CN105754931A - Method for promoting human keratinocytes to activate TNF (tumor necrosis factor) signal pathways - Google Patents
Method for promoting human keratinocytes to activate TNF (tumor necrosis factor) signal pathways Download PDFInfo
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Abstract
The invention discloses a method for promoting human keratinocytes to activate TNF (tumor necrosis factor) signal pathways. The method comprises the step of a 2): isolated human keratinocytes are processed with SDS (sodium dodecyl sulfate), and in the step a 2), the isolated human keratinocytes are processed with SDS with the concentration of 0.550 mu g/mL; the isolated human keratinocytes are processed with SDS at the temperature of 37 DEG C for 1.5 h. The method further comprises the step of a 1): cell suspensions of the isolated human keratinocytes are inoculated to a cell culture plate for culture, and the step a 1) is performed before the step a 2); in the step a 1), the cell concentration of the cell suspensions is 19,000/cm<2>, and culture is performed at 37 DEG C for 24 h. Experiments prove that the method can promote the human keratinocytes to activate the TNF signal pathways and has important application value in various human diseases including tumors, inflammation and the like caused by TNF disorder.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method promoting human keratinized cell to activate tumor necrosis factor signal path.
Background technology
Tumor necrosis factor (tumornecrosisfactor, TNF) it is the class cytokine with multiple biological effect, it is by combining with the specific receptor on cell membrane, it is achieved promote that Growth of Cells, differentiation, tune are died and bring out the biological effects such as inflammation.TNF mediates cell effect widely in vivo, and its imbalance can cause the multiple human diseases such as tumor, inflammation.
TNF signal path is shown in Fig. 1.Gene in TNF signal path refers to table 1.
Gene in table 1.TNF signal path
Summary of the invention
The technical problem to be solved in the present invention is how to promote that human keratinized cell activates tumor necrosis factor signal path.
For solving the problems referred to above, present invention firstly provides a kind of method promoting human keratinized cell to activate tumor necrosis factor signal path, it may include following steps a2): process in vitro human keratinized cell with SDS.
Described step a2) in, the SDS that usable concentration is 0.400～0.600 μ g/mL processes described in vitro human keratinized cell.
Described step a2) in, the SDS that concrete usable concentration is 0.550 μ g/mL processes described in vitro human keratinized cell.
Described step a2) in, the conditional parameter processing in vitro human keratinized cell with SDS can be: 35～39 DEG C, 1h～2h.
Described step a2) in, can be the human keratinized cell in vitro with the medium treatment containing SDS with the SDS in vitro human keratinized cell of process.In the described culture medium containing SDS, the concentration of SDS can be 0.400～0.600 μ g/mL.In the described culture medium containing SDS, the concentration of SDS is 0.550 μ g/mL concretely.Described culture medium concretely DMEM culture medium.
Described SDS processes the in vitro conditional parameter concretely 37 DEG C of human keratinized cell, 1.5h.
Described SDS processes the in vitro conditional parameter concretely 37 DEG C of human keratinized cell, 1h.
Described SDS processes the in vitro conditional parameter concretely 37 DEG C of human keratinized cell, 2h.
Described method also comprises the steps: carrying out described step a2) front, carry out described step a1)；Described step a1) be: by the cell suspension inoculation of in vitro human keratinized cell to Tissue Culture Plate, cultivate.
Described step a2) can be: complete described step a1) after, take described Tissue Culture Plate, add SDS and cultivate.
Described step a1) in, in described cell suspension, the concentration of human keratinized cell can be 18000～20000/cm2。
Described step a1) in, the concentration of human keratinized cell concretely 19000/cm in described cell suspension2。
Described step a1) in, the condition of described cultivation can be: 35～39 DEG C, 18h～26h.
Described step a1) in, the condition of described cultivation concretely: 37 DEG C and 24h.
Described step a1) in, the inoculum concentration of described cell suspension can be 100 μ L/ holes.
In said method, described activation tumor necrosis factor signal path can be activate a1), a2), a3) or a4) in the expression of gene:
A1) CSF2 gene, CSF1 gene, CCL5 gene, IL6 gene, CXCL3 gene, FOS gene, PTGS2 gene, TNFAIP3 gene, LIF gene, MAP3K8 gene, MAPK12 gene and CCL20 gene；
A2) described CSF2 gene, described CSF1 gene, described CCL5 gene, described IL6 gene, described CXCL3 gene, described PTGS2 gene, described TNFAIP3 gene, tnf gene and described CCL20 gene；
A3) described FOS gene, described PTGS2 gene, described MAP3K8 gene and described CCL20 gene；
A4) in described CSF2 gene, described CSF1 gene, described CCL5 gene, described IL6 gene, described CXCL3 gene, described PTGS2 gene, described TNFAIP3 gene, described tnf gene, described CCL20 gene, described FOS gene, described LIF gene, described MAP3K8 gene and described MAPK12 gene appoint several or any one；
Wherein activate the expression of certain gene and refer to more than the twice that the expression of this gene after SDS processes is the expression without SDS this gene processed or less than 1/2nd times.
The GeneID of described CSF2 gene, described CSF1 gene, described CCL5 gene, described IL6 gene, described CXCL3 gene, described PTGS2 gene, described TNFAIP3 gene, described tnf gene, described CCL20 gene, described FOS gene, described LIF gene, described MAP3K8 gene and described MAPK12 gene is specifically shown in table 1.
The addition form of described SDS can be SDS solution.
Described SDS solution can be dissolve the SDS solution obtained by DMEM culture medium.
The preparation method of any of the above-described described cell suspension is specific as follows: with the in vitro human keratinized cell of 0.25% trypsinization, then resuspended by the DMEM culture medium containing 10% hyclone (percent by volume) and dilute.
The preparation method of any of the above-described described DMEM culture medium is specific as follows: by DMEM in high glucose solid medium (Gibco Products) 13.5g and NaHCO33.7g is dissolved in water successively, and pH is adjusted to 7.2～7.4, is then settled to 1L.
Any of the above-described described human keratinized cell can be people's epidermal keratinocyte.
It is demonstrated experimentally that method provided by the invention can promote that human keratinized cell activates tumor necrosis factor signal path, the multiple human diseases such as tumor, inflammation is caused to have important using value TNF imbalance.
Accompanying drawing explanation
Fig. 1 is TNF signal path figure.
Fig. 2 is the survival rate that mtt assay measures SDS solution-treated people's epidermal keratinocyte of variable concentrations.
Fig. 3 is the electrophoresis result of the total serum IgE extracting cell in embodiment 2.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention being further described in detail, the embodiment provided is only for illustrating the present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
Sodium lauryl sulphate (SDS) is SIGMA-ALOAICH Products, and catalog number is #74255；The structural formula of SDS is as follows:
People's epidermal keratinocyte is the rich grand safe Bioisystech Co., Ltd in Beijing product.Hyclone is Hyclone Products, and catalog number is SH30084.03.0.25% trypsin is Hyclone Products, and catalog number is SH30042.01.DMSO is sigma Products, and catalog number is D2650.PBS is hyclone Products, and article No. is SH30256.01B.EastepTM total RNA extraction reagent box is Pu Luomaige (Beijing) Bioisystech Co., Ltd product, and catalog number is CAT#LS1030.CRNA amplification label test kit is Boao Biological Co., Ltd's product, and catalog number is CapitaBio360061.DNA purification column is Germany's MACHEREY-NAGEL Products, and name of product isExtract II, catalog number is Cat.No.740609250.RNA purification column is Germany's MACHEREY-NAGEL Products, and name of product isRNAclean-up, catalog number is Cat.No.740948250.
DMEM culture medium: by DMEM in high glucose solid medium (Gibco Products) 3.5g and NaHCO33.7g is dissolved in water successively, and pH is 7.2～7.4, is settled to 1L, then uses 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 culture medium, is settled to 1L, then uses 0.22 μm of filtering with microporous membrane degerming, in 4 DEG C of preservations.
Embodiment 1, the best inoculating cell density determining people's epidermal keratinocyte and SDS concentration for the treatment of
Arranging test group, carry out three repeated trials, the step of each repeated trials is all as follows:
1, with 0.25% trypsinization people's epidermal keratinocyte, then resuspended by the DMEM culture medium containing 10% hyclone (percent by volume) and dilute, it is thus achieved that cell density is 15600/cm2Cell suspending liquid 1, cell density be 19000/cm2Cell suspending liquid 2, cell density be 22000/cm2Cell suspending liquid 3 and cell density be 25000/cm2Cell suspending liquid 4.
2, SDS is dissolved by DMEM culture medium, it is thus achieved that SDS solution.
3, taking 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, 37 DEG C, 5%CO are inoculated in every hole2Environment is cultivated 24h.
4, after completing step 3, take described 96 orifice plates, add the SDS solution of step 2 preparation, obtain system for handling, the concentration of the SDS in system for handling is 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 3 multiple holes), 5%CO2, 37 DEG C cultivate 44h, be subsequently adding 20 μ LMTT lysates, continue 5%CO2, 37 DEG C cultivate 4h.
5, after completing step 4, taking described 96 orifice plates, inhale the liquid abandoning in hole, then every hole adds PBS cleaning twice.
6, after completing step 5, taking described 96 orifice plates, inhale the liquid abandoning in hole, then every hole adds 150 μ LDMSO, adopts the light absorption value at microplate reader detection 570nm place.
Blank group is set: using the equal-volume DMEM culture fluid containing 10% hyclone (percent by volume) to replace cell suspending liquid, other operates same test group.Each SDS concentration arranges 3 multiple holes.
Cell controls group is set: replacing SDS solution by equal-volume DMEM culture medium, other operates same test group.Every kind of cell suspending liquid arranges 3 multiple holes.
With SDS concentration in system for handling for abscissa, the survival rate of people's epidermal keratinocyte is vertical coordinate, and the SDS comparing variable concentrations processes the survival rate of descendant's epidermal keratinocyte.
Experimental result is shown in Fig. 2 (in Fig. 2, SDS-15600 represent cell suspending liquid 1, SDS-19000 represents cell suspending liquid 2, SDS-22000 represents cell suspending liquid 3, SDS-25000 and represents cell suspending liquid 4) and table 2.It is shown that at cell density respectively 15600/cm2, 19000/cm2, 22000/cm2With 25000/cm2, when cell survival rate is 80%, SDS solution concentration in system for handling respectively 0.575 μ g/mL, 0.523 μ g/mL, 1.16 μ g/mL and 0.517 μ g/mL.Consider that SDS concentration in system for handling is relatively low and relatively low two factors of plating cells density, it is determined that the best inoculating cell density of people's epidermal keratinocyte is 19000/cm2, SDS concentration in system for handling is 0.523 μ g/mL (in practical application 0.400～0.600 μ g/mL).
The experimental result of table 2.SDS solution-treated people's epidermal keratinocyte
Embodiment 2, the optimization of process time
Carrying out three repeated trials, the step of each repeated trials is all as follows:
1, cell suspending liquid 2 prepared by embodiment 1 is inoculated in 6 orifice plates, 37 DEG C, 5%CO2Environment is cultivated 24h.
2, SDS is dissolved by DMEM culture medium, it is thus achieved that SDS solution.
3,6 orifice plates are taken: adding SDS solution in the 1st hole in 6 orifice plates, making the SDS concentration in system for handling is 0.550 μ g/mL, 5%CO2, 37 DEG C cultivate 0.5h, inhale and abandon upper strata culture medium, add the washing of appropriate PBS, suction is abandoned, and collects cell, called after cell A；Adding SDS solution in the 2nd hole in 6 orifice plates, making the SDS concentration in system for handling is 0.550 μ g/mL, 5%CO2, 37 DEG C cultivate 1h, inhale and abandon upper strata culture medium, add the washing of appropriate PBS, suction is abandoned, and collects cell, called after cell B；Adding SDS solution in the 3rd hole in 6 orifice plates, making the SDS concentration in system for handling is 0.550 μ g/mL, 5%CO2, 37 DEG C cultivate 1.5h, inhale and abandon upper strata culture medium, add the washing of appropriate PBS, suction is abandoned, and collects cell, called after cell C；Adding SDS solution in the 4th hole in 6 orifice plates, making the SDS concentration in system for handling is 0.550 μ g/mL, 5%CO2, 37 DEG C cultivate 2h, centrifugal, go upper strata culture medium, add the washing of appropriate PBS, suction is abandoned, and collects cell, called after cell D；Adding SDS solution in the 5th hole in 6 orifice plates, making the SDS concentration in system for handling is 0.550 μ g/mL, 5%CO2, 37 DEG C cultivate 4h, inhale and abandon upper strata culture medium, add the washing of appropriate PBS, suction is abandoned, and collects cell, called after cell E；By the 6th hole in 6 orifice plates, inhaling and abandon upper strata culture medium, add the washing of appropriate PBS, suction is abandoned, and collects cell, called after cell F.
4, extract the total serum IgE of cell (cell A, cell B, cell C, cell D, cell E or cell F) with EastepTM total RNA extraction reagent box, extraction step carries description by test kit to carry out.The electrophoresis result of the total serum IgE of each cell is shown in Fig. 3.
5, chip detection
(1) according toThe total serum IgE of the cell that the operating procedure markers step 4 of cRNA amplification label test kit is extracted.Specifically comprise the following steps that
The total serum IgE of the cell 1. extracted with step 4 is for template, with T7Oligo (dT) Primer for primer, uses 10 × FirstStrandEnzymeMix to synthesize FirststrandcDNA.
2. take described FirststrandcDNA, use SecondStrandEnzymeMix that the RNA chain in DNA RNA hybrid is converted into SecondStrandcDNA.
3. the SecondStrandcDNA 2. synthesized with step, for template, utilizes T7EnzymeMix to synthesize cRNA.
4. RNA purification column purification step (3) cRNA that synthesizes is used, to remove the reagent such as salt in reacting, enzyme.
5. with the cRNA after step 4. purification for template, with RandomPrimer for primer, utilize CbcScript II enzyme to carry out reverse transcription, obtain cDNA.
6. DNA purification column purification step (5) cDNA that obtains is used, to remove the reagent such as salt in reacting, enzyme.
7. with the cDNA after step 6. purification for template, with RandomPrimer for primer, KlenowFragment enzymatic synthesis cDNA complementary strand (there is in amplification system the dCTP of dCTP, Cy5 labelling of Cy3 labelling) is utilized.
8. the cDNA complementary strand that 7. obtains of DNA purification column purification step is used, to remove the reagent such as salt in reacting, enzyme.
In above-mentioned steps, T7Oligo (dT) Primer, 10 × FirstStrandEnzymeMix, SecondStrandEnzymeMix, T7EnzymeMix, RNA purification column, RandomPrimer, CbcScript II enzyme, DNA purification column and KlenowFragment enzyme areAssembly in cRNA amplification label test kit.
(2) the cDNA complementary strand that in step (1), 8. purification obtains is hybridized with chip of expression spectrum, then carry out chip scanning.Utilize the expression in cell (cell A, cell B, cell C, cell D, cell E or cell F) of each gene in TNF signal path described in software analysis table 1.
Experimental result is in Table 3, table 4 and table 5.It is shown that the expression of FOS gene, CCL5 gene and IL6 gene is in cell F more than the twice of the expression of corresponding gene or less than 1/2nd times in cell A；In cell B, the expression of CSF2 gene, CSF1 gene, CCL5 gene, IL6 gene, CXCL3 gene, PTGS2 gene, TNFAIP3 gene, tnf gene and CCL20 gene is in cell F more than the twice of the expression of corresponding gene or less than 1/2nd times；In cell C, the expression of CSF2 gene, CSF1 gene, CCL5 gene, IL6 gene, CXCL3 gene, FOS gene, PTGS2 gene, TNFAIP3 gene, LIF gene, MAP3K8 gene, MAPK12 gene and CCL20 gene is in cell F more than the twice of the expression of corresponding gene or less than 1/2nd times；In cell D, the expression of FOS gene, PTGS2 gene, MAP3K8 gene and CCL20 gene is in cell F more than the twice of the expression of corresponding gene or less than 1/2nd times；In cell E, the expression of CXCL2 gene, MAGI2 gene and BIRC3 gene is in cell F more than the twice of the expression of corresponding gene or less than 1/2nd times.Therefore, in cell A, cell B, cell C, cell D and cell E, the quantity of the gene differential expression of TNF signal path is followed successively by 3,9,12,4 and 3.
The above results shows, promotes that the optimum condition that human keratinized cell activates tumor necrosis factor signal path is: 19000/cm of inoculating cell density2, SDS concentration in system for handling be 0.550 μ g/mL and process time 1.5h.
The gene number gene that relative expression quantity changes in each cell in table 3.TNF signal path
The gene that relative expression quantity changes in each cell in table 4.TNF signal path
| The Gene ID of the gene that the relative expression quantity in TNF signal path changes | |
| Cell A | 2353 6352 3569 |
| Cell B | 1437 1435 6352 2921 3569 5743 7128 7124 6364 |
| Cell C | 1437 1435 6352 3569 2921 2353 5743 7128 3976 1326 6300 6364 |
| Cell D | 2353 5743 1326 6364 |
| Cell E | 2920 9863 330 |
Each gene in table 5.TNF signal path expression in cell
Claims (10)
1. the method promoting human keratinized cell to activate tumor necrosis factor signal path, comprises the steps a2): process in vitro human keratinized cell with SDS.
2. the method for claim 1, it is characterised in that: in described step a2), process described in vitro human keratinized cell with the SDS that concentration is 0.400～0.600 μ g/mL.
3. method as claimed in claim 2, it is characterised in that: in described step a2), process described in vitro human keratinized cell with the SDS that concentration is 0.550 μ g/mL.
4. the method as described in arbitrary in claims 1 to 3, it is characterised in that: in described step a2), the conditional parameter processing in vitro human keratinized cell with SDS is: 35～39 DEG C, 1h～2h.
5. method as claimed in claim 4, it is characterised in that: described conditional parameter is 37 DEG C, 1.5h.
6. such as the method as described in arbitrary in claim 1 to 5, it is characterised in that: described method also comprises the steps: carrying out described step a2) front, carry out step a1)；
Described step a1) be: by the cell suspension inoculation of in vitro Human keratinocytes to Tissue Culture Plate, cultivate.
7. method as claimed in claim 6, it is characterised in that: described step a2) be: complete step a1) after, take described Tissue Culture Plate, add SDS and cultivate.
8. method as claimed in claims 6 or 7, it is characterised in that: in described step a1), in described cell suspension, the concentration of human keratinized cell is 18000～20000/cm2。
9. method as claimed in claim 8, it is characterised in that: in described cell suspension, the concentration of human keratinized cell is 19000/cm2。
10. such as the method as described in arbitrary in claim 1 to 9, it is characterised in that: described activation tumor necrosis factor signal path is activate a1), a2), a3) or a4) in the expression of gene:
A1) CSF2 gene, CSF1 gene, CCL5 gene, IL6 gene, CXCL3 gene, FOS gene, PTGS2 gene, TNFAIP3 gene, LIF gene, MAP3K8 gene, MAPK12 gene and CCL20 gene；
A2) described CSF2 gene, described CSF1 gene, described CCL5 gene, described IL6 gene, described CXCL3 gene, described PTGS2 gene, described TNFAIP3 gene, tnf gene and described CCL20 gene；
A3) described FOS gene, described PTGS2 gene, described MAP3K8 gene and described CCL20 gene；
A4) in described CSF2 gene, described CSF1 gene, described CCL5 gene, described IL6 gene, described CXCL3 gene, described PTGS2 gene, described TNFAIP3 gene, described tnf gene, described CCL20 gene, described FOS gene, described LIF gene, described MAP3K8 gene and described MAPK12 gene appoint several or any one.
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| CN110607370A (en) * | 2019-10-10 | 2019-12-24 | 浙江大学 | Gene combination for human tumor molecular typing and application thereof |
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| CN105111300A (en) * | 2015-08-26 | 2015-12-02 | 中国检验检疫科学研究院 | Method for promoting human keratinocyte to express inflammatory factor IL-alpha |
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| CN105111300A (en) * | 2015-08-26 | 2015-12-02 | 中国检验检疫科学研究院 | Method for promoting human keratinocyte to express inflammatory factor IL-alpha |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110607370A (en) * | 2019-10-10 | 2019-12-24 | 浙江大学 | Gene combination for human tumor molecular typing and application thereof |
| CN110607370B (en) * | 2019-10-10 | 2021-03-26 | 浙江大学 | Gene combination for human tumor molecular typing and application thereof |
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