CN102901817A - Application of Non-B Ig monoclonal antibody RP215 in cell proliferation, migration and stem cell research - Google Patents
Application of Non-B Ig monoclonal antibody RP215 in cell proliferation, migration and stem cell research Download PDFInfo
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Abstract
The invention discloses application of monoclonal antibody RP215 that can specifically identify a Non-B cell-derived immunoglobulin (non-B Ig) heavy chain variable region in judging the proliferation, migration and chemotherapy drug resistance of cancer cells and identifying adult/cancer stem/progenitor cells. The scheme of the invention has an important application value for improvement of cancer cell malignancy, cancer metastasis and prognosis judgement in clinical and scientific research and in-depth study of cancer/adult stem/progenitor cells.
Description
Technical field
The monoclonal antibody RP215 that the present invention relates to specific recognition people Non-B Ig variable region of heavy chain is judging cell in the application aspect propagation, migration, chemotherapeutics resistance and the identification tumour/adult ancestral cells, has important using value to improving clinical and laboratory to the judgement of tumour cell grade of malignancy and transfer, guiding clinical treatment and to the further investigation of tumour/adult ancestral cells.
Background technology
One, non-B cell immunoglobulin present Research
Traditional Immunology thinks that immunoglobulin gene only carries out selectivity and resets in the bone-marrow-derived lymphocyte growth course, so immunoglobulin (Ig) is the characteristic product of bone-marrow-derived lymphocyte.But 1996, Qiu Xiaoyan etc. find to exist protein molecular identical with the immunoglobulin (Ig) antigenicity and that molecular structure is similar by SABC, Western Blot etc. in the endochylema of multiple epithelial malignancy cell, and other normal epithelium cell is negative reaction (Chinese Journal of Immunology; 1996,5:296); A Qiu Xiao man of virtue and ability waits again and expresses the tumour cell of non-B cell derived from protein level and mRNA level confirmation Ig molecule respectively subsequently; But and find to use the special antisense oligonucleotides of Ig and anti-Ig antibody all inducing apoptosis of tumour cell, suppress its growth (October 1,2003 for CANCER RESEARCH 63,6488-6495).At present, our seminar has had very great development for the research of tumour source Ig biologic activity.Our research finds that the external ASODN of utilization or anti-human IgG antibody suppress tumour source IgG can increase programmed cell apoptosis, Cell growth inhibition.In the experiment, anti-human IgG antibody can suppress the growth of the cancerous cell line HelaMR of IgG secretion in nude mouse.And, be not tumour cell, the epithelial cell of some molecular marker for increased proliferation also can be expressed Ig, all has Ig to distribute in the epithelial tissue around the liver cell that breeds in the mammary glandular cell of hyperplasia, the cirrhosis and the cancer nests.The expression of Ig occur to be raised in precancerous lesion, illustrate that the Ig of tumor cell secretion may to the playing an important role of cancer, have the effect that promotes growth of tumour cell and keep its existence.Present above-mentioned discovery has obtained confirmation and approval (Cancer Res, 2006.66 (8): p.3996-4000 of domestic and international several seminars; Cancer Biomark., 2009.5 (4): p.177-188).But in the past 10 years, our seminar and other seminar all use with monoclonal or the polyclonal antibody (commercialization) of circulating Ig molecule as the immunogene preparation, found Ig molecule, particularly IgG molecule high expressed in the tumor tissues of multiple nonimmune cell derived and some normal tissue cells by methods such as SABC, Western blot.But do not find that with this antibody-like the basal cell of IgG epithelium in normal structure and bile duct epithelial cell etc. have high level expression in the cell of strong splitting ability and transfer ability and the tumour liver cell, and participate in the function of cell migration and stem cell.
Two, RP215 source and present Research
The Lee seminar of University of British Columbia is as far back as eighties of last century eighties, in order to obtain the monoclonal antibody of specific recognition oophoroma, once the protein immune animal that extracted with ovarian cancer cell line, nearly 3000 strain of hybridoma have been obtained, in the process of screening, find, one strain of hybridoma is wherein arranged, be referred to as RP215, can identify well ovarian cancer cell and nonrecognition normal ovarian cell, but do not know at that time what its antigen of identifying is, got temporarily called after " CA215 ".Found afterwards that RP215 not only can identify oophoroma well, in the tumour cell identification to other type, also shown good specificity simultaneously, thus CA215 was defined as " pan-cancer marker ".2007, Lee seminar identified CA215.They use the method for affinity chromatography, from the ovarian cancer cell line culture supernatant, obtained a large amount of " CA215 ", 32 peptide sections that Mass Spectrometric Identification provides are the heavy chain of IgG, in order further to confirm this result, they use again purifying " CA215 " to prepare 5 strain specific monoclonal antibodies as antigen, experimental result confirms that all 5 monoclonal antibodies are all identified IgG molecule (Cancer Biol Ther, 2008.7 (12): p.2007-14.).So far, proved that CA215 is exactly IgG (Cancer Biol Ther., 2009.8 (2): p.161-6) of cancer cellular expression.Result subsequently confirms that the IgG that cancer cell is expressed IgG in glycosylation modified and circulation has notable difference.The epi-position that RP215 identifies is the distinctive glycosyl class of this class IgG variable region of heavy chain associated epitope just.Although have been reported serum detection (the Cancer Biomark. that RP215 can be used for the clinical tumor patient, 2009.5 (4): p.177-88.), but this antibody is judging that cell does not also have report in the application aspect propagation, migration, chemotherapeutics resistance and the identification tumour/adult stem cell.
Three, adult/tumor stem cell research background
At the individuality of growing up, the organ of different differentiation and maturations all exists a small amount of multipotential cell with the cell of renewal and alternative aging and apoptosis, keeps the metabolism of histoorgan, and this class cell has the ability of self (self-renewal).But they do not have the totipotence of embryonic stem cell, can only directionally be divided into limited several particular tissue type cells, have the characteristic of pluripotency or monoenergetic, and such as basal cell and the spermatogonium of skin, system is referred to as adult stem cell.It is generally acknowledged, most of ductal epithelium in the normal breast and the basal cell/myoepithelium of lobule of mammary gland and glandular epithelium thereof all have mammary gland stem cell or mammary gland CFU-GM (by the mammary gland Stem cell differentiation but also be not divided into what is called " transition type proliferative cell " or " intermediate cell " of ripe galactophore epithelial cell) characteristic, in addition, there is research to find that normal breast stem cell and musculoepithelia cell are generally expressed the keratin 5 molecule; Cholangiole epithelial cell in the bronchiolar epithelium in the lung tissue in basal cell, eccrine basal cell and the liver organization all belongs to adult stem cell or CFU-GM.Although the research of adult stem cell has had very great development, still also do not find so far the molecular marker of desirable adult stem cell, this has affected the further investigation for adult stem cell greatly.
The at present research of tumor stem cell also is the focus of life science research, the cancer stem-cell hypothesis of the propositions such as Reya is thought: exist the minute quantity oncocyte to serve as the role of stem cell in the tumor tissues, potential with unlimited hyperplasia, in starting tumour formation and growth, play conclusive effect, and remaining most cells, then pass through of short duration differentiation, final dead (Nature, 2001.414 (6859): p.105-11).In recent years research is reported, in the malignant tumours such as leukaemia, brain tumor, breast cancer, prostate cancer, colon cancer, cancer of the stomach, head and neck neoplasm, lung cancer, oophoroma, melanoma, cancer of pancreas and liver cancer, all successfully isolate tumor stem cell, for this theory provides strong evidence (Curr Opin Genet Dev, 2009.19 (1): p.44-50).Tumor stem cell (cancer stem cells, CSC) be to be present in the tumour cell subgroup that has the stem-like cell ability in the tumor tissues, CSCs and adult stem cell are similar, the characteristic that also should have self, CSCs not only has the feature of tumour cell but also have the feature of stem cell, and tumor stem cell can produce (being in different differential periods) all tumour cells of whole tumor tissues.So far, thereby tumor stem cell has self and differentiation capability forms whole knurl body, is considered to the root that tumour produces.Tumor stem cell has very high movement and transfer ability, thereby makes tumour that transfer ability be arranged at the very start, is the major reason that the clinical malignant tumour state of an illness worsens.Studies show that CSCs high expressed Mdr-p, chemotherapeutics can be pumped, cause chemotherapy after CSCs still survive, this may be the important mechanisms of tumor drug resistance, transfer, recurrence.The standard chemotherapy and radiation can significantly dwindle gross tumor volume, but several without effect to tumor stem cell, this is considered to the root of tumor recurrence.Therefore, treat the emphasis for the treatment of tumour after tumour is for CSC.At present, in neoplastic hematologic disorder, breast cancer, brain tumor and prostate cancer, CSC research makes some progress, but most solid tumor all lacks the tumor stem cell specificity marker.Just CD44 (v6) molecule tentatively obtains everybody approval as the breast carcinoma stem cell mark.
Four, experimental technique and principle
1, SABC, it is applied immunology ultimate principle---antigen-antibody reaction, it is the principle of antigen and antibody specific binding, make developer (fluorescein, enzyme, metallic ion, the isotope) colour developing of labelled antibody determine histocyte endoantigen (peptide and protein) by chemical reaction, to its position, qualitative and quantitative research, be called immunohistochemistry technology (immunohistochemistry) or immunocytochemical technique (immunocytochemistry).
Antibody commonly used in the immunohistochemical experiment is monoclonal antibody and polyclonal antibody.Monoclonal antibody is the antibody of bone-marrow-derived lymphocyte clone secretion, and application cell merges the preparation of hybridoma technology immune animal.Polyclonal antibody is with behind the antigen direct immunization animal behind the purifying, and the immune serum that obtains from animal blood is the mixtures of antibodies that a plurality of bone-marrow-derived lymphocyte clones produce.
In recent years, along with the development of immunohistochemistry technology and the appearance of each strain specific antibodies, make many Knotty tumors obtain clarifying a diagnosis.In conventional pathologic diagnosis of tumor, the case of 5%-10% is depended merely on H.E. dyeing and is difficult to make clear and definite Morphologic Diagnosis.Especially the practical value of SABC in diagnosing tumor and antidiastole has been subject to general approval, and it is in low differentiation or not during the antidiastole of differentiation tumor, and accuracy rate can reach 50%-75%.
2, flow cytometry (Flow CytoMeter, FCM) be a kind of on functional level to detection means unicellular or that the other biological particle carries out quantitative test and sorting, it can up to ten thousand cells of high speed analysis, and can from a cell, record a plurality of parameters simultaneously, compare with traditional fluoroscopy, have the advantages such as speed is fast, precision is high, accuracy is good, become contemporary state-of-the-art cell quantitative technology.Flow cytometry combines optics, electronics, and fluid mechanics, cytochemistry, immunology, multi-door subject and the technology such as laser and computing machine are the cell analysis technology, are again accurate sorting technologies.
3, mtt assay introduction
Claiming again the MTT colourimetry, is a kind of method that detects cell survival and growth.It detects principle is that succinate dehydrogenase in the living cells mitochondria can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and is deposited in the cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) the energy dissolved cell is measured its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect living cells quantity.In certain cell number scope, the amount that the MTT crystallization forms is directly proportional with cell number.The method has been widely used in the activity detection of some bioactie agents, large-scale screening anti-tumor medicine, cell toxicity test and tumor radiosensitivity mensuration etc.Its feature is highly sensitive, economical.
4, the cell scratch experiment is introduced
Refer to cell is cultivated on double dish or flat board, scrape at line of middle section picture with cell after Fusion of Cells, the cell in this line has been got rid of by mechanical force, then cell is continued to cultivate, observation of cell is judged the transfer ability of cell to the situation of acellular scored area migration.
Summary of the invention
The present inventor finds that the IgG of non-B cellular expression IgG in glycosylation modified and circulation has notable difference.The epi-position that RP215 identifies is the distinctive glycosyl class of this class IgG variable region of heavy chain associated epitope just.
The present invention is on the basis of early-stage Study, take the monoclonal antibody RP215 of specific recognition people Non-B Ig variable region of heavy chain as main tool, utilize SABC and flow cytometry, having inquired into high expressed Ig is breeding with two groups of cells of the low Ig of expression, the difference of migration and chemotherapeutics resistance aspect, find that the main high level expression of IgG is little at pluripotency ancestral cells those cell volumes that neutralize, form is close to normal cell, it is large not present cell volume, in the typical tumor stem cell such as dyskaryosis, the expression of IgG and cell proliferation, move closely relatedly with the chemotherapeutics resistance, also meet the characteristics of ancestral cells.The solution of the present invention is improving judging that tumour cell has important using value aspect propagation, migration and chemotherapeutics resistance capacity and the identification ancestral cells.The solution of the present invention can also be applied to research work (non-clinical practice) except being applied to clinical position, such as instrument cell in the research work being identified etc.
The solution of the present invention can effectively be judged tumour cell in propagation, migration, chemotherapeutics resistance and effectively identify adult/tumour ancestral cells.Existing research finds that the cholangiole epithelial cell in the bronchiolar epithelium in the lung tissue in basal cell, eccrine basal cell and the liver organization all belongs to adult stem cell or CFU-GM.Although the research of adult ancestral cells has had very great development, still also do not find so far the molecular marker of desirable adult ancestral cells, this has affected the further investigation for the adult ancestral cells greatly.The at present research of tumor stem cell also is the focus of life science research, tumor stem cell (cancer stem cells, CSC) be to be present in the tumour cell subgroup that has the stem-like cell ability in the tumor tissues, CSCs and adult stem cell are similar, the characteristic that also should have self, CSCs not only has the feature of tumour cell but also have the feature of stem cell, and tumor stem cell can produce (being in different differential periods) all tumour cells of whole tumor tissues.So far, thereby tumor stem cell has self and differentiation capability forms whole knurl body, is considered to the root that tumour produces.Tumor stem cell has very high movement and transfer ability, thereby makes tumour that transfer ability be arranged at the very start, is the major reason that the clinical malignant tumour state of an illness worsens.Studies show that CSCs high expressed Mdr-p, chemotherapeutics can be pumped, cause chemotherapy after CSCs still survive, this may be the important mechanisms of tumor drug resistance, transfer, recurrence.At present, in neoplastic hematologic disorder, breast cancer, brain tumor and prostate cancer, CSC research makes some progress, but most solid tumor all lacks the tumor stem cell specificity marker.
Therefore, the object of the present invention is to provide and a kind ofly be applied to clinical and laboratory and judge the kit of cell aspect propagation, migration, chemotherapeutics resistance and identification adult/tumour ancestral cells.
The present invention can realize with following form: provide a kind of antibody RP215 that identifies non-B cell derived immunoglobulin molecules variable region of heavy chain to detect the application of ancestral cells in clinical and research work.A kind of application of antibody RP215 aspect judgement cell proliferation of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is normal breast epithelial cell, bronchial epithelial cell, glandular epithelium, cholangiole epithelial cell or breast cancer cell, colorectal cancer cells, hepatoma carcinoma cell, lung carcinoma cell, esophageal cancer cell, stomach cancer cell, acute myeloid leukemia cell.A kind of application of antibody RP215 aspect the judgement cell migration of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is normal breast epithelial cell, bronchial epithelial cell, glandular epithelium, cholangiole epithelial cell or breast cancer cell, colorectal cancer cells, hepatoma carcinoma cell, lung carcinoma cell, esophageal cancer cell, stomach cancer cell, acute myeloid leukemia cell.A kind of application of antibody RP215 aspect judgement tumour cell chemotherapeutics resistance of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is breast cancer cell, and described medicine is taxol.A kind of application of antibody RP215 in identification tumour ancestral cells of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is breast cancer cell, colorectal cancer cells, hepatoma carcinoma cell, lung carcinoma cell, esophageal cancer cell, stomach cancer cell, acute myeloid leukemia cell.A kind of application of antibody RP215 in being identified as soma/CFU-GM of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is lung adult ancestral cells, mammary gland adult ancestral cells, testis adult ancestral cells, liver adult ancestral cells.A kind of application of antibody RP215 in preparation detection ancestral cells kit of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and wherein, described ancestral cells is adult ancestral cells or tumour ancestral cells.Described tumour ancestral cells is breast cancer cell, colorectal cancer cells, hepatoma carcinoma cell, lung carcinoma cell, esophageal cancer cell, stomach cancer cell, acute myeloid leukemia cell.Described adult ancestral cells is lung adult ancestral cells, mammary gland adult ancestral cells, testis adult ancestral cells, liver adult ancestral cells.A kind of application of antibody RP215 in preparation detection ability of cell proliferation kit of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is normal breast epithelial cell, bronchial epithelial cell, glandular epithelium, cholangiole epithelial cell or breast cancer cell, colorectal cancer cells, hepatoma carcinoma cell, lung carcinoma cell, esophageal cancer cell, stomach cancer cell, acute myeloid leukemia cell.A kind of application of antibody RP215 in preparation detection cell migration ability kit of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is normal breast epithelial cell, bronchial epithelial cell, glandular epithelium, cholangiole epithelial cell or breast cancer cell, colorectal cancer cells, hepatoma carcinoma cell, lung carcinoma cell, esophageal cancer cell, stomach cancer cell, acute myeloid leukemia cell.A kind of application of antibody RP215 in preparation detection cell chemotherapeutics resistance kit of identifying non-B cell derived immunoglobulin molecules variable region of heavy chain is provided, and described cell is breast cancer cell, and described medicine is taxol.
The solution of the present invention is improving judging that tumour cell has important using value aspect propagation, migration and chemotherapeutics resistance capacity and the identification ancestral cells.The solution of the present invention can also be applied to research work (non-clinical practice) except being applied to clinical position, such as instrument cell in the research work being identified etc.
Description of drawings
Fig. 1: at the bottom of the normal breast catheter-based/myoepithelium uses respectively RP215, the result of anti-CD44v6 and anti-CK5/6 antibody staining.
Fig. 2: normal lung tissue's bronchiolar epithelium basal cell and proper mucous membrane exocrine gland basal cell RP215 coloration result.
Fig. 3: the result that normal liver tissue bile duct epithelial cell and testicle spermatogonia (red arrow) and first spermatocyte (green arrow) dye with RP215.
Fig. 4 comparison RP215, CD44v6 and CK5/6 are in the result of the breast ductal cancer tissue staining of same area.
The result that Fig. 5 cancerous lung tissue dyes with RP215.
The result that Fig. 6 Colorectal Carcinoma dyes with RP215
The result that Fig. 7 cancer of the esophagus tissue dyes with RP215.A: strong positive cell in the cancer of the esophagus cancer nests; B: cancer nests edge strong positive cell; C: the cancer cell of cancer nests and towards periphery lymphoid tissue invasion and attack presents obvious RP215 positive reaction.
The former bit organization of Fig. 8 cancer of the stomach and invasion and attack in the relevant lymphoid tissue of mucous membrane stomach organization with the result of RP215 dyeing.Red arrow: former bit organization; Green arrow: the lymphoid tissue that mucous membrane is relevant
The result that Fig. 9 breast cancer primary lesion and Lymph Node Metastasis focus thereof dye with RP215
The expression of Figure 10 mammary gland carcinoma in situ and metastatic carcinoma IgG relatively
Figure 11 MDA-MB-231 P3:RP215-P2:RP215+ of the RP215 sorting cells film IgG positive
Figure 12 RP215 positive and negative MDA-MB-231 cell are in the comparison of external formation single cell clone ability
Positive and the negative MDA-MB-231 cell MTT Comparison of experiment results of Figure 13 RP215
The comparison of Figure 14 RP215 positive and negative MDA-MB-231 cell migration ability
Upper left: RP215+0 hour upper right: RP215-0 hour
Lower-left: RP215+48 hour bottom right: RP215-48 hour
Figure 15 is under the chemotherapeutics effect, and MDA-MB-231RP215 positive cell and negative cells colony formation result are relatively
Positive and the negative cells chemotherapeutics resistance experiment-MTT Comparison of experiment results of Figure 16 MDA-MB-231 RP215
Figure 17 non-B Ig molecule is at thin inner cellular localization
Embodiment
Method: breast cancer and normal galactophore tissue, lung cancer and normal lung tissue, Colorectal Carcinoma, cancer of the stomach and human esophageal carcinoma are taken from PLA General Hospital; The hepatic duct epithelium, testis tissue is taken from Peking University Human disease gene research centre, RP215 is by Canadian Dr.Lee professor laboratory preparation, existing in Peking University Human disease gene research centre, mouse IgG 1, mouse-anti people CD44 antibody is available from company of China fir Golden Bridge in Beijing (U.S. ZATA company product); SABC two anti-kits, mouse-anti people CK5/6 antibody is available from DAKO company.
The SABC operation steps
Dewaxing: paraffin section places dimethylbenzene to soak 20 minutes, renews bright dimethylbenzene and repeats once;
Rehydration: the histotomy that will take off after cured soaks in 100% ethanol 5 minutes 2 times, and 95% ethanol, 80% ethanol, distilled water respectively soak 5 minutes * 1 time;
Antigen retrieval: antigen retrieval liquid is Tris-EDTA (Ph 9.0) damping fluid, and antigen retrieval liquid is heated to boiling with pressure cooker, and section is put into, and is heated to the rear timing 2 minutes that steams, and naturally cools to room temperature;
Remove endogenous peroxydase: fresh configuration 3%H2O2, drip sheet, lucifuge was hatched 10 minutes under the room temperature;
Sealing: with normal sheep serum working fluid room temperature sealing 20 minutes;
Primary antibodie reaction: drip sheet, add respectively 1: 2500 anti-human CA215, antihuman CD 44 antibody (with PBS1: 30 dilutions) and anti-human keratin 5/6 (with PBS1: 100 dilute), 37 ℃ of 1h.
PBS washes 5 minutes * 3 times
Two anti-reaction: DAKO, two anti-chromogenic reagent box (EnVision
TMDetectionKit, Peroxidase/DAB, Rabbit/Mouse), room temperature reaction 25 minutes;
PBS washes 5 minutes * 3 times,
The DAB colour developing is dripped sheet, the Microscopic observation reaction result;
The haematoxylin redyeing nucleus, the tap water oil blackeite;
The up dehydration of ethanol: 80% ethanol 5min, 95% ethanol 5min, 100% ethanol 5min * 2 time;
The neutral gum mounting is observed and the record result in microscopically.
The result: (1) RP215 identification adult, tumor stem cell: in normal structure, the IgG molecule is high level expression in the adult ancestral cells of the pluripotency characteristics such as multiple normal epithelial basal layer cell, bile duct epithelial cell.The RP215 positive signal only is presented on musculoepithelia cell and the part galandular epithelium of mammary gland, and this distribution is consistent (Fig. 1) with present universally recognized breast carcinoma stem cell Marker CD44 molecule and normal breast stem cell Marker keratin 5 molecule; In normal lung tissue, positive reaction only is presented in the bronchiolar epithelium eccrine basal cell (Fig. 2) in the basal layer cell and tracheae lamina propria specifically; Positive reaction mainly is presented on the hepatic duct epithelial cell in hepatic tissue; And at testis, positive cell mainly is arranged in spermatogonium (monoenergetic adult stem cell) and first spermatocyte (Fig. 3).Because the above-mentioned positive cell of present research prompting all is considered to the adult ancestral cells, so prompting IgG molecule is the stem cell marker molecule.In the kinds of tumors tissue, basaloid cells, Partial tumors parenchyma or the move into adenoid tumour cell of IgG molecule around cancer nests presents strong positive reaction.In breast cancer tissue, our result further shows, RP215 strong positive cell mainly is distributed in basal cell, the flat basaloid cells around the infitrating ductal carcinoma cancer nests and the tumor epithelial cell cell in the part cancer nests of DCIS, it distributes and presents close positive correlation with the CD44 signal, and compare with CD44, shown stronger positive degree.In addition, although RP215 presents good correlativity with keratin 5 in normal mammary gland, but the correlativity of the two is not obvious in cancerous tissue, the flat basaloid cells of these RP215 strong positives is not remaining normal musculoepithelia cell around the prompting cancer nests, but has the cancer cell (Fig. 4) of tumor stem cell characteristic.In cancerous lung tissue, adenocarcinoma of lung and squamous cell carcinoma all present positive signal, wherein, also present a group strong positive cell around the cancer nests of adenocarcinoma of lung, and all cells of squamous cell carcinoma all present strong positive, remaining bronchiolar epithelium basal layer cell in the cancerous tissue, active proliferation, and migration (Fig. 5) in the tube chamber; And in colorectal cancer detects, find, most of tumor epithelial cell cell all presents negative reaction, but ironically, tumour cell usually forms asymmetric adenoid structure, wherein a side is usually piled up the cancer cell of multilayer abnormality proliferation, the opposite side of adenoid structure then forms the structure of simple epithelium sample by several forms than cellule, and the RP215 positive cell mainly is limited to the cellule of this class, and its cell characteristic meets the characteristics (Fig. 6) of pluripotent stem cell.
(2) Non B Ig participates in cell proliferation and migration: find in cancer of the esophagus detects, all cancer cells are strong positive reaction all, but the part cell moves to from cancer nests particularly that cancer cell presents extremely strong positive reaction (Fig. 7) the relevant lymphoid tissue of the cell of periphery and local mucous membrane; In the detection of stomach organization, find, in the primary tumor cancerous tissue, do not find the RP215 positive cell, but the stomach cancer cell in transferring to lymph linked groups presents strong positive (Fig. 8).In order further to inquire into the IgG of non-B cell derived and the relation of tumour cell transfer ability, we detect the former bit organization of breast cancer and the lymphatic metastasis tissue (Fig. 9) thereof of 45 example pairings with RP215.Our result shows, in this 45 example pairing tissue, have the expression of 10 routine carcinoma in situ IgG to be better than metastatic carcinoma, and the expression of remaining 35 routine metastatic carcinoma will be better than carcinoma in situ significantly, and both have obvious significant difference (Figure 10).Point out the expression of non-B cell derived IgG and the transfer of breast cancer to be proportionate.
Embodiment 2. uses antibody RP215 judges tumour cell by flow cytometry multiplication capacity, transfer ability, chemotherapeutics resistance and identification tumor stem cell
Breast cancer cell line MDA-MB-231 is available from ATCC, by the cultivation of going down to posterity of this laboratory routine.RP215 is prepared by Canadian professor Dr.Lee, and is existing in Peking University Human disease gene research centre.The Polystat constant water bath box is from U.S. Cole Parmer company, GS-15R Beckman hydro-extractor is from U.S. Beckman company, EL-311SX ELISA Reader is from U.S. Bio-TEK company, and FACSCalibur flow type analyzer and FACS Aria flow cell sorter are from U.S. company BD.
Breast cancer cell line MDA-MB-231, oophoroma primary cell all add 100U/ml penicillin with the DMEM nutrient culture media that contains 10%FBS (4.5g/L glucose, 4mM Pidolidone), and 100 μ g/ml streptomysins are positioned over 37 ℃, the cultivation of going down to posterity in the 5%CO2 incubator.Went down to posterity once with 0.25% pancreatin+EDTA vitellophag in every 2-3 days, and kept the cell index growth.
Use RP215 and detect tumour cell IgG level by flow cytometer showed
Gather in the crops cell to be measured, be prepared as the individual cells suspension, the PBS washed twice adds about PBS 5ml 1,000rpm 5min at every turn; Add the FACS confining liquid of 2%FBS, cell to be measured is made density be approximately 5 * 10
5The single cell suspension of/ml; The specific antibody (RP215 was by dilution in 1: 150) of appropriate dose is added single cell suspension, fully mixing; Hatch 30min on ice; The PBS washed twice adds about PBS 4ml 1,500rpm5min at every turn; Abandon supernatant, with 200 μ l FACS confining liquid re-suspended cells, add fluorescently-labeled two anti-(Dylight488-sheep anti-mouse igg, Jackson Immunoresearch company products), by dilution in 1: 100; Lucifuge is hatched 30min on ice; The PBS washed twice adds about PBS 4ml 1,500rpm 5min at every turn; Do the fluorescent dye analysis with FACSCalibur streaming instrument after 2% paraformaldehyde is fixing.
Reach low tumour cell of expressing by the streaming method with RP215 difference sorting IgG high expressed:
Gather in the crops cell to be measured, sterile working is prepared as the individual cells suspension, and the PBS washed twice adds PBS5ml/1 * 10 at every turn
6Cell, 1,000rpm 5min; Add the FACS confining liquid of 2%FBS, cell to be measured is made density be approximately 1 * 10
6The single cell suspension of/ml; The specific antibody (RP215 was by dilution in 1: 150) of appropriate dose is added single cell suspension, fully mixing; Hatch 30min on ice; The PBS washed twice adds PBS 4ml/10 at every turn
6About cell, 1,500rpm 5min; Abandon supernatant, with 200 μ l FACS confining liquid re-suspended cells, add fluorescently-labeled two anti-(Dylight488-sheep anti-mouse igg, Jackson Immunoresearch company products), by dilution in 1: 100; Lucifuge is hatched 30min on ice; The PBS washed twice, 1,500rpm 5min; Aseptic streaming strainer tube carries out sorting with FACS Aria flow cell sorter after filtering.
Colony formation
Will be through the RP215 mark, the RP215 of FACS sorting
-And RP215
+Cell is inoculated 6 orifice plates with the density of 200 cells/well, 3 every group multiple holes, cellar culture 14 days is fixed 5 minutes with 4% paraformaldehyde that contains 0.1% Triton X-100, use haematoxylin dyeing 1 minute after PBS washes 3 times, tap water washes away excess dyestuff, natural drying rear counting cells clone number.
The mtt assay cell proliferation experiment
The cell that counting is good is with 1 * 10
4The dose inoculation of/ml is to 96 orifice plates, and the cell suspension in every hole is 100 μ l.In incubator, hatched 24 hours, and behind cell attachment, added the taxol that is diluted to variable concentrations with nutrient solution, 0,0.1,0.2,0.4ug/ml, 4 concentration gradients, each concentration are established 3 parallel holes, hatch 24 hours again.The preparation of MTT liquid and colorimetric analysis: MTT is dissolved among the PBS, and making its concentration is 5mg/ml, and 4 ℃ keep in Dark Place after the filtration sterilization.Every hole adds MTT 10 μ l during mensuration, behind the mixing culture plate is put into incubator and continues to cultivate 5 hours, and then every hole adds 100 μ l SDS stopped reactions, 37 ℃ of overnight incubation behind the abundant mixing, the OD value in every hole when measuring the 570nm wavelength with the MTT microplate reader.
The cell scratch experiment
Will be through the RP215 mark, the RP215 of FACS sorting
-And RP215
+Cell is cultivated at 24 orifice plates respectively, after Fusion of Cells, draws a line with yellow rifle head at middle section, then cell is continued to cultivate, and observation of cell is judged the transfer ability difference of two kinds of cells to the situation of acellular scored area migration.
Found that, using RP215 can effectively analyze and sorting (Figure 11) breast cancer cell line MDA-MB-231, the colony formation that after the sorting two groups of cells is carried out, at cellar culture after 14 days, the RP215 positive and negative cells all can form single cell clone, but the plastidogenetic clone's number of positive group is higher than the cell (Figure 12) of negative group.After the sorting two groups of cells have been carried out the MTT experiment, a plurality of time point MTT testing results show that the multiplication capacity of RP215 positive cell is organized cell (Figure 13) apparently higher than feminine gender, and this result has good repeatability.Observe after also two groups of cells being carried out a plurality of time point cuts after the sorting and find, 43 hours RP215 positive cells just merge fully in the cut district behind cut, form obvious difference (Figure 14) with feminine gender group cell.These results suggest, positive (cell membrane) cell of RP215, its multiplication capacity and transfer ability all obviously are better than the RP215 negative cells.
After sorting, carry out the experiment of chemotherapeutics resistance with antineoplastic-taxol again, under the taxol concentration pressure, carried out the detection of cell clonal formation and MTT.Research finds in colony formation, no matter RP215 positive cell group clone number is to clone greatly number or clone's sum, all apparently higher than the negative cells group, has significant difference (Figure 15).In MTT experiment, under the taxol mass action of 0.1ug/ml, 0.2ug/ml, three dosage of 0.4ug/ml, express the multiplication capacity of cell of non-B cell derived Ig all apparently higher than the cell (Figure 16) of not expressing non-B cell derived Ig.Because tumour cell ties up to the phenomenon that exists inevitably inhereditary feature to lose in the incubation, we have also obtained oophoroma (1 example) and carcinoma of endometrium (1 example) ascites with The Third Affiliated Hospital of Peking University cooperation, and sorting wherein RP215+ or RP215+/EpCAM+ and RP215-or RP215-/EpCAM+ ovarian cancer cell of former generation and endometrial carcinoma cell, then in cell cultivation process, observe and become the knurl situation in its form, propagation, migration and the body.Found that, 2 cases are different on cellular morphology, relatively be surprised to find that in RP215+ ovarian cancer cell and the RP215-group process, a lot of pseudopodium (or claiming prominent foot) appear in RP215+ ovarian cancer cell cell after cultivating 48 hours, this phenomenon is more obvious rear 96 hours of cultivation, points out this pseudopodium may be relevant with cell migration.Carry out cell count after 96 hours and find that RP215+ ovarian cancer cell cell number after cultivation is organized apparently higher than RP215-by cultivating, the result has remarkable significant difference.
Embodiment 3. uses antibody RP215 and judges the location of non-B Ig in cell by immunofluorescence experiment
With colorectal cancer cells HT-29, osteosarcoma cell line U2OS creep plate is cultured to proper density, with AML cell rejection tablet to slide, after the PBS washed twice, with the fixing 30min of 2% paraformaldehyde normal temperature, again with the 2% paraformaldehyde permeates cell membranes that contains 0.1%Triton-X100, normal temperature 15min, sealing subsequently and antibody associated methods are with above-mentioned intracellular protein immunofluorescence dyeing, and be last, uses the confocal microscopy record.
Result: in three kinds of clones, all detected the RP215 positive signal, three kinds of intracellular positive signal all show filament (Figure 17), and thread connection signal is arranged between cell, and prompting non-B Ig is relevant with cytoskeleton in intracellular location, participates in the cell migration function.
Utilize the monoclonal antibody of this special IgG molecule for non-B cell derived of RP215, we find to neutralize the main high level expression of IgG in pluripotent stem cell or CFU-GM, and those cell volumes are little, form is close to normal cell, do not present in the typical tumor stem cells such as cell volume is large, dyskaryosis, the expression of IgG and cell mitogen and transfer ability are closely related, also meet the characteristics of ancestral cells.The solution of the present invention is improving judging that tumour cell has important using value aspect propagation, migration and chemotherapeutics resistance capacity and the identification stem cell.The solution of the present invention can also be applied to research work (non-clinical practice) except being applied to clinical position, such as instrument cell in the research work being identified etc.
Claims (10)
1. the antibody RP215 of the non-B cell derived immunoglobulin molecules variable region of heavy chain of identification application in the identification ancestral cells in clinical and research work.
2. the application of the antibody RP215 of the non-B cell derived immunoglobulin molecules variable region of heavy chain of identification aspect judgement cell proliferation.
3. the application of the antibody RP215 of the non-B cell derived immunoglobulin molecules variable region of heavy chain of identification aspect the judgement cell migration.
4. the application of the antibody RP215 of the non-B cell derived immunoglobulin molecules variable region of heavy chain of identification aspect judgement tumour cell chemotherapeutics resistance.
5. application as claimed in claim 1, wherein said ancestral cells is the application of identification tumour ancestral cells aspect.
6. application as claimed in claim 1, wherein said ancestral cells are the application that is identified as soma/CFU-GM aspect.
7. such as the described application of claim 1-5, wherein said cell is normal breast epithelial cell, bronchial epithelial cell, glandular epithelium, cholangiole epithelial cell or breast cancer cell, colorectal cancer cells, hepatoma carcinoma cell, lung carcinoma cell, esophageal cancer cell, stomach cancer cell, acute myeloid leukemia cell.
8. application as claimed in claim 4, wherein said medicine are taxol.
9. application as claimed in claim 6, wherein said cell are lung adult ancestral cells, mammary gland adult ancestral cells, testis adult ancestral cells, liver adult ancestral cells.
10. the application of the antibody RP215 of the non-B cell derived immunoglobulin molecules variable region of heavy chain of identification in preparation detection ancestral cells kit, wherein, described ancestral cells is adult ancestral cells or tumour ancestral cells.
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