CN108588168A - A kind of method that wogonin inhibits SGK1 regulation and control non-small cell lung cancer cell destiny - Google Patents
A kind of method that wogonin inhibits SGK1 regulation and control non-small cell lung cancer cell destiny Download PDFInfo
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- CN108588168A CN108588168A CN201810447557.8A CN201810447557A CN108588168A CN 108588168 A CN108588168 A CN 108588168A CN 201810447557 A CN201810447557 A CN 201810447557A CN 108588168 A CN108588168 A CN 108588168A
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Abstract
The invention discloses a kind of method that wogonin inhibits SGK1 regulation and control non-small cell lung cancer cell destiny, step includes：Human non-small cell lung cancer's cell culture, cell passage, cell viability analysis, cell ageing detection, Apoptosis detection and cell cycle detection；The beneficial effects of the present invention are, pass through different wogonin medications, it can induce the different cell states that non-small cell lung cancer cell enters cell-cycle arrest, cell ageing and Apoptosis, therefore tumour cell can be transferred to another destiny from a kind of destiny, to reduce the toxic side effect blindly come using high-dose chemotherapy medicine band, reference frame is provided for clinical rational drug use.
Description
Technical field
The present invention relates to biotechnologies more particularly to a kind of wogonin to inhibit SGK1 regulation and control non-small cell lung cancers thin
The method of born of the same parents' destiny.
Background technology
The dynamic behavior of p53 albumen have proven to be determine cell fate key factor, as Cyclin-dependent kinase,
Cell ageing, apoptosis etc..There are a negative-feedback regu- lation access, SGK1 expression quantity and p53 eggs between intracellular SGK1 and p53
White level is closely related.The expression of so intracellular SGK1 albumen there is also from p53 fluctuate it is similar can result in it is different thin
The dynamic phenomena of born of the same parents' destiny, influencing the expression of SGK1 also being capable of correspondingly modulate tumor cell fate.
Invention content
The purpose of the present invention：It provides and rational administrated method is found according to the case where tumour, it can ensure that being used twice
The concentration of wogonin slightly above can enter cell ageing with inducing cell in cell or tissue from cell-cycle arrest between medicine
State, or induction tumor cell senescence enter apoptosis status.
To achieve the goals above, the technical scheme is that：A kind of wogonin inhibition SGK1 regulation and control non-small cells
The method of lung carcinoma cell destiny, includes the following steps：
Step 1：Human non-small cell lung cancer's cell culture：Human non-small cell lung cancer's cell culture is contained in addition 3ml
In the RPmI-1640 complete mediums of 10%FBS, RPmI-1641 medium cultures are in 37 DEG C, training that CO2 volumetric concentrations are 5%
It supports in case；
Step 2：Cell passes on：As cell growth fusion degree 80%-90% or so, after Tissue Culture Flask alcohol wipe
Super-clean bench is moved into, old culture solution is discarded, cell is washed twice with PBS buffer solution flushing；After the digestion of 1ml pancreatin is added, in microscope
Lower observation cellular morphology.When cell volume diminution is rounded, gap becomes larger, the RPmI-1640 trainings that 3ml contains 10%FBS are added
Support base；
Step 3：Cell viability is analyzed：The cell of exponential phase is collected, cell density is adjusted, about 5000 are added per hole
96 orifice plates are added in cell, per 100 μ l of hole；When cell fusion degree reaches 80-90%, the drug final concentration point of wogonin is added
It Wei not be 0,5,10,20,40,80 μm；Wogonin acts on 12h and takes out cell, and 10 μ l CCK8 solution are added per hole, after cultivating 2h,
Absorbance (OD) value with multi-function microplate reader in each hole of 450nm wavelength measurements；3 multiple holes of every group of setting repeat experiment 3 times simultaneously
It is averaged；
Step 4：Cell ageing detects：The cell for collecting logarithmic phase growth, is inoculated in six orifice plates, cell fusion degree reaches
Start the wogonin that basal medium dissolving is added by different time points when 80-90%, preparation of samples is completed.Absorb cell training
Nutrient solution is washed 2 times with PBS, and 1ml beta galactosidases are added and dye fixer, room temperature fixes 15 minutes；Dyeing is absorbed to fix
Liquid is washed 3 times, every time 3 minutes with PBS；PBS is absorbed, 1ml is added per hole and dyes working solution.With ParafilmTM 6 orifice plates postposition
In 37 DEG C of incubations；Finally, the aging aspects of cell are observed using ordinary optical microscope；
Step 5：Apoptosis detects：Cell culture medium is sucked out to 15ml centrifuge tubes, and PBS washing attached cells are primary,
0.5ml pancreatin cell dissociation cells are added；Cell suspension is added to the cell culture fluid 1000g collected in step 1 and centrifuges 5min,
Cell is gently resuspended using PBS and counts；It takes the cell of 100000 resuspensions, 1000g to centrifuge 5min, abandons supernatant, be added 195
Cell is gently resuspended in μ l Annexin V-FITC combination buffers；5 μ l Annexin V-FITC liquid blendings are added；It is added 10
μ l propidium iodide stain liquid mixings；Room temperature, which is protected from light, is incubated 20min, is subsequently placed in ice bath.And use flow cytomery pair
Cell carries out withered detection；
Step 6：Cell cycle is detected：It collects spare in cell culture fluid a to centrifuge tube.It is digested using 0.5% pancreatin
Cell is added cell culture fluid and blows and beats lower whole attached cell；Cell is resuspended and collects again into centrifuge tube；1000g
Left and right rotating speed carries out centrifugation in 3-5 minutes with sedimentation cell；The PBS of about 1ml ice baths precooling is added, cell is resuspended, and be transferred to
In the centrifuge tube of 1.5ml；70% ethyl alcohol that 1ml ice baths are pre-chilled is added in cell, and gently piping and druming is uniform, and 4 DEG C of fixations about 2 are small
When；The propidium iodide stain liquid of 0.5ml is added in each solencyte sample, 37 DEG C of incubators are protected from light warm bath 30 minutes；It is thin with streaming
Born of the same parents' instrument detects red fluorescence at excitation wavelength 488nm wavelength, while detecting light scattering situation, using modifit analysis softwares
Carry out cell DNA content analysis and light-scattering analysis.
In conclusion the beneficial effects of the present invention are by different wogonin medications, can induce non-small thin
Born of the same parents' lung carcinoma cell enters the different cell states of cell-cycle arrest, cell ageing and Apoptosis, therefore tumour cell can be with
It is transferred to another destiny from a kind of destiny, to reduce the toxic side effect blindly come using high-dose chemotherapy medicine band, to face
The bed rational use of medicines provides reference frame.
Specific implementation mode
The technical solution further illustrated the present invention in conjunction with specific embodiments.
A kind of method that wogonin inhibits SGK1 regulation and control non-small cell lung cancer cell destiny, includes the following steps：
Step 1：Human non-small cell lung cancer's cell culture：Human non-small cell lung cancer's cell culture is contained in addition 3ml
In the RPmI-1640 complete mediums of 10%FBS, RPmI-1641 medium cultures are in 37 DEG C, training that CO2 volumetric concentrations are 5%
It supports in case；
Step 2：Cell passes on：As cell growth fusion degree 80%-90% or so, after Tissue Culture Flask alcohol wipe
Super-clean bench is moved into, old culture solution is discarded, cell is washed twice with PBS buffer solution flushing；After the digestion of 1ml pancreatin is added, in microscope
Lower observation cellular morphology.When cell volume diminution is rounded, gap becomes larger, the RPmI-1640 trainings that 3ml contains 10%FBS are added
Support base；
Step 3：Cell viability is analyzed：The cell of exponential phase is collected, cell density is adjusted, about 5000 are added per hole
96 orifice plates are added in cell, per 100 μ l of hole；When cell fusion degree reaches 80-90%, the drug final concentration point of wogonin is added
It Wei not be 0,5,10,20,40,80 μm；Wogonin acts on 12h and takes out cell, and 10 μ l CCK8 solution are added per hole, after cultivating 2h,
Absorbance (OD) value with multi-function microplate reader in each hole of 450nm wavelength measurements；3 multiple holes of every group of setting repeat experiment 3 times simultaneously
It is averaged；
Step 4：Cell ageing detects：The cell for collecting logarithmic phase growth, is inoculated in six orifice plates, cell fusion degree reaches
Start the wogonin that basal medium dissolving is added by different time points when 80-90%, preparation of samples is completed.Absorb cell training
Nutrient solution is washed 2 times with PBS, and 1ml beta galactosidases are added and dye fixer, room temperature fixes 15 minutes；Dyeing is absorbed to fix
Liquid is washed 3 times, every time 3 minutes with PBS；PBS is absorbed, 1ml is added per hole and dyes working solution.With ParafilmTM 6 orifice plates postposition
In 37 DEG C of incubations；Finally, the aging aspects of cell are observed using ordinary optical microscope；
Step 5：Apoptosis detects：Cell culture medium is sucked out to 15ml centrifuge tubes, and PBS washing attached cells are primary,
0.5ml pancreatin cell dissociation cells are added；Cell suspension is added to the cell culture fluid 1000g collected in step 1 and centrifuges 5min,
Cell is gently resuspended using PBS and counts；It takes the cell of 100000 resuspensions, 1000g to centrifuge 5min, abandons supernatant, be added 195
Cell is gently resuspended in μ l Annexin V-FITC combination buffers；5 μ l Annexin V-FITC liquid blendings are added；It is added 10
μ l propidium iodide stain liquid mixings；Room temperature, which is protected from light, is incubated 20min, is subsequently placed in ice bath.And use flow cytomery pair
Cell carries out withered detection；
Step 6：Cell cycle is detected：It collects spare in cell culture fluid a to centrifuge tube.It is digested using 0.5% pancreatin
Cell is added cell culture fluid and blows and beats lower whole attached cell；Cell is resuspended and collects again into centrifuge tube；1000g
Left and right rotating speed carries out centrifugation in 3-5 minutes with sedimentation cell；The PBS of about 1ml ice baths precooling is added, cell is resuspended, and be transferred to
In the centrifuge tube of 1.5ml；70% ethyl alcohol that 1ml ice baths are pre-chilled is added in cell, and gently piping and druming is uniform, and 4 DEG C of fixations about 2 are small
When；The propidium iodide stain liquid of 0.5ml is added in each solencyte sample, 37 DEG C of incubators are protected from light warm bath 30 minutes；It is thin with streaming
Born of the same parents' instrument detects red fluorescence at excitation wavelength 488nm wavelength, while detecting light scattering situation, using modifit analysis softwares
Carry out cell DNA content analysis and light-scattering analysis.
Interpretation of result：
The lower cell viability testing result of various concentration wogonin effect, cell is lived between 20 μm and 40 μm of wogonin
Power occurs being decreased obviously (P<0.05)；Under 20 μm of concentration of wogonin the cell viability of different time points as a result, in 8h and
Occur being decreased obviously (P between 10h<0.05).
The result shows that non-small cell lung cancer cell by extraneous medicine irritation occur DNA damage after, the table of SGK1 albumen
Change up to fluctuation dynamics is showed, the cell fate of this dynamic variation and tumour cell is closely related, and SGK1 is to participate in
Determine the important factor of cell fate.Drug enters the pharmacokinetic parameters such as intracellular speed, exposure duration by cellular uptake
Difference determines different SGK1 fluctuation dynamics behaviors, including the fluctuation peak value of SGK1, period of waves, vibration frequency etc., into
And influence the cell fate of tumour cell.The protein expression of SGK1 shows after non-small cell lung cancer (NSCLC) DNA Damage
The dynamic change for going out fluctuation is to be stimulated to generate by external induced drug wogonin；SGK1 is under three kinds of different cell states
The fluctuation of dynamic dynamics there are significant difference but the area under the curve of the protein expression of SGK1 without significant difference, prompt be not
Three kinds of different cell fates caused by protein dynamics level are different due to the difference of the SGK1 Tot Prots of accumulation；
SGK1 is an important factor of the choice for participating in cell fate.
In conclusion the beneficial effects of the present invention are by different wogonin medications, can induce non-small thin
Born of the same parents' lung carcinoma cell enters the different cell states of cell-cycle arrest, cell ageing and Apoptosis, therefore tumour cell can be with
It is transferred to another destiny from a kind of destiny, to reduce the toxic side effect blindly come using high-dose chemotherapy medicine band, to face
The bed rational use of medicines provides reference frame.
The foregoing is merely the preferred embodiment of the present invention, are not intended to limit the scope of the invention, every utilization
Equivalent structure transformation made by present specification, directly or indirectly with the technology neck for being attached to other Related products
Domain is included within the scope of the present invention.
Claims (1)
1. a kind of method that wogonin inhibits SGK1 regulation and control non-small cell lung cancer cell destiny, it is characterised in that：Including as follows
Step：
Step 1：Human non-small cell lung cancer's cell culture：Human non-small cell lung cancer's cell culture is contained 10% in addition 3ml
In the RPMI-1640 complete mediums of FBS, RPMI-1641 medium cultures are in 37 DEG C, CO2The incubator that volumetric concentration is 5%
In；
Step 2：Cell passes on：As cell growth fusion degree 80%-90% or so, will be moved into after Tissue Culture Flask alcohol wipe
Super-clean bench discards old culture solution, and cell is washed twice with PBS buffer solution flushing；After the digestion of 1ml pancreatin is added, see under the microscope
Cellular morphology is examined, when cell volume diminution is rounded, gap becomes larger, the RPMI-1640 culture mediums that 3ml contains 10%FBS are added；
Step 3：Cell viability is analyzed：The cell of exponential phase is collected, cell density is adjusted, is added per hole about 5000 thin
96 orifice plates are added in born of the same parents, per 100 μ l of hole；When cell fusion degree reaches 80-90%, the drug final concentration difference of wogonin is added
It is 0,5,10,20,40,80 μm；Wogonin acts on 12h and takes out cell, and 10 μ l CCK8 solution are added per hole, after cultivating 2h, uses
Absorbance (OD) value of multi-function microplate reader in each hole of 450nm wavelength measurements；3 multiple holes of every group of setting repeat experiment 3 times and take
Average value；
Step 4：Cell ageing detects：The cell for collecting logarithmic phase growth, is inoculated in six orifice plates, cell fusion degree reaches 80-
Start the wogonin that basal medium dissolving is added by different time points when 90%, preparation of samples is completed, and cell culture is absorbed
Liquid is washed 2 times with PBS, and 1ml beta galactosidases are added and dye fixer, room temperature fixes 15 minutes；Dyeing fixer is absorbed,
It is washed 3 times, every time 3 minutes with PBS；PBS is absorbed, 1ml is added per hole and dyes working solution, is placed on ParafilmTM 6 orifice plates
37 DEG C of incubations；Finally, the aging aspects of cell are observed using ordinary optical microscope；
Step 5：Apoptosis detects：Cell culture medium is sucked out to 15ml centrifuge tubes, and PBS washing attached cells are primary, are added
0.5ml pancreatin cell dissociation cells；Cell suspension is added to the cell culture fluid 1000g collected in step 1 and centrifuges 5min, is used
PBS is gently resuspended cell and counts；It takes the cell of 100000 resuspensions, 1000g to centrifuge 5min, abandons supernatant, 195 μ l are added
Cell is gently resuspended in Annexin V-FITC combination buffers；5 μ l Annexin V-FITC liquid blendings are added；10 μ l are added
Propidium iodide stain liquid mixing；Room temperature, which is protected from light, is incubated 20min, is subsequently placed in ice bath, and using flow cytomery to thin
Born of the same parents carry out withered detection；
Step 6：Cell cycle is detected：It is spare in collection cell culture fluid a to centrifuge tube, it is digested using 0.5% pancreatin thin
Born of the same parents are added cell culture fluid and blow and beat lower whole attached cell；Cell is resuspended and collects again into centrifuge tube；1000g is left
Speed of turning right carries out centrifugation in 3-5 minutes with sedimentation cell；The PBS of about 1ml ice baths precooling is added, cell is resuspended, and be transferred to
In the centrifuge tube of 1.5ml；70% ethyl alcohol that 1ml ice baths are pre-chilled is added in cell, and gently piping and druming is uniform, and 4 DEG C of fixations about 2 are small
When；The propidium iodide stain liquid of 0.5ml is added in each solencyte sample, 37 DEG C of incubators are protected from light warm bath 30 minutes；It is thin with streaming
Born of the same parents' instrument detects red fluorescence at excitation wavelength 488nm wavelength, while detecting light scattering situation, using Modifit analysis softwares
Carry out cell DNA content analysis and light-scattering analysis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109797192A (en) * | 2019-02-25 | 2019-05-24 | 安徽古井贡酒股份有限公司 | A kind of fast quantification senile cell detection method based on senescence associated-β-galactosidase |
| CN113533134A (en) * | 2021-08-04 | 2021-10-22 | 山西农业大学 | Circulating detection system and method for detecting pig red blood cell immunoadhesion function |
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2018
- 2018-05-11 CN CN201810447557.8A patent/CN108588168A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109797192A (en) * | 2019-02-25 | 2019-05-24 | 安徽古井贡酒股份有限公司 | A kind of fast quantification senile cell detection method based on senescence associated-β-galactosidase |
| CN109797192B (en) * | 2019-02-25 | 2022-10-14 | 安徽古井贡酒股份有限公司 | Rapid quantitative aging cell detection method based on aging-related beta-galactosidase |
| CN113533134A (en) * | 2021-08-04 | 2021-10-22 | 山西农业大学 | Circulating detection system and method for detecting pig red blood cell immunoadhesion function |
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