CN101930004A - Application of APEX1 as protein molecular marker for detecting hepatocellular carcinoma - Google Patents
Application of APEX1 as protein molecular marker for detecting hepatocellular carcinoma Download PDFInfo
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- CN101930004A CN101930004A CN2009100537237A CN200910053723A CN101930004A CN 101930004 A CN101930004 A CN 101930004A CN 2009100537237 A CN2009100537237 A CN 2009100537237A CN 200910053723 A CN200910053723 A CN 200910053723A CN 101930004 A CN101930004 A CN 101930004A
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Abstract
The invention provides application of DNA (Deoxyribonucleic Acid) apurinic/apyrimidinic site lyase APEX1, and in particular relates to application of DNA apurinic/apyrimidinic site lyase APEX1 as a protein molecular marker for detecting hepatocellular carcinoma, and the protein molecular marker is used for detecting the expression of the APEX1 in hepatic tissue cells. The DNA apurinic/apyrimidinic site lyase APEX1 has obvious differential expression in the cancer cells of the hepatocellular carcinoma and cells adjacent to the cancer, therefore, whether patients have the hepatocellular carcinoma or not can be detected according to the expression of the APEX1 in hepatic cells.
Description
Technical field
The invention belongs to biological technical field, specifically, relate to DNA depurination/take off the application of pyrimidine site lyases APEX1, more particularly, relate to DNA depurination/the take off application of pyrimidine site lyases APEX1 as the protein molecular marker of hepatocellular carcinoma.
Background technology
Hepatocellular carcinoma is the 4th common malignancy in the world wide, second common cancer of China, and annual nearly 250,000 patients die from hepatocellular carcinoma.Hepatocellular carcinoma grade malignancy height recurs easily and shifts, and poor prognosis, and early diagnosis difficulty have often been incured loss through delay optimal treatment period.The incidence of disease of western developed country liver cancer is lower, still comparatively weak to the fundamental research of liver cancer in the world, and China country occurred frequently that is liver cancer, M ﹠ M presents ascendant trend, and age of onset constitutes rejuvenation, the medical expense that is used for liver cancer treatment every year greatly increases, liver cancer has become serious harm China people life property safety's dead enemy, and be a key factor that influences socio-economic development, the fundamental research of going into overdrive to carry out China's liver cancer has strategic importance, and separates and identify that new liver cancer related gene is the advanced subject in the present liver cancer fundamental research.
Up to the present, the gene unconventionality expression that does not have 20 kinds is determined relevant with the generation development of liver cancer, but the unconventionality expression rate of fixed liver cancer related gene in liver cancer is not high, and the pathogenesis of liver cancer is not illustrated so far yet, and the early diagnostic rate of liver cancer still remains to be improved.In addition, traditional operation of liver cancer adds chemotherapy and the several genes methods of treatment that is used does not in recent years still have obviously to improve the survival rate of liver cancer patient, thereby it is significant for the pathogenesis of inquiring into liver cancer to seek new liver cancer related gene, especially liver cance high-expression gene.
Therefore, research and development gene and/or protein of high expressed in hepatocellular carcinoma is significant, seeks the new gene and/or the protein of high expressed in hepatocellular carcinoma and also has the property of pressing for.
DNA depurination/take off pyrimidine site lyases (DNA-(apurinic or apyrimidinic site) lyase, EC4.2.99.18, Apurinic-apyrimidinic endonuclease 1, AP endonuclease 1, APE nuclease, APE, APEN, APEX1, APE, APX, HAP1, REF-1, REF1, REDOX FACTOR 1) accession number of NCBI is NP_001632, and the Swissprot accession number is P27695, is for IPI number: IPI00215911.3 (human3.28version).A large amount of DNA depurination/take off pyrimidine site lyases APEX1 and a lot of related to cancer of studies show that have been arranged.According to existing research, in most of cancer (for example, germinocarcinoma, prostate cancer, oophoroma, non-small cell lung cancer etc.), APEX1 high expressed in cancerous tissue; And in some cancer (for example colonic adenoma and colon cancer), the expression of APEX1 is constant, but variation has taken place in its celluar localization.
The variation of discovery DNA depurinations such as Di Mas/take off the Subcellular Localization of pyrimidine site lyases APEX1 can be used for the Prognosis of human hepatocytes cancer (Subcellular localization of APEX1/Ref-1 in human hepatocellularcarcinoma:possible prognostic significance.Mol Med.2007; 13 (1-2): 89-96).
But do not see as yet in the prior art relevant for adopting DNA depurination/the take off relevant report of the expression of pyrimidine site lyases APEX1 as the human hepatocytes carcinoma marker.
Summary of the invention
Protein by screening differential expression in human hepatocytes cancerous tissue and corresponding cancer beside organism, the present inventor has found a kind of expressed protein (up-regulated expression in cancerous tissue) that there are differences in human hepatocytes cancerous tissue and corresponding cancer beside organism, be accredited as DNA depurination/take off pyrimidine site lyases APEX1 through mass spectrum.Immunoblot experiment confirms, DNA depurination/take off pyrimidine site lyases APEX1 to there are differences expression (up-regulated in cancerous tissue) really in hepatocellular carcinoma tissue and corresponding cancer beside organism.The immunofluorescence experiment that utilizes human liver cell JEG-3 HepG2 and people's normal liver cell strain L02 to carry out proves DNA depurination/take off pyrimidine site lyases APEX1 to there are differences expression (up-regulated in human liver cell JEG-3 HepG2) between these two kinds of cell lines.The immunohistochemical assay that is undertaken by cancerous tissue and cancer beside organism to 62 pairs of human hepatocellular carcinomas has confirmed that further DNA depurination/take off pyrimidine site lyases APEX1 there are differences expression (up-regulated in cancerous tissue) in the cancerous tissue of human hepatocytes cancer and cancer beside organism.
Based on DNA depurination/take off the expression of pyrimidine site lyases APEX1 and the correlativity of hepatocellular carcinoma, therefore, APEX1 can be used as a kind of protein molecular marker thing, and according to its expression in liver cell to determine whether the patient suffers from hepatocellular carcinoma.Accordingly, the anti-DNA depurination of specificity/the take off antibody of pyrimidine site lyases APEX1, comprise various anti-DNA depurinations/take off monoclonal antibody and the polyclonal antibody of pyrimidine site lyases APEX1, because can be used in, it detects DNA depurination/the take off expression of pyrimidine site lyases APEX1, thereby can be used to detect hepatocellular carcinoma, perhaps be used to prepare the preparation or the kit that detect hepatocellular carcinoma.
Therefore, primary and foremost purpose of the present invention is, a kind of DNA depurination/take off the application of pyrimidine site lyases APEX1, to be used for the middle early detection of the detection of hepatocellular carcinoma, particularly hepatocellular carcinoma is provided.
Provided by the invention being applied as the protein molecular marker that detects hepatocellular carcinoma, this detection is to detect the expression of APEX1 in hepatic tissue cell.
According to the present invention, described detection is to detect APEX1 whether all to have up-regulated expression in hepatic tissue cell.
A further object of the invention is, a kind of anti-DNA depurination/take off application of the antibody of pyrimidine site lyases APEX1 is provided.
Being applied as of antibody of anti-DNA depurination provided by the invention/take off pyrimidine site lyases APEX1 is used to prepare the preparation that detects hepatocellular carcinoma, and this detection is to detect the expression of APEX1 in hepatic tissue cell.
Being applied as of antibody of anti-DNA depurination provided by the invention/take off pyrimidine site lyases APEX1 is used to prepare the kit that detects hepatocellular carcinoma, and this detection is to detect the expression of APEX1 in hepatic tissue cell.
According to the present invention, the antibody of anti-DNA depurination/take off pyrimidine site lyases APEX1 comprises monoclonal antibody and polyclonal antibody.
A further object of the invention is, whether unusual expression the method for DNA depurination in a kind of vitro detection hepatic tissue/take off pyrimidine site lyases APEX1 be provided.
Method provided by the invention is for DNA depurination in the antibody test hepatic tissue cell to be measured of the anti-DNA depurination of specificity/take off pyrimidine site lyases APEX1/the take off quantity of pyrimidine site lyases APEX1, and compares with the quantity of DNA depurination/take off pyrimidine site lyases APEX1 in the normal liver tissue.
Description of drawings
Fig. 1 is a DNA depurination/take off the Western blotting testing result of pyrimidine site lyases APEX1, and wherein, swimming lane 1-6 is the testing result of hepatocellular carcinoma tissue, and swimming lane 1 '-6 ' is the testing result of the cancer beside organism corresponding with swimming lane 1-6.
Fig. 2 carries out the relative distribution plan of protein expression amount that data analysis draws for the Western blotting collection of illustrative plates to Fig. 1, and wherein, HCC is hepatocellular carcinoma patient's a cancerous tissue, and non-HCC is a cancer beside organism.
Fig. 3 is DNA depurination/the take off immunofluorescence experiment result of pyrimidine site lyases APEX1, wherein, A1 is the distribution situation of APEX1 in the HepG2 cell, A2 is the nucleus of HepG2, A3 is the stacking diagram of A1 and A2, B1 is the distribution situation of APEX1 in the L02 cell, and B2 is the nucleus of L02, and B3 is the stacking diagram of B1 and B2.
Fig. 4 is a DNA depurination/take off the immunohistochemical assay result of pyrimidine site lyases APEX1, and wherein, A is the testing result of the cancerous tissue of hepatocellular carcinoma, and B is the testing result of cancer beside organism, and object lens magnification is 20 times.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) is carried out.
In following embodiment of the present invention, urea, 3-[(3-courage amido propyl)-the diethyl ammonium]-1-propane sulfonic acid (CHAPS), sodium dodecylsulphonate (SDS), dithiothreitol (DTT) (DTT), three (methylol) aminoethane (Tris), iodo-acetamide (IAA) be available from Bio-Rad company; CLM-2262L-Leu (U-
13C6,98%) available from Cambridge isotopic laboratory; The dialysis hyclone is available from Gibco company; D9785 cellular incubation powder, beta-mercaptoethanol, L-Gln, L-Lys and L-Met are available from Sigma company; Trypsase (Trypsin, sequencing grade) is available from Promega company; SCX post and SAX post and pH damping fluid kit are available from Column Technology company; HepG2 cell line and L02 cell line available from Shanghai Inst. of Life Science, CAS biological chemistry and cell research cell bank.
In following embodiment of the present invention, the solution/culture medium prescription of use is as follows:
The RPMI1640 nutrient culture media that does not contain glutamine: 5% dialysis hyclone, 0.2mM phenylmethylsulfonyl fluoride (PMSF), 1mM EDTA, oxacillin 25mg/mL, gentamicin 50mg/mL, penicillin 100U/mL, streptomysin 100mg/mL, amphotericin B 0.25mg/mL, nystatin 50U/mL.
Lysate: 8mol/L urea, 4%CHAPS, 40mmol/L Tris and 65mmol/L DTT.
Lysis buffer: 8mol/L urea, 40mmol/L Tris, 65mmol/L DTT.
TBST:Tris 2.42g/L, sodium chloride 8g/L, Tween-20 1ml/L regulates pH to 7.6 with HCl.
The present inventor is with non-enzymolysis sample preparation method (nonenzymatic sample preparation, NESP) cancerous tissue of hepatocellular carcinoma and the protein example of cancer beside organism have been prepared, and the proteomics mark quantitative technique that adopts interior enzymolysis of CDIT technology binding soln and negative and positive multidimensional liquid chromatography tandem mass spectrum is identified protein wherein and its expression of comparison, found that DNA depurination/take off pyrimidine site lyases APEX1 up-regulated expression in the hepatocellular carcinoma tissue, in addition, immunoblot experiment, immunofluorescence experiment and immunohistochemical assay all prove DNA depurination/take off pyrimidine site lyases APEX1 to there are differences expression really in hepatocellular carcinoma tissue and corresponding cancer beside organism.
Hospital of liver and gall surgical department obtains 5 routine hepatocellular carcinoma patients' cancerous tissue and cancer beside organism eastwardly, and this 5 routine patient makes a definite diagnosis by 2 doctors of pathology department, suffers from hepatocellular carcinoma, and 5 routine patients' pathological data is as shown in table 1.
Table 1,5 routine hepatocellular carcinoma patients' pathological data
| No. | Sex | Age | HBV | HCV | Grade | AFP | Tumor size |
| P1 | The male sex | 58 | + | - | III | 1000 | 5.2×6.4 |
| P2 | The male sex | 44 | + | - | III | 1000 | 8×8×7 |
| P3 | The male sex | 55 | + | - | III | 1000 | 4×3 |
| P4 | The male sex | 40 | + | - | III | 1000 | 10×8×6 |
| P5 | The male sex | 65 | + | - | III | 1000 | 11.5×6.5 |
Prepare cancerous tissue and cancer beside organism's protein example (used cancerous tissue and cancer beside organism's sample are the paired samples of taking from same hepatocellular carcinoma patient) of above-mentioned 5 routine patients' hepatocellular carcinoma with non-enzymolysis sample preparation method, detailed process is as follows:
The flesh tissue piece of excision places rapidly on ice, is cut into fast that several naked eyes are visible, the fritter of no necrotic zone.After organizing fritter for several times with the RPMI1640 nutrient culture media washing that does not contain glutamine of precooling, in liquid nitrogen, grind to form cell precipitation fast, be dissolved in cell precipitation in the lysate respectively then, then under condition of ice bath, after using ultrasonic cell disintegration instrument (JY92-2D, Ningbo Xin Zhike device research institute) ultrasonication at intermittence 2min, in 15000r/min, 4 ℃ of centrifugal 1h, get supernatant, it is quantitative to carry out gross protein with the Bradford method (seeing Bio-Rad company product description) of improvement.
Containing
13The CLM-2262L-Leu of C mark, 10% dialysis hyclone, L-Gln cultivates HepG2 or L02 cell respectively in the double dish of the 6cr of the D9785 nutrient culture media of L-Lys and L-Met.
After treating that cell grew into for 6 generations, HepG2 cell or L02 cell transfer are cultivated in the 10cm double dish.
Treat that the cell stand density reaches at 80% o'clock, give a baby a bath on the third day after its birth time, add lysate and carry out cracking with the PBS of 4 ℃ of precoolings.Then with scraper with these two kinds
13The cell of C mark scrapes collection respectively, the ice-bath ultrasonic fragmentation, and the centrifugal 1h of 15000g gets supernatant, with quantitative its total protein content of Bradford method (seeing Bio-Rad company product description) of improvement.
Adopt the CDIT technology (with reference to Yasushi Ishihama et al.Nat Biotechnol.2005; 23 (5): 617-621) the interior zymolysis technique of binding soln is (with reference to William M.Old et al.Mol Cell Proteomics.2005; 4:1487-1502) with negative and positive multidimensional liquid chromatography tandem mass spectrum technology (with reference to Jie Dai et al.J Proteome Res.2007; 6 (1): 250-262) carry out the screening of differentially expressed protein, detailed process is as follows:
Get among the embodiment 1 the 5 pairs of hepatocellular carcinoma tissues and each 100ug of cancer beside organism's protein example of obtaining, cancer beside organism's sample with 5 routine hepatocellular carcinoma histone quality samples and 5 routine hepatocellular carcinomas mixes respectively, obtains 500ug cancerous tissue protein example and 500ug cancer beside organism protein example.
Get among the embodiment 2
13The protein example of the HepG2 of C mark and L02 cell mixes the two equal proportion as interior mark.
Get the cancerous tissue of hepatocellular carcinoma of 500ug or cancer beside organism's protein example respectively with 500ug
13The interior mark of C mark mixes, and obtains two kinds of each 1mg of tissue-mixing with cells protein example.
Add lysis buffer respectively to cumulative volume 100 μ l in the mixed protein quality sample, add the 1M DTT of 8ul again, 37 ℃ were reacted 2 hours behind the mixing, added the 1M IAA of 20 μ l then, and the reaction of room temperature lucifuge is 45 minutes behind the mixing.
Acetone with-20 ℃ of precoolings of 4 times of volumes precipitates then ,-20 ℃ of reactions at least 4 hours.Centrifugal 45 minutes of 25000g, remove supernatant, the ammonium bicarbonate 200 μ l that add 50mmol/L, add 20 μ g trypsase (protein and tryptic ratio are 50: 1) again, 37 ℃ of enzyme digestion reactions are after 4 hours, adding trypsase is 25: 1 to gross protein and trypsase ratio, and enzymolysis spends the night to total enzymolysis time 16 hours.After finishing, enzymolysis, collects filtered solution, vacuum freeze drying with the ultra filtration membrane ultrafiltration of Millipore10KD pore size.
Every part of peptide section potpourri behind the enzymolysis is at first analyzed with the SCX/SAX post, uses full automatic multidimensional liquid chromatography tandem mass spectrometer LTQ then
TMOrbitrap
TM(available from Thermo Finnigan company) analyzes, the raw data that obtains adopts SEQUEST software (Thermo Finnigan company) to carry out database search, and adopting Census quantitative analysis software (http://fields.scripps.edu/census/index.php) that the protein that identifies is carried out quantitative test, quantitative result is shown in table 2 and table 3.
Table 2, hepatocellular carcinoma tissue/
13The C labeled cell contains the quantitative result of quantitative information peptide section
| Peptide section sequence | Ratio | R 2Value | X Corr value | Delta Cn value |
| K.EAAGEGPALYEDPPDQK.T | 2.16 | 0.95 | 2.5495 | 0.1826 |
| K.EAAGEGPALYEDPPDQK.T | 2.16 | 0.95 | 2.4941 | 0.2518 |
| K.LPAELQELPGLSHQYWSAPSDK.E | 2.93 | 0.94 | 3.2634 | 0.2486 |
| R.QGFGELLQAVPLADSFR.H | 2.44 | 0.98 | 2.867 | 0.4486 |
| R.QGFGELLQAVPLADSFR.H | 2.44 | 0.98 | 2.0445 | 0.1511 |
| R.QGFGELLQAVPLADSFR.H | 2.5 | 0.98 | 3.2244 | 0.4208 |
| R.QGFGELLQAVPLADSFR.H | 2.5 | 0.98 | 2.6769 | 0.3041 |
| R.QGFGELLQAVPLADSFR.H | 3.29 | 0.99 | 3.4479 | 0.5236 |
| R.QGFGELLQAVPLADSFR.H | 3.53 | 0.99 | 3.5964 | 0.5002 |
The cancer beside organism of table 3, hepatocellular carcinoma/
13The C labeled cell contains the quantitative result of quantitative information peptide section
| Peptide section sequence | Ratio | R 2Value | X Corr value | Delta Cn value |
| K.EAAGEGPALYEDPPDQK.T | 0.62 | 0.98 | 2.94 | 0.4467 |
| K.EAAGEGPALYEDPPDQK.T | 0.62 | 0.98 | 2.721 | 0.2588 |
| K.EAAGEGPALYEDPPDQK.T | 0.63 | 0.98 | 2.7885 | 0.1421 |
| K.EAAGEGPALYEDPPDQK.T | 0.63 | 0.98 | 2.7513 | 0.1759 |
| K.EAAGEGPALYEDPPDQK.T | 0.67 | 0.97 | 3.0567 | 0.3732 |
| K.EAAGEGPALYEDPPDQK.T | 0.67 | 0.97 | 2.6685 | 0.393 |
| K.EAAGEGPALYEDPPDQK.T | 0.67 | 0.97 | 2.6502 | 0.2619 |
| K.EEAPDILCLQETK.C | 3.04 | 0.78 | 2.7801 | 0.3431 |
| K.EEAPDILCLQETK.C | 3.04 | 0.78 | 2.4355 | 0.3141 |
| K.EEAPDILCLQETK.C | 3.04 | 0.79 | 2.2674 | 0.2446 |
| K.EEAPDILCLQETK.C | 3.06 | 0.8 | 2.2861 | 0.3362 |
| R.QGFGELLQAVPLADSFR.H | 0.69 | 0.91 | 2.7705 | 0.1646 |
| R.QGFGELLQAVPLADSFR.H | 0.69 | 0.91 | 2.6038 | 0.4465 |
| R.QGFGELLQAVPLADSFR.H | 0.69 | 0.91 | 2.5854 | 0.3522 |
| R.QGFGELLQAVPLADSFR.H | 0.69 | 0.91 | 2.3455 | 0.2853 |
| R.QGFGELLQAVPLADSFR.H | 0.58 | 0.81 | 2.3615 | 0.2043 |
| R.QGFGELLQAVPLADSFR.H | 0.58 | 0.81 | 2.2533 | 0.2617 |
| R.QGFGELLQAVPLADSFR.H | 0.58 | 0.81 | 2.0513 | 0.3656 |
According to the result of table 2 and table 3, DNA depurination/take off pyrimidine site lyases APEX1 the hepatocellular carcinoma tissue/
13C labeled cell and cancer beside organism/
13Quantitative result in the C labeled cell is respectively 1.802481252 and 0.687241904, high expressed is more than 2.5 times in the hepatocellular carcinoma tissue, express in the hepatocellular carcinoma tissue obviously and raise, wherein, DNA depurination/take off pyrimidine site lyases APEX1 the hepatocellular carcinoma tissue/
13Have 3 non-repetition peptide sections (identifying altogether 9 times) to comprise quantitative information in the C labeled cell, and cancer beside organism/
13There are 3 non-repetition peptide sections (identifying altogether 18 times) to comprise quantitative information in the C labeled cell.
Get the hepatocellular carcinoma tissue of 6 pairs of NESP method preparations and the protein example of corresponding cancer beside organism, its pathological data is as shown in table 4.
The pathological data of table 4,6 routine hepatocellular carcinoma samples
| No. | Sex | Age | HBV | HCV | Grade | AFP | Size |
| P6 | The male sex | 55 | + | - | III | ＞1000 | 4×3 |
| P7 | The male sex | 40 | + | - | III | ＞1000 | 10×8×6 |
| P8 | The male sex | 65 | + | - | III | ＞1000 | 11.5×6.5 |
| P9 | The male sex | 56 | + | - | III | ＞1000 | 7×6 |
| P10 | The male sex | 50 | + | - | III | ＞1000 | 5×6 |
| P11 | The male sex | 55 | + | - | III | ＞1000 | 5×5.5 |
The anti-DNA depurination of use buying/take off pyrimidine site lyases APEX1 antibody carries out immunoblotting assay to the protein example of above-mentioned 6 pairs of hepatocellular carcinoma tissues and corresponding cancer beside organism, and process is as follows:
Each sample is got 10 μ g protein examples, separates with 12%SDS-PAGE, is transferred on the pvdf membrane (available from GEHealthcare company);
One anti-use mouse-anti people DNA depurination/take off pyrimidine site lyases APEX1 monoclonal antibody (available from LifeSpanBiosciences company, 1: 1000), 4 ℃ of overnight incubation were with TBST washing three times, each 5 minutes;
Two anti-are anti-mouse antibody (available from Santa Cruz company, 1: 10000), and incubated at room 1 hour is again with TBST washing three times, each 10 minutes;
Add ECL plus reagent (available from GE Healthcare company), reacted X-mating plate exposure tests 5 minutes.
Testing result adopts Gel-Pro Analyzer gel quantitative analysis software (MediaCybemetic company) to carry out data analysis to the Western blotting collection of illustrative plates of Fig. 1 as shown in Figure 1 then, draws the relative distribution plan of protein expression amount, and the result as shown in Figure 2.
Fig. 1 result shows, the concentration of the hybridization band of DNA depurination in the hepatocellular carcinoma tissue/take off pyrimidine site lyases APEX1 is all apparently higher than corresponding cancer beside organism, in addition, the concentration (the inferior band main band under) of the spliced body hybridization band of DNA depurination in the hepatocellular carcinoma tissue/take off pyrimidine site lyases APEX1 is also apparently higher than corresponding cancer beside organism, the reparation of this spliced body participation mitochondrial DNA.And because the degree height that mitochondrial DNA suffers the damage of active oxy group in the cancer cell, therefore the spliced body concentration of APEX1 rises, and HCC is different with the concentration change of the band of non-HCC under APEX1 master's band, i.e. the variation of the shearing of APEX1 is relevant with HCC.
Fig. 2 result shows, DNA depurination/take off the expression of pyrimidine site lyases APEX1 in the hepatocellular carcinoma tissue all apparently higher than corresponding cancer beside organism, and mean ratio is 1.955, P value (paired t-test) is 0.001583.
According to the result of Fig. 1 and Fig. 2, there is high expressed in DNA depurination/take off pyrimidine site lyases APEX1 in the cancerous tissue of hepatocellular carcinoma, and this result is consistent with the mass spectrum result.
Choose HepG2 cell (human liver cell JEG-3) and L02 cell (strain of people's normal liver cell) and carry out immunofluorescence experiment, process is as follows:
On the circular lid slide, cultivate L02 cell and HepG2 cell with the DMEM that contains 10% dialysis hyclone;
After the PBS washed twice, 100% acetone fixed of-20 ℃ of precoolings; The PBS washed twice, 5% skim milk sealing 30 minutes;
Adding mouse-anti people DNA depurination/take off pyrimidine site lyases APEX1 monoclonal antibody (available from LifeSpanBiosciences company, confining liquid dilution in 1: 30), incubated at room 1h, TBST wash 3 times, each 7 minutes;
The sheep anti-mouse antibody (available from Molecular Probes company, confining liquid dilution in 1: 300) that adds fluorescent dye Alexa 488 marks, incubated at room 45 minutes, TBST washes 3 times, each 7 minutes;
Add DAPI solution (available from Sigma company, distilled water dilution in 1: 1000), hatched 1 minute, PBS washed 10 minutes;
Mounting, 4 ℃ keep in Dark Place.
The use laser confocal microscope is observed, and the result as shown in Figure 3.
According to Fig. 3 result, DNA depurination/take off the expression of pyrimidine site lyases APEX1 in the HepG2 cell apparently higher than its expression in the L02 cell, this is consistent with the result who obtains from tissue.
In order further to confirm DNA depurination/the take off differential expression of pyrimidine site lyases APEX1 between hepatocellular carcinoma tissue and cancer beside organism, the cancerous tissue of 62 pairs of liver cancer of picked at random and corresponding cancer beside organism's sample, use two liver cancer tissue chips (to be respectively OD-CT-DgLiv03-002 liver cancer tissue chip and OD-CT-DgLiv03-003 liver cancer tissue chip, all available from Xinchao Biotech Co., Ltd., Shanghai) carry out immunohistochemistry research, the physical resource of the sample of wherein, choosing is respectively shown in table 5 and table 6:
Table 5, OD-CT-DgLiv03-002 liver cancer tissue chip data and marking situation
Table 6, OD-CT-DgLiv03-003 liver cancer tissue chip data and marking situation
The experimentation of immunohistochemistry research is as follows:
Place 60 ℃ of constant temperature ovens to toast about 3 hours histotomy, use dimethylbenzene, dimethylbenzene/ethanol (1: 1), 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% second alcohol and water after the taking-up successively, the aquation that dewaxes is handled;
PBS washes 3 times, each 5 minutes; 0.3%H
2O
2(methyl alcohol dilution) soaks half an hour, and PBS washes 3 times, each 5 minutes;
High pressure is repaired antigen, and distilled water is washed 2 times, each 5 minutes; PBS washes 2 times, each 5 minutes; 10% serum room temperature sealing 20 minutes;
Adding mouse-anti people DNA depurination/take off pyrimidine site lyases APEX1 monoclonal antibody (available from LifeSpanBiosciences company, dilution in 1: 200), 4 ℃ are spent the night, and PBST washes 3 times, each 5 minutes;
Add biotin labeled horse anti-mouse antibody (from the ABC kit, available from VECTOR company), incubated at room 30 minutes, PBST washes 3 times, each 5 minutes; PBS washes 2 times, each 5 minutes;
Add ABC solution (from the ABC kit, available from VECTOR company), incubated at room 30 minutes, PBS washes 3 times, each 5 minutes;
DAB solution (available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) colour developing; (available from VECTOR company, H3404) dyeing is 20 seconds for haematine;
Histotomy uses successively: 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, dimethylbenzene/ethanol (1: 1), dimethylbenzene, the transparent processing of dewatering.
Use the neutral resins mounting then, in microscopic examination, the result as shown in Figure 4.
Organization chip is assessed, the result shows: totally 59 pairs of effective samples (remove pair of sample and destroy serious and 2 pairs of samples that are cancerous tissue) in the organization chip, and according to staining power and positive rate marking, wherein, staining power marking standard is: feminine gender is that 0 minute, the weak positive are that 1 minute, the medium positive are that 2 minutes, strong positive are 3 minutes; Positive rate marking standard is: being lower than 5% is 0 minute, and 5%～30% is 1 minute, and 31%～60% is 2 minutes, is 3 minutes more than 60%.With the score of staining power and the score addition of positive rate, obtain the integrate score of histotomy, the score situation is respectively shown in table 5 and table 6.
Result according to table 5 and table 6: the information in comprehensive tenuigenin and the nucleus, the average integrate score of hepatocellular carcinoma tissue is 4.88, the average integrate score of cancer beside organism is 2.66, both have utmost point significant difference, P value (Wilcoxon rank sumtest) is 4.011E-07, the sample centering of about 75% (44 pairs), DNA depurination/take off pyrimidine site lyases APEX1 high expressed in hepatocellular carcinoma.Pathological grading I, there is significant difference between the sample group of I-II and the sample group of pathological grading II, the P value is less than 0.01, pathological grading I, also there is significant difference between the sample group of I-II and the sample group of pathological grading II-III, the P value is less than 0.01, DNA depurination/take off the expression of pyrimidine site lyases APEX1 and the differentiation degree of cell is inversely proportional to.
If only utilize the information in the nucleus, the average integrate score of hepatocellular carcinoma tissue is 2.92, the average integrate score of cancer beside organism is 1.03, both have utmost point significant difference, P value (Wilcoxon rank sum test) is 1.924E-07, the sample centering of about 75% (44 pairs), DNA depurination/take off pyrimidine site lyases APEX1 high expressed in hepatocellular carcinoma.Pathological grading I, there is significant difference between the sample group of I-II and the sample group of pathological grading II, the P value is less than 0.01, pathological grading I, also there is significant difference between the sample group of the sample group of I-II and case classification II-III, the P value is less than 0.01, DNA depurination in the nucleus/take off the expression of pyrimidine site lyases APEX1 and the differentiation degree of cell is inversely proportional to.
If only utilize cytoplasmic information, pathological grading I, there is significant difference between the sample group of I-II and the sample group of pathological grading II, the P value is less than 0.01, pathological grading I, also have significant difference between the sample group of the sample group of I-II and case classification II-III, the P value is less than 0.01, DNA depurination in the tenuigenin/take off the expression of pyrimidine site lyases APEX1 and the differentiation degree of cell is inversely proportional to.
This result is consistent with mass spectrum result, Western blotting result and the immunofluorescence result of front.
Therefore, can express, be used for the early diagnosis of hepatocellular carcinoma, and not only be the evaluating prognosis standard according to the evident difference that DNA depurination/take off pyrimidine site lyases APEX1 exists in the cancerous tissue of hepatocellular carcinoma and cancer beside organism.
In sum, DNA depurination/take off pyrimidine site lyases APEX1 exists evident difference to express in the cancerous tissue of hepatocellular carcinoma and cancer beside organism, generation development obvious and hepatocellular carcinoma has close correlativity, so its expression can be used to detect hepatocellular carcinoma.Accordingly, the anti-DNA depurination of specificity/the take off antibody of pyrimidine site lyases APEX1, comprise various anti-DNA depurinations/take off monoclonal antibody and the polyclonal antibody of pyrimidine site lyases APEX1, because can be used in, it detects DNA depurination/the take off expression of pyrimidine site lyases APEX1, thereby can be used to detect hepatocellular carcinoma, perhaps be used to prepare the preparation or the kit that detect hepatocellular carcinoma, this is conspicuous for a person skilled in the art.
Though relative dna depurination/take off the dynamic biological function of pyrimidine site lyases APEX1 and the tumour related mechanism is still waiting further research but is sure with it as the label that detects hepatocellular carcinoma.DNA depurination/take off the mark that pyrimidine site lyases APEX1 can be used as hepatocellular carcinoma, depurination/taking off pyrimidine site lyases APEX1 may be as the prognosis molecule mark of liver cancer and the target molecule of clinical treatment and its biological function in born of the same parents is pointed out DNA.
Claims (10)
1.DNA depurination/take off the application of pyrimidine site lyases APEX1 as the protein molecular marker that detects hepatocellular carcinoma is characterized in that described detection is to detect the expression of APEX1 in hepatic tissue cell.
2. application as claimed in claim 1 is characterized in that, whether the expression of described detection APEX1 in hepatic tissue cell all exists up-regulated expression for detecting APEX1 in hepatic tissue cell.
3. an anti-DNA depurination/take off the application of the antibody of pyrimidine site lyases APEX1 is characterized in that be used to prepare the preparation that detects hepatocellular carcinoma, described detection is to detect the expression of APEX1 in hepatic tissue cell.
4. application as claimed in claim 3 is characterized in that, the antibody of described anti-DNA depurination/take off pyrimidine site lyases APEX1 comprises monoclonal antibody and polyclonal antibody.
5. application as claimed in claim 3 is characterized in that, whether the expression of described detection APEX1 in hepatic tissue cell all exists up-regulated expression for detecting APEX1 in hepatic tissue cell.
6. an anti-DNA depurination/take off the application of the antibody of pyrimidine site lyases APEX1 is characterized in that be used to prepare the kit that detects hepatocellular carcinoma, described detection is to detect the expression of APEX1 in hepatic tissue cell.
7. application as claimed in claim 6 is characterized in that, the antibody of described anti-DNA depurination/take off pyrimidine site lyases APEX1 comprises monoclonal antibody and polyclonal antibody.
8. application as claimed in claim 6 is characterized in that, whether the expression of described detection APEX1 in hepatic tissue cell all exists up-regulated expression for detecting APEX1 in hepatic tissue cell.
9. the whether unusual method of the expression of DNA depurination in the vitro detection hepatic tissue/take off pyrimidine site lyases APEX1, it is characterized in that, described method is for DNA depurination in the antibody test hepatic tissue cell to be measured of the anti-DNA depurination of specificity/take off pyrimidine site lyases APEX1/the take off quantity of pyrimidine site lyases APEX1, and compares with the quantity of DNA depurination/take off pyrimidine site lyases APEX1 in the normal liver tissue.
10. method as claimed in claim 9 is characterized in that, the antibody of described anti-DNA depurination/take off pyrimidine site lyases APEX1 comprises monoclonal antibody and polyclonal antibody.
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