CN109557317A - Application of the ATXN2L as the marker of aided assessment gastric cancer oxaliplatin secondary resistance - Google Patents
Application of the ATXN2L as the marker of aided assessment gastric cancer oxaliplatin secondary resistance Download PDFInfo
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Abstract
The invention belongs to the functions and application field of gene and albumen, are related to new application of plain -2 sample albumen (ATXN2L) of incoordination as the marker of aided assessment gastric cancer oxaliplatin secondary resistance.The present invention confirms the Patients with Gastric Cancer prognosis mala of high ATXN2L expression by clinical samples.After silencing ATXN2L, stomach cancer cell enhances oxaliplatin sensitivity.Oxaliplatin-resistant strain is obtained by external evoked method, MTT experiment confirms that oxaliplatin is resisted in Oxaliplatin-resistant strain, and grade malignancy is higher.Western blot detection discovery expression of ATXN2L in the persister of oxaliplatin is higher than the expression in sensitive strain.In the experiment of flow cytometer detection apoptosis, after silencing ATXN2L, oxaliplatin-resistant cells strain increases the sensibility of oxaliplatin.Therefore, it can use marker of the ATXN2L as aided assessment gastric cancer oxaliplatin secondary resistance, so as to provide the kit of aided assessment gastric cancer oxaliplatin secondary resistance using the preparation process of available reagent box.
Description
Technical field
The invention belongs to the functions and application field of gene and albumen, are related to plain -2 sample albumen (ATXN2L) of incoordination
New application, the purposes of the drug of reversing stomach Oxaliplatin-resistant is used to prepare more particularly to ATXN2L.
Background technique
Gastric cancer is the common malignant tumour in China, and chemotherapy is treatment means important in Comprehensive Therapy on Gastric Carcinoma.Tumour cell
It is drug resistant to chemotherapeutics to limit chemotherapeutic efficacy, it can frequently result in patient's treatment failure.Chemotherapy resistance includes that primary is resistance to
Medicine and secondary resistance, primary drug resistance refer to tumour itself resistance to drug before receiving drug therapy；Secondary resistance
Refer to tumour script not drug resistance, the emerging drug resistance during chemotherapy.It solves the problems, such as gastric cancer initial drug-resistant and secondary drug resistance is existing
For a big difficulty in tumor research field.
Clinically being usually used in the drug of gastric cancer now includes following a few major class: fluorouracil (fluorouracil, Ka Peita
Shore and S-1), platinum class (cis-platinum and oxaliplatin), taxanes (taxol, docetaxel), the soft ratio of table in anthracyclines
Star and topoisomerase enzyme inhibitor Irinotecan.Wherein, oxaliplatin is third generation platinum-like compounds, its effect and other
Platinum medicine action principle is similar, using intracellular DNA as site of action, in two adjacent guanine residues or guanine and gland
Adduct in chain is formed between purine, to inhibit DNA replication dna and transcription, plays antineoplastic action.Existing research confirms difficult to understand
Husky benefit platinum can significantly improve the prognosis of advanced gastric carcinoma patient, have critical role in chemotherapy of gastric cancer.However, Oxaliplatin-resistant
The case where it occur frequently that, influence chemotherapeutic efficacy.The molecular marker for finding energy aided assessment Oxaliplatin-resistant has important face
Bed meaning.
Incoordination -2 sample albumen (Ataxin-2like, ATXN2L) of element are to cause hereditary spinocerebellar ataxia
Plain (Ataxin-2, the ATXN2) homologous protein of incoordination.It is considerably less about the research report of ATXN2L at present, it is only known
ATXN2L is only expressed in the cell height of the immortalizations such as embryonic tissue, adult testis and candidate stem cell；ATXN2L can be tied with EpoR
It closes；ATXN2L is overexpressed the generation for promoting stress granules.Whether ATXN2L has an effect in tumour generation, influences tumour progression
Specific mechanism it is not yet clear, need further to be studied.
Summary of the invention
The purpose of the present invention is to provide the new applications of plain -2 sample albumen (ATXN2L) of incoordination.
It is difficult to understand as aided assessment gastric cancer that the first aspect of the invention provides plain -2 sample albumen (ATXN2L) of incoordination
The application of the marker of husky benefit platinum secondary resistance.
The second aspect of the invention provides plain -2 sample albumen (ATXN2L) of incoordination in preparation for aided assessment
Purposes in the kit of gastric cancer oxaliplatin secondary resistance.
The further feature of purposes according to the present invention, the kit are the contents according to ATXN2L in sample
Or the variation tendency of content carrys out the state of aided assessment gastric cancer oxaliplatin secondary resistance.
The further feature of purposes according to the present invention, the kit are expressing quantity detection kit.
Preferably, the expressing quantity detection kit detects expressing quantity using Western blot method
Kit.
The present invention confirms the Patients with Gastric Cancer prognosis mala of high ATXN2L expression by clinical samples.After silencing ATXN2L, stomach
Cancer cell enhances oxaliplatin sensitivity.
Oxaliplatin-resistant strain is obtained by external evoked method, MTT experiment confirms Oxaliplatin-resistant strain to difficult to understand husky
Sharp platinum is resisted, and grade malignancy is higher.The expression ratio of Western blot detection discovery ATXN2L in the persister of oxaliplatin
Expression in sensitive strain is higher.In the experiment of flow cytometer detection apoptosis, after silencing ATXN2L, oxaliplatin-resistant cells strain is to difficult to understand husky
The sensibility of sharp platinum increases.Therefore, it can use mark of the ATXN2L as aided assessment gastric cancer oxaliplatin secondary resistance
Object, so as to provide the reagent of aided assessment gastric cancer oxaliplatin secondary resistance using the preparation process of available reagent box
Box.
Detailed description of the invention
Fig. 1 is expression of the ATXN2L in human gastric cancer sample and the influence to clinical prognosis.Wherein, (a) figure is shown
Differential expression of the ATXN2L of TCGA database analysis stomach organization and the gastric epithelial tissue of pairing in mRNA level in-site；(b)
Figure shows differential expression of the ATXN2L in protein level of the gastric epithelial tissue of fresh stomach organization sample and pairing；(c)
Figure is the immunohistochemistry representative figure of follow-up patient；(d) figure show gastric cancer by stages, recurrence, existence and ATXN2L expression phase
It closes；(e) figure shows influence of the expression of ATXN2L to IV phase patients with gastric cancer Overall survival；(f) figure shows the expression pair of ATXN2L
The influence of I-III phase patients with gastric cancer recurrence-free survival phase；(g) figure shows the interim difference of I-III by stages to the recurrence-free survival phase
It influences；(h) figure shows influence of the expression of ATXN2L to the I-II phase patients with gastric cancer recurrence-free survival phase；(i) figure shows ATXN2L
Influence of the expression to the III phase patients with gastric cancer recurrence-free survival phase.
Fig. 2 is the experimental result picture that ATXN2L resists Apoptosis caused by oxaliplatin.Wherein, (a) figure shows gastric cancer
Cell is stimulated form to change to spindle shape by low concentration oxaliplatin, and this morphologic change is suppressed after silencing ATXN2L；(b)
Figure shows that silencing ATXN2L can reverse EMT caused by low concentration oxaliplatin；(c) figure shows Flow cytometry silencing
Apoptosis in gastric cancer ratio increases compared with control group after oxaliplatin is handled after ATXN2L, the then apoptosis handled through 5 FU 5 fluorouracil
Ratio variation is little；(d) figure shows MTT experiment detection stomach cancer cell cell survival rate after the processing of various concentration oxaliplatin,
Cell survival rate is decreased obviously after silencing ATXN2L；(e) figure shows colony formation, and Ao Shali is added after silencing ATXN2L
Platinum Colony forming number is substantially less than control group；(f) figure shows Flow cytometry active oxygen, is added after silencing ATXN2L
Oxaliplatin active oxygen generates more compared with control group.
Fig. 3 is the experimental result picture that the cell strain ATXN2L of the secondary Oxaliplatin-resistant of gastric cancer increases.Wherein, (a) figure
Display MTT experiment verifying Oxaliplatin-resistant strain constructs successfully；(b) figure shows that scratch experiment finds oxaliplatin-resistant cells strain
Transfer ability it is strong compared with sensitive cells strain；(c) figure, which is shown, finds that Oxaliplatin-resistant strain has more by EMT marker immunofluorescence
Strong EMT tendency；(d) figure is Western blot detection, and sensitive cells is compared in the expression of ATXN2L in Oxaliplatin-resistant strain
Plant height；(e) figure is FCM analysis, and the silencing ATXN2L in silencing Oxaliplatin-resistant strain causes Apoptosis to increase；(f)
Figure is shown under oxaliplatin stimulation, and the ATXN2L of silencing persister promotes active oxygen to generate.
Specific embodiment
One, experimental material
1. tissue specimen
Histotomy: the stomach that Hospital of Southern Medical University row in Guangzhou includes radical correction and palliative resection is chosen
Cancer operation Operated Specimens.
Pathological section is included in standard are as follows: 1. through definitive pathological diagnosis be gastric cancer；2. pathological section comes from stomach primary tumor.
Pathological section exclusion criteria are as follows: 1. pathological diagnosis have dispute；2. clinical treatment is not complete.
According to situation, imageological examination in patient's art, TNM stage is carried out to patient.According to the above standard, it is included in 48 altogether
IV phase and 119 I-III phase stomach organization samples are analyzed, and sample is fixed with 10% formalin, and paraffin embedding, are cut
Piece.
Fresh gastric cancer sample: Hospital of Southern Medical University general surgery Surgery for Gastric Carcinoma sample is chosen.Every takes stomach
Cancerous tissue and each two pieces of cancer beside organism, sample collect put into rapidly in cryopreservation tube it is quick-frozen in liquid nitrogen after be transferred to -80 DEG C of Low-temperature Ices
Cryo-conservation in case.
2. stomach cancer cell line and immortalization gastric epithelial cells strain
Human stomach cancer cell line AGS, BGC-823, MKN-45, MGC-803, SGC7901, people immortalize gastric epithelial cell
Strain GES-1 is all from Hospital of Southern Medical University oncology laboratory, is purchased from Foleibao Bioisystech Co., Ltd.
3. main agents and consumptive material
Fetal calf serum, 1640 culture mediums are the production of Hyclone company.2000 × PBS powder is purchased from Biosharp company.
It EDTA, is the production of solarbio company without EDTA pancreatin, Tris-Hcl (PH8.8 and PH6.8) is from Shanghai White match biotechnology
Co., Ltd.The reagent that TEMED, 30% polyacrylamide, ammonium persulfate, 10%SDS etc. prepare gel is public purchased from Biosharp
Department.Tris alkali is purchased from Biosharp company.Glycine, SDS powder are the production of Sigma company.Methanol, isopropanol, anhydrous second
Alcohol, chloroform, tween are the production of Guangdong Province's chemical reagent engineering and technological research development centre.Skimmed milk power buying is certainly
Biosharp company.Lowlenthal serum is purchased from Guangzhou Zhuo Sheng Biotechnology Co., Ltd.2000 × TBS powder, primary antibody dilution
Purchased from Biosharp company.Holoprotein extracts kit is the production of Co., Ltd, Nanjing Keygen Biotech.5 × albumen loading buffer
Liquid is purchased from Hangzhou Fu De Biotechnology Co., Ltd.PCR kit for fluorescence quantitative, RNAiso plus, Reverse Transcriptase kit are purchased from
Takara company.DEPC water is the production of Shanghai Sheng Gong bio-engineering corporation.DMSO is the production of Mike woods company.Pvdf membrane is
The production of Millipore company.GAPDH monoclonal antibody, ATXN2L polyclonal antibody are the production of Proteintech company.Sheep
Anti-rabbit secondary antibody is the production of Li-COR company.Dimethylbenzene, sodium citrate buffer, hydrogenperoxide steam generator, DAB working solution, haematoxylin,
Hydrochloric acid, neutral gum are purchased from Guangdong Province's chemical reagent engineering and technological research development centre.SiRNA is the synthesis of Rui Bo company.Slow disease
Malicious shRNA is the building of Ji Kai company.ATXN2L, GAPDH upstream and downstream primer are the synthesis of Shanghai Sheng Gong bio-engineering corporation.It is various
Model Tissue Culture Dish, 6 orifice plates, 12 orifice plates, 96 orifice plates are Corning Incorporated's production.BCA determination of protein concentration kit is Jiangsu
Green skies biotech firm production.Apoptosis detection kit is three arrow biological productions.ROS detection kit is that the green skies in Jiangsu are raw
The production of object company.EDU kit is sharp rich production.The anti-mountant Mounting Medium that is quenched is solarbio company.
Western blot glass plate is the production of U.S. Bio-rad company.
Two, experimental method
1.MTT experiment
(1) it plants plate: by the cell dissociation tested, centrifugation, obtaining precipitating, be added suitable 10% into precipitating
Quan Pei carries out cell count, according to needing 6 × 10 in each experimental port3Cell and experiment hole count cell total required for calculating
Suspension always needs liquid volume according still further to needing 100 μ l to train calculating entirely in each experimental port, calculated cell suspension is added
Into 15ml centrifuge tube, into centrifuge tube be added 10% full Cilostazol to each experimental port be 100 μ l liquid.By liquid after being sufficiently mixed
Body is transferred in 96 orifice plates, and the liquid in each hole is 100 μ l.96 orifice plates are put into overnight incubation in deposited case.
(2) after overnight incubation cell is adherent according to experiment arrange and packet transaction cell after continue to be put into deposited case and be incubated for.
(3) plus MTT is detected: blotting cell conditioned medium, 10 μ l of 0.5%MTT is added in each hole under the conditions of being protected from light, and is protected from light and puts back to
It applies in case after continuing culture 4 hours and blots MTT, 150 μ l dimethyl sulfoxides are added, concussion is until bluish violet knot crystalline substance formazan is sufficiently molten
Solution.The light absorption value of each experimental port is measured at microplate reader OD 490nm.
2. colony formation
(1) it plants plate: by the cell dissociation tested, centrifugation, obtaining precipitating, be added suitable 10% into precipitating
Quan Pei carries out cell count, needs 1 × 10 according to each hole4Cell suspension volume required for cell calculates simultaneously is added to 6 holes
In plate, finally liquid trim in each hole to 2ml is placed in deposited case and is incubated for 7-10 days, centre is changed liquid 1 time with 10% full training.
(2) fixed: the supernatant that will form six orifice plates of colony abandons, and is washed 2 times with PBS, blots PBS, toward each
4% paraformaldehyde of 800 μ l is added in hole, fixes 30 minutes.
(3) it dyes: after the completion of fixed, being washed 2 times with PBS, 800 μ l0.1% crystal violet solutions are added into each hole, contaminate
Color 30 minutes
(4) it takes pictures: gently washing away extra crystal violet solution after the completion of dyeing under tap water, dry, taken pictures with camera.
3.EDU tests (cell proliferation experiment)
(1) it plants plate: the cell dissociation needed in experiment, centrifugation being precipitated, cell is resuspended, carries out cell count.It presses
According to addition 4 × 10 in each hole396 orifice plate of cell kind, and the liquid in each hole uniformly uses 10% to train the body for being fitted on 100 μ l entirely
Product.It is put into overnight incubation in deposited case and waits for that cell is adherent.
(2) it needs to handle cell according to experiment after cell is adherent.Continue culture experiment 24 hours.
(3) EDU indicates: blotting the full training in 96 orifice plates with liquid-transfering gun, the EDU of 50 μM of concentration is prepared using complete medium
The prepared full training containing EDU of 100 μ l is added into each hole for culture medium, is put into deposited case after continuing culture 2 hours and discards
Clearly, PBS is added, is washed 2 times on shaking table, each time is 5 minutes.
(3) fixed: to wash 96 orifice plates finished and blot PBS, 50 μ l, 4% paraformaldehyde is added into each hole and is consolidated
It is 30 minutes fixed.Paraformaldehyde is blotted after the completion of fixed, the glycine solution of 50 μ l 2mg/ml is added into each hole, by 96 holes
Plate is placed in decolorization swinging table, is continued to be incubated for and is abandoned glycine solution after five minutes, and PBS is added and washs on decolorization swinging table 1 time.Each
The triton solution of 100 μ l 0.5% is added in hole, 96 orifice plates are placed in decolorization swinging table and abandon triton solution after ten minutes, is added
PBS is washed 1 time on decolorization swinging table.
(4) dye: 100 μ l Apollo reaction solutions are added into each hole and carry out to the cell in 96 orifice plates by exhaustion PBS
Dyeing, is then incubated for 30 minutes on decolorization swinging table under conditions of being protected from light.
(5) supernatant is abandoned after the completion of dyeing, and the triton solution of 100 μ l 0.5% is added into each hole, is protected from light and shakes in decoloration
It is washed on bed 3 times, every time 10 minutes.100 μ l methanol are added into each hole to continue to rinse 2 times, are finally washed 1 time with PBS.With
Upper washing carries out on decolorization swinging table, and the time is 5 minutes.
(6) Hoechst33342 working solution is added into each hole, volume is 100 μ l, in the condition for being protected from light and be room temperature
Lower stationary incubation 30 minutes, PBS is washed after the completion of incubation.
(7) 96 orifice plates are placed under fluorescence inverted microscope and are observed, and taken pictures and save photo data, used
ImageJ carries out cell count.
4. Flow Cytometry detects apoptosis
(1) cell conditioned medium for needing to detect is completed in processing to collect, digests adherent cell with the pancreatin not comprising EDTA,
Pay attention to observing cellular morphology under the microscope, works as when the gap between cell rounding and cell and cell increases and use immediately
The supernatant collected before carries out termination digestion, collects cell suspension.It is then centrifuged for, obtains cell precipitation.With 4 degrees Celsius of PBS
Washing cell 1 time.Cell precipitation after being washed.
(2) Bing Buffer solution for later use is prepared according to the specification deionized water of the kit of detection apoptosis.
(3) the Bing Buffer solution that 1ml has diluted is added into cell precipitation, centrifugation is precipitated, again toward thin
100 μ l of Bing Buffer solution is added in born of the same parents' precipitating, it is anti-that 5 μ l of AnnexinV-FITC dye liquor then is added in the condition being protected from light
It answers 10 minutes, is eventually adding 5 μ l of PI dye liquor and reacts 10 minutes.Being eventually adding 390 microlitres of PBS solutions and mixing to system is 500 μ l.
It is protected from light cryo-conservation.
Upper machine testing apoptosis rate in (4) 1 hours.
5. oxaliplatin-resistant cells strain constructs
This experimental construction be MGC803 oxaliplatin-resistant cells strain.It is handled with the oxaliplatin of gradient concentration
MGC803 replaces culture solution and chemotherapeutics every other day, and oxaliplatin pharmaceutical concentration decides whether to increase according to cell state, to thin
Had digestive transfer culture when born of the same parents' degrees of fusion is 80% or so, the had digestive transfer culture same day are generally added without oxaliplatin stimulation, continue sand difficult to understand later
Sharp platinum stimulation, the sensibility of the oxaliplatin of periodic detection cell.When sensibility of the cell to oxaliplatin is substantially reduced and surely
Fixed, drug-resistant cell strain constructs successfully when having statistical difference.
6. immunofluorescence
(1) cell state has been raised, in the previous day plantation in being copolymerized in burnt capsule, has made cell density 30-40% or so.
(2) it is transiently transfected in 24 hours by cell seeding after capsule.
(3) culture medium of 10% different fetal calf serums is replaced within 24 hours after transiently transfecting according to experiment purpose difference.
(4) culture medium in capsule is sucked after the completion of processing, filtration grade PBS is cleaned 2 times, sufficiently to wash away culture medium.
(5) the fixed filtration grade PBS after twenty minutes of 4% paraformaldehyde that 300 μ L are added into each capsule is cleaned three times, often
Secondary 5 minutes, upper decolorization swinging table.
(6) filtration grade PBS is cleaned three times the perforation of 0.5%Triton rupture of membranes after ten minutes, and 5 minutes every time, upper decolorization swinging table.
(7) exhaust PBS, and 4 degree of incubation primary antibodies are stayed overnight after 1%BSA is closed 30 minutes.
(8) it is stored at room temperature rewarming 30 minutes, sucks primary antibody, filtration grade PBS is cleaned three times, and 5 minutes every time, upper decoloration was shaken
Bed.
(9) exhaust PBS, stationary incubation secondary antibody 1 hour under the conditions of room temperature is protected from light.
(10) secondary antibody is sucked, filtration grade PBS is cleaned three times, and 5 minutes every time, upper decolorization swinging table.
(11) PBS is sucked, DAPI (methanol dilution) is dyed five minutes, and rear filtration grade PBS is cleaned three times, and 5 minutes every time, on
Decolorization swinging table.
(12) it takes pictures under Laser Scanning Confocal Microscope, detects intracellular fluorescence intensity.
7. cell activity oxygen detects
(1) after needing to handle cell according to experiment, former supernatant is abandoned, the Austria for being 50 μ g/ml containing concentration is added into cell
The 10% of husky benefit platinum trains base, continues culture 6 hours in cell incubator.
(2) dilute DCFH-DA: according to 1640 training bases: DCFH-DA=1000:1 prepares DCFH- in 15ml centrifuge tube
DA。
(3) cell abandons supernatant after handling 6 hours, is washed cell 2 times, in addition with the PBS by high pressure sterilization processing
Prepared fluorescence probe DCFH-DA is stated, 500 μ l fluorescence probe DCFH-DA solution are added in each hole in general 6 orifice plates.
It is protected from light, applies in case and continue culture 20 minutes.
(4) it is protected from light and abandons fluorescence probe DCFH-DA solution, washed cell 3 times with 4 DEG C of PBS, it is thin sufficiently to rinse well
It is extracellular not enter intracellular DCFH-DA probe.Then collected by trypsinisation cell is resuspended cell with 500 μ Lpbs, is protected from light
Under the conditions of fluorescence in time in flow cytomery cell it is strong and weak, i.e., the amount of the active oxygen generated in expression cell.
Three, ATXN2L high expression is an important factor for influencing gastric cancer prognosis
Inventor is analyzed by cancer to the gastric cancer in TCGA database and cancer beside organism, finds the mRNA of ATXN2
Level is highly expressed (a of Fig. 1 schemes) in cancerous tissue.Then, it collects fresh stomach organization sample and carries out expressing quantity
Analysis finds that the expressing quantity of the ATXN2L of stomach organization is higher than cancer beside organism (b of Fig. 1 schemes).Further, south is had collected
The tissue specimen and clinical data of 167 Patients with Gastric Cancer of hospital are analyzed, wherein 48 are to receive to appease to control for the IV phase by stages
The patient for the treatment of, 119 are for the I-III phase and to receive the Patients with Gastric Cancer of Radical Gastrectomy by stages.Inventor has possessed this
167 patients of whole Follow-up Data carry out immunohistochemical analysis (c of Fig. 1 schemes) discovery, and the highly expressed patient of ATXN2L is later
Staging of Gastric Cancer patient in proportion it is higher, in I-III phase Patients with Gastric Cancer, trouble of the highly expressed patient of ATXN2L in recurrence
Proportion is higher compared to not recurring in person, in IV phase Patients with Gastric Cancer, patient of the highly expressed patient of ATXN2L in survival
Middle proportion is compared to dead low (d of Fig. 1 schemes).This prompt ATXN2L and gastric cancer by stages, existence, recurrence exist it is close
Relationship.Further, follow-up discovery, in the patient of IV phase palliative treatment, ATXN2L are carried out to 48 IV phase patients with gastric cancer
Highly expressed IV phase patients overall survival is considerably shorter than ATXN2L low expression person the time (e of Fig. 1 schemes).It is eradicated in the I-III phase
Follow-up discovery, ATXN2L highly expressed patients with gastric cancer recurrence-free survival phase (figure short compared with ATXN2L low expression are carried out in the patient of art
1 f figure).Layering survival analysis discovery is carried out to I-III phase Patients with Gastric Cancer, when the middle position recurrence-free survival of III phase Patients with Gastric Cancer
Between (g of Fig. 1 schemes) is obviously shortened compared with I, II phase.According to disease it is specific by stages, further, for the I-II phase (h of Fig. 1
Figure) and III phase (i of Fig. 1 schemes) patient's progress subgroup analysis, equally it can be found that the highly expressed patients with gastric cancer of ATXN2L is easier
Recurrence.
It is worth noting that being all made of oxaliplatin+fluorouracil in the patient of radical-ability postoperative adjuvant therapy and controlling
Treatment scheme.Cell experiment, which has proven to interference ATXN2L expression, influences less the cytotoxicity of fluorouracil, and to Ao Shali
Platinum has an impact.It is therefore contemplated that the highly expressed prognosis of ATXN2L that shows of clinical data is due to having occurred than low expression difference
Caused by Oxaliplatin-resistant.
Four, ATXN2L resists Apoptosis caused by oxaliplatin
It has been reported that the oxaliplatin stimulation of low concentration can cause liver cancer and colon carcinogenesis Epithelial and stromal to turn
Change, and experiment of the invention is, it was also found that low concentration (10 μ g/ml) oxaliplatin handles microscopically observation cellular morphology after cell
It was found that oxaliplatin can significantly be such that cellular morphology changes to spindle shape direction, and oxaliplatin is added after silencing ATXN2L
Then this morphologic change is unobvious (a of Fig. 2 schemes).EMT marker is dyed and is found, low concentration oxaliplatin inhibits E-
Cadherin and the expression for promoting vimentin albumen, and silencing ATXN2L can reverse this process.Show silencing ATXN2L energy
Reverse EMT caused by oxaliplatin.(b of Fig. 2 schemes).
There is document report cell that EMT occurs related to sensibility of the cell to drug.Therefore, the present invention considers
ATXN2L may take part in stomach cancer cell and resist Apoptosis caused by chemotherapeutics.Inventor is in shN, shATXN2L cell
The 5 FU 5 fluorouracil of the middle oxaliplatin that 30 μ g/ml are added and 50 μ g/ml continue Annexin V-FITC/ after culture 24 hours
The bis- transfection reagent box staining for flow of PI detect apoptosis rate, as a result, it has been found that apoptosis rate is aobvious after oxaliplatin group silencing ATXN2L
It writes and is higher than control group, and apoptosis rate and control group difference less (c of Fig. 2 schemes) after 5 FU 5 fluorouracil group silencing ATXN2L.
It thereby confirms ATXN2L and takes part in Apoptosis caused by stomach cancer cell resistance oxaliplatin.
It is completed with the oxaliplatin processing shATXN2L building of 0,10,15,20,30,40,50 μ g/ml concentration gradients steady
Turn cell strain, MTT is added after processing 24 hours and carries out MTT detection (d of Fig. 2 schemes), after as a result prompting silencing ATXN2L, gastric cancer is thin
Born of the same parents' strain increases the sensibility of oxaliplatin compared with control group.
Similarly, the stable cell strain passage kind plate that shATXN2L building is completed is done into colony formation, each hole kind 1
×104Cell is added 2 μ g/ml oxaliplatin of low concentration after cell is adherent in 24 hours and acts on 7 days, and primary training was replaced in centre
Base and drug, 7 days poststaining observation Colony forming situations (e of Fig. 2 schemes), as a result, it has been found that oxaliplatin group silencing is added
Cell colony forms number and is substantially less than control group after ATXN2L.
It is lost after oxaliplatin processing 6 hours of 50 μ g/ml are added in shN, shATXN2L#1, shATXN2L#2 cell
Supernatant is abandoned, is changed to and the anteserum-less substrate culture of the fluorescence probe DCFH-DA flow cytometer detection f of Fig. 2 (scheme) after twenty minutes, knot is added
The amount of the active oxygen generated in stomach cancer cell after fruit display silencing ATXN2L is higher than control group.
To sum up, the experimental results showed that ATXN2L, which participates in stomach cancer cell, resists Apoptosis caused by oxaliplatin.
Five, the cell strain ATXN2L of the secondary Oxaliplatin-resistant of gastric cancer increases
The problem of in order to inquire into oxaliplatin secondary drug resistance, inventor constructs oxaliplatin with Gastric cancer line
Secondary drug resistant cell strain OxaResist-#1 and OxaResist-#2.Inventor is with 0,10,15,20,30,40,50 μ g/ml's
Concentration acts on cell strain OxaResist-#1 and OxaResist-#2 and sensitive cells MGC803, and row MTT is detected, as a result
OxaResist#1 compares parental cell MGC803 (a of Fig. 3 schemes) more insensitive to oxaliplatin with OxaResist#2.Invention
People has carried out scratch experiment, it is found that the transfer ability of two Oxaliplatin-resistant strains of building is strong compared with sensitive strain (b of Fig. 3 schemes).
(c of Fig. 3 schemes) is remarkably reinforced in Oxaliplatin-resistant strain EMT ability.Inventor extracts resistance to oxaliplatin-resistant cells strain
The albumen row Western blot of OxaResist-#1 and OxaResist-#2 and sensitive cells MGC803 is detected, ATXN2L
Expression in Oxaliplatin-resistant strain than parental cell plant height the d of Fig. 3 (scheme).
In order to further inquire into value of the ATXN2L in the secondary drug resistance of oxaliplatin, inventor uses two different sequences
ShRNA (shATXN2L-#1 and shATXN2L-#2) silencing ATXN2L of column.Flow cytometer detection apoptosis discovery, in OxaResist-#1
Compared to the control group in OxaResist-#2 cell strain non-disclosre ATXN2L, the apoptosis ratio of cell, which has no, substantially change,
And be added after 30 μ g/ml oxaliplatins handle 24 hours, the apoptosis rate of cell obviously increases, and in silencing ATXN2L group, it is secondary
The apoptosis ratio of drug-resistant cell strain increases more obvious (e of Fig. 3 schemes) compared to the control group.Similarly, it after silencing ATXN2L, enhances
Oxaliplatin causes the generation of active oxygen (f of Fig. 3 schemes).Thus prompt in oxaliplatin-resistant cells strain that silencing ATXN2L can
Partially to restore cell to the drug susceptibility of oxaliplatin.
Six, the kit for aided assessment gastric cancer oxaliplatin secondary resistance
Based on the studies above as a result, existing preparation process in conjunction with this field about detection kit, can prepare and be used for
The kit of aided assessment gastric cancer oxaliplatin secondary resistance.
Specifically, which is according to the content of ATXN2L in sample or the variation tendency of content come aided assessment
The state of gastric cancer oxaliplatin secondary resistance, therefore it is prepared as expressing quantity detection kit.
Previous experiments are that expressing quantity is detected using Western blot method, and discovery ATXN2L can be with aided assessment gastric cancer
Oxaliplatin secondary resistance, it is therefore preferable that expressing quantity detection kit is to detect egg using Western blot method
The kit of white expression quantity.
Claims (5)
1. marker of incoordination -2 sample albumen (ATXN2L) of element as aided assessment gastric cancer oxaliplatin secondary resistance
Using.
2. the examination that incoordination -2 sample albumen (ATXN2L) of element are used for aided assessment gastric cancer oxaliplatin secondary resistance in preparation
Purposes in agent box.
3. purposes according to claim 2, it is characterised in that: the kit be according to the content of ATXN2L in sample or
The variation tendency of content carrys out the state of aided assessment gastric cancer oxaliplatin secondary resistance.
4. purposes according to claim 2, it is characterised in that: the kit is expressing quantity detection kit.
5. purposes according to claim 4, it is characterised in that: the expressing quantity detection kit is to utilize
The kit of Western blot method detection expressing quantity.
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