CN107384962A - MGST1 genes are bred and the application in apoptosis in lung adenocarcinoma cell - Google Patents
MGST1 genes are bred and the application in apoptosis in lung adenocarcinoma cell Download PDFInfo
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Abstract
The present invention relates to a kind of application of people source MGST1 genes in promoting A549 lung adenocarcinoma cells system propagation and suppressing A549 lung adenocarcinoma cells system apoptosis.The present invention is struck by slow virus MGST1 subtracts A549 lung adenocarcinoma cells system, the detection of MTS propagation, the dyeing of Jim Sa plate clone, the detection of flow cytometer apoptosis, nude mice by subcutaneous into being influenceed the methods of knurl on MGST1, breed and apoptosis judges by A549 lung adenocarcinoma cells system, as a result show that the expression for suppressing MGST1 can suppress the propagation and Cell clonality of A549 cells, promote the early apoptosis of A549 cells.Experiment of nude mouse shows that the gross tumor volume of adenocarcinoma of lung can be substantially reduced by suppressing MGST1 expression, suppress tumour growth.
Description
Technical field
The present invention relates to a kind of people source MGST1 new application, more particularly, to people source MGST1 genes in lung adenocarcinoma cell
Propagation and the application in apoptosis.
Background technology
Lung cancer is a kind of very high tumour of grade malignancy, very high morbidity and mortality in the world all be present,
Serious threat the life and health of people.In China, because suffer from lung cancer and caused by the death rate be also to occupy all malignant tumours
First place, and the emphasis and difficult point of tumor research person research at present.Adenocarcinoma of lung is one of most common organization type of lung cancer, closely
The effect of a little years, does not improve significantly, and 5 year life cycle of patient is still very low, the prognosis of patient or very poor, matter of living
Amount is very low.Therefore the pathogenic driving gene of adenocarcinoma of lung is studied, the new treatment target gene of adenocarcinoma of lung is sought and is significant.
Microsome glutathione s-transferase 1 (Microsomal glutathione S transferase 1, MGST1)
It is encoding proteins matter gene, positioned at No. 12 bands of 12nd area of the short arm of a chromosome 3, MGST1 albumen belongs to II phase metabolic enzyme, catalyzed combination
Reaction, metabolism exogenous material especially medicine excrete.Endoplasmic reticulum, particulate are distributed mainly in subcellsular level MGST1
Body and mitochondrial outer membrane, there is the characteristic that is oxidized and stress activate, protect these films from oxidative stress.In recent years, oxidation should
Swash the focus for being increasingly becoming the formation of research tumour and development factors.Oxidative and anti-oxidative is unbalance to pass through cellular signal transduction, regulation and control
Oncogene and the expression of tumor suppressor gene, promote the Proliferation, Differentiation of cell, cause Apoptosis to reduce, so as to trigger tumour.And
Existing lot of documents report, MGST1 participate in the occurrence and development of melanoma, the oxidative stress regulation tumour of neuroblastoma.
In order to study the effect that MGST1 is played in adenocarcinoma of lung, the present invention is using the high people for expressing MGST1 of slow-virus infection
Lung adenocarcinoma cell, with MTS, plate clone dyeing, flow cytometer, nude mice by subcutaneous is into detection MGST1 the methods of knurl to lung gland
The influence of cancer cell multiplication and apoptosis.
The content of the invention
In view of this, it is an object of the invention to：(1) people's source MGST1 genes are provided and are suppressing A549 lung adenocarcinoma cells system
Application in propagation；(2) application of people's source MGST1 genes in A549 lung adenocarcinoma cells system apoptosis is suppressed is provided；(3) people is provided
Application of the source MGST1 genes in tumour growth is suppressed.
In order to realize the above object the present invention is realized by following technical solution：
1. cultivate A549 lung adenocarcinoma cells system.
2. using slow-virus infection A549 cells, control group is unrelated sequences Scramble, experimental group MGST1-85, MGST1-
86 two sequences, control group and experimental group carry GFP green fluorescence labels.Wherein Scramble sequences, shMGST1-85 sequences
Row, shMGST1-86 sequence table are as follows：
Scramble sequences：5’-TTCTCCGAACGTGTCACGT-3’
ShMGST1-85 sequences：5’-ACTGCAACTGCATTCTATA-3’
ShMGST1-86 sequences：5’-GAAGACTGTGTAGCATTTG-3’
3. study the effect that MGST1 is played in adenocarcinoma of lung by the following method：
(1) slow-virus infection A549 MGST1 protein expressions are detected with Western；
(2) struck with MTS detections and subtract ability of cell proliferation after MGST1；
(3) struck with plate clone detection and subtract Cell clonality after MGST1；
(4) struck with flow cytomery and subtract Apoptosis after MGST1；
(5) effect with nude mice by subcutaneous into knurl technology, in vivo layer viewpoint MGST1 gene pairs tumour growth.
As a result show：After striking the MGST1 genes for subtracting A549 cells, the expression of MGST1 albumen is notable in A549 cells
Decline, the multiplication capacity of cell is remarkably decreased, and cell plates clonality significantly lowers, and early stage Apoptosis significantly increases
Add, be subcutaneously substantially reduced into knurl knurl volume.
Brief description of the drawings
Fig. 1 is：Slow-virus infection A549 cell efficiency, figure A and B is respectively unrelated sequences Scramble light fields and fluorescence,
Figure C and D is respectively shMGST1-85 light fields and fluorescence, and figure E and F is respectively shMGST1-86 light fields and fluorescence.(multiplication factor：
50 times；Scale 20um)
Fig. 2 is：Western detection slow virus suppresses the expression of MGST1 albumen.
Fig. 3 is：MTS detections show that the expression for suppressing MGST1 can significantly reduce the multiplication capacity of A549 cells, P<0.05.
Fig. 4 is：Plate clone detection shows that the expression for suppressing MGST1 can significantly inhibit the Clone formation energy of A549 cells
Power, P<0.01.
Fig. 5 is：Flow cytomery suppression MGST1 expression dramatically increases the early apoptosis rate of A549 cells, P<
0.05。
Fig. 6 is：Tumor formation in nude mice result shows that the expression for suppressing MGST1 can be substantially reduced the volume of adenocarcinoma of lung tumour,
Suppress tumour growth.
Design, concrete structure and the caused technique effect of the present invention are described further below with reference to accompanying drawing, with
It is fully understood from the purpose of the present invention, feature and effect.
Embodiment 1：MGST1 slow-virus infections
A549 cell lines spread 6 orifice plate after 10 hours, changed the serum-free RPMI- of the polybree containing 5ug/ul into
1640Medium, corresponding viral volume (number of cells * MOI/ virus titers) is added, after cultivating 8 hours, culture medium is changed into
Containing 10% hyclone RPMI-1640Medium.72 hours detection fluorescence radiation situations (Fig. 1), can be observed to compare after transfection
Group and experimental group slow-virus infection efficiency are all higher than 80%.
Embodiment 2：Western detects protein expression
Cell is collected in centrifuge tube and centrifuged, 2000rpm, 4min, the PBS of precooling are washed twice, are added according to cell concentration
The RIPA (10 μ l/ml protease inhibitors and inhibitors of phosphatases) of suitable volumes, piping and druming mix, after EP channel closure membrane closures
It is put in 4 DEG C of cell fragmentation Ultrasound Instruments, 500watt, cracks 20min；14000g, 20min are centrifuged, sedimentation cell fragment, supernatant
As cell holoprotein, BCA methods measure concentration.It is 50ug per PFP applied sample amount.Deposition condition 5% concentrates glue 80V, 50 points
Clock；12% separation gel 120V, 100 minutes.Transferring film condition 100V, 100 minutes.MGST1 primary antibodies dilution ratio 1:1000, secondary antibody is dilute
Release ratio 1:8000.
Shown in testing result (Fig. 2)：MGST1 protein expression is bright in experimental group shMGST1-85, shMGST1-86 cell
It is aobvious lower than control group Scramble.
Case 3 is embodied：The propagation of human umbilical vein endothelial cell can be significantly inhibited by suppressing MGST1 expression
The proliferative conditions of human A459 lung cancer cell line are detected using MTS experiments.Take the control in the growth index phase
Group Scramble and experimental group shMGST1-85, shMGST1-86 cell, are inoculated in 96 porocyte culture plates, often after cell count
2000, hole cell, OD values, data analysis are measured at full wavelength scanner instrument 490nm wavelength.
Experimental result (Fig. 3) is shown：Compared with control group Scramble, experimental group shMGST1-85, shMGST1-86 is thin
Born of the same parents' growth rate substantially slows down, and starts with the passage of time, the difference of viable count with statistical significance, p<0.05,
The propagation of human pulmonary epithelial cells can be significantly inhibited after the expression for illustrating to suppress MGST1.
Case 4 is embodied：The cell clonal formation energy of human umbilical vein endothelial cell can be significantly inhibited by suppressing MGST1 expression
Power
Experiment is formed using plate clone to detect the clonality situation of human A459 lung cancer cell line.Take and be in
The control group Scramble and experimental group shMGST1-85, shMGST1-86 cell of growth index phase, 6 are inoculated in after cell count
Porocyte culture plates, per 200, hole cell, 3 parallel multiple holes of every group of setting, put 37 DEG C of 5%CO214 are cultivated in constant incubator
It or so, cultivate Clone formation visible to naked eyes；After Clone formation, culture is terminated, abandons culture medium, PBS carefully cleans 2
It is secondary, dry, add 1ml methanol to fix 30min, abandon after methanol dries and add 1ml Giemsa stains dyeing 30min, wash away dye liquor, dry in the air
It is dry；The clone's number for being more than 50 cells is counted, does histogram analysis data.
As a result (Fig. 4) is shown：Experimental group shMGST1-85, shMGST1-86 cell clonal formation number is substantially than control
Group Scramble is few, p<0.01.Illustrate to suppress the cell clonal formation that can significantly inhibit human umbilical vein endothelial cell after MGST1 is expressed
Ability.
Case 5 is embodied：The early apoptosis rate of human umbilical vein endothelial cell can be dramatically increased by suppressing MGST1 expression
The apoptosis situation of A549 cells is detected using flow cytometer.Take the control group in the growth index phase
Scramble and experimental group shMGST1-85, shMGST1-86 cell, single cell suspension is digested to the pancreatin without EDTA,
2000rpm, centrifuge 5min；PBS liquid is washed twice, abandons supernatant, and 500 μ l BindingBuffer are resuspended cell, add 5 μ l Annexin
After V-APC (triumphant base biology) is mixed, 5 μ l 7-ADD (triumphant base biology) dye liquor is added, lucifuge is incubated at room temperature 15min after mixing；1 is small
When interior upper machine testing sample.Arrange data, analysis result.
Testing result (Fig. 5) is shown：Experimental group shMGST1-85, shMGST1-86 early apoptosis of cells rate substantially compares
According to a group Scramble height, (4.43 ± 0.84), (4.37 ± 1.92) %vs (1.30 ± 0.10) %, p<0.05, but late apoptic
Rate no significant difference.
Embodiment 6：The volume of nude mice by subcutaneous tumour can be substantially reduced by suppressing MGST1 expression
Control group Scramble and experimental group shMGST1-85 cells in the growth index phase are taken, list is digested to pancreatin
Cell suspension, 2000rpm, centrifuge 5min；PBS liquid is washed twice, abandons supernatant, and is resuspended with PBS liquid, cell count, is injected in hero
Property 4 weeks BALB/c nude mices on the left of oxter, every nude mice injects 5*106Individual cell, every group of 7 nude mices, nude mice into knurl 7 weeks or so,
Knurl body is dissected, arranges data, analysis result.
As a result (Fig. 6) is shown：Experimental group shMGST1-85 knurl volume and knurl body weight is substantially smaller than control group Scramble.
Embodiment described above, its description is more specific and detailed, but therefore can not be interpreted as to patent of the present invention
The limitation of scope.It should be pointed out that for the person of ordinary skill of the art, the premise of present inventive concept is not being departed from
Under, various modifications and improvements can be made, these belong to protection scope of the present invention.Therefore, the protection of patent of the present invention
Scope should be determined by the appended claims.
Claims (6)
1. a kind of method for the effect that research MGST1 genes play in adenocarcinoma of lung, it comprises the following steps：
Step1. A549 lung adenocarcinoma cells system is cultivated.
Step2. slow-virus infection A549 cells are used, control group is unrelated sequences Scramble, experimental group MGST1-85, MGST1-
86 two sequences, control group and experimental group carry GFP green fluorescence labels.
Step3. the effect that MGST1 is played in adenocarcinoma of lung is studied by certain method.
2. the method for the effect that research MGST1 genes play in adenocarcinoma of lung as claimed in claim 1, it is characterised in that：Institute
Scramble sequences, shMGST1-85 sequences, shMGST1-86 sequence table are as follows in the step Step2 stated：
Scramble sequences：5’-TTCTCCGAACGTGTCACGT-3’
ShMGST1-85 sequences：5’-ACTGCAACTGCATTCTATA-3’
ShMGST1-86 sequences：5’-GAAGACTGTGTAGCATTTG-3’.
3. the method for the effect that research MGST1 genes play in adenocarcinoma of lung as claimed in claim 1, it is characterised in that：Institute
The method for the effect that MGST1 is played in adenocarcinoma of lung is studied in the step Step3 stated to be included：(1) slow virus is detected with Western
Infect A549 MGST1 protein expressions；
(2) struck with MTS detections and subtract ability of cell proliferation after MGST1；
(3) struck with plate clone detection and subtract Cell clonality after MGST1；
(4) struck with flow cytomery and subtract Apoptosis after MGST1；
(5) effect with nude mice by subcutaneous into knurl technology, in vivo layer viewpoint MGST1 gene pairs tumour growth.
, can be with 4. the method for the effect that the research MGST1 genes as described in any in claims 1 to 3 play in adenocarcinoma of lung
For studying application of the people source MGST1 genes in A549 lung adenocarcinoma cells system propagation is promoted.
, can be with 5. the method for the effect that the research MGST1 genes as described in any in claims 1 to 3 play in adenocarcinoma of lung
For studying application of the people source MGST1 genes in A549 lung adenocarcinoma cells system apoptosis is suppressed.
, can be with 6. the method for the effect that the research MGST1 genes as described in any in claims 1 to 3 play in adenocarcinoma of lung
For studying application of the people source MGST1 genes in adenocarcinoma of lung tumour growth is suppressed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108373999A (en) * | 2017-11-28 | 2018-08-07 | 信雅生物科技（苏州）有限公司 | A kind of construction method and its application that specific can strike the slow virus for subtracting RBM47 genes |
| CN114533878A (en) * | 2020-11-25 | 2022-05-27 | 中国科学院大连化学物理研究所 | Application of MGST1 inhibitor and medicine mixture for treating liver cancer |
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| WO2008108993A1 (en) * | 2007-03-02 | 2008-09-12 | Sidney Kimmel Cancer Center | Ectopic, orthotopic model for revascularization and tumor assessment |
| CN101230387A (en) * | 2007-04-28 | 2008-07-30 | 廖凌虹 | Use of human MGST1 gene mononucleotide polymorphism in diagnosing and treating breast cancer |
| CN104789563A (en) * | 2015-01-27 | 2015-07-22 | 上海市胸科医院 | HSG related anti lung adenocarcinoma kit and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108373999A (en) * | 2017-11-28 | 2018-08-07 | 信雅生物科技（苏州）有限公司 | A kind of construction method and its application that specific can strike the slow virus for subtracting RBM47 genes |
| CN114533878A (en) * | 2020-11-25 | 2022-05-27 | 中国科学院大连化学物理研究所 | Application of MGST1 inhibitor and medicine mixture for treating liver cancer |
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Application publication date: 20171124 |