CN101250498A - Method for culturing liver cell by hepatocyte-carrying growth factor polylactic acid-0-carboxymethyl shellglycan nano partical - Google Patents
Method for culturing liver cell by hepatocyte-carrying growth factor polylactic acid-0-carboxymethyl shellglycan nano partical Download PDFInfo
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- CN101250498A CN101250498A CNA2008100234742A CN200810023474A CN101250498A CN 101250498 A CN101250498 A CN 101250498A CN A2008100234742 A CNA2008100234742 A CN A2008100234742A CN 200810023474 A CN200810023474 A CN 200810023474A CN 101250498 A CN101250498 A CN 101250498A
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- carboxymethyl chitin
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Abstract
The invention discloses a method for culturing hepatocyte through using carrier hepatocyte growth factor polylactic acid-O-carboxymethyl chitosan nanoparticels and the carrier hepatocyte growth factor polylactic acid-O-carboxymethyl chitosan nanoparticels and the hepatocyte are mixed to vaccinate and culture when the hepatocyte is cultured. The method of the invention can effectively promote the growth of the hepatocyte, the hepatocyte activity is increased, and the method must play a great promoting role for developing the hepatocytes cultured technique.
Description
Technical field:
The present invention relates to a kind of hepatocellular cultural method.
Background technology:
The acute hepatic failure mortality ratio is up to 70%.Hepatocyte transplantation can play certain liver function support and promote the liver regeneration effect.Because it has advantages such as simple to operate, little to the organism physiology ambient interference, that donor source is extensive, has caused numerous investigators' extensive concern.Yet, how to improve the liver transplantation cell activity, reduce immunological rejection and promote that the acceptor liver regeneration is three big problems of being badly in need of solution.
Summary of the invention:
The object of the present invention is to provide and a kind ofly can effectively promote liver cell growth, improve the liver cell culture method that the usefulness of liver cell vigor is carried pHGF polylactic acid-0-carboxymethyl chitin nanometer.
Technical solution of the present invention is:
A kind of usefulness is carried the liver cell culture method of pHGF (HGF) polylactic acid-0-carboxymethyl chitin nanometer, it is characterized in that: when liver cell culture, will carry pHGF polylactic acid-0-carboxymethyl chitin nanometer and mix with liver cell and carry out inoculation culture.
PHGF polylactic acid-0-carboxymethyl chitin nanometer was to make through the following steps in described year: will carry pHGF and join in the polylactic acid-0-carboxymethyl chitin nanometer aaerosol solution, fully reaction 4 hours under 4 ℃, the condition of 800rpm induction stirring, 10000rpm high speed centrifugation, distilled water wash, freezing draining obtain solidified and carry HGF polylactic acid-0-carboxymethyl chitin nanometer then.
During liver cell culture, culture temperature is 35～38 ℃, and 5%CO
2Cultivate under the condition, cultivate in 12 hours, vibration in per 30 minutes once continues 5 minutes at every turn.
Liver cell with year pHGF polylactic acid-0-carboxymethyl chitin nanometer with magnitude relation is: 5 * 10
5Individual/ml liver cell and 40 μ g/ml's carries the combined inoculation mutually of HGF polylactic acid-0-carboxymethyl chitin nanometer.
The present invention can effectively promote liver cell growth, improves the liver cell vigor, must play huge pushing effect to the development of liver cell culture technology.Experimental results show that, compare with common liver cell culture, the time that the liver cell that the present invention cultivates forms sphere aggregates obviously shortens, the ratio of sphere aggregates obviously increases, cellular form is good, cultivated the back the 2nd day to the 7th day, and adopted CCK-8 body outer cell proliferation test kit to detect the hepatocellular vigor of cultivating, find to carry the amount that HGF nanometer cultivation group generates yellow Jia Za dyestuff and be higher than common cultivation group (P＜0.05).Show and carry HGF nanometer cultivation group viable cell number, carry the HGF nanoparticle and can promote rat hepatocytes growth and active maintenance constantly external more than common cultivation group.
The invention will be further described below in conjunction with drawings and Examples.
Fig. 1 carries pHGF polylactic acid-0-carboxymethyl chitin nanometer transmission electron microscope photo.
Fig. 2 carries pHGF polylactic acid-0-carboxymethyl chitin nanometer stereoscan photograph.
The invention will be further described below in conjunction with embodiment.
Embodiment:
The preparation of polylactic acid-0-carboxymethyl chitosan (PLA-O-CMC) nanoparticle:
With 10ml concentration be 0.03% PLA (poly(lactic acid)) dichloromethane solution and 10ml concentration be 0.15% O-CMC solution in fully reaction (about about 25 minutes) under the ultrasonic situation, treat to obtain the heavier even suspension liquid of opalescence after the organic solvent volatilization fully wherein.With this suspension liquid earlier with 2000rpm low-speed centrifugal 5 minutes, the supernatant liquor of getting gained behind the low-speed centrifugal was with 14000rpm high speed centrifugation 10 minutes, obtain the part white precipitate, the part supernatant liquor is removed in suction, to precipitate with behind twice of the distilled water centrifuge washing, will precipitate freezing drain take out behind cobalt-60 irradiation sterilization that walks abreast standby.
Carry the preparation of the polylactic acid-0-carboxymethyl chitin nanometer of HGF:
Take by weighing 5mg PLA-O-CMC nanoparticle and put into the 5ml deionized water and carry out ultrasonic dispersing, make the nanoparticle aaerosol solution.Getting 5 μ g HGF adds in the 5ml PLA-O-CMC nanoparticle aaerosol solution, fully reaction 4 hours under 4 ℃, the condition of 800rpm induction stirring, centrifugal, the distilled water wash of 10000rpm, freezing draining obtain the polylactic acid-0-carboxymethyl chitin nanometer that solidified carries HGF then.
Carry the sign of HGF polylactic acid-0-carboxymethyl chitin nanometer:
Transmission electron microscope observation: get PLA-O-CMC nanoparticle that the 5mg solidified carries HGF and put into the 5ml deionized water and carry out ultrasonic dispersing, make the nanoparticle aaerosol solution.Then a small amount of aaerosol solution is dripped on the copper mesh of film carrier, inhale with filter paper and go excess liquid, promptly make sample after drying, transmission electron microscope observation carries structure, particle diameter and the surface topography of the PLA-O-CMC nanoparticle of HGF.
Laser particle analyzer/zeta potentiometer test: the particle diameter and the surface potential that carry the HGF nanoparticle in the PLA-O-CMC nanoparticle aaerosol solution with laser particle analyzer/above-mentioned year HGF of zeta potentiometer detection.
Carry the PLA-O-CMC nanoparticle carrying drug ratio of HGF and the mensuration of encapsulation rate: carrying drug ratio (drug loading, LD) be meant the mass percent of particulate Chinese traditional medicine, (embeddingratio ER) is meant particulate Chinese traditional medicine amount and the mass percent that drops into the medication amount in the preparation system to encapsulation rate.Calculate carrying drug ratio and encapsulation rate by following formula respectively: HGF content/nanoparticle quality * 100% in carrying drug ratio=nanoparticle, HGF content/HGF input amount * 100% in encapsulation rate=nanoparticle.The PLA-O-CMC nanoparticle that carries HGF that adopts the improvement supersound method to make has comparatively slick surface topography and distributes with relative homogeneous particle diameter, its median size is 139.82nm, the size distribution index is 0.108, the surface potential of particle is 32.8eV, carrying drug ratio is 0.12665%, and encapsulation rate is 76.32%.Have comparatively significantly nucleocapsid structure existence, microparticle surfaces has superpolymer parcel comparatively uniformly, and the surface potential of particle is 32.8eV.This shows that this nanoparticle surface has stronger positive charge, is convenient to carry HGF nanoparticle and electronegative liver cell and contacts and realize the targeted therapy of medicine.
Take by weighing a series of years HGF nanoparticles, every part of 1mg is respectively charged in the centrifuge tube, adds the PBS solution of 1ml pH 7.4; Centrifuge tube is placed in 37 ℃ the constant temperature water bath vibrator (frequency 60rpm), and timing, certain interval of time takes out a centrifuge tube centrifugation (14000rpm) and carries the HGF nanoparticle, and supernatant liquor and precipitation are collected respectively.Detect the content of HGF in the supernatant liquor with the ELLISA method, and measure the pH value that discharges liquid, also accurately weigh with the freezing nanoparticle of draining after the machine lyophilize release with acidometer.Revision test 3 times is averaged, and draws to carry HGF particulate release profiles.
Find that by dynamic observing year HGF nanoparticle vitro degradation properties just year HGF nanoparticle of system is spherical in shape, have slick surface topography and relative homogeneous particle diameter, and have tangible nucleocapsid structure existence that there is one deck superpolymer parcel on the surface.Through 15 days releases and degraded, particle surface shrinkage distortion, volume become greatly, the avalanche of part particle loses spherical shapes.After 45 days, carry the HGF nanoparticle and further discharge and degraded, most of particles are degraded to flocculent substance, and the small part particle still has nucleocapsid structure.Mass loss will appear in the continuous degraded along with nanoparticle, and bulk degradation mainly takes place.Nanoparticle is discovered at external drug release behavior: carry the preceding 24 hours degraded of HGF nanoparticle and can be divided into the high speed release, the middling speed release, in 3 stages of low speed release, preceding 24 hours release kinetic equation is Q=148.4266+189.0493 t
1/2(R=0.97589), meet the Huguchi equation.Carry the HGF nanoparticle in preceding 30 days drug release behavior can be divided into that early stage medicine is prominent to be released the stage, the later stage is stablized the release stage.Release dynamics equation Q=1086.28966+58.23938t (R=0.99716) meets zero level and discharges equation.
The separation of rat hepatocytes and cultivation:
Adopt Seglen in-situ two-step perfusion method to separate rat hepatocytes.Separating obtained liver cell is cultivated liver cell with 5 * 10
5Individual/ml and 40 μ g/ml's carries the combined inoculation (1ml/ hole) to culture plate mutually of HGF polylactic acid-0-carboxymethyl chitin nanometer, places 37 ℃, 5%CO
2Leave standstill cultivation under the condition, cultivate in 12 hours, vibration in per 30 minutes once continues 5 minutes at every turn, with promote to carry nanoparticle with hepatocellular combine and help cell gather the formation spheroplast.Cultivate after 12 hours, cell is combined closely with a year HGF nanoparticle.Cultivate after 48 hours, form cell adhesion accumulative spheroplast, improved cell density.Cultivated 24 hours, most of cell still keeps spherical shape, and part of hepatocytes changes to Polygons, and most liver cells are inter-adhesive, the fasciculation growth.After cultivating 48 hours approximately, most of liver cell reconstituted cell polarity presents typical case, uniform Polygons morphological specificity.
In order to analyze effect of the present invention better, set up common cultivation group simultaneously liver cell cultivated: with liver cell with 5 * 10
5The concentration of individual/ml is inoculated into (1ml/ hole) on the culture plate, places 37 ℃, and concentration is 5% CO
2Leave standstill cultivation under the condition, cultivate in 12 hours, vibration in per 30 minutes once continues 5 minutes at every turn, is beneficial to cell and gathers the formation spheroplast.
The liver cell of common cultivation, be sunken at the bottom of the culture plate after the inoculation very soon and adherent growth, its form stretches the further attenuation of cell after 48～72 hours by firm isolating spheroidal to the flat Polygons of individual layer immediately, gradually lose the Polygons feature, liver cell connects growth in flakes.
Cultivating the liver cell vigor detects: adopt CCK-8 body outer cell proliferation detection kit to detect the liver cell vigor of cultivating.The contained WST-8 of CCK-8 can be reduced into the yellow first Za dyestuff (Formazen) with high water soluble by the desaturase in the viable cell plastosome under the electron carrier effect, the amount of the first Za dyestuff of generation is directly proportional with the viable cell number.To be called " carrying HGF nanoparticle cultivation group " below in the table with the cultivation group of the inventive method, make comparisons with common cultivation group.
Hepatocellular vigor (mean ± SD) is cultivated in two groups of table 1CCK-8 method comparisons
Learn to handle by statistics, cultivate one day two groups of cell viability and do not have than big-difference (P＞0.05), this may with the liver cell separation and Culture after to be in latent period in first day relevant.Cultivated 2～7 days, and carried HGF nanoparticle group cell viability, illustrate that year HGF nanoparticle has the promoter action that significantly continues to the propagation of vitro culture rat hepatocytes apparently higher than common cultivation group (P＜0.05).
Carry HGF polylactic acid-0-carboxymethyl chitin nanometer by research the vitro culture rat hepatocytes is found, this year, the HGF nanoparticle can promote the rat hepatocytes growth constantly external.
Claims (4)
1, a kind of usefulness is carried the liver cell culture method of pHGF polylactic acid-0-carboxymethyl chitin nanometer, it is characterized in that: when liver cell culture, will carry pHGF polylactic acid-0-carboxymethyl chitin nanometer and mix with liver cell and carry out inoculation culture.
2, usefulness according to claim 1 is carried the liver cell culture method of pHGF polylactic acid-0-carboxymethyl chitin nanometer, it is characterized in that: pHGF polylactic acid-0-carboxymethyl chitin nanometer was to make through the following steps in described year: will carry pHGF and join in the polylactic acid-0-carboxymethyl chitin nanometer aaerosol solution, at 4 ℃, fully reacted 4 hours under the condition of 800rpm induction stirring, 10000rpm high speed centrifugation then, distilled water wash, freezing draining obtains solidified and carries HGF polylactic acid-0-carboxymethyl chitin nanometer.
3, usefulness according to claim 1 and 2 is carried the liver cell culture method of pHGF polylactic acid-0-carboxymethyl chitin nanometer, it is characterized in that: during liver cell culture, culture temperature is 35～38 ℃, and 5%CO
2Cultivate under the condition, cultivate in 12 hours, vibration in per 30 minutes once continues 5 minutes at every turn.
4, usefulness according to claim 1 and 2 is carried the liver cell culture method of pHGF polylactic acid-0-carboxymethyl chitin nanometer, it is characterized in that: liver cell with year pHGF polylactic acid-0-carboxymethyl chitin nanometer with magnitude relation is: 5 * 10
5Individual/ml liver cell and 40 μ g/ml's carries the combined inoculation mutually of HGF polylactic acid-0-carboxymethyl chitin nanometer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110165095A1 (en) * | 2008-03-26 | 2011-07-07 | Fundacao Sao Francisco Xavier | Composition and method for the inhibition of postoperative adhesions severity |
| CN102321569A (en) * | 2011-10-19 | 2012-01-18 | 山东省分析测试中心 | Method for constructing Kareius bicoloratus liver cell line |
| CN110437338A (en) * | 2019-08-08 | 2019-11-12 | 厦门大学 | A kind of preparation method of tumor vascular targeting magnetic nanometer accelerator protein |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL165392A0 (en) * | 2002-06-05 | 2006-01-15 | Genentech Inc | Compositions and methods for liver growth and liver protection |
| CN100460505C (en) * | 2005-11-04 | 2009-02-11 | 南通大学附属医院 | Method for culturing liver cell by polylactic acid-0-carboxymethyl shellglycan nano partical |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110165095A1 (en) * | 2008-03-26 | 2011-07-07 | Fundacao Sao Francisco Xavier | Composition and method for the inhibition of postoperative adhesions severity |
| US8889114B2 (en) * | 2008-03-26 | 2014-11-18 | Jackson Brandao Lopes | Composition and method for the inhibition of postoperative adhesions severity |
| US9226948B2 (en) | 2008-03-26 | 2016-01-05 | Jackson Brandao Lopes | Composition and method for the inhibition of postoperative adhesions severity |
| CN102321569A (en) * | 2011-10-19 | 2012-01-18 | 山东省分析测试中心 | Method for constructing Kareius bicoloratus liver cell line |
| CN110437338A (en) * | 2019-08-08 | 2019-11-12 | 厦门大学 | A kind of preparation method of tumor vascular targeting magnetic nanometer accelerator protein |
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