO
2Concentration is to the influence of test result.
Summary of the invention
In order to overcome the deficiencies of the prior art, the present invention is to provide a measuring surface of endothelial cells of live performance of the method of friction, this method for in vitro direct, quantitative and can accurately determine the in vitro for a long time, or Human endothelial cells of live performance of the method of surface friction, enhance hydrogel stent cell surface adhesion forces, resulting in the test cell can not easily be peeled off from the hydrogel, to facilitate accurate detection of living cells friction time; the Different human endothelial cells were expanded to find out suitable for their single synthetic polymer hydrogel membrane holder, and having close to vascular endothelial cells in vivo endothelial glycocalyx structure and anti-coagulation, and the method The polymer used in the hydrogel will be "soft" hydrogel cell culture scaffolds and "soft" hydrogel friction between the substrate "sandwich" state, the viscoelastic polymer hydrogel will effectively reduce the stress on cells, thereby protecting cell survival status was stripped easy to overcome cell damage shortcomings, with an accurate assessment of the advantages of friction properties of living cells.
In order to achieve the above object, the technical scheme that the present invention adopted is:
A kind of method of measuring vascular endothelial cell mantle friction performance alive, step is following:
Step 1: at first macromolecule hydrogel is cut into macromolecule hydrogel support and macromolecule hydrogel rubbed substrate respectively according to preset support framework and substrate framework, this macromolecule hydrogel support and macromolecule hydrogel rubbed substrate were carried out high-temperature sterilization 15-20 minute;
Step 2: after subsequently human umbilical cord's vein endothelial cell directly being seeded in a side surface of the macromolecule hydrogel support behind the high-temperature sterilization, be 37 ℃ and inner CO at internal temperature
2Percent by volume be to cultivate in 5% the incubator, be the individual layer endothelial cellular membrane up to the vascular endothelial cell amplification, have amplification like this and just be built into soft material living cells artificial blood vessel model for the macromolecule hydrogel support of cell monolayer film;
Step 3: Subject to the endothelial cell monolayer friction polymer hydrogel substrate bonded to the bottom surface of the rheometer solution ponds and tanks in the rheometer was added to the corresponding cell culture medium, and then the polymer hydrogel plastic stent with endothelial cell layer on one surface of the substrate facing the hydrogel polymer, the polymer hydrogel and the other side surface of the bracket is bonded to the upper part of the rheometer geometry measuring head, start flow Instrument landing system control variable geometry probe decreases until soft material of living cells, artificial blood vessel model of vascular endothelial cell monolayer with a polymer hydrogel contact with the substrate in contact with the vertical pressure P reaches a predetermined value range 1 ~ 50Pa when balance for 10 minutes, subsequently the rheometer according to a preset torque T (ω) the polymer hydrogel to the substrate along the center of rotation a predetermined maximum angular velocity ω, when the maximum angular velocity ω, the friction test point force F (ωR) and torque T (ω) the relationship F (ωR) = [4T (ω)] / 3πR
3 , wherein R is a test point and the center of rotation The distance between the under friction F (ωR) perpendicular to the applied pressure P in addition to the value of the coefficient of friction measured point derived μ.
Described macromolecule hydrogel is polyvinyl alcohol hydrogel, polyoxyethylene hydrogel, polyglycol hydrogel, polyacrylic acid hydrogel, polymethylacrylic acid hydrogel, kayexalate hydrogel or gathers 2-acrylamido-2-methyl propane sulfonic acid hydrogel.
The present invention adds that to have macromolecule hydrogel with physiology blood vessel similar structures and function the individual layer endothelial cellular membrane constructs soft material living cells artificial blood vessel model; This model has following advantage: the puppet of (1) vascular endothelial cell can stretch into the inside of tridimensional network completely; Strengthened the adhesion of cell at the hydrogel rack surface; Thereby cell is difficult for being peeled off from hydrogel surface in test, is convenient to long-time accurate detection of living cells friction; (2) to different human vas endothelial cells (artery, vein, microtubules); Can seek out respectively and be fit to their amplifications and be the synthetic high polymer hydrogel support of cell monolayer film, and have with body in the approaching endothelium glycocalyx structure and the anticoagulation function of vascular endothelial cell.Above-mentioned advantage has guaranteed that " soft material living cells artificial blood vessel model " approaches the physiology blood vessel on 26S Proteasome Structure and Function.And macromolecule hydrogel is brought into play two vital role in the method: timbering material is cultivated as the human vas endothelial cell in (1), in order to construct " soft material living cells artificial blood vessel model "; (2) the rubbed substrate material of conduct and individual layer human vas endothelial cell membrane friction.Therefore; Vascular endothelial cell will be in " sandwich " state between " softness " hydrogel cell culturing bracket and " softness " hydrogel rubbed substrate; Viscoelastic macromolecule hydrogel will effectively reduce the stress that cell bears; Thereby protection cells survival state; Overcome the shortcoming that cell is easy to be stripped from damage, have the advantage of accurate evaluation living cells frictional behaviour.
Embodiment
Below in conjunction with embodiment the present invention is done more detailed explanation.
Measure the method for the vascular endothelial cell mantle friction performance of living, step is following:
Step 1: at first macromolecule hydrogel is cut into macromolecule hydrogel support and macromolecule hydrogel rubbed substrate respectively according to preset support framework and substrate framework, this macromolecule hydrogel support and macromolecule hydrogel rubbed substrate were carried out high-temperature sterilization 15-20 minute;
Step 2: after subsequently human umbilical cord's vein endothelial cell directly being seeded in a side surface of the macromolecule hydrogel support behind the high-temperature sterilization, be 37 ℃ and inner CO at internal temperature
2Percent by volume be to cultivate in 5% the incubator, be the individual layer endothelial cellular membrane up to the vascular endothelial cell amplification, have amplification like this and just be built into soft material living cells artificial blood vessel model for the macromolecule hydrogel support of cell monolayer film;
Step 3: Subject to the endothelial cell monolayer friction polymer hydrogel adhesive substrate solution in a rotary rheometer bottom of the pool and the pool was added in the rheometer corresponding cell culture medium, and then the polymer hydrogel stent with endothelial cell layer on one surface of the substrate facing the hydrogel polymer, the polymer hydrogel and the other side surface of the bracket is bonded to the upper part of the rheometer geometry measurement head, Start lifting system control rheometer geometry probe decreases until soft material of living cells, artificial blood vessel model of vascular endothelial cell monolayer with a polymer hydrogel contact with the substrate in contact with the vertical pressure P reaches a predetermined value range 1 ~ 50Pa When balance for 10 minutes, subsequently the rheometer according to a preset torque T (ω) the polymer hydrogel substrate along a center of rotation to a predetermined maximum angular velocity ω, when the maximum angular velocity ω, the test point The friction force F (ωR) and torque T (ω) the relationship F (ωR) = [4T (ω)] / 3πR
3 , where R is the target point, and The distance between the center of rotation, based on friction F (ωR) perpendicular to the applied pressure P in addition to the value of the coefficient of friction measured point derived μ.
Described macromolecule hydrogel is polyvinyl alcohol hydrogel, polyoxyethylene hydrogel, polyglycol hydrogel, polyacrylic acid hydrogel, polymethylacrylic acid hydrogel, kayexalate hydrogel or gathers 2-acrylamido-2-methyl propane sulfonic acid hydrogel; Such synthetic high polymer hydrogel is neutral, weak electrolyte and strong electrolyte, has that chemical constitution is clear and definite, a stable performance and be easy to, do not have foreign matter and infect, be easy to sterilize and cheap advantage.
During friction force of the present invention changes in time; Can make friction force in seconds at first reach a peak-peak; Change in time then and reduce gradually; Reach an equilibrium value at last, even test 2000s like this, the cell greater than 98% all is kept at the surface of " soft material living cells artificial blood vessel model "; The cell friction factor can maintain 0.006-0.008 simultaneously; Illustrate that mantle friction is very low, cell does not almost have impaired, can test for a long time.
Claims (2)
1. method of measuring vascular endothelial cell mantle friction performance alive is characterized in that step is following:
Step 1: at first macromolecule hydrogel is cut into macromolecule hydrogel support and macromolecule hydrogel rubbed substrate respectively according to preset support framework and substrate framework, this macromolecule hydrogel support and macromolecule hydrogel rubbed substrate were carried out high-temperature sterilization 15-20 minute;
Step 2: after subsequently human umbilical cord's vein endothelial cell directly being seeded in a side surface of the macromolecule hydrogel support behind the high-temperature sterilization, be 37 ℃ and inner CO at internal temperature
2Percent by volume be to cultivate in 5% the incubator, be the individual layer endothelial cellular membrane up to the vascular endothelial cell amplification, have amplification like this and just be built into soft material living cells artificial blood vessel model for the macromolecule hydrogel support of cell monolayer film;
Step 3: Subject to the endothelial cell monolayer friction polymer hydrogel substrate bonded to the bottom surface of the rheometer solution ponds and tanks in the rheometer was added to the corresponding cell culture medium, and then the polymer hydrogel plastic stent with endothelial cell layer on one surface of the substrate facing the hydrogel polymer, the polymer hydrogel and the other side surface of the bracket is bonded to the upper part of the rheometer geometry measuring head, start flow Instrument landing system control variable geometry probe decreases until soft material of living cells, artificial blood vessel model of vascular endothelial cell monolayer with a polymer hydrogel contact with the substrate in contact with the vertical pressure P reaches a predetermined value range 1 ~ 50Pa when balance for 10 minutes, subsequently the rheometer according to a preset torque T (ω) the polymer hydrogel to the substrate along the center of rotation a predetermined maximum angular velocity ω, when the maximum angular velocity ω, the friction test point force F (ωR) and torque T (ω) the relationship F (ωR) = [4T (ω)] / 3πR
3 , wherein R is a test point and the center of rotation The distance between the under friction F (ωR) perpendicular to the applied pressure P in addition to the value of the coefficient of friction measured point derived μ.
2. the method for the vascular endothelial cell mantle friction performance that mensuration according to claim 1 is lived is characterized in that: described macromolecule hydrogel is polyvinyl alcohol hydrogel, polyoxyethylene hydrogel, polyglycol hydrogel, polyacrylic acid hydrogel, polymethylacrylic acid hydrogel, kayexalate hydrogel or gathers 2-acrylamido-2-methyl propane sulfonic acid hydrogel.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108761004A (en) * | 2018-05-21 | 2018-11-06 | 北京工商大学 | A kind of evaluation method of the rice viscosity based on frictional index |
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| US5785054A (en) * | 1989-06-06 | 1998-07-28 | Kelly; Patrick D. | Genital lubricant with zinc salt, labelled as anti-viral agent |
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Cited By (2)
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