CN103923214A - Anti-hepatoma stem cell monoclonal antibody and application thereof - Google Patents
Anti-hepatoma stem cell monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-hepatoma stem cell monoclonal antibody which is secreted by a mouse hybridoma cell 4P-120 with the collection number of CGMCC No.8800. Proven by experiments, the monoclonal antibody can be specifically combined with hepatoma cells and is capable of specifically recognizing hepatoma tissues, antigens recognized by the monoclonal antibody are highly expressed in cancer tissues of human hepatoma, but are not expressed or slightly expressed in para-carcinoma tissues, and positive-to-negative differentiation and negative-to-positive defifferentiation exist in a cell multiplication process. Compared with cells with low antigen expression, cells with high antigen expression have higher proliferation capability, clone formation capability, balling capability, scratch transfer capability and invasion transfer capability; and the invention provides a functional monoclonal antibody with an application prospect for treating hepatoma with targeted tumor stem cells, and the monoclonal antibody can be used for preparing medicines for treating hepatoma.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of monoclonal antibody.
Background technology
Liver cancer is one of common malignant tumour of China, and its case fatality rate occupies the 3rd of malignant tumour, and 5 years survival rates of liver cancer patient only have 14%～30%.Primary hepatocarcinoma has the advantages that morbidity is hiddenly attacked, case fatality rate is high, early diagnosis and prevent that recurrence is great to prognosis meaning.After monoclonal antibody technique comes out, people attempt to improve by application liver cancer-resisting monoclone antibody early diagnostic rate and the result for the treatment of of liver cancer, but the specificity of the liver cancer-resisting monoclone antibody obtaining is at present still unsatisfactory, due to the existence of Tumor Heterogeneity, make again its clinical application be under some influence.Therefore, be necessary to find better anti-liver cancer-specific monoclonal antibody, to improve the specificity to liver cancer diagnosis and treatment.In recent years, tumor stem cell (cancer stem cell, CSC) has become the new focus of tumor research.Because CSC is the root of metastases, recurrence and resistance, therefore, the treatment New Policy of setting up target CSC is expected to overcome the defect of existing treatment means clinically, improves the prognosis of liver cancer patient.Contriver place seminar is the similarity with embryonic stem cell based on tumor stem cell in earlier stage, using Nanog as sorting indicia, built and take slow virus reporting system Lv-Nanog (p)-GFP that Nanog is that promoter regulation GFP expresses, the liver cancer cell of the Nanog positive that sorting obtains confirms to have tumor stem cell characteristic (Hepatology2012) through internal and external test.
Summary of the invention
In view of this, the object of the invention is to using the liver cancer cell of the Nanog positive as immunogen and screening antigen, by monoclonal antibody technique, filter out the monoclonal antibody for liver-cancer stem cell, and to this monoclonal antibody for antigen study, to provide the functional monoclonal antibody of application prospect for the liver cancer treatment of targeting tumor stem cells.
After deliberation, the invention provides following technical scheme:
1. anti-liver-cancer stem cell monoclonal antibody, is the mouse hybridoma cell 4P-120 secretion generation of CGMCC No.8800 by deposit number.
2. the application of anti-liver-cancer stem cell monoclonal antibody in preparing cancer treatment drug.
Beneficial effect of the present invention is: the present invention using the Nanog positive liver cancer cell as immunogen and screening antigen, by monoclonal antibody technique filter out can stably excreting monoclonal antibody mouse hybridoma cell strain 4P-120, the monoclonal antibody of its secretion confirms through experiment, can specific binding liver cancer cell specific recognition liver cancer tissue; The antigen that this monoclonal antibody is identified is not expressed or low expression in high expression level Er cancer beside organism in the cancerous tissue of people's liver cancer, and in during proliferation process, has the positive contrary differentiation to negative differentiation and negative heliotropism; Compare the low express cell of antigen, antigen high expressing cell has stronger multiplication capacity, clonality, balling-up ability, cut transfer ability and Invasion and Metastasis ability; Therefore, the liver cancer treatment that the present invention is targeting tumor stem cells provides the functional monoclonal antibody that has application prospect, and it can be for the preparation of cancer treatment drug.
Biomaterial preservation
Mouse hybridoma cell 4P-120 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 5th, 2014 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica), deposit number is CGMCC No.8800.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing and describe:
Fig. 1 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe response situation of monoclonal antibody 4P-120 and different clones.
Fig. 2 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe response situation of monoclonal antibody 4P-120 and people's liver cancer tissue and normal liver tissue.
Fig. 3 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe expression of the antigen that monoclonal antibody 4P-120 identifies in people's liver cancer tissue and cancer beside organism.
Fig. 4 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe expression changing conditions of the antigen that monoclonal antibody 4P-120 identifies in during proliferation process.Wherein, A, B are respectively sorting 4P-120 antigen and Nanog jack to jack adapter sexual cell (P9), 4P-120 antigen list positive cell (P8), 4P-120 antigen and Nanog pair of positive cells (P6), these four cell colonys of 4P-120 antigen list negative cells (P7) from PLC-Nanog cell; C, D, E, F are for to carry out to the P9 sub-electing, P8, P7, P6 cell colony the result that streaming is returned survey; G, H, I, J carry out to P9, P8, P7, P6 cell colony the flow cytometer detection result that 4P-120 antigen breaks up situation after cultivating 7d.
Fig. 5 is that 4P-120 antigen high expressing cell has stronger multiplication capacity.
Fig. 6 is that 4P-120 antigen high expressing cell has higher clonality.
Fig. 7 is that 4P-120 antigen high expressing cell has higher balling-up ability.
Fig. 8 is that 4P-120 antigen high expressing cell has higher cut transfer ability.
Fig. 9 is that 4P-120 antigen high expressing cell has higher Invasion and Metastasis ability.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
One, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe preparation of monoclonal antibody
1, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe acquisition of hybridoma cell strain
By the PLC-Nanog cultivating
pOSnanog
negcell (the liver cancer cell PLC that the Nanog of take expresses as promoter regulation GFP) airflow classification goes out PLC-Nanog
pOScell (being the PLC cell of the Nanog positive), as immunogen; Get the healthy BALB/c mouse in 4～6 week age of body weight 14～18g, in every mouse peritoneal, inject 1 * 10
6individual PLC-Nanog
pOScell carries out first immunisation, afterwards at interval of 2 weeks booster immunizations 2 times in the same way again.After last booster immunization the 10th day, get mouse tail blood, separation of serum, adopts the indirect Enzyme-linked Immunosorbent Assay of cell (ELISA) method to detect antibody titer.
It is specific as follows that ELISA method detects antibody titer: by PLC-Nanog
pOScell is inoculated in 96 porocyte culture plates, every hole 1 * 10
4individual cell, take out Tissue Culture Plate next day, after abandoning supernatant, PBS washes 3 times, with 4% paraformaldehyde solution, under room temperature, fix 20 minutes, PBS rinsing 3 times, use again 10% foetal calf serum (FBS) in 37 ℃ of sealings 30 minutes, abandon supernatant, different cell cultures hole adds respectively the immune serum of continuous 10 times of dilutions, every hole 100ul, the mice serum of usining before first immunisation as negative control simultaneously, PBS is as blank, hatch after 1 hour for 37 ℃ and abandon supernatant, PBST washes plate 5 times, add again with confining liquid by the goat anti-mouse IgG of the HRP mark of 1:1000 dilution, every hole 100ul, hatch 30 minutes for 37 ℃, discard traget antibody solution, PBST washes plate 5 times, add again OPD nitrite ion, hatch after 20 minutes for 37 ℃, by 2mol/L sulfuric acid termination reaction, measure and respectively organize OD
490nmvalue, experimental group OD
490nmvalue is more than or equal to 2.1 times of persons of negative control and is judged to the positive, usings and is judged to positive highly diluted multiple as antibody titer.
Choose the mouse that antibody titer is the highest, with PLC-Nanog
pOScell carries out spleen reinforcement, after 3 days, gets its spleen and prepares splenocyte suspension, and SP2/0 merges with murine myeloma cell.Gained fused cell is with on average assigning in 5-7 96 porocyte culture plates and cultivate containing the RPMI-1640 of HAT, in cultivating about the 7th day, by the RPMI-1640 full dose containing HAT, change liquid once, within the 10th day, collect culture supernatant, adopt ELISA method to carry out positive cell screening.
ELISA method screening positive cell is specific as follows: in fused cell, cultivate the 9th day, by PLC-Nanog
pOScell is inoculated in another 96 porocyte culture plate, every hole 1 * 10
4individual cell, within the 10th day, take out Tissue Culture Plate, after abandoning supernatant, PBS washes 3 times, with 4% paraformaldehyde solution, under room temperature, fix 20 minutes, PBS rinsing 3 times, with 10%FBS, in 37 ℃, seal 30 minutes again, abandon supernatant, each hole adds respectively different fused cell culture supernatant, every hole 100ul, the immune serum that the 1:100 of usining dilutes is simultaneously as positive control, using SP2/0 cells and supernatant as negative control, hatch 1 hour for 37 ℃, abandon supernatant, PBST washes plate 5 times, add again with confining liquid by the goat anti-mouse IgG of the HRP mark of 1:1000 dilution, every hole 100ul, hatch 30 minutes for 37 ℃, discard traget antibody solution, PBST washes plate 5 times, add again OPD nitrite ion, hatch after 20 minutes for 37 ℃, by 2mol/L sulfuric acid termination reaction, measure and respectively organize OD
490nmvalue, experimental group OD
490nmvalue is more than or equal to 2.1 times of persons of negative control and is judged to the positive.
Positive porocyte is changed to liquid with the RPMI-1640 containing HAT and cultivate, adopt next day aforementioned ELISA method to recheck, detected positive porocyte further carries out mono-clonal screening.
The acquisition of monoclonal hybridoma strain: adopt limiting dilution assay to carry out subclone to positive porocyte, after each subclone about the 7th day, pick out monoclonal cell hole and adopt aforementioned ELISA method to detect, retain 1-2 positive hole, repeat subclone more than three times, until all subclones are all positive, this cell strain is carried out to enlarged culturing frozen, obtain monoclonal hybridoma strain.
The present invention is merged screening through four times, obtains altogether 64 strain monoclonal hybridoma strains, through identifying, confirms, the mouse hybridoma cell of called after 4P-120 can stably excreting has the monoclonal antibody of liver cancer-specific.
2, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe acquisition of monoclonal antibody
Healthy BALB/c mouse more than choosing body weight 20g, more than 8 week age, abdominal injection by whiteruss with lanolin in mass ratio for 3:1 mixes the adjuvant making, every 0.3ml, after 7-10 days, the mouse hybridoma cell 4P-120 of cultivation is washed and is once injected into afterwards mouse peritoneal with PBS or physiological saline, after 7-10 days, get mouse ascites, with ProteinG post by specification, carry out purifying, obtain mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120, measures antibody concentration ,-20 ℃ of preservations.
Two, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe specificity identification of monoclonal antibody
1, flow cytometer detects antibodies specific
Choose different tumor cell lines: three kinds of hepatoma cell line (PLC, HU-7, T1224), two kinds of gastric carcinoma cell lines (Xn0422, SGC7901), two kinds of glioma cell line (U87, U251), three kinds of colon carcinoma cell line (SW480, SW620, HCT-116), and two kinds of normal cell systems (293, L02) the normal CIK cell (CIK-1 of and three example, CIK-2, CIK-3), culturing cell is washed 1 time with PBS, add mouse hybridoma cell 4P-120 culture supernatant, hatch 30 minutes for 4 ℃, PBS washes 3 times, add again with PBS by the goat anti-mouse IgG (Sigma company) of fluorescein isothiocyanate (FITC) mark of 1:500 dilution, hatch 30 minutes for 4 ℃, PBS washes three times, after redissolving with PBS400ul, flow cytometer detects.
Result as shown in Figure 1, mouse hybridoma cell 4P-120 culture supernatant is all obvious high reaction to three kinds of hepatoma cell line, other tumor cell line is to low reaction, to the reaction that is negative of normal clone, the mouse-anti people liver-cancer stem cell PLC-Nanog being secreted by mouse hybridoma cell 4P-120 is described
pOSmonoclonal antibody 4P-120 can specific binding liver cancer cell.
2, identified by immunofluorescence antibodies specific
Cut respectively people's liver cancer tissue and normal liver tissue, with cold acetone, fix 20 minutes, PBS washes 4 times, then hatches 30 minutes with 10%BSA in 37 ℃, then adds 10ug/ml mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120,4 ℃ of refrigerator overnight, PBS washes 5 times, then adds with PBS by the goat anti-mouse IgG of the FITC mark of 1:500 dilution, hatch 50 minutes for 37 ℃, then add the DAPI of 1:500 dilution, hatch 10 minutes for 37 ℃, PBS washes 5 times, 40% glycerine mounting, and laser co-focusing detects.
Result as shown in Figure 2, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120 and people's liver cancer tissue are strong positive reaction, and to the reaction that is negative of people's normal liver tissue, illustrate that this monoclonal antibody can specific recognition liver cancer tissue.
3, immunohistochemical methods is identified antibodies specific
Choose respectively cancerous tissue and cancer beside organism's paraffin section of people's liver cancer, under lamp, bake 20 minutes, with dimethylbenzene, soak 2 times (each 10 minutes), use successively again dehydrated alcohol, dehydrated alcohol, 95% ethanol, 85% ethanol, 75% ethanol respectively soaks 5 minutes, use successively again distilled water, PBS respectively washes 3 times, add antigen retrieval liquid, 800W microwave heating 3 minutes and 15 seconds, 150W microwave heating 16 minutes, cooling rear distillation washing 3 times, add again 3% hydrogen peroxide, hatch 15 minutes for 37 ℃, PBS washes 3 times, add again sealing serum, hatch 15 minutes for 37 ℃, add again 5ug/ml mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120,4 ℃ of refrigerator overnight, PBS washes 5 times, add the goat anti-mouse IgG (Sigma company) of 1:500 dilution again, hatch 25 minutes for 37 ℃, PBS washes 5 times, add horseradish peroxidase again, hatch 15 minutes for 37 ℃, PBS washes 3 times, DAB dyeing, tap water termination reaction, Hematorylin is redyed approximately 10 seconds, then with 75% ethanol, 85% ethanol, 95% ethanol, dehydrated alcohol, dehydrated alcohol, respectively soaks 5 minutes successively, dry mounting, micro-Microscopic observation.
Result as shown in Figure 3, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe antigen that monoclonal antibody 4P-120 identifies is high expression level in the cancerous tissue of people's liver cancer, does not express or low expression in cancer beside organism; Statistic analysis result also shows, the expression of the antigen that monoclonal antibody 4P-120 identifies in cancerous tissue is apparently higher than cancer beside organism.
Three, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe functional performance of the antigen presentation of monoclonal antibody and different antigen presentation amount cells detects
1, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe expression of the antigen that monoclonal antibody is identified in during proliferation process
By the PLC-Nanog cultivating
pOSnanog
negcell trysinization, PBS washes 1 time, and centrifugal 3 minutes of 1000rpm, abandons supernatant, adds 50ug/ml mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120100ul, incubated at room 15 minutes, uses PBS re-suspended cell 1000rpm centrifuge washing 3 times, then adds the anti-mouse IgG of the Alexa Fluo647 mark of 1:1000 dilution, hatch 15 minutes for 4 ℃, with selected by flow cytometry apoptosis, go out mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe low express cell group of the antigen high expressing cell group of monoclonal antibody 4P-120 and antigen, with cultivating respectively containing the DMEM substratum of 10%FBS, and by cell antigen expression before cultivations and return survey result after 7 days and compare.
Result as shown in Figure 4, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSduring proliferation process, there is the positive contrary differentiation to negative differentiation and negative heliotropism in the antigen that monoclonal antibody 4P-120 identifies.
2, the functional performance of the cell colony of different antigen presentation amounts detects
The mouse-anti people liver-cancer stem cell PLC-Nanog that get respectively unsorted PLC cell, sub-elects from PLC clone
pOSeach 1000 of the antigen high expressing cell of monoclonal antibody 4P-120, the low express cells of antigen, be inoculated in 96 porocyte culture plates, by the DMEM culture medium culturing containing 10%FBS, within 1～4 day, measure ability of cell proliferation difference afterwards with cell tilter blue cell proliferation detecting kit, data detect 560nm/590nm ratio by the long microplate reader of all-wave and obtain.As shown in Figure 5,4P-120 antigen high expressing cell has and is significantly better than the not multiplication capacity of sorting PLC cell, the low express cell of 4P-120 antigen result.
The mouse-anti people liver-cancer stem cell PLC-Nanog that get respectively unsorted PLC cell, sub-elects from PLC clone
pOSeach 50 of the antigen high expressing cell of monoclonal antibody 4P-120, the low express cells of antigen, be inoculated in 24 porocyte culture plates, by the DMEM culture medium culturing containing 10%FBS, within 2 days, changes liquid once, after 14 days, clone formed to number and count and add up.As shown in Figure 6, compared to the low express cell of antigen of monoclonal antibody 4P-120,4P-120 antigen high expressing cell has higher clonality to result.
The mouse-anti people liver-cancer stem cell PLC-Nanog that get respectively unsorted PLC cell, sub-elects from PLC clone
pOSeach 20 of the antigen high expressing cell of monoclonal antibody 4P-120, the low express cells of antigen, be inoculated in 96 porocyte culture plates, by serum-free balling-up culture medium culturing, after 14 days, one-tenth nodule number counted and added up.As shown in Figure 7, compared to not sorting cells and the low express cell of 4P-120 antigen, 4P-120 antigen high expressing cell has higher balling-up ability to result.
The mouse-anti people liver-cancer stem cell PLC-Nanog that get respectively unsorted PLC cell, sub-elects from PLC clone
pOSthe antigen high expressing cell of monoclonal antibody 4P-120, the low express cell of antigen each 10
5individual, be inoculated in 24 porocyte culture plates, with the low serum free culture system liquid containing 2%FBS, cultivate, after being paved with orifice plate, carry out cut and take pictures, after 24 hours, again take pictures, the width average healing state of statistics cut.As shown in Figure 8, compared to not sorting cells and the low express cell of 4P-120 antigen, 4P-120 antigen high expressing cell has higher cut transfer ability to result.
The mouse-anti people liver-cancer stem cell PLC-Nanog that get respectively unsorted PLC cell, sub-elects from PLC clone
pOSthe antigen high expressing cell of monoclonal antibody 4P-120, the low express cell of antigen each 10
5individual, be inoculated in 8um transwell cell, in cell, be 200ul serum-free DMEM substratum, in lower floor's 24 orifice plates, be the DMEM substratum of 1300ul containing 10%FBS, after 24 hours, perforation cell count is dyeed, statistics perforation cell count.As shown in Figure 9, compared to the low express cell of 4P-120 antigen, 4P-120 antigen high expressing cell has higher Invasion and Metastasis ability to result.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can to it, make various changes in the form and details, and not depart from the claims in the present invention book limited range.
Claims (2)
1. anti-liver-cancer stem cell monoclonal antibody, is characterized in that, the mouse hybridoma cell 4P-120 secretion that is CGMCC No.8800 by deposit number produces.
2. the application of anti-liver-cancer stem cell monoclonal antibody as claimed in claim 1 in preparing cancer treatment drug.
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| CN105132445A (en) * | 2015-10-23 | 2015-12-09 | 上海麦禾生物科技有限公司 | Receptor protein, T lymphocyte and NK cells for specifically identifying cancer cells |
| CN106366190A (en) * | 2015-07-22 | 2017-02-01 | 中国医学科学院肿瘤医院 | Anti-human-liver-cancer-stem-cell monoclonal antibody |
| CN107163146A (en) * | 2017-06-23 | 2017-09-15 | 苏州博聚华生物医药科技有限公司 | A kind of monoclonal antibody for targetting human tumour stem cell and its application |
| CN107474139A (en) * | 2017-06-23 | 2017-12-15 | 苏州博聚华生物医药科技有限公司 | A kind of monoclonal antibody for targetting human tumour stem cell and its application |
| WO2018233333A1 (en) * | 2017-06-23 | 2018-12-27 | 苏州博聚华生物医药科技有限公司 | Monoclonal antibody of targeted human tumor stem cells and application of monoclonal antibody |
| CN111303290A (en) * | 2020-05-11 | 2020-06-19 | 北京广未生物科技有限公司 | Antibody specifically binding to liver cancer stem cells and application of antibody in preparation of liver cancer detection kit |
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| CN106366190A (en) * | 2015-07-22 | 2017-02-01 | 中国医学科学院肿瘤医院 | Anti-human-liver-cancer-stem-cell monoclonal antibody |
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| WO2018233333A1 (en) * | 2017-06-23 | 2018-12-27 | 苏州博聚华生物医药科技有限公司 | Monoclonal antibody of targeted human tumor stem cells and application of monoclonal antibody |
| CN107163146B (en) * | 2017-06-23 | 2021-04-23 | 苏州博聚华生物医药科技有限公司 | Monoclonal antibody targeting human tumor stem cells and application thereof |
| CN107474139B (en) * | 2017-06-23 | 2021-04-27 | 苏州博聚华生物医药科技有限公司 | Monoclonal antibody targeting human tumor stem cells and application thereof |
| CN111363034A (en) * | 2018-12-25 | 2020-07-03 | 上海市第十人民医院 | NANOG Ser68 antibody, inhibitor and application |
| CN111303290A (en) * | 2020-05-11 | 2020-06-19 | 北京广未生物科技有限公司 | Antibody specifically binding to liver cancer stem cells and application of antibody in preparation of liver cancer detection kit |
| CN111303290B (en) * | 2020-05-11 | 2020-12-01 | 绍兴百奥尼科技有限公司 | Antibody specifically binding to liver cancer stem cells and application of antibody in preparation of liver cancer detection kit |
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