CN109536567A - The detection method of composition, the detection reagent of cell activity containing the composition and cell activity - Google Patents
The detection method of composition, the detection reagent of cell activity containing the composition and cell activity Download PDFInfo
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- CN109536567A CN109536567A CN201811549285.9A CN201811549285A CN109536567A CN 109536567 A CN109536567 A CN 109536567A CN 201811549285 A CN201811549285 A CN 201811549285A CN 109536567 A CN109536567 A CN 109536567A
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- 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
- G01N33/5067—Liver cells
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- 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
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Abstract
The present invention relates to field of biotechnology, in particular to the detection method of composition, the detection reagent of cell activity containing the composition and cell activity.The composition and reagent of cytoactive detection provided by the invention, with making hepatocyte growth promoting factor show certain stimulus index.RPMI-1640 culture medium (+Gln) is substituted with RPMI-1640 culture medium (- Gln), the presence of+Gln can promote the growth metabolism of cell, influence the effect of drug itself.By changing the type of detection serum, hepatocyte growth promoting factor is made to show apparent stimulus index.With the fetal calf serum of the newborn bovine serum substitution Gibco of Chinese holly, stimulus index is promoted from 1.2 to 4.6 or more.The experimental results showed that cell concentration is 2.5 × 104/ mL~4.0 × 104/ mL is cultivated 3.5 hours, and the activity test method that hepatocyte growth-promoting factor enteric-coated capsule acts on L02 normal liver cell shows very high stimulus index.
Description
Technical field
The present invention relates to field of biotechnology, in particular to the detection examination of composition, cell activity containing the composition
The detection method of agent and cell activity.
Background technique
In bioactivity context of detection, since cancer cell has the ability of infinite multiplication, and stability is preferable, relative to just
Normal cell difficulty culture, passage number are few, therefore it has a wide range of applications in terms of Activity determination in vitro.Promoting liver class drug body
In terms of outer Activity determination, the more cell applied at present has SMMC7721, MHCC-97L, HepG2, Huh7, Bel7402 etc.
Deng, these liver cancer cells genes, there is a large amount of mutation, and in heredity, metabolic pathway, habit, cellular morphology etc. is all
Differ larger with normal liver cell.Although liver cancer cells have big advantage in bioactivity context of detection, drug is to liver
Cancer cell may not be able to illustrate that it has effect to normal liver cell with effect, and liver cancer cells are not in terms of Activity determination
Normal liver cell can be substituted well to illustrate effect that a certain class drug has liver.
L02 cell is a kind of Human normal hepatocyte of adherent growth, is easy culture, stability is good, and passage number is more, phase
Than in liver cancer cells promote cell Proliferation, activity mechanism research, in terms of closer to normal hepatic tissue, from
And illustrate effect of the drug to liver.
L02 cell is widely used in terms of studying liver function, in hepatic injury mechanism, hepatocellular apoptosis access, glycolipid metabolism
Etc. have pertinent literature report, but have no promote liver class drug promote hepatocyte growth in terms of research.
Summary of the invention
In view of this, the present invention provides the detection reagent and cell of a kind of composition, cell activity containing the composition
Active detection method.The effect for having selected suitable cell strain to study drug to liver.L02 cell is a kind of adherent growth
Human normal hepatocyte, effect of the drug to liver is verified with L02 normal liver cell, and it is normal to establish a kind of suitable L02
The activity test method of liver cell.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of compositions, by the new life of RPMI-1640 culture medium and Chinese holly without glutamine
Cow's serum composition.
In some specific embodiments of the invention, the newborn bovine serum of Chinese holly and the RPMI- for being free of glutamine
The volume ratio of 1640 culture mediums is 1:9.
The present invention also provides application of the composition in the detection preparation of preparation cell activity.
In some specific embodiments of the invention, the cell is L02 Human normal hepatocyte.
In some specific embodiments of the invention, the cell is the drug work of prevention and/or treatment liver diseases
L02 Human normal hepatocyte after.
In some specific embodiments of the invention, the drug is hepatocyte growth-promoting factor enteric-coated capsule.
On this basis, the present invention also provides the detection reagents of cell activity, including in the composition and preparation
Available auxiliary material.
The present invention also provides kits, including the detection reagent.
In some specific embodiments of the invention, the kit further includes trypsase-ethylenediaminetetraacetic acid two
Sodium digestive juice.
The present invention also provides application of the kit in detection cell activity.
In some specific embodiments of the invention, the cell is L02 Human normal hepatocyte.
In some specific embodiments of the invention, the cell is the drug work of prevention and/or treatment liver diseases
L02 Human normal hepatocyte after.
In some specific embodiments of the invention, the drug is hepatocyte growth-promoting factor enteric-coated capsule.
The present invention also provides a kind of detection method of cell activity, take the composition, the detection reagent or
Detection reagent and mixing with cells to be measured in the kit, culture, detection activity.
In some specific embodiments of the invention, it is described mixing obtain cell culture fluid in cell number be 2.5 ×
104/ mL~4.0 × 104/ mL, the time of the culture are 3.5h.
The present invention provides a kind of composition of cytoactive detection and reagent, with making hepatocyte growth promoting factor show one
Fixed stimulus index.RPMI-1640 culture medium (+Gln) is substituted with RPMI-1640 culture medium (- Gln), the presence of+Gln can promote
Into the growth metabolism of cell, the effect of drug itself is influenced.By changing the type of detection serum, make hepatocyte growth promoting factor table
Reveal apparent stimulus index.With the fetal calf serum of the newborn bovine serum substitution Gibco of Chinese holly, stimulus index is promoted from 1.2
To 4.6 or more.The experimental results showed that cell concentration is 2.5 × 104/ mL~4.0 × 104/ mL is cultivated 3.5 hours, hepatocyte
The activity test method that auxin capsulae enterosolubilis acts on L02 normal liver cell shows very high stimulus index.
Specific embodiment
The invention discloses a kind of composition, the inspection of the detection reagent and cell activity of the cell activity containing the composition
Survey method, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.Method and application of the invention is described by preferred embodiment, and related personnel can obviously not depart from this hair
Method described herein and application are modified or appropriate changes and combinations in bright content, spirit and scope, to realize and answer
Use the technology of the present invention.
The detection side of composition provided by the invention, the detection reagent of cell activity containing the composition and cell activity
Raw materials used and reagent is available on the market in method.
0.01mol/L phosphate buffer (pH7.3)
Sodium chloride 8.0g, potassium chloride 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g are taken, ultrapure water 1000ml is molten
Xie Hou, 121 DEG C of sterilizing 20min filtration sterilizations.
RPMI-1640 culture solution
The RPMI-1640 culture medium (+Gln) of Gibco
10% calf serum culture solution
Above-mentioned RPMI-1640 culture solution 45ml is taken, the Chinese holly calf serum 5ml dispensed is added.
RPMI-1640 detects liquid
The RPMI-1640 culture medium (- Gln) of Gibco
10% calf serum detects liquid
Above-mentioned RPMI-1640 detection liquid 45ml is taken, the Chinese holly calf serum 5ml dispensed is added.
0.25% trypsase-Calcium Disodium Versenate digestive juice
0.25g trypsase and 0.02g Calcium Disodium Versenate are taken, adds 0.01mol/L phosphate buffer 1 00ml molten
Xie Hou adjusts pH value to 7.2 with 5.6% sodium bicarbonate, and filtration sterilization, -20 DEG C save.
Thiazolyl blue (MTT) solution
MTT50mg is taken, after adding 0.01mol/L phosphate buffer 1 0ml to dissolve, 60 DEG C of water-bath hydrotropies, filtration sterilization.Point
Dress is kept in dark place in Leng Chu, uses no more than two weeks (be protected from light bag or black paper, masking foil encase)
Cultivate the cleaning and sterilizing of articles:
Glassware: new vessel are first thoroughly cleaned with detergent, are dried, and the glassware after use should immerse clearly immediately
In water, it is adhered on glass and is difficult to clean after avoiding vessel internal protein from drying up.Vessel should be completely immersed in water, be made when immersion
Water enters in vessel and bubble-free gap is left.It is thoroughly cleaned, is dried with detergent.
Vessel after drying are put into soaked overnight in chromic acid lotion, minimum to be not less than 6 hours.It should will be inside vessel when immersion
It is completely filled with washing lotion, does not stay bubble.
Vessel cleaning after immersion: the first step is filled, is outwelled using flowing water (tap water) cleaning down, must be repeated ten times
More than, until washing lotion all rinses well, until not staying any vestiges, then rinsed 6 to 8 times with purified water, drying in oven.
Sterilizing: 180 DEG C of hot air sterilization 2 hours.
The sundries such as rubber plug and lid cannot be impregnated with washing lotion, and new is first rinsed well with tap water, then be carried out conventional clear
It washes, the rubber plug, lid after use should be immersed in clear water in time, then be scrubbed with detergent, then impregnated with 1% dilute hydrochloric acid
30min, tap water rinse, then are rinsed 4 to 6 times with purified water.It dries.It is wrapped with brown paper.
Sterilizing: 121 DEG C of 20min of high pressure sterilization.
Below with reference to embodiment, the present invention is further explained:
The detection of 1 cell activity of embodiment
Sample used is hepatocyte growth-promoting factor enteric-coated capsule, and sample source is Hangzhou Huajin Pharmaceutical Co., Ltd..
The preparation of test solution takes test sample (to prevent and/or treat the drug of liver diseases, be to promote liver in the present embodiment
Archusia capsulae enterosolubilis) in right amount, it is diluted with water to 10mg/ml, after the filtering of aseptic filtration head, is detected with RPMI-1640
The solution of the 100 μ g containing polypeptide in every 1ml is made in liquid.
Measurement:
Superclean bench is wiped with 75% ethyl alcohol or 0.2% bromogeramine before test, then opens the ultraviolet of superclean bench
Lamp half an hour.
(1) the RPMI-1640 culture solution (2) 1640 of refrigeration in refrigerator is detected (3) 0.25% trypsase of liquid-second
Four acetic acid disodium digestive juice of diamines is placed on rewarming in 37 DEG C of water-bath and heats.
Liquid-transfering gun, pipette tips, 96 porocyte culture plates, 0.01mol/L phosphate buffer (pH7.3), syringe and disposable
0.22 filter membrane and PBS is placed on superclean bench after 75% ethanol, opens ultraviolet light irradiation 30min.
Into measuring cell, band muzzles, and puts on lab-gown, wears gloves.With 75% alcohol wipe gloves.Open titanium dioxide
Carbon incubator, takes out cell bottle, is put on inverted microscope and how sees cell growth, and bottom of bottle covers with 80% and is preferred (it is required that using
10% calf serum culture solution culture L-02 cell is to increased logarithmic phase), culture solution is discarded, it is first slow with 0.01mol/L phosphate
Fliud flushing (PH7.3) is cleaned cell 2 times, and with the digestion of 0.25% trypsase-Calcium Disodium Versenate digestive juice, (digestion time is big
About 5min, bottom of bottle occur netted) add the detection liquid termination digestion of 10% calf serum to be transferred in 15ml centrifuge tube 1500 turns
Centrifugation 3 minutes.Supernatant is abandoned, 10% calf serum detection liquid is added and is diluted in every 1ml containing (2.5 × 104/ mL~4.0 × 104/
ML) (the block plaid quadrangle among tally is corresponding with 16 medium squares to a cell, and amounting to the sum of four horn cells is 10~16
It is a), by above-mentioned cell suspension in 96 porocyte culture plates bed board, every 100 μ l of hole, wherein 3 holes is stayed to add 10% small ox blood
Clear detection 100 μ l of liquid sets 37 DEG C, cultivating 3.5 hours in 5% carbon dioxide saturation vapour incubator makes its patch as blank control
Wall.Test sample group after 3.5 hours, every hole add 100 μ l of test solution, and every batch of test sample does 3 holes, cell controls group and blank
The every hole of control group adds RPMI-1640 to detect 100 μ l of liquid respectively, sets 37 DEG C, cultivates 44 in 5% carbon dioxide saturation vapour incubator
Hour, terminate to take out culture plate after cultivating, suck culture solution, every hole is added 0.01mol/L phosphate buffer (pH7.3) and washes one
It is secondary, 100 μ l and MTT solution of above-mentioned phosphate buffer (pH7.3), 20 μ l is then added in every hole again, continues to cultivate 4h.Training
After supporting, culture solution to the greatest extent is abandoned, 100 μ l dimethyl sulfoxides are added in every hole, and low speed shakes up 10min on shaking table, in microplate reader
Its trap A value is measured at the wavelength of 570nm respectively.
It calculates:
Preparation method: taking 2 capsules (50mg polypeptide every), and 20ml purified water, which is added, to be made to dissolve, and after filtering, uses RPMI-
The solution of the 100 μ g containing polypeptide in every 1ml is made in 1640 detection liquid.
It is detected according to L02 normal liver cell activity test method, result such as following table.
Experimental result is shown in Table 1.
Table 1
The result shows that the stimulus index of L02 normal liver cell is higher, this method is feasible.
Embodiment 2
Change cell concentration, keeps other conditions of embodiment 1 constant, with the same sample, compare when cell concentration changes
Its active variation when change.
Experimental result is shown in Table 2.
Table 2
By changing cell concentration, for stimulus index 3 or more, the L02 normal liver cell is living for promoting liver class drug
Property detection have preferable effect.
Embodiment 3
It keeps other conditions of embodiment 1 constant, changes the culture medium used.
Experimental result is shown in Table 3.
Table 3
Note:*Show that compared with the control group there is significant difference (P < 0.05)；#Show has extremely significant difference compared with the control group
(P < 0.01).
When with RPMI-1640 culture medium (+Gln), cell growth is fast, and the absorbance value of test sample and control group is higher than
RPMI-1640 culture medium (- Gln), but gap is little between the test sample and control group of RPMI-1640 culture medium (+Gln), thorn
It is lower to swash index, there is significant difference (P < 0.05) compared with RPMI-1640 culture medium (- Gln), therefore culture medium is selected
RPMI-1640 culture medium (- Gln).
Embodiment 4
It keeps other conditions of embodiment 1 constant, changes serum used in detection culture medium, use the new of Chinese holly respectively
The fetal calf serum of raw cow's serum and Gibco.
Experimental result is shown in Table 4.
Table 4
Note:*Show that compared with the control group there is significant difference (P < 0.05)；#Show has extremely significant difference compared with the control group
(P < 0.01).
Fetal calf serum group stimulus index is smaller than Chinese holly newborn bovine serum as can be known from Table 4, has significant difference (P <
0.05).All long very fast of fetal calf serum group cell controls group and test sample group cell, Chinese holly calf serum cell controls group is thin
Intracellular growth is slower, shows that hepatocyte growth promoting factor contains some active materials and plays imitate consistent with the nutritional ingredient of fetal calf serum
Fruit compensates for the active constituent that Chinese holly newborn bovine serum lacks.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. composition, which is characterized in that by the newborn bovine serum group of RPMI-1640 culture medium and Chinese holly without glutamine
At.
2. composition as described in claim 1, which is characterized in that the newborn bovine serum of Chinese holly with without glutamine
The volume ratio of RPMI-1640 culture medium is 1:9.
3. application of the composition as claimed in claim 1 or 2 in the detection preparation of preparation cell activity.
4. application as claimed in claim 3, which is characterized in that the cell is the drug of prevention and/or treatment liver diseases
L02 Human normal hepatocyte after effect.
5. the detection reagent of cell activity, which is characterized in that including available in composition as described in claim 1 and preparation
Auxiliary material.
6. kit, which is characterized in that including detection reagent as claimed in claim 5.
7. application of the kit as claimed in claim 6 in detection cell activity.
8. the use as claimed in claim 7, which is characterized in that the cell is the drug of prevention and/or treatment liver diseases
L02 Human normal hepatocyte after effect.
9. a kind of detection method of cell activity, which is characterized in that composition as claimed in claim 1 or 2, such as right is taken to want
Detection reagent described in asking 5 or the detection reagent in kit as claimed in claim 6 and mixing with cells to be measured, are cultivated, inspection
Survey activity.
10. detection method as claimed in claim 9, which is characterized in that cell number in the cell culture fluid that the mixing obtains
It is 2.5 × 104/ mL~4.0 × 104/ mL, the time of the culture are 3.5h.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811549285.9A CN109536567B (en) | 2018-12-18 | 2018-12-18 | Composition, reagent for detecting cell activity containing the composition, and method for detecting cell activity |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811549285.9A CN109536567B (en) | 2018-12-18 | 2018-12-18 | Composition, reagent for detecting cell activity containing the composition, and method for detecting cell activity |
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| CN109536567B CN109536567B (en) | 2022-04-22 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101226138A (en) * | 2008-02-04 | 2008-07-23 | 杭州华锦药业有限公司 | Method for detecting activity of hepatocyte auxin |
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2018
- 2018-12-18 CN CN201811549285.9A patent/CN109536567B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101226138A (en) * | 2008-02-04 | 2008-07-23 | 杭州华锦药业有限公司 | Method for detecting activity of hepatocyte auxin |
Non-Patent Citations (1)
| Title |
|---|
| 陈男 等: "促肝细胞生长素对H2O2损伤人LO2细胞的保护作用和机制探究", 《海峡药学》 * |
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Effective date of registration: 20230828 Address after: No. 156, Guagang 1st Road, Guali Town, Xiaoshan District, Hangzhou City, Zhejiang Province, 311241 Patentee after: HANGZHOU HUAJIN PHARMACEUTICAL Co.,Ltd. Address before: 313300 No. 296 Xigang Road, Anji District, Huzhou Interprovincial Industrial Transfer Demonstration Zone, Huzhou City, Zhejiang Province Patentee before: ZHEJIANG HUADI PHARMACEUTICAL GROUP Co.,Ltd. |