CN109536567B - Composition, reagent for detecting cell activity containing the composition, and method for detecting cell activity - Google Patents
Composition, reagent for detecting cell activity containing the composition, and method for detecting cell activity Download PDFInfo
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Abstract
The present invention relates to the field of biotechnology, and in particular, to a composition, a reagent for detecting cell activity containing the composition, and a method for detecting cell activity. Hair brushThe composition and reagent for detecting cell activity is used to make the liver cell growth promoting factor to show certain stimulation index. The presence of the (+ Gln) culture medium by replacing the RPMI-1640 culture medium with the RPMI-1640 culture medium (-Gln) can promote the growth and metabolism of cells and influence the action of the medicament per se. By changing the type of the detected serum, the hepatocyte growth-promoting factor shows obvious stimulation index. The fetal bovine serum of Gibco is replaced by the newborn calf serum of the ilex purpurea, and the stimulation index is improved from 1.2 to more than 4.6. The results of the experiments show that the cell concentration is 2.5X 104/mL～4.0×104The activity detection method that the hepatocyte growth-promoting factor enteric-coated capsule acts on L02 normal liver cells shows very high stimulation index after being cultured for 3.5 hours.
Description
Technical Field
The present invention relates to the field of biotechnology, and in particular, to a composition, a reagent for detecting cell activity containing the composition, and a method for detecting cell activity.
Background
In the aspect of biological activity detection, cancer cells have the capability of unlimited proliferation, good stability, difficult culture compared with normal cells and less passage times, so the cancer cells have wide application in the aspect of in vitro activity detection. In the aspect of in-vitro activity detection of liver-promoting drugs, most of the currently applied cells include SMMC7721, MHCC-97L, HepG2, Huh7, Bel7402 and the like, and the liver cancer cell genes have a large amount of mutations, and the differences of heredity, metabolic pathways, growth habits, cell morphology and the like from normal liver cells are large. Although liver cancer cells have great advantages in the aspect of biological activity detection, the effect of the drug on liver cancer cells does not necessarily indicate that the drug has an effect on normal liver cells, and liver cancer cells cannot well replace normal liver cells in the aspect of activity detection to indicate that a certain class of drugs has an effect on livers.
The L02 cell is a human normal hepatocyte growing adherently, is easy to culture, has good stability and more passage times, and is closer to normal liver tissue in the aspects of promoting cell proliferation, researching activity mechanism, regulating metabolism and the like compared with liver cancer cells, thereby illustrating the effect of the drug on liver.
The L02 cell has wide application in the aspect of researching liver function, and has related literature reports in the aspects of liver injury mechanism, hepatocyte apoptosis pathway, glycolipid metabolism and the like, but no research in the aspect of promoting liver drugs to promote hepatocyte proliferation is found.
Disclosure of Invention
In view of the above, the present invention provides a composition, a reagent for detecting cell activity comprising the composition, and a method for detecting cell activity. Appropriate cell lines were selected to study the effect of the drug on the liver. The L02 cell is a human normal hepatocyte growing adherently, the L02 normal hepatocyte is used for verifying the effect of the medicament on the liver, and a method for detecting the activity of the L02 normal hepatocyte is established.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a composition, which consists of an RPMI-1640 culture medium without glutamine and a new-born calf serum of ilex purpurea.
In some embodiments of the invention, the ratio of the volume of the ilex neogenesis bovine serum to the glutamine-free RPMI-1640 medium is 1: 9.
the invention also provides application of the composition in preparing a cell activity detection preparation.
In some embodiments of the invention, the cell is an L02 human normal hepatocyte.
In some embodiments of the invention, the cell is an L02 human normal hepatocyte after the action of a drug for the prevention and/or treatment of liver disease.
In some embodiments of the invention, the medicament is a hepatocyte growth-promoting factor enteric capsule.
On the basis, the invention also provides a detection reagent for cell activity, which comprises the composition and auxiliary materials available in the preparation.
The invention also provides a kit comprising the detection reagent.
In some embodiments of the invention, the kit further comprises trypsin-edetate disodium digest.
The invention also provides application of the kit in detecting cell activity.
In some embodiments of the invention, the cell is an L02 human normal hepatocyte.
In some embodiments of the invention, the cell is an L02 human normal hepatocyte after the action of a drug for the prevention and/or treatment of liver disease.
In some embodiments of the invention, the medicament is a hepatocyte growth-promoting factor enteric capsule.
The invention also provides a cell activity detection method, which comprises the steps of mixing the composition, the detection reagent or the detection reagent in the kit with cells to be detected, culturing and detecting activity.
In some embodiments of the invention, the number of cells in the cell culture fluid obtained by said mixing is 2.5X 104/mL～4.0×104mL, the incubation time was 3.5 h.
The invention provides a composition and a reagent for detecting cell activity, which are used for leading hepatocyte growth-promoting factor to show a certain stimulation index. The presence of the (+ Gln) culture medium by replacing the RPMI-1640 culture medium with the RPMI-1640 culture medium (-Gln) can promote the growth and metabolism of cells and influence the action of the medicament per se. By changing the type of the detected serum, the hepatocyte growth-promoting factor shows obvious stimulation index. The fetal bovine serum of Gibco is replaced by the newborn calf serum of the ilex purpurea, and the stimulation index is improved from 1.2 to more than 4.6. The results of the experiments show that the cell concentration is 2.5X 104/mL～4.0×104The activity detection method that the hepatocyte growth-promoting factor enteric-coated capsule acts on L02 normal liver cells shows very high stimulation index after being cultured for 3.5 hours.
Detailed Description
The invention discloses a composition, a cell activity detection reagent containing the composition and a cell activity detection method, and a person skilled in the art can use the content for reference and appropriately improve the process parameters to realize the detection. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The composition, the reagent for detecting cell activity containing the composition and the raw material and the reagent used in the method for detecting cell activity provided by the invention are all commercially available.
0.01mol/L phosphate buffer (pH7.3)
Dissolving 8.0g of sodium chloride, 0.2g of potassium chloride, 1.15g of disodium hydrogen phosphate and 0.2g of potassium dihydrogen phosphate in 1000ml of ultrapure water, sterilizing at 121 ℃ for 20min, and filtering for sterilization.
RPMI-1640 culture solution
RPMI-1640 medium (+ Gln) from Gibco
10% calf serum culture solution
Taking 45ml of the RPMI-1640 culture solution, and adding 5ml of subpackaged ilex purpurea Hassk calf serum.
RPMI-1640 detection liquid
RPMI-1640 medium (-Gln) from Gibco
10% calf serum detection liquid
Taking 45ml of the RPMI-1640 detection solution, and adding 5ml of the subpackaged ilex purpurea Hassk calf serum.
0.25% trypsin-disodium edetate digestive juice
Dissolving 0.25g trypsin and 0.02g disodium ethylene diamine tetraacetate in 100ml of 0.01mol/L phosphate buffer solution, adjusting pH to 7.2 with 5.6% sodium bicarbonate, filtering, sterilizing, and storing at-20 deg.C.
Thiazole blue (MTT) solution
Dissolving MTT50mg in 0.01mol/L phosphate buffer solution 10ml, dissolving in water bath at 60 deg.C, filtering and sterilizing. Subpackaging in dark place, storing in cold place, and using for no more than two weeks (dark bag or black paper, tin foil paper package)
Cleaning and sterilizing culture articles:
glass ware: the new ware is thoroughly cleaned by detergent and dried, and the used glassware is immediately immersed in clear water, so that the problem that the protein in the ware is adhered to the glass and is difficult to clean after being dried is avoided. When in soaking, the vessel is completely immersed in water, so that water enters the vessel without leaving bubble gaps. Thoroughly washing with detergent, and air drying.
And (3) soaking the dried vessel in chromic acid washing liquor overnight for at least 6 hours. When soaking, the inside of the utensil should be completely filled with the washing liquid without air bubbles.
Washing the soaked utensils: the first step, using running water (tap water) to wash thoroughly, filling and pouring, repeating more than ten times until the washing liquid is completely washed and no residue is left, then washing with purified water for 6 to 8 times, and drying in a drying oven.
And (3) sterilization: dry heat sterilization at 180 ℃ for 2 hours.
The used rubber plug and the used cover are soaked in clear water in time, then scrubbed by a detergent, soaked for 30min by 1% diluted hydrochloric acid, washed by tap water and washed by purified water for 4-6 times. And (5) drying. Wrapping with kraft paper.
And (3) sterilization: autoclaving at 121 deg.C for 20 min.
The invention is further illustrated by the following examples:
example 1 detection of cell Activity
The used sample is hepatocyte growth-promoting factor enteric-coated capsule, and the sample source is Hangzhou Huajin pharmaceutical industry GmbH.
Preparation of test solution A suitable amount of test substance (a drug for preventing and/or treating liver diseases, in this example, hepatocyte growth-promoting factor enteric-coated capsule) is diluted with water to 10mg/ml, filtered with a sterile filter head, and prepared into a solution containing 100 μ g of polypeptide per 1ml with RPMI-1640 detection solution.
And (3) determination:
before the test, the clean bench is wiped with 75% ethanol or 0.2% benzalkonium bromide, and the ultraviolet lamp of the clean bench is turned on for half an hour.
The RPMI-1640 culture solution (1) and the detection solution (2)1640, and the 0.25% trypsin-disodium edetate digestive juice (3) which are refrigerated in a refrigerator are put into a water bath kettle at 37 ℃ for rewarming and heating.
The pipette, the pipette tip, the 96-well cell culture plate, 0.01mol/L phosphate buffer (pH7.3), the syringe, the disposable 0.22 filter membrane and PBS are wiped by 75% ethanol and then placed on an ultra-clean workbench, and the ultraviolet lamp is turned on to irradiate for 30 min.
Entering a measuring room, wearing a mask, wearing a lab coat and wearing gloves. The gloves were wiped with 75% alcohol. Opening a carbon dioxide incubator, taking out a cell bottle, putting the cell bottle on an inverted microscope to see how the cells grow, preferably, the bottom of the bottle grows to be full of 80% (10% calf serum culture solution is required to culture the L-02 cells to a logarithmic growth period), discarding the culture solution, firstly washing the cells for 2 times by using 0.01mol/L phosphate buffer solution (PH7.3), digesting by using 0.25% trypsin-disodium ethylene diamine tetraacetate digestive juice (the digestion time is about 5min, and the bottom of the bottle is just meshed), adding 10% calf serum detection solution to terminate digestion, transferring the solution into a 15ml centrifuge tube, and centrifuging for 3 minutes at 1500 times. The supernatant was discarded and diluted to 1ml (2.5X 10) with 10% calf serum test solution4/mL～4.0×104and/mL) cells (sixteen medium squares are corresponding to four corners of a large square grid in the middle of the counting plate, and the sum of the cells at the four corners is 10-16), spreading the cell suspension on a 96-well cell culture plate, wherein 100 mu l of the cell suspension is kept in each well, 3 wells are reserved, 100 mu l of 10% calf serum detection solution is added as a blank control, and the cell suspension is cultured in a 5% carbon dioxide saturated water vapor incubator at 37 ℃ for 3.5 hours to be attached to the wall. Adding 100 μ l of test solution into each well of the test group after 3.5 hr, preparing 3 wells of each test, adding 100 μ l of RPMI-1640 detection solution into each well of the cell control group and the blank control group, culturing at 37 deg.C in 5% carbon dioxide saturated water vapor incubator for 44 hr, and collecting the culture solutionThe plate was removed, the culture medium was aspirated, and the plate was washed once with 0.01mol/L phosphate buffer (pH7.3) and then 100. mu.l of the above phosphate buffer (pH7.3) and 20. mu.l of MTT solution were added to each well, followed by further culture for 4 hours. After the culture was completed, the culture medium was discarded, 100. mu.l of dimethyl sulfoxide was added to each well, the mixture was shaken on a shaker at a low speed for 10min, and the absorbance A was measured at a wavelength of 570nm on a microplate reader.
And (3) calculating:
the preparation method comprises the following steps: 2 capsules (50mg of each polypeptide) were dissolved in 20ml of purified water, filtered, and prepared into a solution containing 100. mu.g of the polypeptide per 1ml using RPMI-1640 detection solution.
The results of the assay were as shown in the following table, according to the L02 method for detecting the activity of normal hepatocytes.
The results are shown in Table 1.
TABLE 1
The result shows that the stimulation index of the L02 normal liver cells is higher, and the method is feasible.
Example 2
The same samples were used to compare the changes in activity when the cell concentration was changed, while changing the cell concentration, keeping the other conditions of example 1 unchanged.
The results are shown in Table 2.
TABLE 2
By changing the cell concentration, the stimulation indexes are all above 3, and the L02 normal liver cells have better effect on the detection of the activity of liver-promoting medicaments.
Example 3
The medium used was changed while keeping the other conditions of example 1 unchanged.
The results are shown in Table 3.
TABLE 3
Note:*shows significant difference (P < 0.05) compared with the control group;#shows a very significant difference (P < 0.01) compared to the control group.
When the RPMI-1640 medium (+ Gln) is used, the cells grow fast, the absorbance values of the test sample and the control group are higher than that of the RPMI-1640 medium (-Gln), but the difference between the test sample and the control group of the RPMI-1640 medium (+ Gln) is not large, the stimulation index is lower, and the difference is significant (P is less than 0.05) compared with that of the RPMI-1640 medium (-Gln), so that the RPMI-1640 medium is selected as the medium.
Example 4
The other conditions of example 1 were kept constant, and serum used in the test medium was changed to a newborn calf serum of ilex purpurea and a fetal calf serum of Gibco, respectively.
The results are shown in Table 4.
TABLE 4
Note:*shows significant difference (P < 0.05) compared with the control group;#shows a very significant difference (P < 0.01) compared to the control group.
From Table 4, it can be seen that the fetal calf serum group stimulation index is smaller than that of the ilex purpurea Hassk newborn calf serum, and has significant difference (P is less than 0.05). The cells of the fetal calf serum cell control group and the test sample group grow faster, and the cells of the ilex purpurea Hassk calf serum cell control group grow slower, so that the hepatocyte growth promoting factor contains active substances which have the same effect with the nutrient components of fetal calf serum, and the active components which are lacked by the ilex purpurea Hassk newborn calf serum are compensated.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. The composition is applied to the preparation of a preparation for detecting the hepatocyte growth-promoting factor by utilizing the normal liver cells of the L02 human; the composition consists of an RPMI-1640 culture medium without glutamine and a new-born calf serum of the ilex purpurea; the volume ratio of the new-born calf serum of the ilex purpurea Hassk to the RPMI-1640 culture medium without glutamine is 1:9, and the number of the L02 human normal liver cells is 2.5 multiplied by 104/mL～4.0×104/mL。
2. The application of the kit in detecting the hepatocyte growth-promoting factor by using the normal liver cells of the L02 human; the kit comprises the composition of claim 1 and excipients useful in the formulation.
3. A method for detecting cell activity, which comprises mixing the composition of claim 1 or the kit of claim 2 with L02 human normal liver cells, wherein the number of cells is 2.5X 104/mL～4.0×104and/mL, culturing and detecting the activity of the L02 human normal liver cells.
4. The assay of claim 3, wherein the incubation time is 3.5 hours.
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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. |
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