CN105126098A - Application of monoclonal antibody NJ001-1 to preparation of drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer) - Google Patents
Application of monoclonal antibody NJ001-1 to preparation of drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer) Download PDFInfo
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Abstract
The invention discloses application of a monoclonal antibody NJ001-1 to preparation of a drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer). The monoclonal antibody NJ001-1 is secreted by a hybridoma cell strain NM001-1 with the preservation number CCTCC NO: C201172. The monoclonal antibody NJ001-1 is capable of inhibiting migration and invasion of lung adenocarcinoma cells effectively and increasing TIMP-3mRNA and protein expression remarkably. Under the action of the monoclonal antibody NJ001-1, specific antigen of the monoclonal antibody NJ001-1 can be combined with transcription factors FOXP1 (forkhead box protein P1 ) in lung adenocarcinoma cell nucleuses to inhibit transcription inhibition effect on a TIMP-3 gene promoter region.
Description
Technical field
The invention belongs to field of biological medicine, relate to the application of monoclonal antibody NJ001-1 in preparation suppression NSCLC metastasis.
Background technology
The invasion inhibition of pulmonary carcinoma is its pernicious mark and feature, is also the main reason affecting treatment in patients with lung cancer effect He cause death.Pulmonary carcinoma can be divided into small cell lung cancer and nonsmall-cell lung cancer (Non-small-cellLungCancer, NSCLC), and wherein NSCLC accounts in 85%, NSCLC the most common with adenocarcinoma of lung again.NSCLC onset is hidden, and rapidly, poor prognosis, the median survival interval of therapist is not only 4 ~ 5 months, and 1 year survival rate is that about 10%, 5 years survival rates are less than 5% in development.There is metastasis (IV phase) in most NSCLC patient, chemotherapeutic drug therapy effect very undesirable when clinical first visit.At present, the molecular mechanism that transfer occurs non-small cell lung cancer cell is illustrated not yet completely, and also lacks the medicine of specificity suppression lung carcinoma cell transfer high, with strong points clinically.Therefore, explore the molecular mechanism of invasion of lung cancer transfer, finding and develop the active drug and therapy approach for the treatment of invasion of lung cancer transfer, is the key point of pulmonary carcinoma molecular biology research.
The Invasion and Metastasis of tumor is a multi-step, the multistage, multipath, relate to polygenes change complex process.There are some researches show, in cancer cell invasion and transfer process, multiple signal path can be activated, as RAS/MAPK path, and Akt/PI3K path and ERK path etc., thus make cell produce biological characteristic and the behavior of tumor cell.Under normal circumstances, invasion and metastasis of tumor is the process of an active, needs tumor cell and tumor mechanism composition jointly to participate in, often comprises three steps: sticking of tumor cell and extracellular matrix; Tumor cell release or induction release multiple protein hydrolytic enzyme, degradation of cell epimatrix; The tumor cell of degraded areas shifts under the guiding of chemotactic factor, and tumor cell induction of vascular in target tissue is formed on this basis, and antagonism Host Anti-tumor Immunity, finally forms metastasis a long way off.
Expression of TIMP-3 (TIMP-3) is the insoluble proteins of one in conjunction with extracellular matrix (ECM).TIMP-3 participates in growth and the process of reconstruction of normal structure, also take part in the generation of numerous disease.TIMP-3 mono-aspect can stimulate fibroblast proliferation, on the other hand the apoptosis of but inducing malignant tumor cell.Because TIMP-3 can with 1: 1 molecular ratios and matrix metalloproteinase (MMPs) Non-covalent binding, suppress the activity of MMPs, therefore TIMP-3 has that Tumor suppression grows, the effect of Infiltration and metastasis, the downward of TIMP-3 expression or the process of losing will promote tumor in tumor tissues.As can be seen here, matrix metalloproteinase (MMPs) and inhibitor (TIMP) thereof are the key factors of regulation and control nonsmall-cell lung cancer invasive ability.
The process that malignant cell sends out other positions from original site through approach such as lymph, blood, body cavitys is the transfer of tumor.The transfer of malignant cell is that it is different from one of Normocellular feature, is the biological property of itself.The transfer of tumor is the main cause that it jeopardizes host's life and affects therapeutic effect.Malignant cell can shift, it is all many-sided different that its reason is that it and normal cell have, as: forfeiture, the fluidity of plasma membrane of adjusting and controlling growth mechanism change, the change, cell atypia increase, adhesiveness change, cell polarity change etc. of cell motility.In different tumors, its cell characteristics is incomplete same, and therefore its transfer ability is not identical yet, even if in same tumor body, also may there is the cell subsets with Metastatic potential.The research of current anti-metastasis drug has carried out many-sided research mainly for the various changes of tumor cell, body tissue in the mechanism of neoplasm metastasis and transfer process, but due to polytropy, the complexity of neoplasm metastasis itself, the multiformity of influence factor, and the restriction of research means, up to the present, not yet find that the definite Malignant tumor of bonal metastasis that can control moves medicine.
At present, can grow by Tumor suppression to a certain extent although be applied to clinical antitumor drug, promote apoptosis of tumor cells, but significant inhibitory action be there is no to the Infiltration and metastasis of tumor cell.Apoptosis and the Invasion and Metastasis of tumor cell are two pathophysiological processes that molecular mechanism is different.The approach of regulating cell apoptosis mainly comprises mitochondria pathway, endoplasmic reticulum-induced and death receptor pathway *.But tumor cell invasion transfer is mostly relevant to the gene expression, Epithelial and stromal etc. of matrix metalloproteinase and mortifier thereof.For the platinum series antineoplastic medicament of clinical practice-cisplatin, carboplatin etc., although platinum medicine can remarkable Tumor suppression propagation, anticancer spectrum is comparatively wide, and it cannot the Infiltration and metastasis of Tumor suppression.Therefore, it is possible to promote that the medicine of apoptosis of tumor cells, Tumor suppression growth or antibody might not can be used in Tumor suppression invasion inhibition.The medicine developed for Malignant tumor of bonal metastasis will become the important channel extending tumor patient life span, raising oncotherapy effect.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, provide monoclonal antibody NJ001-1 to suppress the application in the medicine of NSCLC metastasis in preparation.
Object of the present invention realizes by following technical scheme:
Monoclonal antibody NJ001-1 suppresses the application in the medicine of NSCLC metastasis in preparation.The hybridoma cell strain NM001-1 that described monoclonal antibody NJ001-1 is CCTCCNO:C201172 by preserving number secretes.
Monoclonal antibody NJ001-1 is preparing the application in the medicine suppressing NSCLC metastasis at the key gene suppressing to act in NSCLC metastasis as target spot.
Described monoclonal antibody NJ001-1 is at the key gene optimized encoding transcription factor FOXP1 suppressing to act in NSCLC metastasis.
The preparation method of monoclonal antibody NM001-1 of the present invention is shown in CN102391992B.
Beneficial effect:
Monoclonal antibody NJ001-1 specific recognition can be positioned at SP70 antigen on NSCLC cytoplasm and cell membrane, has the apoptotic effect of induction NSCLC, and also there is potential effects to the Infiltration and metastasis of tumor cell.Monoclonal antibody NJ001-1 effectively can suppress migration (Fig. 1) and the invasion and attack (Fig. 2) of lung adenocarcinoma cell, and significantly can increase TIMP-3mRNA and protein expression (Fig. 3).Under monoclonal antibody NJ001-1 effect, in lung adenocarcinoma cell core, NJ001-1 specific antigen can be combined with transcription factor FOXP1, suppresses it to the transcripting suppressioning action of TIMP-3 gene promoter area.
Accompanying drawing explanation
Fig. 1 cell scratch experiment detects lung adenocarcinoma cell transfer ability
A: monoclonal antibody NJ001-1 treatment S PC-A1 cell 0h and 48h " cut " figure;
B: monoclonal antibody group compares with matched group SPC-A1 cell migration distance;
* compare between monoclonal antibody group and corresponding matched group, * P<0.05, * * P<0.01, * * * P<0.001.
Fig. 2 Transwell tests and detects lung adenocarcinoma cell invasive ability
Fig. 3 monoclonal antibody NJ001-1 induces TIMP-3 to express
Fig. 4 monoclonal antibody NJ001-1 specific antigen (50kDa) and FOXP1 interact
The preservation information of biological material specimens
Hybridoma cell strain NM001-1 is preserved in China typical culture collection center on August 31st, 2011, and preservation address is Wuhan, China, Wuhan University, and preserving number is CCTCCNO:C201172.
Detailed description of the invention
Embodiment 1
1, cell migration detects
Collect the lung adenocarcinoma cell SPC-A-1 being in exponential phase and make single cell suspension, with 2 × 10
5cells/well adds in 6 porocyte culture plates, incubated overnight, inhales and abandons culture supernatant, and wash one time with Hank ' s liquid 500 μ l/ hole.Antibody group (50 μ g/mL, 100 μ g/mL and 150 μ g/mL tri-groups): every hole adds monoclonal antibody NJ001-1 solution, and final concentration is respectively 50 μ g/mL, 100 μ g/mL and the 150 every holes of μ g/mL; Matched group (0 μ g/mL): every hole adds 20ml culture fluid.Cultivate 24h and 48h after intervening, detect for cell migration.Often group establishes 3 parallel holes, experiment repetition 3 times.
2, cell migration ability (scratch experiment) detects
Principle: be fused into monolayer state when cell grows to be, artificially on the cell monolayer merged manufactures a white space, is called " cut " (Wound).The cell of scratching edge can progress into white space makes " cut " heal (Healing).Basic step comprises the manufacture of " cut ", the acquisition of image and the process of later data during cell migration.
Operating process:
(1) by 2 × 10
5cells/well adds in 6 porocyte culture plates, and incubated overnight, treats that cell grows to Fusion Strain.Quantity is advisable to be paved with at the bottom of plate after adherent;
(2) four roads orthogonal " well " stroke trace is marked with the cell surface that 10 μ l import rifle heads after sterilization are being cleaned;
(3) suck cell culture fluid, use PBS buffer solution cell 3 times, wash away the cell debris that cut produces;
(4) select the cut visual field, carry out labelling, and under microscope, carry out observation take pictures;
(5) 6 porocyte culture plates being put into cell culture incubator to cultivate, taking pictures respectively at taking out after 24h and 48h;
(6) cut healing state calculates with the spacing ratio of cell wound healing spacing and cell cut.
Result:
After the monoclonal antibody NJ001-1 of 50 μ g/mL, 100 μ g/mL and 150 μ g/mL concentration acts on adenocarcinoma of lung SPC-A-1 cell 48h, " cut " healing ability reduces, namely, compared with matched group (without monoclonal antibody NJ001-1 process), the distance of monoclonal antibody group cut blank space is wider.By measuring " cut " width of monoclonal antibody group and matched group 0h and 48h, calculating SPC-A-1 cell migration distance, respectively organizing the difference of SPC-A-1 cell migration ability.Result shows, monoclonal antibody group SPC-A-1 cell migration ability is subject to suppression in various degree, after wherein 100 μ g/mL and 150 μ g/mL monoclonal antibody NJ001-1 act on SPC-A1, cell migration ability reduces, and has significant difference (P value is all less than 0.05) compared with matched group; After 50 μ g/mL monoclonal antibody NJ001-1 act on SPC-A1, cell migration distance diminishes, but compared with matched group no difference of science of statistics.The above results shows, monoclonal antibody NJ001-1 can suppress lung adenocarcinoma cell to move.Low dosage monoclonal antibody NJ001-1 is not obvious to the inhibitory action of lung adenocarcinoma cell transfer ability, but along with the increase of monoclonal antibody NJ001-1 concentration, the transfer ability of lung adenocarcinoma cell reduces gradually, and prompting monoclonal antibody NJ001-1 moving lung adenocarcinoma cell of dose dependent can play inhibitory action.
Embodiment 2
(1) cell invasion detects
Collect SPC-A-1 and A549 being in exponential phase and make single cell suspension, with 8 × 10
4cells/well adds in 24 porocyte culture plates.Antibody group (50 μ g/mL, 100 μ g/mL and 150 μ g/mL tri-groups): every hole mistress adds monoclonal antibody NJ001-1 solution, and final concentration is respectively 50 μ g/mL, 100 μ g/mL and the 150 every holes of μ g/mL; Matched group (0 μ g/mL): every hole adds 500 μ l culture fluid.Cultivate 24h after intervention, detect for cell invasion.Often group establishes 3 parallel holes, experiment repetition 3 times.
(2) cell invasion ability (Transwell experiment) detects
Principle: transwell cell is put into culture plate, little indoor are called room, are called lower room in culture plate, upper indoor interpolation culture supernatants, and lower indoor interpolation lower floor culture fluid, levels culture fluid is separated by with film.By cell kind in upper indoor, because film has permeability, the composition in lower floor's culture fluid can have influence on the cell of indoor, thus can study the impact of the composition cell growth, motion etc. in lower floor's culture fluid.Select film and the aperture of different materials, the research of the multiple aspects such as Dual culture, cell chemotaxis, cell migration, cell invasion can be carried out.
Operating process:
(1) in the cell of Transwell24 porocyte culture plate, add 60 μ l matrigel (ECMgel) diluents, the dilution ratio of matrigel and serum-free RPMI-1640 culture fluid is 1:9;
(2) Transwell24 porocyte culture plate is put into 37 DEG C of cell culture incubators, make matrigel natural coagulation;
(3) lung adenocarcinoma cell is resuspended in serum-free RPMI-1640 culture fluid, every cell adds 1 × 10
5~ 1 × 10
6cell (specifically changing because cell-penetrating ability is different), upper indoor serum-free RPMI-1640 culture fluid cumulative volume is 200 μ l;
(4) according to cell grouping, add the RPMI-1640 culture fluid of 500 μ l containing 10%FBS in lower indoor, wherein the concentration of monoclonal antibody NJ001-1 is respectively 0 μ g/mL, 50 μ g/mL, 100 μ g/mL and 150 μ g/mL;
(5) Transwell24 porocyte culture plate is put into cell culture incubator to cultivate, take out after 24h;
(6) wipe with the cell of cotton swab by little for transwell chamber internal surface, cell outer surface cellular 20min fixed by 95% ethanol;
(7) with 1% Crystal Violet Dye, cell outer surface cellular is dyeed, 20min;
(8) the PBS buffer solution for cleaning cell outer surface cellular 3 times of pre-cooling, sucks residual liquid from little chamber internal surface;
(9) Olympus Optical basis of microscopic observation penetrates the cell of matrigel, and counting of taking pictures.
Result:
After the monoclonal antibody NJ001-1 of 50 μ g/mL, 100 μ g/mL and 150 μ g/mL concentration acts on lung adenocarcinoma cell (SPC-A1 and A549) 24h, the lung adenocarcinoma cell quantity that invasion and attack occur obviously reduces, and has significant difference (P value is all less than 0.05) compared with matched group (without monoclonal antibody NJ001-1 process); Along with the increase of monoclonal antibody NJ001-1 concentration, the lung adenocarcinoma cell quantity with invasive ability significantly reduces.The above results shows, monoclonal antibody NJ001-1 significantly can suppress the invasive ability of lung adenocarcinoma cell, and this inhibitory action has obvious dose dependent.
Embodiment 3TIMP-3 detection of expression
RT-PCR：
1) sample prepares
Discard culture fluid in culture plate, every hole adds 700 μ lTrizol reagent, repeatedly blows and beats cell respectively with Trizol, carries out digesting, cracking.Solution is collected in going in enzyme EP pipe ,-70 DEG C of preservations after cell dissolves completely.
2) RNA extracts
(1) desk-top high-speed refrigerated centrifuge to 4 DEG C is regulated;
(2) cell mass having added Trizol is taken out from-70 DEG C of refrigerators;
(3) to blow and beat gently with rifle head after cell mass dissolves or whirlpool concussion mixing, whirlpool shakes 1min;
(4) room temperature (15 ~ 25 DEG C) places 5min;
(5) add 140 μ l chloroforms, cover tightly pipe lid gently, concuss 15s;
(6) room temperature leaves standstill 2 ~ 3min;
(7) 4 DEG C of 12000g × 15min, are adjusted to room temperature by centrifuge;
(8) upper strata aqueous phase is carefully inhaled new EP pipe (providing) is provided, add the dehydrated alcohol of 1.5 times of volumes (about 525 μ l), blow and beat mixing gently with rifle head, carry out next-step operation immediately;
(9) inhale 700 μ l in post, froth pipe lid, (15 DEG C ~ 25 DEG C) ，≤10000rpm × 15sec abandon liquid to room temperature, if total liquid volume is greater than 700 μ l, repeat step (9);
(10) add 700 μ lBufferRWT on pillar film, build lid gently, (15 DEG C ~ 25 DEG C) ，≤10000rpm × 15sec abandon liquid to room temperature;
(11) add 500 μ lBufferRPE on pillar film, build lid gently, (15 DEG C ~ 25 DEG C) ，≤10000rpm × 15sec abandon liquid to room temperature;
(12) add 500 μ lBufferRPE on pillar film, build lid gently, (15 DEG C ~ 25 DEG C) ，≤10000rpm × 2min abandon liquid to room temperature;
(13) pillar is carefully shifted out to new 2mlEP pipe, high speed centrifugation 1min;
(14) pillar is moved to new 1.5mlEP pipe, draw 30 ~ 50 μ lddH2O on pillar film, build pipe Gai ，≤10000rpm × 1min gently;
(15) if Nong Du≤30 μ g, step (15) is repeated.
3) cDNA synthesis (uses Japanese TAKAR company PrimeScript
tMrTMasterMix (PerfectRealTime) Reverse Transcription box)
(1) reverse transcription reaction system:
(2) reverse transcription reaction is carried out in PCR amplification instrument: 16 DEG C of 30min, 42 DEG C of 30min, 85 DEG C of 5min; Stand-by or-70 DEG C of preservations are on ice placed it in after reaction terminates.
4) PCR detects
(1) reaction system (10 μ l):
Primer sequence: TIMP-3 forward primer: 5 '-CCTGCTGACAGGTCGCGTCT-3 '; Downstream primer: 5 '-TCCAGAGACACTCGTTCTTG-3 '.MMP-7 forward primer: 5 '-TCGAGACTTACCGCATATTAC-3 '; Downstream primer: 5 '-TCCAGCGTTCATCCTCAT-3 '; β-actin as reference gene, forward primer: 5 '-TGGCCCCAGCACAATGAA-3 '; Downstream primer: 5 '-CTAAGTCATAGTCCGCCTAGAAGCA-3 '.
(2) amplified reaction is carried out in PCR amplification instrument: 95 DEG C 10min1 circulation, 95 DEG C of 15sec, 60 DEG C of 1min, 45 circulations; Reaction terminates rear according to gained CT value application 2
-△ △ CTmethod calculates the relative expression quantity of miRNA.
WesternBlot method detects protein expression
(1) glue plate is prepared
Clean glass plate gently with detergent liquid, use distilled water flushing again after clean with tap water, align bottom the glass plate of thickness both sides after drying, isostasy, is installed on gum-making rack;
(2) according to the form below preparation separation gel and concentrated glue
When joining glue, by table order reagent adding, TEMED to add after mixing the slow implantation glass plate holder layer along side immediately.The separation gel amount of recording is 4ml/ block, stays concentrated glue desired height (1cm longer than comb length), and adding distil water flattens, and leaves standstill 60min (room temperature comparatively low time delay is long-time) polymerization.Topple over glass plate and remove distilled water, filter paper blots, and can not touch gel.Concentrate glue by upper table preparation and pour into, concordant with outer field thin glass, insert comb from lopsidedness, prevent bubble from producing, standing 30min makes it be polymerized, and preservative film adds appropriate ddH
2o puts 4 DEG C of refrigerator overnight after wrapping up gel;
Glass plate is moved into electrophoresis tank, and inside groove fills it up with electrophoretic buffer, observes whether leakage, and removes the bubble attached to bottom gel, otherwise it is unimpeded to affect electric current, takes out comb, avoids loading wells to tear;
(3) sample treatment and loading
Add 2 × electrophoresis sample buffer of equivalent in protein sample, boil 5min after mixing, 4 DEG C, the centrifugal 5min of 12000rpm, sample thief adds loading wells, and each porin amount is impartial.Add molecular weight standard protein Marker for contrast simultaneously;
(4) electrophoresis
Under 60V voltage, electrophoresis to sample all leaves loading wells to changing voltage during separation gel interface is 80V, and after continuing electrophoresis appropriate time, power-off stops electrophoresis;
(5) transferring film
The preparation (prepare ice and ice chest, the pre-cooling of transferring film buffer, pvdf membrane, puts into transferring film buffer by sponge and filter paper and soak) of transferring film is carried out at the end of SDS-PAGE is fast
↓
Pry open glass plate gently, according to the position that Marker judges object fragment, cut glue, immerse transferring film buffer
↓
Cut the pvdf membrane of corresponding size according to the size of glue, film is bigger, can not be little and shear angle carries out labelling
↓
Pvdf membrane methanol activates 1min, then uses transferring film buffer rinsing 1min
↓
Install the interlayer of film and glue by the order of black glue tunica albuginea, install electrophoresis transferring groove by the order of glue negative electrode film positive pole
↓
Electrophoresis transferring groove is put in ice, puts the transferring film buffer that ice bag adds pre-cooling in groove, 100mA transferring film under constant current state, and transferring film electric current and time can regulate according to molecular size range
↓
Transferring film terminates carefully to take off film afterwards, and 5% defatted milk powder (TBST preparation) room temperature closes 3h
↓
Add primary antibodie reaction (antibody is by respective description dilution), 4 DEG C are spent the night.
↓
1 × TBST washes film, and 15min changes and once washes film buffer, totally 3 times
↓
Add two to resist, room temperature reaction 1h, period slowly shakes on shaking table
↓
1 × TBST washes film, and 15min changes and once washes film buffer, totally 3 times
↓
After blotting film buffer with filter paper, film is put on sealed membrane
↓
Add the luminous AB mixing material of ECL, reaction 3min, notes lucifuge in operating process
↓
Put into magazine to expose in darkroom, time of exposure from 10s to 1h not etc.
↓
Development, notes holding developing time, rinsing, fixing transparent to film
↓
Gel imaging system is utilized to gather image and preserve
Result:
After the monoclonal antibody NJ001-1 of 50 μ g/mL, 100 μ g/mL and 150 μ g/mL concentration acts on lung adenocarcinoma cell (SPC-A1 and A549) 24h, can significantly increase TIMP-3mRNA and protein expression, compared with matched group (without monoclonal antibody NJ001-1 process), there is significant difference (P value is all less than 0.05).
The interaction of embodiment 4 monoclonal antibody NJ001-1 specific antigen and nuclear factor FOXP1
Co-immunoprecipitation:
After monoclonal antibody NJ001-1 treatment S PC-A1, hatch with the SCP-A1 Nuclear extract of total amount 1mg and POXP1 antibody (3 μ g) or rabbit igg and proteinA-Agarose.FOXP1 complex SDS buffer solution elution, obtains albumen and carries out WesternBlot detection.
Result:
Under monoclonal antibody NJ001-1 effect, in lung adenocarcinoma cell core, monoclonal antibody NJ001-1 specific antigen can be combined with transcription factor FOXP1, suppresses its transcripting suppressioning action to TIMP-3 gene promoter area (Fig. 4).
Claims (3)
1. monoclonal antibody NJ001 suppresses the application in the medicine of NSCLC metastasis in preparation; Wherein, the hybridoma cell strain NM001-1 that described monoclonal antibody NJ001 is CCTCCNO:C201172 by preserving number secretes.
2. monoclonal antibody NJ001 is preparing the application in the medicine suppressing NSCLC metastasis at the key gene suppressing to act in NSCLC metastasis as target spot.
3. application according to claim 2, is characterized in that described monoclonal antibody NJ001 is suppressing the key gene acted in NSCLC metastasis to be encoding transcription factors FOXP1.
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| CN102391992A (en) * | 2011-11-03 | 2012-03-28 | 潘世扬 | Monoclonal antibody for resisting human NSCLC and application of monoclonal antibody |
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| SHIYANG PAN, 等: "The Study on Newly Developed McAb NJ001 Specific to Non-Small Cell Lung Cancer and Its Biological Characteristics", 《PLOS ONE》 * |
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