CN101893630B - Method for detecting expression level of annexin A3 - Google Patents
Method for detecting expression level of annexin A3 Download PDFInfo
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- CN101893630B CN101893630B CN201010121278.6A CN201010121278A CN101893630B CN 101893630 B CN101893630 B CN 101893630B CN 201010121278 A CN201010121278 A CN 201010121278A CN 101893630 B CN101893630 B CN 101893630B
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Abstract
The invention generally relates to the field of medicament resistance of chemotherapeutic medicaments, in particular to a method for detecting the expression level of annexin A3 in the supernate of the culture medium of platinum-based chemotherapeutic medicament resisting tumor cells, a method for adjusting the excretion amount of tumor cell annexin A3, a method for evaluating the medicament resistance of patients with tumor to the platinum-based chemotherapeutic medicament, a method for adjusting the platinum releasing of the platinum-based chemotherapeutic medicament resisting tumor cells, a method for combining deoxyribonucleic acid (DNA) and the platinum in the platinum-based chemotherapeutic medicament resisting tumor cells and a method for adjusting the quantity of vesicles in the platinum-based chemotherapeutic medicament resisting tumor cells.
Description
Technical field
Relate generally to chemotherapeutics drug resistance of the present invention field, particularly, relate to following aspect: a kind of method that detects annexin A 3 (Annexin A3) expression in platinum-based chemotherapy Drug-resistant tumor cell culture base supernatant, a kind of regulate tumor cell annexin A 3 method of the amount of secreting outward, a kind of method of tumor patient to platinum-based chemotherapy drug resistance of assessing, a kind of method that regulates platinum-based chemotherapy Drug-resistant tumour cell to discharge platinum, a kind ofly regulate the method that in platinum-based chemotherapy Drug-resistant tumour cell, DNA is combined with platinum, a kind of and method that regulates vesica quantity in platinum-based chemotherapy Drug-resistant tumour cell.
Background technology
Oophoroma is the first fatal disease of gynecological tumor, and 5 years survival rates of patients with terminal are hovered all the time at 15%-20%, and it is to cause the one of the main reasons of this situation that tumour cell produces drug resistance for platinum-based chemotherapy medicine.Platinum-based chemotherapy medicine comprises cis-platinum, carboplatin, JM-216, Nedaplatin, Lobaplatin etc., being the First-line chemotherapy medicine of current the most frequently used treatment oophoroma, is also the common drug that other entity tumor for the treatment of comprises breast cancer, lung cancer, orchioncus, head and neck cancer, osteosarcoma, melanoma, the cancer of the esophagus etc.
Platinum-based chemotherapy medicine is combined with DNA and is formed cross key, thereby the function of destruction DNA can not copy again, also can suppress the synthetic of RNA and protein when high concentration.But platinum-based chemotherapy medicine, in the time of the above-mentioned entity tumor for the treatment of, has all occurred resistance phenomenon, recurring just appears in the treatment later stage or the treatment initial stage is just reactionless for tumour control is good to treat the initial stage.
Resistance for oophoroma to platinum-based chemotherapy medicine, clinically existing clear and definite understanding and definition.Meanwhile, also obtain certain achievement for the research of cisplatin resistance mechanism, as MDR-1, LRP-1, MRP-1, GST-pi etc. have become well-known drug resistance related gene.
But numerous results of study show, the expression of several drug resistance related gene in oophoroma be predicting tumors ground resistance and prognosis well, and it may be one of the main reasons that its corresponding protein level of abundance of mRNA has inconsistency.DNA is the carrying person of hereditary information, and protein is only the executor of function.The inventor is by comparison protein omics technology, protein expression in platinum-based chemotherapy sensitive cells and its corresponding platinum-based chemotherapy mdr cell is compared, and 5 kinds of platinum-based chemotherapy drug resistance-associated proteins are found, they are respectively annexin A 3 (Annexin A3), IDHc, cofilin 1, GSTO1-1, destrin, and by RT-PCR and Western blot technology, platinum-based chemotherapy resistance and sensitive cells are carried out respectively verifying (Yan XD at RNA and protein level, Pan LY, et al.J Proteome Res.2007, 6 (2): 772-780).According to the difference degree of 5 kinds of albumen, selection differences the most significantly annexin A 3 carries out relevant platinum class resistance mechanism discussion experiment, the preferred ovarian cancer cell of tumour cell.
Annexin A 3 is a member in annexin superfamily, there is the function of inhibition of phospholipase A 2 and anti-freezing, it can also cut off inositol 1,2-cyclic phosphate forms inositol 1-phosphate, at (the Ross TS that plays a role aspect control cell proliferation, etal.J Biol Chem.1991,266 (14): 9086-9092.).Annexin A 3 is at certain C a
2+under concentration, can be attached on the phosphatidylserine of vesica the effect between the films such as mediation phagosome and lysosomal fusion and particle disappearance and film.Connect albumen as calcium, annexin A 3 has the inside and outside function of a series of born of the same parents, comprises that film transhipment, lymphocyte migration, cell movement, calcium current go out and signal transduction.It is one of critical function of annexin A 3 (Gerke V, MossSE.Physiol.Rev.2002,82:331-371) in conjunction with negative electrification membrane phospholipid that the annexin A 3 of Gao Fengliang produces calcium dependent expansion vesicle.
A series of experiment shows, annexin A 3 is one of mechanism of oophoroma platinum-based chemotherapy medicine chemotherapy resistance.The inventor is the expression of the interior annexin A 3 of means reduction ovarian cancer cell by experiment, and oophoroma increases platinum-based chemotherapy drug susceptibility; Otherwise if improve the expression of annexin A 3 in ovarian cancer cell, oophoroma reduces platinum-based chemotherapy drug susceptibility.This experimental result has all obtained effective checking (YanXD, Pan LY, et al.J Proteome Res.2007,6 (2): 772-780 in human ovarian cancer tissue and animal subject; XuedongYan, Jie Yin, Huiyu Yao, Ning Mao, Yili Yang, Lingya Pan.CancerRes, 2010,70 (4): OF1-9.).
In addition, in the cancerous tissue of the colorectal cancer patients about 2/3rds, find that high-caliber annexin A 3 concentrates on film calmodulin binding domain CaM (Madoz-Gurpide J, Lopez-Serra P, etal.Mol Cell Proteomics.2006,5:1471-1483).Recently, in the urine of other patients with prostate cancer same period not, can detect the annexin A 3 that changes content by Western blot method, and compare with present clinical specific marker thing prostate specific antigen used, discovery annexin A 3 points out prostate cancer to have more advantage in early days.
In sum, annexin A 3 has the hope that becomes human tumor mark.Based on the correlativity of annexin A 3 and platinum-based chemotherapy Drug-resistant, the invention is intended to carry out clinical assessment tumour platinum-based chemotherapy drug susceptibility by the level of annexin A 3 in human body fluid, this still belongs to the first time in chemotherapy of tumors resistance field.
In view of lacking clinically at present the protein marker that can indicate ahead of time tumour platinum-based chemotherapy medicine, for giving full play to the medical science advantage that transforms, the inventor is finding on this platinum-based chemotherapy Drug-resistant associated protein basis of annexin A 3, further affirm by experiment the status of its platinum-based chemotherapy Drug-resistant Specific marker, and inquired into the molecular mechanism that it causes platinum-based chemotherapy Drug-resistant.For example, on the phenomenon basis of finding tumour cell (ovarian cancer cell) secretion annexin A 3, further seek simultaneously and outside it, secrete approach, and by building the enzyme-linked immunosorbent assay scheme of specific detection mankind annexin A 3 albumen, successfully in ovarian cancer patients serum, detect the existence of annexin A 3, and on this basis, assess in oophoroma platinum-based chemotherapy Drug-resistant patients serum the level difference of annexin A 3 in annexin A 3 level and oophoroma platinum-based chemotherapy medicaments insensitive patients serum.Prove by a large amount of clinical samples, in serum, high-caliber annexin A 3 can indicate the generation of platinum-based chemotherapy Drug-resistant exactly.Find early platinum-based chemotherapy Drug-resistant, can change in time oncotherapy scheme, improve 5 years survival rates, improve prognosis.
Summary of the invention
One aspect of the present invention is to provide a kind of method that detects expression level of annexin A 3, and described method is used platinum-based chemotherapy Drug-resistant tumor cell culture base supernatant as detected sample.
Wherein, preferably, described platinum-based chemotherapy medicine is cis-platinum; Preferably, described tumour cell is ovarian cancer cell; Preferably, described nutrient culture media is serum free medium, and DMEM more preferably; Preferably, described detection is preferably immunology detection, and more preferably Westernblot or ELISA detection.
In one embodiment of the invention, in the time using Western blot to detect, described method comprises the steps:
1) collect the tumor cell culture supernatant of the same type of cleer and peaceful platinum-based chemotherapy medicaments insensitive on platinum-based chemotherapy Drug-resistant tumor cell culture base and concentrate respectively, cycles of concentration is 75～100 times, obtains two concentrated liquids;
2) detect annenxin A3 expression in described two concentrated liquids;
3) annenxin A3 expression in described two concentrated liquids is compared, expression level of annexin A 3 in platinum-based chemotherapy Drug-resistant tumor cell culture supernatant is carried out to sxemiquantitative.
Another aspect of the present invention is to provide a kind of regulate tumor cell annexin A 3 method of the amount of secreting outward, and described method comprises the step that changes annenxin A3 expression in tumour cell, and wherein, preferably, described platinum-based chemotherapy medicine is cis-platinum; Preferably, described tumour cell is ovarian cancer cell.
In one embodiment of the invention, improve the tolerance of tumour cell to platinum-based chemotherapy medicine by the annexin A 3 expression improving in tumour cell, preferably, improve the annexin A 3 expression in cell by express annexin A 3 gene in tumour cell, the plasmid of more preferably, expressing just annexin A 3 gene by use improves the content of the annexin A 3 in described cell.
In one embodiment of the invention, express and reduce the tolerance of tumour cell to platinum-based chemotherapy medicine by the annexin A 3 in minimizing and/or inhibition tumor cell, preferably, reduce and/or the annexin A 3 that suppresses in cell is expressed by the ribozyme of expressing the antisensenucleic acids of annexin A 3 and/or being specific to annexin A 3 in tumour cell, more preferably, reduce the content of the annexin A 3 in cell by the DNA construct with antisence annexin A 3 or expression vector.
Another aspect of the present invention is to provide a kind of method of tumor patient to platinum-based chemotherapy drug resistance of assessing, and comprises the steps:
1) detect annexin A 3 level in patient's humoral sample;
2) if step 1) in annexin A 3 level higher than 1.34ng/ml, judge that tumor patient is to platinum-based chemotherapy Drug-resistant;
Wherein, preferably, described body fluid is serum; Preferably, step 1) in detection be ELISA detect.Described cancer is to use the cancer of platinum-based chemotherapy drug therapy, as oophoroma, breast cancer, lung cancer, orchioncus, head and neck cancer, osteosarcoma, melanoma, the cancer of the esophagus etc., and preferably oophoroma.
In research of the present invention, the inventor uses enzyme-linked immunosorbent assay to detect the content of annexin A 3 in also quantitative human serum first, by comparing expression level of annexin A 3 in platinum-based chemotherapy Drug-resistant patient and platinum-based chemotherapy medicaments insensitive patients serum, obtain the annexin A 3 indication best susceptibility of platinum-based chemotherapy Drug-resistant and specific expression simultaneously.Set up clinical in detecting the platform of serum annexin A 3 level prompting platinum-based chemotherapy resistance.
First, by further experimental results show that annexin A 3 is platinum-based chemotherapy Drug-resistant specificity marker protein, and cause that by the detection explanation annexin A 3 of platinum content in cell the mechanism of platinum-based chemotherapy Drug-resistant may be to realize by the release of platinum in cell.Platinum-based chemotherapy medicaments insensitive tumour cell in by transfection justice annexin A 3 rise cell after expression level of annexin A 3 strengthens the drug resistance of cis-platinum, but to the drug resistance of taxol and Epi-ADM change, this explanation annexin A 3 is the specific related protein of platinum-based chemotherapy Drug-resistant, and the expression of annexin A 3 has determined the susceptibility of tumour cell to platinum-based chemotherapy medicine.A desirable mark is can be secreted in other body fluid of peripheral blood or human body, is convenient to detect.The inventor collects the nutrient culture media supernatant of platinum-based chemotherapy medicaments insensitive and drug-resistant tumor cell, and whether detect tumour cell can exocrine annexin A 3 and the regulative mode of protein secretion.For avoiding high-abundance proteins impact, medium optimization serum free medium.Adopt centrifugal ultrafiltration technology, the liquid in nutrient culture media is concentrated, adopt in the immune marking technology for detection concentrate annexin A 3 content and carry out sxemiquantitative.The outer ability of secreting annexin A 3 of tumour cell (ovarian cancer cell) of finding platinum-based chemotherapy Drug-resistant is obviously better than drug sensitive cell.Change the annexin A 3 amount of secreting outward, can realize by changing annexin A 3 content in cell.The annexin A 3 level expression of the interior annexin A 3 of reacting cells exactly in the outer liquid of this explanation, and can judge that cell is to platinum-based chemotherapy drug susceptibility.
Secondly, the inventor find and verified ovarian cancer cell annexin A 3 secrete phenomenon and annexin A 3 is secreted approach outward outward, and a kind of mark of good indication platinum-based chemotherapy Drug-resistant is can be to this material detected in the exocrine body fluid of human body or blood, and expression and the platinum-based chemotherapy drug susceptibility of this material in human body liquid has certain correlativity.Use mankind's annexin A 3 enzyme-linked immunosorbent assay kits (ANXA3 ELISAkit) successfully to detect the content of annexin A 3 in human serum also quantitative, the average expression of normal women serum annexin A 3 is 0.859 ± 0.0744, the average expression of ovarian cancer patients serum annexin A 3 is 1.6898 ± 2.6563, two groups are carried out Mann-Whitney check, P < 0.0001.All ovarian cancer patients are divided into platinum-based chemotherapy Drug-resistant group and platinum-based chemotherapy medicaments insensitive group, resistance group annexin A 3 average level is 2.1145 ± 3.3833, responsive group annexin A 3 average level is 1.0528 ± 0.1178, two groups of relatively P=0.0003 < 0.05.In the time that annexin A 3 level is greater than 1.13ng/ml, the susceptibility of this albumen prediction oophoroma platinum-based chemotherapy Drug-resistant reaches 63%, specificity 80%.Ovarian cancer patients serum is all collected in before chemotherapy, not affected by chemotherapy, and it is main combined chemotherapy that whole ovarian cancer patients have all been accepted platinum-based chemotherapy medicine after surgery.To sum up, in Serum of Cancer Patients, expression level of annexin A 3 can be predicted the susceptibility of ovarian cancer patients to platinum-based chemotherapy medicine well.
Another aspect of the present invention is to provide a kind of method that regulates platinum-based chemotherapy Drug-resistant tumour cell to discharge platinum, and described method comprises the step that changes annenxin A3 expression in tumour cell, and wherein, preferably, described platinum-based chemotherapy medicine is cis-platinum; Preferably, described tumour cell is ovarian cancer cell.
Another aspect of the present invention is to provide a kind of method that in platinum-based chemotherapy Drug-resistant tumour cell, DNA is combined with platinum that regulates, described method comprises the step that changes annenxin A3 expression in tumour cell, wherein, preferably, described platinum-based chemotherapy medicine is cis-platinum; Preferably, described tumour cell is ovarian cancer cell.
Another aspect of the present invention is to provide and a kind of method that regulates vesica quantity in platinum-based chemotherapy Drug-resistant tumour cell, described method comprises the step that changes annenxin A3 expression in tumour cell, wherein, preferably, described platinum-based chemotherapy medicine is cis-platinum; Preferably, described tumour cell is ovarian cancer cell.
Secrete annexin A 3 albumen in order to illustrate that better tumour cell (ovarian cancer cell) is can be enduringly outer, the present patent application people uses electron microscopy to observe the outer approach of secreting of annexin A 3.With two kinds of cisplatin resistance ovarian cancer cell SKOV3/Cis and A2780/Cis, the responsive ovarian cancer cell SKOV3 of two kinds of cis-platinums and A2780, two kinds of transfections annexin A 3 justice plasmid raise cell SKOV3/Ann and the A2780/Ann that in cis-platinum sensitive cells, annexin A 3 is expressed, two kinds of transfections annexin A 3 antisense plasmid to lower cell SKOV3/Cis/R and the A2780/Cis/R that annexin A 3 is expressed in cisplatin-resistant cell be research object, use immunofluorescence technique and immunoelectron microscopic method observe annexin A 3 albumen cell submicroscopic structure distribute, inquire into annexin A 3 and secrete approach outward.Under transmission electron microscope, observe above-mentioned cis-platinum sensitivity and drug-resistant ovarian carcinoma cell submicroscopic structure, compared with cis-platinum sensitive cells, find to occur more vesica spline structure in cisplatin-resistant cell endochylema, this structure is dispersed in and is distributed in endochylema, smooth surface, be different from rough surfaced endoplasmic reticulum (RER) and golgiosome, in vesica, there is no the ridge of similar mitochondria sample yet, in some vesica, also have some not clear particulate matter, be different from the homogeneous in lysosome.Prolong cell membrane observe vesica, can observe some vesica from after birth very close to, some with after birth merge cut, some has been broken through outside after birth.Use transmission electron microscope observing transfection annexin A 3 justice plasmid to raise after the interior annexin A 3 level of cis-platinum sensitive cells, find in endochylema, also to have occurred compared with before transfection at sensitive cells the intracytoplasmic imitated vesicle structure of a lot of similar cisplatin-resistant cells; On the contrary, transfection annexin A 3 antisense plasmid is lowered after the interior annexin A 3 level of cisplatin-resistant cell, and cisplatin-resistant cell endochylema intracellular vesicle has reduced in a large number, and the annexin A 3 raising in above explanation cell can cause the generation of born of the same parents' intracellular vesicle structure.Use immunofluorescence technique, the intracytoplasmic annexin A 3 albumen of the above-mentioned 8 kinds of cell karyons of specific dyeing, finds that ovarian cancer cell kind annexin A 3 is mainly distributed in endochylema, and minority is distributed in karyon, main or a kind of plasmosin.Use immunoelectronmicroscopy, annexin A 3 cell distribution is carried out to subcellular organelle location, annexin A 3 in above-mentioned 8 kinds of cells is carried out to immunostaining, adopt colloid gold particle mark annexin A 3 albumen, find that annexin A 3 is distributed in the interior membrane structure of endochylema in a large number, comprises the film surface of the vesica of finding under transmission electron microscope, simultaneously at indivedual vesicas, particularly near the existence that can find annexin A 3 in the vesica of after birth, illustrate that annexin A 3 secretes by the form of vesica outward.
Above-mentioned adjusting platinum-based chemotherapy Drug-resistant tumour cell discharge platinum method, regulate the method for vesica quantity in method that in platinum-based chemotherapy Drug-resistant tumour cell, DNA is combined with platinum and adjusting platinum-based chemotherapy Drug-resistant tumour cell, all comprise the step that changes annenxinA3 expression in tumour cell, this step can realize by method below, for example:
Express realization by the annexin A 3 improving in tumour cell, preferably, improve the annexin A 3 expression in cell by express annexin A 3 gene in tumour cell, the plasmid of more preferably, expressing just annexin A 3 gene by use improves the content of the annexin A 3 in described cell; Or
Express and realize by the annexin A 3 in minimizing and/or inhibition tumor cell, preferably, reduce and/or the annexin A 3 that suppresses in cell is expressed by the ribozyme of expressing the antisensenucleic acids of annexin A 3 and/or being specific to annexin A 3 in tumour cell, more preferably, reduce the content of the annexin A 3 in cell by the DNA construct with antisence annexin A 3 or expression vector.
Of the present invention all aspect, if applicable, preferably, described tumour cell includes but not limited to the cell of following cancer, as the cell of oophoroma, breast cancer, lung cancer, orchioncus, head and neck cancer, osteosarcoma, melanoma, the cancer of the esophagus etc., preferably ovarian cancer cell.
Of the present invention all aspect, if applicable, preferably, described platinum-based chemotherapy medicine includes but not limited to cis-platinum, carboplatin, JM-216, Nedaplatin, Lobaplatin etc., preferably cis-platinum.
In the present invention, term " mdr cell " refers to respect to similar parental cell, is difficult for accepting some stimulations and cell that corresponding change occurs.In the present invention, correspond to the cell that is difficult for accepting platinum-based chemotherapy medicine irritation and occurs corresponding change.
In the present invention, term " sensitive cells " refers to more acceptant stimulations and the cell of corresponding change occurs.In the present invention, correspond to the cell that corresponding change easily platinum-based chemotherapy medicine irritation is occurred.
In the present invention, term " drug resistance " refers to that prolong drug effect reduces in time, or needs escalated dose guarantee drug effect not subtract.The drug resistance of phalangeal cell of the present invention to platinum-based chemotherapy medicine, logical common drug median lethal dose (IC50) and Resistance index (RI) represent.
In the present invention, for term " susceptibility ", during in the time carrying mutually with specificity or as concept statistically, refer to the percent that laboratory examination result is " true positives "; Other situation refers to the concept relative with " drug resistance ".
In the present invention, term " specificity " refers to the percent that laboratory examination result is " true negative ".
In the present invention, if there is no specified otherwise, SKOV3 and A2780 are SKOV3 and the A2780 of transfection empty plasmid, SKOV3/Cis is the SKOV3 cisplatin-resistant cell subbreed of transfection empty plasmid, A2780/Cis is the A2780 cisplatin-resistant cell subbreed of transfection empty plasmid, and above-mentioned empty plasmid is pcDNA3.1 (+) plasmid.
The beneficial effect of the invention
First passage platinum-based chemotherapy Drug-resistant tumor cell culture base supernatant of the present invention has been realized the detection of expression level of annexin A 3, and provide first the method for assessment tumor patient to platinum-based chemotherapy drug susceptibility, make it possible to find early platinum-based chemotherapy Drug-resistant, change in time oncotherapy scheme, improve 5 years survival rates, and improve prognosis.
Accompanying drawing explanation
In accompanying drawing below, SKOV3 and A2780 are SKOV3 and the A2780 cell of pcDNA3.1 (+) plasmid of transfection sky; SKOV3/Cis is the SKOV3 cisplatin-resistant cell subbreed of pcDNA3.1 (+) plasmid of transfection sky, and A2780/Cis is the A2780 cisplatin-resistant cell subbreed of pcDNA3.1 (+) plasmid of transfection sky; SKOV3/Ann and A2780/Ann clone are respectively the stable transfection clones who obtains after SKOV3 and A2780 transfection justice annexin A 3 plasmid; SKOV3/Cis/R and A2780/Cis/R are the stable transfection clones who obtains after SKOV3/Cis and A2780/Cis transfection antisense annexin A 3 plasmid.
Fig. 1: the platinum concentration ratio in eight kinds of ovarian cancer cells,
*p < 0.01,
*p < 0.001.
Fig. 2: SKOV3, the comparison of SKOV3/Ann cell Pt discharge rate.
Fig. 3: eight kinds of ovarian cancer cell Pt-DNA concentration ratios,
*p < 0.05,
*p < 0.01.
Fig. 4: in ovarian cancer cell nutrient culture media supernatant, annexin A 3 detects.Parental represents parental cell, i.e. the tumour cell of untransfected plasmid; Transfectant represents the tumour cell after transfection plasmid.The equal transfection justice of figure A:SKOV3 and A2780 annexin A 3 plasmid, intracellular membrane connection albumin A 3 levels raise; Figure B:SKOV3/Cis and the equal transfection antisense of A2780/Cis annexin A 3 plasmid, intracellular membrane connection albumin A 3 expressions are lowered; β-actin is actin, is this experiment internal reference.
Fig. 5: A: the change (50,000x) of submicroscopic structure under the responsive ovarian cancer cell of cis-platinum (SKOV3 and A2780) and cisplatin-resistant cell (SKOV3/Cis and A2780/Cis) transmission electron microscope thereof.B: prolong cell membrane and observe the imitated vesicle structure that increases in endochylema and the relation (100,000x) of cell membrane.C: after the positive and negative adopted plasmid of transfection annexin A 3, the variation of ovarian cancer cell endochylema intracellular vesicle.Arrow is depicted as abnormal imitated vesicle structure in endochylema.
Fig. 6: immunofluorescence technique detects the location of annexin A 3 in ovarian cancer cell.
Fig. 7: immunoelectronmicroscopy detects annexin A 3 subcellular organelle location (50,000x and 100,000x paired observation).A: contrast (PBS replaces annexin A 3 antibody); What B, C, D showed is the location of annexin A 3 in vesica surface and vesica.Intracytoplasmic black particle is colloid gold particle, and each particle represents annexin A 3 albumen; What small arrow was indicated is the position at annexin A 3 albumen place.Large arrow indication vesica.100,000x figure is the enlarged drawing of square frame in 50,000x figure.
Fig. 8: the expression of annexin A 3 exosome (cell intracellular vesicle is discharged into extracellular a kind of existence form) in mdr cell SKOV3/Cis culture supernatant.Figure A: the form of transmission electron microscope observing exosome, mostly be circular or oval, diameter is between 40-100nm; Figure B and C: the expression of immune electron microscopy annexin A 3 in exosome; Figure D:DMEM1 represents normal SKOV3/Cis culture supernatant concentrate (cycles of concentration 75x), the SKOV3/Cis culture supernatant concentrate (cycles of concentration 75x) of exosome has been removed in DMEM2 representative, the exosome upper strata sucrose liquid in exosome process is extracted in sucrose representative, and Hsp70 is exosome internal reference (exosome multilist reaches Hsp70).
Fig. 9: the expression scatter diagram of annexin A 3 in figure A:30 example chemotherapy resistance ovarian cancer patients, 20 routine chemosensitivity ovarian cancer patients and 30 routine normal women serum; Figure B: the ovarian cancer patients that the level of annexin A 3 in serum is greater than to 1.13ng/ml is classified A group as, the ovarian cancer patients that is less than 1.13ng/ml is classified B group as, draw Kaplan Meier curve, two groups without platinum interval (combined chemotherapy containing platinum-based chemotherapy medicine of first course for the treatment of finish after to time of Ovarian cancer) length there is remarkable significant difference (P=0.009 < 0.05).IQR: range interquartile number.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples are only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (for example show with reference to J. Pehanorm Brooker etc. according to the described technology of the document in this area or condition, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: annexin A 3 regulates the in vitro study of oophoroma epithelial cell platinum-based chemotherapy Drug-resistant mechanism
1. experimental technique
1) clone and condition of culture thereof
People's epithelial ovarian cancer cell SKOV3 clone is purchased from Chinese Academy of Medical Sciences's preclinical medicine cell centre, SKOV3/Cis is that the SKOV3 cisplatin-resistant cell subbreed that the inventor induces (can be with reference to Yan Xuedong, Zhang Mingwei, Pan Lingya. preclinical medicine and clinical, 2006,26 (7): 739-744).This experiment is also used A2780 and cisplatin-resistant cell subbreed A2780/Cis (can be with reference to LuanYZ, Li L.et al.Zhonghua Fu Chan Ke Za Zhi, 2004,39 (6): 403-407.) thereof.PcDNA3.1 (+) plasmid that the equal transfection of these cells is free.SKOV3/Ann and A2780/Ann clone are respectively the stable transfection clones who obtains after SKOV3 and A2780 transfection justice annexin A 3 plasmid, SKOV3/Cis/R and A2780/Cis/R are that the stable transfection clone who obtains after SKOV3/Cis and A2780/Cis transfection antisense annexin A 3 plasmid (can be with reference to Xuedong Yan, Jie Yin, Huiyu Yao, Ning Mao, Yili Yang, Lingya Pan.Cancer Res, 2010,70 (4): OF1-9.), all stable transfection clones pass through Western blot technical identification.Adopt simultaneously SKOV3 and A2780 transfection empty plasmid cell (can be with reference to Xuedong Yan, Jie Yin, Huiyu Yao, Ning Mao, Yili Yang, Lingya Pan.Cancer Res, 2010,70 (4): OF1-9.) in contrast.
All adherent growth in the nutrient culture media of the DMEM (HG) containing 10% hyclone of above-mentioned all cells system, are placed in 37 ℃, 5%CO
2in the incubator of saturated humidity, cultivate, while growing to 80% density, adopt 0.25% trypsinization to go down to posterity.
2) drug sensitivity assay
Detect the susceptibility of cell to cis-platinum (Cisplatin, CDDP), carboplatin (Carboplatin, CBP), taxol (Taxol) and Epi-ADM (epirubicin).
Get cell exponential phase, after trypsinization, make single cell suspension, by 2000/ hole/100 μ L, every kind of each concentration of medicine is established 6 parallel holes, 12 of control wells, do not add medicine, wherein 6 negative contrasts in hole (add cell, do not add medicine), 6 holes are blank (only adding nutrient culture media).37 ℃, 5%CO
2cultivate and add chemotherapeutics after 24 hours, be diluted to 7 gradients, continue to cultivate 72 hours, add 5mg/mL MTT20 μ L, 37 ℃, 5%CO
2continue to cultivate 4 hours, control clean nutrient culture media, add 100 μ L lysates (containing 20%SDS, 50%N-N dimethyl formamide, pH 4.7), spend the night, full-automatic microplate reader is measured light absorption value (OD value) with 540nm, can ask the mean value of calculating every kind of drug concentration light absorption value, calculate cell survival rate and inhibiting rate, according to drug concentration and inhibiting rate, use SPSS11.5 software to calculate IC50 (suppressing the drug concentration of 50% Growth of Cells) and Resistance index (RI).
Inhibiting rate (inhibition rate)=[(OD contrast-OD experiment)/OD contrast] × 100%
The IC50 of the IC50/ sensitive cell line of RI=medicine-resistant cell line
3) platinum (Pt) concentration determination in cell
Treat that each cell grows to exponential phase, digestion, counting, with 2 × 10
6cell kind, to 100mm double dish, adds CDDP to final concentration 10 μ M, 37 ℃, 5%CO after 24 hours
2in saturated humidity incubator, cultivate 24 hours (control group does not add medicine), discard nutrient culture media, PBS washes 3 times, scrapes cell is scraped with cell, is collected in Ep pipe.Use 50-100 μ L 70% nitric acid cell lysis, put 65 ℃ of water-baths 2 hours (now extraction portion divides pyrolysis product to utilize BCA method to detect protein concentration), in pyrolysis product, add distilled water, be diluted to 5% concentration of nitric acid.Utilize the intracellular platinum of inductance coupling plasma Mass Spectrometer Method (Pt) content.Discharge situation for detecting CDDP, add CDDP to final concentration 10 μ M, 37 ℃, 5%CO in cell
2in saturated humidity incubator, cultivate 24 hours, discard nutrient culture media, change into without medicine nutrient culture media and hatch 2 hours, as front collection step and processing cell, detect Pt content.Pt discharge rate=(1-cultivates in 2 hour cells Pt concentration in Pt concentration/dosing 24 hour cells without medicine) × 100%.
4) DNA is in conjunction with the detection of Pt
Treat that each cell grows to exponential phase, digestion, counting, with 2 × 10
6cell kind, to 100mm double dish, adds CDDP to final concentration 10 μ M, 37 ℃, 5%CO after 24 hours
2in saturated humidity incubator, cultivate 24 hours (control group does not add medicine), discard nutrient culture media, PBS washes 3 times, scrapes cell is scraped with cell, is collected in Ep pipe.Extract kit instructions according to Qiagen DNA and extract DNA, by 300 μ L Buffer FG1 cell lysis, piping and druming mixes, add 300 μ L Buffer FG2/protease, put upside down and mix 3 times, put 65 ℃ of water-baths 10 minutes, add 600 μ L isopropyl alcohols, put upside down and mix, until there is DNA wire or block precipitation, centrifugal 3 minutes of 10000g, abandon supernatant, Ep pipe is upside down in to suck dry moisture on filter paper, add 600 μ l 70% ethanol, vortex 5s, centrifugal 3 minutes of 10000g, abandon supernatant, Ep pipe is upside down in to suck dry moisture on filter paper, the air-dry DNA of air precipitates until evaporate, add 50-100 μ L 70% nitric acid cell lysis, put 65 ℃ of water-baths 2 hours (now extraction portion divides pyrolysis product to utilize spectrophotometer to detect DNA concentration), in pyrolysis product, add distilled water, be diluted to 5% concentration of nitric acid.The Pt content that utilizes inductance coupling plasma Mass Spectrometer Method to be combined with DNA.
5) statistical analysis
Experimental data is used the processing of SPSS11.5 statistical software, carries out statistical study through t check and variance test (Oneway Anova).
2. experimental result
1) cell detects the drug resistance of cis-platinum, carboplatin, taxol, Epi-ADM
CDDP mdr cell SKOV3/Cis and A2780/Cis, not only to CDDP resistance, and also produced identical drug resistance (table 1) to CBP.Detect in addition the susceptibility of each transfectional cell to taxol and Epi-ADM.We can see, compare with A2780 with sensitive cells SKOV3, and mdr cell SKOV3/Cis and A2780/Cis have not only produced resistance to platinum class, and crossing drug resistant has been appearred in taxol and Epi-ADM, and their RI is presented at respectively in table 2.Express or the cell of not expressing contrasts (cell of transfection empty plasmid) and compares with it and annexin A 3 is crossed, to the susceptibility of taxol and Epi-ADM without obviously change (table 2).
The drug resistance comparison of table 1 platinum class mdr cell to cis-platinum and carboplatin
*p < 0.05,
*p < 0.01, compared with parental cell
Cell and compared with control cells thereof the drug resistance to taxol, Epi-ADM after table 2 stable transfection.
2) mensuration of Pt concentration
Each cell after 24 hours, detects intracellular Pt concentration through 10 μ M CDDP effects.Compared with the sensitive cells (SKOV3, A2780) of transfection empty plasmid, at the cell (SKOV3/Ann of annexin A 3 overexpression, A2780/Ann), in, Pt concentration significantly reduces (P < 0.001, P=0.002).Compared with the mdr cell (SKOV3/Cis, A278/Cis) of transfection empty plasmid, in the cell (SKOV3/Cis/R, A2780/Cis/R) of annexin A 3 down-regulated expression, Pt concentration obviously raises (P=0.008, P=0.009) (Fig. 1).In the experiment of detection Pt discharge rate, SKOV3 cell is (31.4 ± 4.0) %, SKOV3/Ann cell is (59.2 ± 2.2) %, and in SKOV3/Ann cell, Pt discharges obviously increases (P < 0.001) (Fig. 2).In addition, detecting in the result of Pt-DNA combination, find that, in SKOV3/Ann, A2780/Ann, the Pt concentration of being combined with DNA reduces (P=0.007, P=0.011).In SKOV3/Cis/R, A2780/Cis/R, the Pt concentration of being combined with DNA obviously raises (P=0.003, P=0.026) (Fig. 3).
3. conclusion
In cell, drug accumulation minimizing is one of important mechanisms causing platinum class resistance, and a critical function of annexin A 3 is contacting between mediation film and film, it can be attached on plasma membrane by phosphatidylserine, the permeability of cell membrane is changed, and participate in the activities such as the engulfing of cell, secretion.Therefore, we detected annexin A 3 cross express and the cell of annexin A 3 downward in whether there is the change of accumulating of platinum.Experimental result shows, in annexin A 3 overexpressing cell, platinum concentration obviously reduces, Pt-DNA is in conjunction with obviously reducing, and obtained consistent therewith result in the cell of lowering at annexin A 3, illustrate in cell that it is the key factor that annexin A 3 causes platinum class resistance that platinum medicine is accumulated minimizing.Platinum medicine carrys out damage dna by forming adduct with DNA, mismatch repair system identification adduct can cause p53 activation, activate MAPK path, induction BAX expresses, finally cause apoptosis (Hartwell LH, et al.Science, 1994,266 (5192): 1821-1828).This experiment has also confirmed this point.With transfection the parental cell of empty pcDNA3.1 (+) plasmid compare, in annexin A 3 overexpressing cell, give after cisplatin treated, platinum concentration obviously reduces, and Pt-DNA is in conjunction with obviously reducing, and the degree that p53 increases is less than parental cell, therefore apoptosis reduces, and causes resistance.Because the plasma peak concentration of CDDP chemotherapy dosage in clinical treatment is 10 μ M, in therefore testing in this section, the activity of CDDP all adopts this dosage.
Embodiment 2: the evaluation of annexin A 3 is secreted in ovarian epithelial carcinoma extracellular
1. experimental technique
1) clone and cell are cultivated
With embodiment 1.
2) collection of ovarian epithelial carcinoma cell line culture medium supernatant and concentrated
Get 2 × 10
5individual cell is inoculated in 25cm
2adherent growth in Tissue Culture Flask, changes serum-free DMEM, approximately every 1 × 10 when growing to 70%
6cell gives 5ml nutrient culture media, put in cell culture incubator and continue to cultivate 48 hours, then this serum-free DMEM is collected and put in 10ml centrifuge tube, centrifugal 10 minutes of 1000g, to remove the visible component in DMEM, centrifugal rear careful collection supernatant, be placed in Amicon Ultra-15 super filter tube (Millipore company of the U.S.), 3000g horizontal centrifugal 15 minutes, collects liquid after concentrated in Ep pipe ,-70 ℃ of preservations.
3) content of annexin A 3 in Western blot half-quantitative detection nutrient culture media
A. detect the preparation of sample
Be this experiment testing sample through total protein in cleer and peaceful cell on concentrated cell culture medium, nutrient culture media supernatant sample and laemmli sample-loading buffer (Bio-Rad company of the U.S.) are mixed and made into loading sample at 2: 1, be placed in ice-water bath, about 20 microlitres of volume are for well.Non-resistance and the mdr cell of taking the logarithm growth period, trypsinization, centrifugal 5 minutes of 1500rpm, supernatant discarded, add aseptic PBS (pH 7.4) resuspended, carry out cell count, centrifugal 5 minutes of 1500rpm, abandon supernatant, wash cell twice with cold PBS, exhaustion supernatant.Approximately 10
6individual cell adds laemmli lysate (Bio-Rad company of the U.S.), and in the time of cell lysis, other cell is in ice-water bath.Add after lysate, 99 ℃ are boiled 10 minutes, centrifugal 10 minutes of 10000g, 4 ℃, ice-water bath after taking out.Take out and measure after the sample of protein concentration, every remaining sample 100 μ l are added to 2-ME (2 mercapto ethanol) 5 μ l, centrifugal 10 minutes of 10000g, 4 ℃ ,-20 ℃ of preservations.
B.BCA method is surveyed total protein concentration in cell
Get BCA quantification of protein kit (Pierce) A liquid, B liquid with 50: 1 (v: v) mix, getting above-mentioned sample adds in 500 μ l AB mixed liquors, mix, blank is that 2 μ l lysates join 500 μ l AB mixed liquors, 37 ℃ of water-baths 30 minutes, with blank zeroing, at Smartspec
tMplus spectrophotometer (Bio-Rad) surveys the OD value of 562nm wavelength.Use Excel drawing standard curve, ask calculation sample concentration.
C.SDS-PAGE electrophoresis
Glue: first prepare 10% separation gel 5ml: deionized water 1.9ml, 30% acrylamide 1.7ml, 1.5%Tris 1.3ml (pH 8.8), 10%SDS 0.05ml, 10% Ammonium Persulfate 98.5 0.05ml, TEMED 0.002ml.After 30 minutes separation gels solidify, then prepare 5% concentrated glue 2ml: deionized water 1.4ml, 30% acrylamide 0.33ml, 1.0%Tris0.25ml (pH 6.8), 10%SDS 0.02ml, 10% Ammonium Persulfate 98.5 0.02ml, TEMED0.002ml.Application of sample: after gelling to be concentrated is solid, gets total protein in 20 μ l enrichment medium loading samples, 30 μ g cells and add in sample cell.Electrophoresis: add Tris-glycocoll electrophoretic buffer (25mmol/L Tris, 250mmol/L glycocoll, the 0.1%SDS) 80-100v of q.s, electrophoresis 2 hours.
D. transferring film
Prepare transfering buffering liquid: 48mM Tris-HCl, 39mM glycocoll, 0.037%SDS, 20% methyl alcohol.Shear 6 and gel filter paper of the same size and a pvdf membrane (Millipore company of the U.S.), filter paper is placed in transfering buffering liquid and soaks 15 minutes, and pvdf membrane is soaked in transfering buffering liquid after activating 10 seconds with methyl alcohol again.Filter paper, gel and pvdf membrane stack according to the order of three metafiltration paper-pvdf membrane-gel-tri-metafiltration paper, and whole operating process completes in transfering buffering liquid, and attention can not have bubble.Lower right corner mark.Electrotransfer 1 hour in 270mA, ice-water bath.
E. Western blotting
Take off the pvdf membrane that turns upper albumen, protein powder upward, TBST (200mM Tris-HCl, 1.5mM NaCl, 0.05%Tween-20) shake and wash 3 times, after each 5 minutes, taking out containing after sealing 1 hour in the TBST of 5% skimmed milk power, add the primary antibodie (the anti-human annexin A 3 specific polyclonal antibody of rabbit) of confining liquid preparation, jiggle for 4 ℃ and spend the night.Shake and wash 3 times with TBST, each 5 minutes, add afterwards two anti-(the goat anti-rabbit igg antibody of horseradish peroxidase-labeled) of the HRP mark that sealing also prepares, incubated at room 1 hour, TBST shakes and washes three times, Chemoluminescent substrate ECL (Thermo company of the U.S.) A, B liquid are mixed for by volume 1: 1, by pvdf membrane effect 5 minutes, hint X-ray exposure imaging.
2. experimental result
Tumour cell serum free medium protein content is very low, after supernatant is concentrated through super filter tube, volume can be concentrated to 500 μ l by 15ml, has so greatly improved the content of albumen in nutrient culture media, provides the foundation to detecting annexin A 3 by Western blot method.Use Western blot technology successfully in concentrated nutrient culture media supernatant, to detect annexin A 3, and annexin A 3 in the nutrient culture media supernatant of above-mentioned 8 kinds of ovarian cancer cell lines is carried out to sxemiquantitative.Sxemiquantitative is mainly the thickness of relative immunity trace annexin A 3 protein band.On above-mentioned 2 kinds of cisplatin resistance ovarian cancer cell line nutrient culture media in cleer and peaceful cell expression level of annexin A 3 all higher than it, the expression level of annexin A 3 in corresponding cis-platinum sensitive cell line, especially supernatant raises more obvious.In transfection annexin A 3 antisense plasmid is lowered cisplatin-resistant cell, after expression level of annexin A 3 (SKOV3/Cis/R and A2780/Cis/R clone), in cell culture medium, annexin A 3 level obviously declines.On the contrary, in transfection in the SKOV3/Ann of just annexin A 3 plasmid and the nutrient culture media of A2780/Ann cell expression level of annexin A 3 obviously raise (Fig. 4).
3. conclusion
In ovarian cancer cell nutrient culture media supernatant, contain a certain amount of annexin A 3, illustrate that ovarian cancer cell has the function of secretion annexin A 3, by sxemiquantitative comparison, illustrate ovarian cancer cell annexin A 3 outward the amount of secreting number become positive correlation with the content of annexin A 3 in cell, in nutrient culture media, high-caliber annexin A 3 can react the drug resistance of ovarian cancer cell to cis-platinum exactly.Based on more than, annexin A 3 has the basis of the biological markers that becomes effective indication platinum-based chemotherapy Drug-resistant.
Embodiment 3: outer the secrete approach of annexin A 3 in ovarian cancer cell
1. experimental technique
1) clone and cultivation
It is in full accord that this tests cell used and condition of culture and " embodiment bis-" thereof, and this is repeated description not.
2) cellular immunofluorescence and laser confocal imaging thereof
With 2 × 10
4cell kind is to completing in 24 porocyte culture plates of slide, at 37 ℃, 5%CO
2in saturated humidity incubator after overnight incubation, discard nutrient culture media, PBS washes 3 times, 4% paraformaldehyde is fixed 20 minutes, and PBS washes 3 times, 0.5%TritonX-100 permeable membrane 20 minutes, PBS washes 3 times, 2%BSA sealing 30 minutes, 1: 50 the anti-human annexin A 3 polyclonal antibody of rabbit (primary antibodie) hatch, 4 ℃ are spent the night.PBS washes 3 times, and goat antirabbit FITC bis-is anti-hatches 1 hour, and PBS washes after 3 times 1: 1000 containing the PI dyeing liquor incubated at room of 0.1%RNase 20 minutes, and PBS washes 3 times, utilizes the PBS mounting containing 90% glycerine.Under Radiance 2100 laser confocal microscopes, observe, take pictures.
3) transmission electron microscope
A. cell (total cellular score>=1 × 10 of logarithmic phase growth
6), abandon nutrient culture media, PBS washing 2 this.
B. add PBS 10ml, send Military Medical Science Institute (No. 27, TaiPing Road, Haidian District, BeiJing) instrument inspection center Electron Microscopy Room to prepare cell transmission electron microscope sample.
C. preparation of specimen's process is as follows: cell grows to exponential phase, and PBS washes twice, cell is scraped by cytobrush, centrifugal 10 minutes of 1000g, 3% glutaraldehyde (1/15M PBS pH 7.4), fixes 2 hours at 4 ℃, 1/15M PBS+0.19M sucrose damping fluid rinsing 15 minutes at 4 ℃.4 ℃ of Gradients of ethanol dehydration (each 10 minutes of 50% ethanol, 70% ethanol, 90% ethanol, 90% ethanol+90% acetone, 90% acetone, 100% acetone), 100% acetone at room temperature dewaters 10 minutes.100% acetone: embedding medium (1: 1) room temperature is soaked into 30 minutes, and pure embedding medium soaks into and spends the night.Carry out embedding: polymerization time is 35 ℃, 12 hours: 45 ℃, 12 hours; 60 ℃, 24 hours.ULTRACUT E/S type microtome (RMC company of the U.S.) carries out ultra-thin section, acetic acid uranium dye liquor lucifuge dyeing 10 minutes, lead citrate dyeing 10 minutes.
D. voltage 75kV, EM400T Electronic Speculum (Dutch Philips company) is observed, and selects × 8000, × 22000 electron microscope photographings, observation of cell submicroscopic structure.
4) immuno-electron microscope
A. cell (total cellular score>=1 × 10 of logarithmic phase growth
6), abandon nutrient culture media, PBS washing 2 times.
B. add PBS 10ml, send Chinese Academy of Medical Sciences's Electron Microscopy Room to prepare cell ultra-thin section (70-80nm is thick).Process is as follows: cell grows to exponential phase, PBS washes twice, cell is scraped by cytobrush, centrifugal 10 minutes of 1000g, the immobile liquid that 4% phosphoric acid paraformaldehyde and 0.1% glutaraldehyde mix at 1: 1, at 4 ℃, fix 4-6 hour, after Gradient elution using ethanol, adopt special white resin (Sigma company of the U.S.) embedding cell.After embedding medium cohesion, ULTRACUT E/S type microtome (RMC company of the U.S.) carries out ultra-thin section.
C. immunostaining: deionized water rinsing ultra-thin section three times, the confining liquid that is placed in 1%BSA seals 30 minutes, overnight incubation in 4 ℃ of wet boxes of 1: 200 anti-human annexin A 3 polyclonal antibody of rabbit, 0.1M PBS cleans 3 times, the anti-incubated at room of the anti-rabbit two of donkey of 1: 40 colloid gold particle mark 1 hour, washed with de-ionized water 3 times.
D. dyeing is observed: after immunostaining, cell ultra-thin section is again through peracetic acid uranium dye liquor lucifuge dyeing 10 minutes, and lead citrate dyes 10 minutes.Put the annexin A 3 albumen of observing colloid gold grain mark under Electronic Speculum, under 50000 × Electronic Speculum, take a picture.
5) exosome extraction in SKOV3/Cis clone culture supernatant, observation and annexin A 3 expression identification
A. get approximately 5 × 10
6individual SKOV3/Cis cell is on average inoculated in 2 T75 Tissue Culture Flasks, after cell attachment, is replaced by serum free medium (DMEM), puts 5%CO
2in cell culture incubator, cultivate 48 hours.Collect the about 60ml of nutrient culture media supernatant.
B. will collect the nutrient culture media supernatant coming in 1000 × g, 10min is centrifugal, removes cell and fragment composition, collects after centrifugal and gets supernatant.Again supernatant is placed in to Centricon Plus-20filter capsule (Millipore, USA) in, centrifugal 30 minutes of 4000 × g, 60ml supernatant simmer down to 1.5ml concentrate the most at last, again concentrate is diluted to 15ml with PBS, is placed in 100kDa Amicon Ultra-15 (Milipore, USA), centrifugal 30 minutes of 4000 × g, simmer down to 200 μ l concentrates.
C. collect 200 μ l concentrates, PBS is diluted to 5ml, and is transferred to 5ml and surpasses in pipe, simultaneously with 300 μ l 30%sucrose/D
2o is rebasing, 100,000 × g, 4 ℃ centrifugal 40 minutes, collect 350 μ l bottom glucose pads, and be diluted to 15ml with PBS, again use 100kDaAmicon Ultra-15 (Milipore, USA) to be concentrated into 200 μ l (this is the liquid containing exosome).Collect upper strata 350 μ l low concentration glucose liquid (using " sucrose " to represent this sample) below, as the contrast liquid without exosome ,-20 ℃ of preservations, analyze for westernblot simultaneously.The fluid preservation that 100 μ l are contained to exosome is in-20 ℃ of refrigerators.Low concentration glucose liquid and containing exosome liquid in the time that western blot analyzes, first liquid sample is mixed in laemmli sample-loading buffer (Bio-Rad company of the U.S.) in 1: 1 ratio, detect the expression (western blot method is described in detail in embodiment 2, and this is not repeated in this description) of annexin A 3 in sucrose and exosome.
D. in this 200 μ l concentrate, contain a large amount of exosome, concentrate is dropped on 400 order nickel screens of carbon membrane covering, naturally dry, now exosome has been adsorbed on nickel screen, 1% glutaraldehyde is fixed exosome, then with 1% uranium acetate negative staining, observes exosome form under transmission electron microscope.
E. when immune electron microscopy, first exosome is adsorbed to after nickel screen, PBS rinses 3 times repeatedly, overnight incubation in 4 ℃ of wet boxes of 1: 200 anti-human annexin A 3 polyclonal antibody of rabbit, 0.1MPBS cleans 3 times, the anti-incubated at room of the anti-rabbit two of donkey of 1: 40 colloid gold particle mark 1 hour, fixes with 1% glutaraldehyde thereafter again, under transmission electron microscope, observes.
6) collect without the cell culture medium supernatant of exosome
A. by 5 × 10
6individual SKOV3/Cis cell is on average inoculated in 2 T75 Tissue Culture Flasks, changes serum free medium after cell attachment, continues to cultivate after 48 hours and collects honest and upright and thrifty 30ml on nutrient culture media.
B. will collect next nutrient culture media supernatant 1000 × g centrifugal 10 minutes, collect supernatant 30ml.
C. divide the supernatant of collection equally two parts, the a 100kDa Amicon Ultra-15 (Millipore that uses, USA) under 4000 × g condition centrifugal 30 minutes, collect the non-concentrated liquid of lower floor, the about 15ml of volume, then 15ml liquid is used to 10kDa AmiconUltra-15 (Millipore, USA) under similarity condition centrifugal 30 minutes, the concentrate of collecting upper strata approximately 200 μ l, this concentrate is the supernatant of having removed exosome, this sample called after DMEM2.
D. the other a supernatant of dividing equally uses 10kDa Amicon Ultra-15 (Millipore, USA) under 4000 × g condition centrifugal 30 minutes, the concentrated liquid of collecting the about 200 μ l in upper strata, this liquid is the concentrated supernatant that contains exosome, this sample called after DMEM1.
E. all Sample preservations are in-20 ℃ of refrigerators, its 1: 1 is mixed with laemmli sample-loading buffer (Bio-Rad company of the U.S.), use western blot method to detect the expression (western blot experimental procedure can reference example 2) of annexin A 3.
2. experimental result
Under transmission electron microscope, observe above-mentioned cis-platinum sensitivity and drug-resistant ovarian carcinoma cell submicroscopic structure, compared with cis-platinum sensitive cells, the structure of finding to occur in cisplatin-resistant cell endochylema more vesica sample, this structure is dispersed in and is distributed in endochylema, smooth surface, be different from rough surfaced endoplasmic reticulum (RER) and golgiosome, in vesica, there is no the ridge of similar mitochondria sample yet, in some vesica, can also see and have some not clear particulate matter, be different from the homogeneous in lysosome.Prolong cell membrane and observe vesica, can observe some vesica from after birth very close to, some merges cut with after birth, some has broken through after birth outer (Fig. 5 A～B).Use transmission electron microscope observing transfection annexin A 3 justice plasmid to raise after the interior annexin A 3 level of cis-platinum sensitive cells, find in endochylema, also to have occurred compared with before transfection at sensitive cells the intracytoplasmic imitated vesicle structure of a lot of similar cisplatin-resistant cells; On the contrary, transfection annexin A 3 antisense plasmid is lowered after the interior annexin A 3 level of cisplatin-resistant cell, cisplatin-resistant cell endochylema intracellular vesicle has reduced (Fig. 5 C) in a large number, and the annexin A 3 raising in above explanation cell can cause the generation of born of the same parents' intracellular vesicle structure.Use immunofluorescence technique, the intracytoplasmic annexin A 3 albumen of the above-mentioned 8 kinds of cell karyons of specific dyeing, finds that ovarian cancer cell kind annexin A 3 is mainly distributed in endochylema, and minority is distributed in karyon, main or a kind of plasmosin (Fig. 6).Use immunoelectronmicroscopy, annexin A 3 cell distribution is carried out to subcellular organelle location, annexin A 3 in above-mentioned 8 kinds of cells is carried out to immunostaining, adopt colloid gold particle mark annexin A 3 albumen, find that annexin A 3 is distributed in the interior membrane structure of endochylema in a large number, comprise the film surface of the vesica of finding under transmission electron microscope, simultaneously at indivedual vesicas, particularly near exist (Fig. 7) that can find annexin A 3 in the vesica of after birth, illustrate that annexin A 3 secretes by the form of vesica outward.Use western blot method, detect annexin A 3 expression contents difference between DMEM1 and DMEM2, illustrate that exosome has carried a large amount of annexin A 3 albumen.Detect annexin A 3 level between exosome and sucrose, so that the purification degrees (Fig. 8) of exosome to be described simultaneously.
3. conclusion
Annexin A 3 is present in the endochylema and karyon of ovarian cancer cell, but is mainly present in endochylema, is a kind of plasmosin.The annexin A 3 raising can produce imitated vesicle structure in ovarian cancer cell endochylema, it both unlike endoplasmic reticulum also unlike golgiosome and lysosome, immuno-electron microscope has been verified in this vesica surface and vesica and has been had annexin A 3 albumen, observe vesica along cell membrane, can find that vesica has with cell membrane merges and the trend of giving prominence to cell, it can be said that bright annexin A 3 secretes cell outward by this approach.This mechanism of secretion is that annexin A 3 becomes clinical indication oophoroma platinum-based chemotherapy Drug-resistant biological markers as a kind of secretory protein, and more solid theoretical foundation is provided.
Embodiment 4: the detection of expression level of annexin A 3 in human peripheral
1. experimental technique
1) clinical case Data acquisition,
2007-2009 accepts the healthy women (30 example) of health check-up and treats ovarian cancer patients (50 example) preoperative (or before postoperative chemotherapy) in gynemetrics in Beijing Union Medical College BJ Union Hospital of the Chinese Academy of Medical Sciences is all research objects of this experiment.Health examination women need meet condition: (1) is in child-bearing period, climacteric and menopausal women; (2) do not suffer from any chronic disease, infectious diseases and innocent and malignant tumour disease; (3) within nearly one week, do not take any medicine and health products medicine.Healthy women serum is placed in Ep pipe after collecting, and is stored in-70 ℃, to be detected.The ovarian cancer patients that enters anthology experiment need meet: (1) postoperative histopathology is diagnosed as primary ovarian epithelial malignancy; (2), for the first time after surgical cytoreduction, all patients have all accepted regular single medicine or combined chemotherapy take platinum-based chemotherapy medicine as main (cis-platinum or carboplatin); (3) postoperative and for the first time after end of chemotherapy, all patients have all accepted regular following up a case by regular visits to, follow up a case by regular visits to the time approximately for the first time chemotherapy complete after 6 months to 1 year.After operation in patients, First Year is monthly followed up a case by regular visits to 1 time, and Second Year is followed up a case by regular visits to once for every 3 months, after the 3rd year, within every 6 months, follows up a case by regular visits to once.Follow up a case by regular visits at every turn and all detect peripheral blood CA125, gynecologial examination, and corresponding imaging examination, if desired biopsy.Include research according to entering group standard, collect patient's age, histological typing, by stages, classification, chemotherapy situation (table 3).Platinum-based chemotherapy Drug-resistant is defined as to reach after satisfied surgical cytoreduction and standard chemotherapy alleviates (CR), duration < 6 months completely; The best curative effect of chemotherapeutic period tumour is partial rcsponse (PR), stable (SD) or progress (PD) person.Platinum-based chemotherapy medicaments insensitive is defined as after satisfied surgical cytoreduction and standard chemotherapy and reaches CCR (CR), duration > 6 months.According to above standard, 50 routine ovarian cancer patients meet resistance standard through having followed up a case by regular visits to 30 patients, remain 20 and classify responsive group (table 3) as.
The routine ovarian cancer patients clinical case of table 3 50 statistics.A and b: pathological staging and clinical stages all carry out by stages according to international uniform standard.
2) ELISA (enzyme-linked immunosorbent assay)
A. experimental specimen: meet above-mentioned enter the healthy women of set condition and the serum of the ovarian cancer patients detected object that is this experiment.Blood sample collection is in 5 milliliters of collection tubes, and after blood natural coagulation, centrifugal 5 minutes of 3000g, collects supernatant (serum), in Ep pipe, is stored in-70 ℃.
B. adopt human annexin-V A3 ELISA detection kit (Cusabio company of the U.S.) quantitatively to detect expression level of annexin A 3 in serum.Kit contains ELISA Plate, standard items, sample diluting liquid, biotin labeling antibody diluent, Horseradish peroxidase-conjugated avidin dilution, biotin labeling antibody, Horseradish peroxidase-conjugated avidin, substrate solution, cleansing solution and stop buffer.Specifically, according to the operation of ELISA detection kit instructions, step is as follows:
(1) dilution standard product.Concentration is followed successively by 0ng/ml, 0.4ng/ml, 0.8ng/ml, 1.6ng/ml, 3.2ng/ml, 6.2ng/ml, 12.5ng/ml and 25ng/ml, and standard items are in experiment preparation in first 15 minutes;
(2) application of sample.Establish respectively blank well, gauge orifice, testing sample hole.Testing sample and standard items add in the ELISA Plate that is embedded with annxin A3 antibody, and every hole 100 microlitres add 100 microlitre standard items dilutions in blank well, the multiple hole of every Kong Junshe.Application of sample is complete by ELISA Plate overlay film, is placed in wet box, and 37 ℃ are reacted 120 minutes;
(3) mark.Discard liquid, dry, need not wash, every hole adds biotin labeling antibody working fluid 100 microlitres, 37 ℃, 60 minutes; Discard liquid again, dry, cleansing solution is washed plate 3 times, and each 2 minutes, every hole 200 microlitres, dried, and add Horseradish peroxidase-conjugated avidin working fluid 100 microlitres, and 37 ℃, 60 minutes;
(4) colour developing.Incubation is complete, discards liquid, dries, and washes plate 5 times, and every hole adds substrate solution 90 microlitres successively, and 37 ℃ of colour developings of lucifuge are after 30 minutes, and in hole, liquid color part becomes blue, and every hole adds 50 microlitre stop buffers successively, and now liquid transfers yellow to;
(5) detect.Add after stop buffer, in 15 minutes, upper microplate reader detects, and absorbing wavelength is made as 450nm, records every hole OD value.
C. data processing: use Excel take OD value as Y-axis, the 1g value of concentration be X-axis production standard curve, acquisition calcium formula, calculates the concentration value of bringing formula into after each sample mean OD value and just can draw the interior annexin A 3 of each sample.
3) statistical analysis
Data acquisition is analyzed with GraphPad Prism 5.0 softwares, all data are made annexin A 3 and are expressed scatter diagram according to normal group, oophoroma group (two groups of point platinum-based chemotherapy Drug-resistant group and responsive groups), group difference relatively uses Mann-Whitney to analyze, in the time of P > 0.05, difference has statistical significance.
3. experimental result
Use mankind's annexin A 3 enzyme-linked immunosorbent assay kits (ANXA3 ELISAkit) successfully to detect the content of annexin A 3 in human serum also quantitative, the expression of annexin A 3 in human peripheral is lower.The average expression of normal women serum annexin A 3 is 0.8590 ± 0.0744, and the average expression of ovarian cancer patients serum annexin A 3 is 1.6898 ± 2.6563, two groups and carries out Mann-Whitney check, P < 0.0001.All ovarian cancer patients are divided into platinum-based chemotherapy Drug-resistant group and platinum-based chemotherapy medicaments insensitive group, resistance group annexin A 3 average level is 2.1145 ± 3.3833, responsive group annexin A 3 average level is 1.0528 ± 0.1178, two groups of relatively P=0.0003 < 0.05 (Fig. 9).In the time that annexin A 3 level is greater than 1.13ng/ml, the susceptibility of this albumen prediction oophoroma platinum-based chemotherapy Drug-resistant reaches 63.3%, specificity 80%.In the time being greater than 1.34ng/ml, susceptibility and the specificity of this albumen prediction oophoroma platinum-based chemotherapy medicine all reach 100% (table 4).
4. conclusion
Annexin A 3, as a kind of platinum-based chemotherapy Drug-resistant associated protein, confirms to have exocrine function in ovarian cancer cell through experiment in vitro, and uses the success of ELISA method to detect annexin A 3 at human peripheral.And resistance group and responsive group expression level of annexin A 3 are compared, resistance group expression level of annexin A 3 is apparently higher than responsive group, when annexin A 3 level is higher than 1.13ng/ml, susceptibility and the specificity of its prediction platinum-based chemotherapy Drug-resistant all exceed 50%, when higher than 1.34ng/ml, its susceptibility and specificity reach 100%.This experiment is chosen human serum as research object, is to consider than being easier to acquisition and clinical conventional detected object based on it, also can adopt in theory other human secretions such as ascites, urine, saliva, leukorrhea as detected object.In this experiment, for the consideration of quality control, all pattern detection all has single disposable operation to complete, and double-blind study analysis is taked in data analysis.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (4)
1. the purposes of the annexin A 3 gene of expressing in tumour cell in the medicine of preparation rise platinum-based chemotherapy Drug-resistant tumour cell release platinum, wherein, described platinum-based chemotherapy medicine is cis-platinum, described tumour cell is ovarian cancer cell.
2. express the plasmid of just annexin A 3 gene in the purposes of preparing in the medicine that raises platinum-based chemotherapy Drug-resistant tumour cell release platinum, wherein, described platinum-based chemotherapy medicine is cis-platinum, and described tumour cell is ovarian cancer cell.
3. the ribozyme of expressing the antisensenucleic acids of annexin A 3 and/or be specific to annexin A 3 in tumour cell is in the purposes of preparing in the medicine of lowering platinum-based chemotherapy Drug-resistant tumour cell release platinum, wherein, described platinum-based chemotherapy medicine is cis-platinum, and described tumour cell is ovarian cancer cell.
4. the DNA construct of antisence annexin A 3 or the expression vector purposes in the medicine of preparation downward platinum-based chemotherapy Drug-resistant tumour cell release platinum, wherein, described platinum-based chemotherapy medicine is cis-platinum, described tumour cell is ovarian cancer cell.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1915433A (en) * | 2006-09-06 | 2007-02-21 | 中国医学科学院北京协和医院 | Relativity between AnnexinA3 and drug resistance of platinum type chemical curing medication for cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1915433A (en) * | 2006-09-06 | 2007-02-21 | 中国医学科学院北京协和医院 | Relativity between AnnexinA3 and drug resistance of platinum type chemical curing medication for cancer |
Non-Patent Citations (2)
| Title |
|---|
| Increased Expression of Annexin A3 Is a Mechanism of Platinum Resistance in Ovarian Cancer;Yan, XD et al.;《CANCER RESEARCH 》;20100126;第70卷(第4期);1616-1624 * |
| Yan, XD et al..Increased Expression of Annexin A3 Is a Mechanism of Platinum Resistance in Ovarian Cancer.《CANCER RESEARCH 》.2010,第70卷(第4期),1616-1624. |
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