CN105111300A - Method for promoting human keratinocyte to express inflammatory factor IL-alpha - Google Patents
Method for promoting human keratinocyte to express inflammatory factor IL-alpha Download PDFInfo
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- CN105111300A CN105111300A CN201510531599.6A CN201510531599A CN105111300A CN 105111300 A CN105111300 A CN 105111300A CN 201510531599 A CN201510531599 A CN 201510531599A CN 105111300 A CN105111300 A CN 105111300A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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Abstract
The invention discloses a method for promoting isolated human keratinocytes to express an inflammatory factor IL-alpha, which comprises the following step: a2) treating isolated human keratinocytes with SDS (sodium dodecylsulfate), wherein the concentration of the SDS for treating the isolated human keratinocytes is 0.357 mu g/ml, and the condition parameters for SDS treatment of the isolated human keratinocytes is 37 DEG C and 6 hours. The method also comprises the following step: a1): inoculating a cell suspension of the isolated human keratinocytes into a cell culture plate, and culturing, wherein the step a1) is performed before the step a2); and in the step a1), the cell concentration in the cell suspension is 19000/cm<2>, and the culture conditions are 37 DEG C and 24 hours. The experiment proves that the method disclosed by the invention can obviously promote the expression of the inflammatory factor IL-1alpha in the human epidermal keratinocytes, and thus, can be used for screening drugs for treating inflammation.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method that human keratinized cell expresses inflammatory factor IL-1 α that promotes.
Background technology
Inflammatory factor is small molecule polypeptide and glycoprotein, has to regulate and mediated immunity reacts, the effect of inflammatory reaction, comprises interleukin class, interferons, tumor necrosis factor subclass, somatomedin class and chemokine class.Produce a large amount of inflammatory factors after cell is upset, directly or indirectly affect lymphocyte or peripheral cell, play an important role in immunomodulatory, inflammatory reaction, apoptosis.
Epidermic cell or inoblast release inflammatory factor IL-1 α, be IL-1A or IL1F1 again, it and other 8 interleukin family genes are all positioned on the mankind's No. 2 karyomit(e), and it can in conjunction with cell surface receptor, performance immune inflammation regulates, and promotes the biological actions such as cytodifferentiation.
Keratinocyte secretion inflammatory factor interleukin-11 (IL-1 α) promotes fibroblasts to secrete IL-6 on the one hand, cause inoblast lots of genes to express to change, promote that neutrophil adhesion is on endotheliocyte on the other hand, the reaction thus stimulation body local is inflamed.
Summary of the invention
Technical problem to be solved by this invention promotes that human keratinized cell expresses inflammatory factor IL-1 α.
For solving the problem, the present invention provide firstly a kind of method that in vitro human keratinized cell expresses inflammatory factor IL-1 α that promotes, can comprise the steps a2): with the human keratinized cell that SDS process is in vitro.
In aforesaid method, described step a2) in, usable concentration is the described in vitro human keratinized cell of SDS process of 0.3-0.4 μ g/ml.
In aforesaid method, described step a2) in, concrete usable concentration is the described in vitro human keratinized cell of SDS process of 0.357 μ g/ml.
In aforesaid method, described step a2) in, can be with the conditional parameter of the in vitro human keratinized cell of SDS process: 35-39 DEG C, 5h-7h.
In aforesaid method, the conditional parameter of the human keratinized cell that described SDS process is in vitro specifically can be 37 DEG C and 6h.
In aforesaid method, also comprise the steps a1): by the cell suspension inoculation of in vitro human keratinized cell to Tissue Culture Plate, cultivate; Carrying out described step a2) frontly first carry out described step a1).
In aforesaid method, described step a2) can be: complete described step a1) after, get described Tissue Culture Plate, add SDS solution and cultivate.
In aforesaid method, described step a1) in, in described cell suspension, the concentration of human keratinized cell can be 18000-20000/cm
2.
In aforesaid method, in described cell suspension, the concentration of human keratinized cell specifically can be 19000/cm
2.
In aforesaid method, described step a1) in, the condition of described cultivation can be: 35-39 DEG C, 18h-26h.
In aforesaid method, described step a1) in, the condition of described cultivation specifically can be: 37 DEG C and 24h.
In aforesaid method, described step a1) in, the inoculum size of described cell suspension can be 100 μ L/ holes.
The gene order of above-mentioned arbitrary described inflammatory factor IL-1 α can as shown in sequence in sequence table 1.
Above-mentioned arbitrary described SDS solution can be and dissolves SDS acquisition with DMEM substratum.
The preparation method of above-mentioned arbitrary described cell suspension is specific as follows: with the human keratinized cell that 0.25% tryptic digestion is in vitro, then uses the DMEM substratum containing 10% foetal calf serum (volume percent) resuspended and dilutes.
The preparation method of above-mentioned arbitrary described DMEM substratum is specific as follows: by DMEM in high glucose solid medium (Gibco Products) 13.5g and NaHCO
33.7g is water-soluble successively, and pH is adjusted to 7.2-7.4, is then settled to 1L.
Above-mentioned arbitrary described human keratinized cell can be people's epidermal keratinocyte.
Experiment proves, method provided by the invention significantly can promote the expression of inflammatory factor IL-1 α in people's epidermal keratinocyte, thus may be used for the medicine screening treatment inflammation.
Accompanying drawing explanation
Fig. 1 is the survival rate that mtt assay measures the SDS solution-treated people epidermal keratinocyte of different concns.
Fig. 2 is the expression amount of inflammatory factor IL-1 α under the SDS solution different treatment time.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Quantitative experiment in following embodiment, all arranges three repetitions, results averaged if no special instructions.
People's epidermal keratinocyte is the rich grand safe Bioisystech Co., Ltd in Beijing product; SDS is sigma Products, and catalog number is L5750; Foetal calf serum is Hyclone Products, and catalog number is SH30084.03; 0.25% trypsinase is Hyclone Products, and catalog number is SH30042.01; DMSO is sigma Products, and catalog number is D2650.
DMEM substratum: by DMEM in high glucose solid medium (Gibco Products) 13.5g and NaHCO
33.7g is water-soluble successively, and pH is 7.2-7.4, is settled to 1L.Use 0.22 μm of filtering with microporous membrane degerming, in 4 DEG C of preservations.
MTT lysate: MTT (sigma Products) 5g is dissolved in above-mentioned DMEM substratum, is settled to 1L.Use 0.22 μm of filtering with microporous membrane degerming, in 4 DEG C of preservations.
PBS damping fluid: buy from hyclone company, article No. SH30256.01B.
Embodiment 1, the best inoculating cell density determining people's epidermal keratinocyte and SDS concentration for the treatment of
Arrange test group, carry out three revision tests, the step of each revision test is all as follows:
1, with 0.25% tryptic digestion people epidermal keratinocyte, then use the DMEM substratum containing 10% foetal calf serum (volume percent) resuspended and dilute, obtaining cell density is 15600/cm
2cell suspending liquid 1, cell density be 19000/cm
2(18000-20000/cm in practical application
2) cell suspending liquid 2, cell density be 22000/cm
2cell suspending liquid 3 and cell density be 25000/cm
2cell suspending liquid 4.
2, dissolve SDS with DMEM substratum, obtain SDS solution.
3, get 96 well culture plates, the cell suspending liquid (cell suspending liquid 1, cell suspending liquid 2, cell suspending liquid 3 or cell suspending liquid 4) that 100 μ L steps 1 obtain is inoculated in every hole, at 37 DEG C (in practical application 37 ± 2 DEG C), 5%CO
224h (in practical application 18h-26h) is cultivated in environment.
4, after completing steps 3, get described 96 orifice plates, add SDS solution prepared by step 2, obtain system for handling, the concentration of the SDS in system for handling be 0.05 μ g/mL, 0.15 μ g/mL, 0.46 μ g/mL, 1.37 μ g/mL, 4.11 μ g/mL, 12.35 μ g/mL, 37.03 μ g/mL, 111.1 μ g/mL, 333.3 μ g/mL or 1000 μ g/mL (each SDS concentration arranges 5 multiple holes).5%CO
2, 37 DEG C cultivate 44h (in practical application 40h-48h), then add 20 μ LMTT lysates, continue 5%CO
2, 37 DEG C cultivate 4h.
5, after completing steps 4, get described 96 orifice plates, inhale the liquid abandoned in hole, then every hole adds 150 μ LDMSO, adopts microplate reader to detect the light absorption value at 570nm place.
Blank group is set: with the DMEM nutrient solution replacement cell suspending liquid of equal-volume containing 10% foetal calf serum (volume percent), other operates same test group.Each SDS concentration arranges 5 multiple holes.
Cell controls group is set: replace SDS solution with equal-volume DMEM substratum, other operates same test group.Often kind of cell suspending liquid arranges 5 multiple holes.
With the concentration of SDS in system for handling for X-coordinate, the survival rate of people's epidermal keratinocyte is ordinate zou, compares the survival rate of the SDS process descendant epidermal keratinocyte of different concns.
Experimental result is shown in Fig. 1.15600/cm is respectively at cell density
2, 19000/cm
2, 22000/cm
2with 25000/cm
2, cell survival rate is under the condition of 85%, and the concentration of SDS solution in system for handling is respectively 0.432 μ g/ml, 0.357 μ g/ml, 0.96 μ g/ml, 0.286 μ g/ml.Consider lower and lower two factors of plating cells density of the concentration of SDS in system for handling, determine that the best inoculating cell density of people's epidermal keratinocyte is 19000/cm
2, the concentration of SDS in system for handling is 0.357 μ g/ml.
The optimization in embodiment 2, treatment time
Carry out three revision tests, the step of each revision test is all as follows:
1, cell suspending liquid 2 prepared by embodiment 1 is inoculated in 6 orifice plates, 37 DEG C, 5%CO
224h (in practical application 18h-26h) is cultivated in environment.
2, dissolve SDS with DMEM substratum, obtain SDS solution.
3, get 6 orifice plates: in the hole of first in 6 orifice plates, add SDS solution, make the SDS concentration in system for handling be 0.357 μ g/ml (in practical application 0.3-0.4 μ g/ml), 5%CO
2, 37 DEG C cultivate 1h, inhale and abandon upper strata substratum, add appropriate PBS buffer solution, suction is abandoned, and collects cell, called after cell A; In the hole of second in 6 orifice plates, add SDS solution, make the SDS concentration in system for handling be 0.357 μ g/ml (in practical application 0.3-0.4 μ g/ml), 5%CO
2, 37 DEG C cultivate 2h, inhale and abandon upper strata substratum, add appropriate PBS buffer solution, suction is abandoned, and collects cell, called after cell B; In the hole of the 3rd in 6 orifice plates, add SDS solution, make the SDS concentration in system for handling be 0.357 μ g/ml (in practical application 0.3-0.4 μ g/ml), 5%CO
2, 37 DEG C cultivate 4h, inhale and abandon upper strata substratum, add appropriate PBS buffer solution, suction is abandoned, and collects cell, called after cell C; In the hole of the 4th in 6 orifice plates, add SDS solution, make the SDS concentration in system for handling be 0.357 μ g/ml (in practical application 0.3-0.4 μ g/ml), 5%CO
2, 37 DEG C cultivate 6h (in practical application 5-7h), centrifugal, remove upper strata substratum, add appropriate PBS buffer solution, suction is abandoned, and collects cell, called after cell D; In the hole of the 5th in 6 orifice plates, add SDS solution, make the SDS concentration in system for handling be 0.357 μ g/ml (in practical application 0.3-0.4 μ g/ml), 5%CO
2, 37 DEG C cultivate 8h, inhale and abandon upper strata substratum, add appropriate PBS buffer solution, suction is abandoned, and collects cell, called after cell E; By the hole of the 6th in six orifice plates, inhale and abandon upper strata substratum, add appropriate PBS buffer solution, suction is abandoned, and collects cell, called after cell F.
4, with EastepTM total RNA extraction reagent box (Pu Luomaige (Beijing) Bioisystech Co., Ltd product, catalog number is CAT#LS1030) extract the total serum IgE of cell (cell A, cell B, cell C, cell D, cell E or cell F), extraction step carries specification sheets by test kit to carry out.
5, total serum IgE FastQuantRTKit (WithgDNase) (TIANGEN Biotech's product step 4 obtained, catalog number is KR106-01) carry out cDNA Article 1 chain synthesis reaction, obtain cDNA, add ddH to above-mentioned cDNA
2o dilutes, and obtains the template solution that cDNA concentration is 1000ng/ μ L.
6, fluorescent quantitative PCR:
The template solution obtained with step 5 is template, by the relative expression quantity (with GADPH gene for reference gene) of inflammatory factor IL-1 α in fluorescence quantitative PCR detection cell.The gene order of inflammatory factor IL-1 α is as shown in sequence in sequence table 1.
Identify that the primer of inflammatory factor IL-1 α is 5 '-AGTAGCAACCAACGGGAAGG-3 ' and 5 '-AAGGTGCTGACCTAGGCTTG-3 ', object fragment is if sequence in sequence table 1 is from shown in 5 ' end the 1179th to the 1306th.
Internal reference is house-keeping gene GADPH, and the primer of internal reference is actin-F:5 '-ACCACAGTCCATGCCATCAC-3 ' and actin-R:5 '-TCCACCACCCTGTTGCTGTA-3 '.
Wherein quantitative fluorescent PCR loading system is according to OneStep
primeScript
tMthe specification sheets loading of RT-PCRKitII (PerfectRealTime) (TaKaRa Products), reaction system is totally 20 μ l, 10.0 μ lMIX, 0.4 μ lDye II, 0.5 μ l forward primer (containing 5ng primer), 0.5 μ l reverse primer (containing 5ng primer), 1 μ l template solution and 7.6 μ lddH
2o.
Using the relative expression quantity of inflammatory factor IL-1 α in cell F as 1, in each cell (cell A, cell B, cell C, cell D or cell E), the relative expression quantity of inflammatory factor IL-1 α is shown in Fig. 2.
In cell A, cell B, cell C, cell D and cell E, the expression amount of inflammatory factor IL-1 α is respectively 0.89 times, 0.96 times, 0.92 times, 1.19 times and 1.13 times of the expression amount of inflammatory factor IL-1 α in cell F.Visible, the optimum handling time of SDS is 6h.
Result shows, the top condition improving the expression amount of inflammatory factor IL-1 α in people's epidermal keratinocyte is: inoculating cell density 19000/cm
2, the concentration of SDS in system for handling is 0.357 μ g/ml and treatment time 6h.
Claims (10)
1. promote that in vitro human keratinized cell expresses a method of inflammatory factor IL-1 α, comprises the steps a2): with the human keratinized cell that SDS process is in vitro.
2. the method for claim 1, is characterized in that: described step a2) in, be the described in vitro human keratinized cell of SDS process of 0.3-0.4 μ g/ml by concentration.
3. method as claimed in claim 2, is characterized in that: described step a2) in, be the described in vitro human keratinized cell of SDS process of 0.357 μ g/ml by concentration.
4., as the method as described in arbitrary in claims 1 to 3, it is characterized in that: described step a2) in, with the conditional parameter of the in vitro human keratinized cell of SDS process be: 35-39 DEG C, 5h-7h.
5. method as claimed in claim 4, is characterized in that: described conditional parameter is 37 DEG C and 6h.
6., as the method as described in arbitrary in claim 1 to 5, it is characterized in that: described method also comprises the steps a1): by the cell suspension inoculation of in vitro human keratinized cell to Tissue Culture Plate, cultivate;
Carrying out described step a2) frontly first carry out described step a1).
7. method as claimed in claim 6, is characterized in that: described step a2) be: completing steps a1), get described Tissue Culture Plate, add SDS solution and cultivate.
8. method as claimed in claims 6 or 7, is characterized in that: described step a1) in, in described cell suspension, the concentration of human keratinized cell is 18000-20000/cm
2.
9. method as claimed in claim 8, is characterized in that: in described cell suspension, the concentration of human keratinized cell is 19000/cm
2.
10., as the method as described in arbitrary in claim 1 to 9, it is characterized in that: the gene order of described inflammatory factor IL-1 α is as shown in sequence in sequence table 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105368900A (en) * | 2015-12-11 | 2016-03-02 | 中国检验检疫科学研究院 | Method capable of promoting human keratinocytes to express inflammatory factor IL-8 |
| CN105754931A (en) * | 2016-03-25 | 2016-07-13 | 中国检验检疫科学研究院 | Method for promoting human keratinocytes to activate TNF (tumor necrosis factor) signal pathways |
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Cited By (2)
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| CN105368900A (en) * | 2015-12-11 | 2016-03-02 | 中国检验检疫科学研究院 | Method capable of promoting human keratinocytes to express inflammatory factor IL-8 |
| CN105754931A (en) * | 2016-03-25 | 2016-07-13 | 中国检验检疫科学研究院 | Method for promoting human keratinocytes to activate TNF (tumor necrosis factor) signal pathways |
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Application publication date: 20151202 |