CN103743909B - The influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts - Google Patents
The influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The present invention relates to a kind of influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts, specifically comprise the following steps: (1) mtt assay detects the propagation of cell; (2) A549 Cell clonality detects; (3) the two dye method of Hochest33342/PI detects Apoptosis; (4) cell monolayer migration experiment (wound? healing); (5) Transwell Matrigel; (6) Western? blot detects cell shifting related protein expression; (7) enzyme linked immunosorbent assay; (8) statistical study.The impact that the present invention is grown by research furin inhibitor Lung Adenocarcinoma A 549 Cell and shifts, can utilize Furin inhibitor a1-PDX to suppress Furin to express in Lung Adenocarcinoma A 549 Cell better, thus effectively for treatment of cancer provides foundation.
Description
Technical field
The present invention relates to a kind of influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts, belong to biological technical field.
Background technology
Lung cancer is the malignant tumour of serious threat human health and life in the world today, and the incidence of disease also rises just year by year, and lung cancer morbidity rate and mortality ratio all occupy first of malignant tumour in many countries.According to estimates, the patient numbers dying from lung cancer in a year exceedes prostate cancer, breast cancer, colorectal cancer total number of persons.About 85% patients with lung cancer is non-small cell lung cancer (non-smallcelllungcancer, NSCLC), and it is divided into gland cancer, squama cancer, large cell carcinoma etc. by Histopathology.Most patients is Local advancement or DISTANT METASTASES IN when diagnosing.Transfer and recurrence are patients with lung cancer main causes of death, and the patients with lung cancer of about 90% dies from metastases.Thus deeply grind adenocarcinoma of lung infiltrate, transfer molecular mechanism necessary.
Furin is the important member in amyloid protein precursor processive enzyme family, and many important physiology courses need the participation of Furin, as the synthesis of polypeptide and proteohormone and secretion, the maturation of membrane receptor, the activation etc. of plasma protein precursors; Simultaneously the generation of various diseases is also closely related with Furin, as the transfer etc. of the processing activated and tumour of virus capsid protein and bacterial exotoxin.These protein needed to cut amyloid protein precursor through convertase before performance activity, then just can become the protein having function.Comprise the many of Notch, Wnt, MT1-MMP, VEGF etc. with the closely-related protein of tumor development in vivo maturation, have to pass through the convertases such as Furin and its precursor is sheared, competence exertion biologic activity.And in these protein few members and tumour generation, develop closely related, Furin high expressed in kinds of tumors, can as the Marker in tumour progression process, to a certain extent can as the index of tumor prognosis.
Summary of the invention
The object of the present invention is to provide a kind of influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts, to utilize Furin inhibitor a1-PDX to suppress Furin to express in Lung Adenocarcinoma A 549 Cell better, thus effectively for treatment of cancer provides foundation.
To achieve these goals, technical scheme of the present invention is as follows.
The influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts, specifically comprises the following steps:
(1) mtt assay detects the propagation of cell:
Exponential phase A549 cell is inoculated in 96 orifice plates, every hole 5 × 10
3, the a1-PDX starting to add 200nM or 400nM variable concentrations after 24h continues to cultivate 24h-96h; Every hole adds 20 μ l5 mg/ml MTT reagent solutions, and in 37 DEG C of incubations 4 hours.Every hole adds the DMSO Rong Xie formazan crystal of 150 μ L, and concussion detects the optical density at 490nm place after dissolving.
(2) A549 Cell clonality detects:
The single-layer culturing cell of taking the logarithm growth period, blows and beats into individual cells with 0.25% Trypsin Induced, and cell is suspended in the RPMI-1640 containing 10% hyclone, with 1 × 10
3the cell density of/ml is inoculated in double dish; The a1-PDX (200nM, 400nM) adding variable concentrations puts 37 DEG C, under the saturated humidity environment of 5%CO2, quiescent culture 2 weeks.Abandoning supernatant, PBS carefully embathes 2 times; Methyl alcohol fixes 15min.Remove immobile liquid, Giemsa stain dyeing 10min, after flowing water slowly rinses rear air oxygen detrition, naked eyes directly count clone and statistical study.
(3) the two dye method of Hochest33342/PI detects Apoptosis:
Exponential phase cell A549 is inoculated on the cover plate in advance with poly-D-lysine process by 1 × 104/ml concentration.After the a1-PDX process 48h of variable concentrations, incline nutrient solution, adds precooling PBS washed cell 2 times; Adjust Hoechst33342/PI dye liquor concentration to final concentration 5 μ g/ml, 37 DEG C of lucifuges dyeing 15min; Discard dye liquor, add 4% paraformaldehyde, 4 DEG C of fixing 5min.Take at fluorescence microscopy Microscopic observation.
(4) cell monolayer migration experiment (woundhealing):
A549 cell is seeded in 6 orifice plates, in time growing to degree of converging and reach 100%, makes cut (wound) with the Tip head point of 200 aseptic μ l, and with PBS washed cell fragment.Cell process is the same.In the cell migration situation of the digital camera shooting injured area that the time of specifying is equipped with inverted microscope.
(5) Transwell Matrigel:
The A549 cell (1 × 10 of variable concentrations a1-PDX process
5) be inoculated in the upper chamber of Transwell cell, the RPMI1640 nutrient culture media containing 200 μ l, but not containing 10%FBS.Transwell cell lower chamber is filled with the complete RPMI1640 nutrient culture media of 500 μ l, containing 10%FBS.Make cell migration 48 hours, then with 4% formaldehyde, cell is fixed, incubated at room temperature 15 minutes.After deionized water washing, 0.1% violet staining.Take the clone of migration under an optical microscope.
(6) Westernblot detects cell shifting related protein expression:
Collecting cell adds RIPA damping fluid (TrispH7.4 of 50mM, 150mM sodium chloride, the TritonX-100 of 1%, 0.1%SDS, 1% NaTDC, 5mMEDTA, the sodium fluoride of 100mM) and protease inhibitors, hatch 30 minutes on ice, the centrifugal 30min of 13200rpm.Collect supernatant BCA method (Pierce, USA) measures protein concentration.Total protein of cell transfers to pvdf membrane after 12%SDS-PAGE gel electrophoresis, and under room temperature, 1h closed by 5% skim milk.The antisera overnight of MT1-MMP, VEGF-C, VEGF-D and GAPDH (1: 1000) is hatched at 4 DEG C.The sheep anti mouse two adding HRP mark after PBST washing resists, and after incubated at room temperature 1h, PBST washing, after super quick luminescent solution (Pierce company, the U.S.) is hatched, LAS3000 imager (Fuji, Japan) is taken pictures.
(7) enzyme linked immunosorbent assay:
Described in the process ditto of A549 cell, collecting cell culture supernatant also carries out follow-up detection according to MMP-9, MMP-2, VEGF-cELISA detection kit instructions.Each sample repeats 5 times.
(8) statistical study:
Application SPSS13.0 software carries out statistical procedures, and measurement data adopts paired t-test and one-way analysis of variance, and enumeration data adopts Chi-square Test, and P < 0.05 has statistical significance.
This beneficial effect of the invention is: the impact that the present invention is grown by research furin inhibitor Lung Adenocarcinoma A 549 Cell and shifts, Furin inhibitor a1-PDX can be utilized better to suppress Furin to express in Lung Adenocarcinoma A 549 Cell, thus effectively for treatment of cancer provides foundation.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention a1-PDX to the proliferative effect figure of A549 cell.
Fig. 2 be in the embodiment of the present invention a1-PDX to the Colony forming effect diagram of A549 cell.
Fig. 3 be in the embodiment of the present invention a1-PDX to the apoptotic effect diagram of A549 (control group).
Fig. 4 be in the embodiment of the present invention a1-PDX to the apoptotic effect diagram of A549 (200nMa1-PDX processed group).
Fig. 5 be in the embodiment of the present invention a1-PDX to the apoptotic effect diagram of A549 (400nMa1-PDX processed group).
Fig. 6 be in the embodiment of the present invention a1-PDX to the apoptotic effect diagram of A549 (statistical study of three independent experiment apoptosis rates).
Fig. 7 is the effect diagram of cell monolayer scratch detection a1-PDX on cell migration in the embodiment of the present invention.
Fig. 8 be in the embodiment of the present invention a1-PDX on three of the impact of A549 cell migration independent experiment statistical study figure.
Fig. 9 be in the embodiment of the present invention a1-PDX to the effect diagram (control group) of A549 wetting capacity.
Figure 10 be in the embodiment of the present invention a1-PDX to the effect diagram (200nMa1-PDX processed group) of A549 wetting capacity.
Figure 11 be in the embodiment of the present invention a1-PDX to the effect diagram (400nMa1-PDX processed group) of A549 wetting capacity.
Figure 12 be in the embodiment of the present invention a1-PDX to A549 tri-independent experiment cellular infiltration ability statistical study figure.
Figure 13 is the effect diagram that in the embodiment of the present invention, a1-PDX expresses A549 cell shifting related protein.
Figure 14 is the effect diagram that in the embodiment of the present invention, a1-PDX expresses A549 cell MMP-9.
Figure 15 is the effect diagram that in the embodiment of the present invention, a1-PDX expresses A549 cell MMP-2.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described, better to understand the present invention.
Embodiment
The influence research method that furin inhibitor grows lung adenocarcinoma cell and shifts, specifically comprises following aspect:
(1) cell cultivation process: human lung adenocarcinoma cell line A549 to coordinate cell bank purchased from the Chinese Academy of Medical Sciences.Grow containing 10% hyclone (FBS) and 100 units/mL penicillin, in the RPMI1640 nutrient culture media (Gibco, USA) of 100 μ g/mL streptomysins, in 37 DEG C, 5%CO2, to cultivate under saturated humidity.
(2) main agents used comprises: Furin inhibitor a1-PDX (Merck article No.: 126850-2.5MG) is dissolved in DMSO, makes 5mM mother liquor.Mouse-anti people VEGF-C, VEGF-D, MT1-MMP and GAPDH antibody is all purchased from SantaCruz company (SantaCruz, the U.S.).Dynamics-HRP and anti-rabbit IgG-HRP, purchased from Sigma (Sigma, USA); MTT, Hochest33342, Transwell purchased from American Promega company; MMP2, MMP9ELISA detection kit builds up biological reagent company purchased from Nanjing.It is pure that other reagent is domestic analysis.
(3) specific implementation process and methods experiment method:
(3a) mtt assay detects the propagation of cell:
Exponential phase A549 cell is inoculated into (every hole 5 × 10 in 96 orifice plates
3), the a1-PDX (200nM, 400nM) starting to add variable concentrations after 24h continues to cultivate 24h-96h.Every hole adds 20 μ lMTT reagent (5 mg/ml) solution, and in 37 DEG C of incubations 4 hours.Every hole adds the DMSO Rong Xie formazan crystal of 150 μ L, and concussion detects the optical density at 490nm place after dissolving.
(3b) A549 Cell clonality detects:
The single-layer culturing cell of taking the logarithm growth period, blows and beats into individual cells with 0.25% Trypsin Induced, and cell is suspended in the RPMI-1640 containing 10% hyclone, with 1 × 10
3the cell density of/ml is inoculated in double dish.The a1-PDX (200nM, 400nM) adding variable concentrations puts 37 DEG C, under the saturated humidity environment of 5%CO2, quiescent culture 2 weeks.Abandoning supernatant, PBS carefully embathes 2 times.Methyl alcohol fixes 15min.Remove immobile liquid, Giemsa stain dyeing 10min, after flowing water slowly rinses rear air oxygen detrition, naked eyes directly count clone and statistical study.
(3c) the two dye method of Hochest33342/PI detects Apoptosis:
Exponential phase cell A549 is by 1 × 10
4individual/ml concentration is inoculated on the cover plate in advance with poly-D-lysine process.After the a1-PDX process 48h of variable concentrations, incline nutrient solution, adds precooling PBS washed cell 2 times; Adjust Hoechst33342/PI dye liquor concentration to final concentration 5 μ g/ml, 37 DEG C of lucifuges dyeing 15min; Discard dye liquor, add 4% paraformaldehyde, 4 DEG C of fixing 5min.Take at fluorescence microscopy Microscopic observation.
(3d) cell monolayer migration experiment (woundhealing):
A549 cell is seeded in 6 orifice plates, in time growing to degree of converging and reach 100%, makes cut (wound) with the Tip head point of 200 aseptic μ l, and with PBS washed cell fragment.Cell process is the same.In the cell migration situation of the digital camera shooting injured area that the time of specifying is equipped with inverted microscope.
(3e) Transwell Matrigel:
The A549 cell (1 × 10 of variable concentrations a1-PDX process
5) be inoculated in the upper chamber of Transwell cell, the RPMI1640 nutrient culture media containing 200 μ l, but not containing 10%FBS.Transwell cell lower chamber is filled with the complete RPMI1640 nutrient culture media of 500 μ l, containing 10%FBS.Make cell migration 48 hours, then with 4% formaldehyde, cell is fixed, incubated at room temperature 15 minutes.After deionized water washing, 0.1% violet staining.Take the clone of migration under an optical microscope.
(3f) Westernblot detects cell shifting related protein expression:
A549 cell chulture and process are ditto.Collecting cell adds RIPA damping fluid (TrispH7.4 of 50mM, 150mM sodium chloride, the TritonX-100 of 1%, 0.1%SDS, 1% NaTDC, 5mMEDTA, the sodium fluoride of 100mM) and protease inhibitors, hatch 30 minutes on ice, the centrifugal 30min of 13200rpm.Collect supernatant BCA method (Pierce, USA) measures protein concentration.Total protein of cell transfers to pvdf membrane after 12%SDS-PAGE gel electrophoresis, and under room temperature, 1h closed by 5% skim milk.The antisera overnight of MT1-MMP, VEGF-C, VEGF-D and GAPDH (1: 1000) is hatched at 4 DEG C.The sheep anti mouse two adding HRP mark after PBST washing resists, and after incubated at room temperature 1h, PBST washing, after super quick luminescent solution (Pierce company, the U.S.) is hatched, LAS3000 imager (Fuji, Japan) is taken pictures.
(3g) enzyme linked immunosorbent assay:
Described in the process ditto of A549 cell, collecting cell culture supernatant also carries out follow-up detection according to MMP-9, MMP-2, VEGF-cELISA detection kit instructions.Each sample repeats 5 times.
(4) statistical study:
Application SPSS13.0 software carries out statistical procedures, and measurement data adopts paired t-test and one-way analysis of variance, and enumeration data adopts Chi-square Test, and P < 0.05 has statistical significance.
(5) result of implementation:
(5a) propagation of a1-PDX on A549 cell and the impact of Colony forming:
After the a1-PDX effect A549 different time of variable concentrations, mtt assay detects the survival of cell, as shown in Figure 1.Result shows, and during a1-PDX effect A549 more than cell 48h, the growth of cell is significantly inhibited.Colony forming method detects the Clone formation of A549 cell.Result shows, compared with control group, the Colony forming ability of the A549 cell of a1-PDX process significantly reduces, as shown in Figure 2.
(5b) a1-PDX is on the apoptotic impact of A549:
For inquiring into a1-PDX to the cytostatic mechanism of A549, the two dye of Hochest33342/PI is first used to detect A549 Apoptosis.Result shows, a1-PDX can induce the apoptotic generation of A549, as shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6.
(5c) a1-PDX is on the impact of A549 cell migration and invasion and attack:
Whether there is regulating action to the migration and invasion of A549 cell for detecting a1-PDX, have detected the situation of A549 cell migration with woundHealing and Transwell experiment further.Two kinds of experimental results all show that a1-PDX significantly reduces A549 cell migration and invasive ability, as shown in Fig. 7, Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12.
(5d) impact of the expression of a1-PDX on cell migration associated protein:
Numerous protein participates in the adjustment of tumor cell migration in vivo.Regulating the molecular mechanism of A549 cell migration for fully understanding a1-PDX, have detected cell MT1-MMP, MMP2, MMP9, VEGF-c, VEGF-d protein expression level by WesternBlot and ELISA method.As shown in figure 13, a1-PDX significantly reduces the expression of MT1-MMP, VEGF-c, VEGF-d in cell.In cell culture supernatant, MMP2 and MMP9 concentration is also starkly lower than control group, as shown in Figure 14, Figure 15 simultaneously.These results show, a1-PDX suppression A549 cell migration may be relevant with the expression reducing MT1-MMP, MMP2/9, VEGF-C/D.
In embodiments of the present invention, Furin inhibitor a1-PDX can induce A549 cell inhibitory effect and apoptosis.In addition, A549 cell is after a1-PDX is hatched, and transfer ability significantly reduces.The expression of these matrix metalloproteinases, reflects the wetting capacity of tumour cell effectively.A1-PDX can not only suppress the expression of MMP2 and MMP9, and can reduce the expression of MT1-MMP.A1-PDX reduces Furin enzymatic activity, and then reduces MT1-MMP maturation and activate, and further suppress the maturation of MMP2, MMP9.Cell VEGE-C and the VEGF-D protein expression of a1-PDX process significantly reduce.Lower Furin enzymatic activity, thus suppress the expression of MMPs and the VEGFs albumen of A549 cell may be the mechanism that it suppresses A549 growth and moves, infiltrates.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (1)
1. furin inhibitor to lung adenocarcinoma cell growth and transfer an influence research method, it is characterized in that: specifically comprise the following steps:
(1) mtt assay detects the propagation of cell: exponential phase A549 cell is inoculated in 96 orifice plates, every hole 5 × 10
3, the a1-PDX starting to add 200nM or 400nM variable concentrations after 24h continues to cultivate 24h-96h; Every hole adds 20 μ l5 mg/ml MTT reagent solutions, and in 37 DEG C of incubations 4 hours; Every hole adds the DMSO Rong Xie formazan crystal of 150 μ L, and concussion detects the optical density at 490nm place after dissolving;
(2) A549 Cell clonality detects: the single-layer culturing cell in growth period of taking the logarithm, and blows and beats into individual cells, cell is suspended in the RPMI-1640 containing 10% hyclone, with 1 × 10 with 0.25% Trypsin Induced
3the cell density of/ml is inoculated in double dish; The a1-PDX adding 200nM or 400nM variable concentrations puts 37 DEG C, 5%CO
2saturated humidity environment under, quiescent culture 2 weeks; Abandoning supernatant, PBS carefully embathes 2 times; Methyl alcohol fixes 15min; Remove immobile liquid, Giemsa stain dyeing 10min, after flowing water slowly rinses rear air oxygen detrition, naked eyes directly count clone and statistical study;
(3) the two dye method of Hochest33342/PI detects Apoptosis: exponential phase cell A549 is by 1 × 10
4individual/ml concentration is inoculated on the cover plate in advance with poly-D-lysine process; After the a1-PDX process 48h of variable concentrations, incline nutrient solution, adds precooling PBS washed cell 2 times; Adjust Hoechst33342/PI dye liquor concentration to final concentration 5 μ g/ml, 37 DEG C of lucifuges dyeing 15min; Discard dye liquor, add 4% paraformaldehyde, 4 DEG C of fixing 5min; Take at fluorescence microscopy Microscopic observation;
(4) cell monolayer migration experiment: A549 cell is seeded in 6 orifice plates, in time growing to degree of converging and reach 100%, makes cut with the Tip head point of 200 aseptic μ l, and with PBS washed cell fragment; In the cell migration situation of the digital camera shooting injured area that the time of specifying is equipped with inverted microscope;
(5) Transwell Matrigel: the A549 cell of the a1-PDX process of variable concentrations is inoculated in the upper chamber of Transwell cell, and upper chamber contains the RPMI1640 nutrient culture media of 200 μ l, but not containing 10%FBS; Transwell cell lower chamber is filled with the complete RPMI1640 nutrient culture media of 500 μ l, containing 10%FBS; Make cell migration 48 hours, then with 4% formaldehyde, cell is fixed, incubated at room temperature 15 minutes; After deionized water washing, 0.1% violet staining; Take the clone of migration under an optical microscope;
(6) Westernblot detects cell shifting related protein expression: collecting cell adds RIPA damping fluid and protease inhibitors, this RIPA damping fluid be 50mM TrispH7.4,150mM sodium chloride, 1% TritonX-100,0.1%SDS, 1% NaTDC and 5mMEDTA, the sodium fluoride potpourri of 100mM, hatch 30 minutes on ice, the centrifugal 30min of 13200rpm; Collect supernatant and BCA method mensuration protein concentration; Total protein of cell transfers to pvdf membrane after 12%SDS-PAGE gel electrophoresis, and under room temperature, 1h closed by 5% skim milk; The antisera overnight of MT1-MMP, VEGF-C, VEGF-D and GAPDH is hatched at 4 DEG C; The sheep anti mouse two adding HRP mark after PBST washing resists, and after incubated at room temperature 1h, PBST washing, super quick luminescent solution is hatched rear employing LAS3000 imager and taken pictures;
(7) enzyme linked immunosorbent assay: collecting cell culture supernatant also carries out follow-up detection according to MMP-9, MMP-2, VEGF-cELISA detection kit instructions; Each sample repeats 5 times;
(8) statistical study: application SPSS13.0 software carries out statistical procedures, and measurement data adopts paired t-test and one-way analysis of variance, and enumeration data adopts Chi-square Test, and P < 0.05 has statistical significance.
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| CN101120959A (en) * | 2006-08-08 | 2008-02-13 | 阿尔贝拉医药(中国)有限公司 | Application of guhong injection for treating lung cancer |
| CN101346397A (en) * | 2005-10-24 | 2009-01-14 | 杜门蒂斯有限公司 | Agents that bind a target in pulmonary tissue for treating respiratory diseases |
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| CN101120959A (en) * | 2006-08-08 | 2008-02-13 | 阿尔贝拉医药(中国)有限公司 | Application of guhong injection for treating lung cancer |
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| Title |
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| A Small-Molecule Furin Inhibitor Inhibits Cancer Motility and Invasiveness.;Julia M.Coppola, et al.;《Neoplasia》;20080430;第10卷(第4期);第363-370页 * |
| Effect of Furin inhibitor on lung adenocarcinoma cell growth and metastasis.;YongChao Ma, et al.;《Cancer Cell International》;20140522;第14卷(第43期);第1-6页 * |
| siRNA沉默MIF基因抑制大细胞肺癌H460细胞增殖;侯俊娜，等;《中国病理生理杂志》;20110531;第27卷(第5期);第853-858页 * |
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