CN1920570B - Application of thioredoxin protein 2 - Google Patents
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- CN1920570B CN1920570B CN2005100291671A CN200510029167A CN1920570B CN 1920570 B CN1920570 B CN 1920570B CN 2005100291671 A CN2005100291671 A CN 2005100291671A CN 200510029167 A CN200510029167 A CN 200510029167A CN 1920570 B CN1920570 B CN 1920570B
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Abstract
The invention selects the proteins with different expressions in cancer organization and cancer beside organism, to find one protein with high expression in cancer organization of liver cell cancer, wherein the immunity print test has proved that the thioredoxin 2 has different expressions in cancer organism and cancer beside organism. Based on said relation, the protein can be used in protein molecule mark to detect liver cancer.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to of the application of a kind of thioredoxin protein 2 as the protein molecular marker that detects liver cancer.
Background technology
Liver cancer is a kind of serious harm human diseases.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 especially liver cance high-expression gene is significant for the pathogenesis of inquiring into liver cancer to seek new liver cancer related gene.
Therefore, to research and develop in liver cancer the gene and/or the albumen of high expressed significant for treatment and diagnostic purpose.This area press for new in liver cancer the gene and/or the albumen of high expressed.
(Thioredoxin-like protein2 TXNL2), claims PKC-interacting cousinof thioredoxin again to thioredoxin protein 2, PKC-theta-interacting protein, PKC θ-interacting protein, HUSSY-22.Its Genebank accession number is gi|6840953|, and the accession number of NCBI is AAF28844, and the Swissprot accession number is O76003, is for IPI number: IPI00216136.1.Thioredoxin protein 2, claim that again (PKC interacting cousin of thioredoxin finds by yeast two-hybrid when PICOT) being Stephan Witte research protein kinase C interaction protein the earliest the same albuminoid of protein kinase C interaction thioredoxin.They are by instantaneous high expressed thioredoxin protein 2 in leucocyte, find that it can suppress c-JunN and hold kinase whose activation and transcription factor AP-1 and NF-kB, they find thioredoxin protein 2 sequence high conservative simultaneously, disclose the function (J.Biol.Chem.Vol.275 that thioredoxin protein 2 and protein kinase C interact and can regulate thioredoxin system, No.3, Issue of January21, pp.1902-1909,2000).
Thioredoxin protein 2 (TXNL2, TXL2) be the albumen that belongs to the member of thioredoxin family and nucleoside diphosphokinase family simultaneously, the albumen that it is made up of 330 amino acid, comprise N end thioredoxin typical structure territory and C end nucleoside diphosphokinase domain, the centre is a little boundary domain.It is the highest that original research person discloses thioredoxin protein 2 expression in testis and lung by PCR in real time, and find that by Electronic Speculum thioredoxin protein 2 and micro-tubular structure have interaction (J.Biol.Chem.Vol.278, No.15, Issue of April11, pp.13133-13142,2003).
But up to the present, do not see report in the prior art relevant for the correlativity of thioredoxin protein 2 and hepatocellular carcinoma.
Summary of the invention
Protein by screening differential expression in hepatocellular carcinoma cancerous tissue and hepatocellular carcinoma cancer beside organism, the present inventor found a kind of in hepatocellular carcinoma cancerous tissue and cancer beside organism in there are differences expressed protein (up-regulated expression in cancerous tissue), be accredited as thioredoxin protein 2 through mass spectrum.Further immunoblot experiment confirms, thioredoxin protein 2 there are differences expression (up-regulated expression in cancerous tissue) really in the cancerous tissue of hepatocellular carcinoma and cancer beside organism.
Based on this correlativity of thioredoxin protein 2 and hepatocellular carcinoma, its expression is detected as a protein molecular marker with this albumen and can be used to detect liver cancer.
Therefore, primary and foremost purpose of the present invention promptly is to provide the application of a kind of thioredoxin protein 2 as the protein molecular marker that detects liver cancer.
Another object of the present invention is to provide a kind of antibody of anti-thioredoxin protein 2, comprises monoclonal antibody and polyclonal antibody, is used to prepare the application of the preparation that detects liver cancer.
A further object of the present invention also is to provide a kind of antibody of anti-thioredoxin protein 2, comprises monoclonal antibody and polyclonal antibody, is used to prepare the application of the kit that detects liver cancer.
Whether unusual another purpose of the present invention be to provide expression the method for thioredoxin protein 2 in a kind of vitro detection liver cell tissue, and this method may further comprise the steps:
A, with the quantity of thioredoxin protein 2 in the antibody test of the anti-thioredoxin protein 2 of the specificity liver cell to be measured;
The quantity of B, thioredoxin protein 2 that steps A is recorded and the quantity of the thioredoxin protein 2 in the normal liver tissue compare, as the albumen quantity that records is higher than normal value, then represents the abnormal expression of thioredoxin protein 2 in the detected hepatic tissue.
In view of up to the present, also there is not the report of the correlativity of thioredoxin protein 2 and hepatocellular carcinoma, therefore, this discovery of the present invention will provide a brand-brand-new way for the diagnosis and/or the treatment of hepatocellular carcinoma.
Description of drawings
Fig. 1 is the quantitative change synoptic diagram of thioredoxin protein 2 in the 2-DE of cancerous tissue and cancer beside organism collection of illustrative plates, shown that the expression that the expression of protein spots SSP2307 (thioredoxin protein 2 of identifying through mass spectrum) in hepatocellular carcinoma patient's cancerous tissue compared in cancer beside organism obviously raises.
Fig. 2 is the immunoblotting assay result schematic diagram of thioredoxin protein 2.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The present inventor is with non-enzymolysis sample preparation method (nonenzymatic sample preparation, NESP) cancerous tissue of Zhi Bei hepatocellular carcinoma and cancer beside organism's protein example, protein spots and mass spectrum with two dimensional gel electrophore-sis (2-DE) technology screening differential expression in cancerous tissue and cancer beside organism are identified, found that thioredoxin protein 2 up-regulated expression in the hepatocellular carcinoma cancerous tissue.Immunoblot experiment confirms that further thioredoxin protein 2 there are differences expression really in the cancerous tissue of hepatocellular carcinoma and cancer beside organism.
Therefore, its expression is detected as a protein molecular marker with thioredoxin protein 2 and can be used to detect liver cancer, promptly thioredoxin protein 2 can be as the protein molecular marker that detects liver cancer.
Embodiment 1, hepatocellular carcinoma cancerous tissue and cancer beside organism's protein example preparation
Employed urea, 3-[(3-courage amido propyl in the present embodiment)-the diethyl ammonium]-1-propane sulfonic acid (CHAPS), phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT) (DTT) be all available from Sigma company.
Present embodiment with non-enzymolysis sample preparation method (nonenzymatic sample preparation, NESP) preparation hepatocellular carcinoma cancerous tissue and cancer beside organism's protein example, specific 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.RPMI1640 nutrient culture media (5% hyclone that does not contain glutamine with precooling, 0.2mM PMSF, 1mM EDTA, oxacillin 25mg/mL, gentamicin 50mg/mL, penicillin 100U/mL, streptomysin 100mg/mL, amphotericin B 0.25mg/mL, nystatin 50U/mL) after washing organizes fritter for several times, in liquid nitrogen, grind to form cell precipitation fast, cell precipitation is dissolved in an amount of lysate (8mol/L urea respectively, 4%CHAPS, 40mmol/L Tris and 65mmmol/L DTT) in, ultrasonic cell disintegration instrument (Soniprep150, Britain, MSE) ice bath ultrasonic 2min at intermittence, 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, the hepatocellular carcinoma cancerous tissue for preparing and the protein example packing of corresponding adjacent tissues, and-80 ℃ of preservations are standby.
With 16 pairs of hepatocellular carcinoma cancerous tissues of method for preparing and cancer beside organism's protein example.16 routine hepatocellular carcinoma samples clearly are hepatocellular carcinoma all from east hospital of liver and gall surgical department by 2 doctors of pathology department.Be the male sex, 49.4 years old mean age (31～65 years old), serum detects the hepatitis B virus infections positive, and 16 examples (100%) belong to clinical scale (TNM classification) III level.Wherein, alpha-fetoprotein (AFP) is higher than 15 examples (93.75%) of 25 μ g/L; 14 routine tumours are greater than 5cm.The pathological data of 16 routine hepatocellular carcinoma samples sees following table 1 for details.
The pathological data of table 1,16 routine hepatocellular carcinoma samples
| No. | Sex | Age | HBV | HCV | Grade | AFP | Size |
| f31 | The male sex | 56 | + | - | III | >1000 | 7×6 |
| f32 | The male sex | 51 | + | III | >1000 | 14×12×12 | |
| f33 | The male sex | 50 | + | - | III | >1000 | 5×6 |
| f39 | The male sex | 55 | + | - | III | >1000 | 5×5.5 |
| 327 | The male sex | 44 | + | - | III | >1000 | 8×8×7 |
| 328 | The male sex | 45 | + | - | III | >1000 | 7.5×6 |
| 415 | The male sex | 40 | + | - | III | >1000 | 10×8×6 |
| 418 | The male sex | 31 | + | - | III | 3.7 | 8×5×8 |
| 422 | The male sex | 57 | + | - | III | >1000 | 3.5×4 |
| 429 | The male sex | 44 | + | - | III | >1000 | 7.2×6 |
| 317 | The male sex | 58 | + | - | III | >1000 | 5.2×6.4 |
| 42 | The male sex | 45 | + | - | III | >1000 | 7.7×5.4 |
| 45 | The male sex | 51 | + | - | III | >1000 | 5.5×4.0 |
| 48 | The male sex | 55 | + | - | III | >1000 | 4×3 |
| 49 | The male sex | 43 | + | - | III | >1000 | 12×12 |
| 424 | The male sex | 65 | + | - | III | >1000 | 11.5×6.5 |
The used cancerous tissue of present embodiment and cancer beside organism's sample are the paired samples of taking from same hepatocellular carcinoma patient, all 16 routine hepatocellular carcinoma cases have the case diagnosis index of fairly similar: be the male sex, 49.4 years old mean age (31～65 years old), serum detects the hepatitis B virus infections positive, and 16 examples (100%) belong to TNM classification III level.Wherein, AFP is higher than 15 examples (93.75%) of 25 μ g/L; 14 routine tumours are greater than 5cm.This sampling method helps reducing between individuality difference to the influence of experimental analysis work.
Embodiment 2, differentially expressed protein screening
The urea that uses in the present embodiment, 3-[(3-courage amido propyl)-the diethyl ammonium]-1-propane sulfonic acid (CHAPS), sodium dodecylsulphonate (SDS), dithiothreitol (DTT) (DTT) be available from Sigma company; Iodoacetamide (IAA), acrylamide, N, N-methylene diacrylamide etc. are available from Fluka company.
Ammonium Persulfate 98.5 (AP), Tri-n-butylphosphat (TBP), PDQuest software etc. are the Bio-Rad product.
MALDI TOF REFLEX
TMIII system is available from Bruker-Daltonics company.
The prefabricated adhesive tape of non-linear solid phase pH gradient (IPG immobilized ph gradient strip, pH3-10NL, 130 * 3 * 0.5mm), IPG damping fluid, IPGphore isoelectric focusing system (Amersham Pharmacia Biotech), Amersham PharmaciaEttan Dalt II systems etc. be Amersham Bioscience company product.
Get cancerous tissue and 10 couple in cancer beside organism's protein example (f31, f32, f33, f39,327,328,415,418,422 and 317 in the table 1) of 16 pairs of hepatocellular carcinomas that embodiment 1 obtains, employing two dimensional gel electrophore-sis method is screened differentially expressed protein wherein, two dimensional gel electrophore-sis is mainly by people's such as the Sanchez (Sanchez that improves one's methods, J.C.et al.Electrophoresis1997,18,324-327) carry out, specific as follows:
At first adopt the analytic type two dimensional gel electrophore-sis: 60 μ g protein examples and the Chong Pao liquid (8mol/L urea, 2%CHAPS, 0.5%IPG damping fluid, 18mmol/L DTT and trace bromophenol blue) that rises is mixed, cumulative volume 250 μ l, use 130 * 3 * 0.5mm pH3-10NL adhesive tape, carry out one to separation in IPGphore isoelectric focusing system, total voltage hour is about 80000Vhrs.Adhesive tape is successively at equilibrium liquid I (6M urea, 30% glycerine, 2%SDS, 1%DTT) and the middle balance of equilibrium liquid II (DTT replaces with 2.5%IAA among the equilibrium liquid I), each 15min after the isoelectric focusing.Adhesive tape is transferred to sodium dodecylsulphonate-polyacrylamide gel (SDS-PAGE, gum concentration 12%) upper limb, and deposition condition is 15mA/ glue 30min, and 30mA/ glue is retained to bromophenol blue from glue lower edge 0.5cm then.
Then, adopt silver to dye and make adhesive tape generate image, and with GS-710 image reading apparatus (Bio-Rad) transmission mode scanning glue image, resolution is 84.7 μ m/pixel.(Version7.0 Bio-Rad) analyzes with PDQuest software for the detection of point and coupling.The isoelectric point pI of protein spots and molecular weight Mr with 2-DE standard protein (Bio-Rad) as Marker, Input Software is used to analyze the pI and the Mr of other albumen.
Present embodiment prepares 10 couples of hepatocellular carcinoma patients' the cancerous tissue and the protein example of corresponding adjacent tissues altogether with non-enzymolysis sample preparation method, obtain the width of cloth surplus the 2-DE collection of illustrative plates 40 altogether, wherein the 5 pairs of samples repeat the differentially expressed analysis of spectrum of protein that 3 PDQuest softwares have carried out cancerous tissue and cancer beside organism, and analysis result is seen following table 2.
Table 2, PDQuest software analysis result (part)
Annotate: more in T=protein spots of up-regulated in liver cancer tissue;
More in N=protein spots of down-regulated expression in liver cancer tissue;
Only in T=detected protein spots in liver cancer tissue only;
Only in N=detected protein spots in the non-liver cancer tissue only;
Related coefficient between the T-T=tumour;
Related coefficient between the paired non-tumour of N-N=;
Related coefficient between T-N=tumor tissues and the paired nonneoplastic tissue.
On the pH3-10 adhesive tape, silver dyes that two class tissue samples show that all about 1200 silver dye a little under the condition.To dye a matching rate be 0.75～0.86 to silver between cancerous tissue, and to dye a matching rate be 0.60～0.76 to silver between cancerous tissue and cancer beside organism, and the science of two class tissue sample sampling methods is described to a certain extent.
After the atlas analysis, present embodiment adopts preparation type two dimensional gel electrophore-sis again, and applied sample amount is 1.5mg, and total Vhrs is about 90000, and employing can detect with the Kao Masi light blue method of mass spectrum compatibility, and other is identical with analytic type two dimensional gel electrophore-sis method.
Enzymolysis and mass spectrum qualification process are as follows in the ensuing glue: protein spots is by manual cutting on the preparative electrophoresis gel of the Kao Masi light blue dyeing of mass spectrum compatibility, at 100mM NH
4HCO
3, decolour vacuum freeze drying, 5 μ l50mmol/L NH in 30% acetonitrile
4HCO
3(pH8.3, protein: trypsase=1:5, w/w) in 4 ℃ place 2hr, add 20 μ l50mmol/L NH
4HCO
3(pH8.3), 37 ℃ of enzymolysis spend the night.Extracting albumen (60% acetonitrile, 0.1% trifluoroacetic acid), vacuum freeze drying.(Karlsruhe Germany) identifies the good sample of enzymolysis, MASCOT research tool (http://www.matrix science.com with Bruker Reflex III MALDI-TOF mass spectrum; Matrix Science, London UK) carries out database search.
Use zymolysis technique, mass-spectrometric technique in two dimensional gel electrophore-sis technology, the glue, present embodiment has identified 116 differential points, corresponding 102 kinds of differentially expressed protein.Wherein, high expressed or what only express therein is 61 differential points, corresponding 54 kinds of protein in the hepatocellular carcinoma cancerous tissue; High expressed or what only express therein is 55 differential points, corresponding 48 kinds of protein in the hepatocellular carcinoma cancer beside organism.
In the differential expression spectrum, point SSP2307 (Fig. 1) expresses in cancerous tissue obviously and raises, enzymolysis in point of contact, glue, point SSP2307 identifies with the MALDI-TOF mass spectrum and database search get 5 non-repetition peptide sections conform to thioredoxin protein 2 and satisfy MASCOT give a mark condition (single isotopic peak search library standard: the MASCOT score is greater than 63, p＜0.05): score 67, p=0.019; The sour coverage rate of base is 20%, and concrete outcome sees following table 3 for details:
The mass spectrum qualification result of table 3, some SSP2307
| The sequence start stop bit | Peptide section sequence |
| 115-136 | HASSGSFLPSANEHLKEDLNLR |
| 172-188 | HNIQFSSFDIFSDEEVR |
| 172-192 | HNIQFSSFDIFSDEEVRQGLK |
| 295-308 | AYSNWPTYPQLYVK |
| 320-332 | ELKENGELLPILR |
And, according to of the analysis of PDQuest software to existing 2-DE collection of illustrative plates, by the expression of comparison point SSP2307 in the 2-DE collection of illustrative plates of different hepatocellular carcinoma patients' cancerous tissue and cancer beside organism, find all to be high expressed (table 4) in cancerous tissue in the following 6 routine different cases of SSP2307:
Table 4, the repetition situation of the some up-regulated expression of SSP2307 in cancerous tissue in 6 routine different cases
| SSPNo. | Case 429 (by cancer/cancer) | Case 327 | Case 328 | Case 415 | Case 418 | Case average proportions (by cancer/cancer) 422 ± STDEV |
| SSP2307 | 3 | 2.1 | 2.6 | 3.4 | 2.3 | 3.62.8±0.6 |
Thereby the 2-DE atlas analysis shows: the up-regulated expression of some SSP2307 in cancerous tissue obtains good repetition (60%) in the 2-DE collection of illustrative plates of different hepatocellular carcinoma patients' cancerous tissue and cancer beside organism
Embodiment 3, the thioredoxin protein 2 differential expression Western blotting checking
For confirming the differential expression of thioredoxin protein 2, get 10 hepatocellular carcinoma patients' cancerous tissue and corresponding adjacent tissues protein example (in the table 1 317,327,42,45,48,49,415,418,422 and 424), carry out immunoblotting assay with the anti-thioredoxin protein 2 antibody of buying, detailed process is summarized as follows: each sample is got 20 μ g protein examples and is separated with 12%SDS-PAGE, be transferred on the pvdf membrane (available from Amersham Biosciences company), the one anti-anti-Trx's sample of chicken albumen 2 polyclonal antibodies that use are (available from GenWay Biotech, Inc. company, 1:500), 4 ℃ of overnight incubation, (every liter contains Tris2.42g with TBST, sodium chloride 8g, Tween20ml, regulate pH to 7.6 with HCI) wash three times, each 5 minutes, two anti-for anti-chicken antibody (available from Santa Cruz company, 1:10000), incubated at room 1 hour, again with TBST washing three times, each 10 minutes, use ECL plus reagent (Amersham Biosciences) reaction after 5 minutes at last, with X-mating plate exposure tests, testing result as shown in Figure 2.
The Western blotting result of Fig. 2 shows, substantially all present such phenomenon in the 10 pairs of cancerous tissues and the cancer beside organism: the concentration of the hybridization band of thioredoxin protein 2 is all apparently higher than corresponding cancer beside organism in the cancerous tissue; As seen there is high expressed in thioredoxin protein 2 in the cancerous tissue of hepatocellular carcinoma, and this result is consistent with the two dimensional gel electrophore-sis result.
In sum, thioredoxin protein 2 there are differences expression in the cancerous tissue of hepatocellular carcinoma and cancer beside organism, generation development obvious and hepatocellular carcinoma has close correlativity, therefore, its expression is detected as a protein molecular marker with thioredoxin protein 2 and can be used to detect hepatocellular carcinoma.Accordingly, the antibody of the anti-thioredoxin protein 2 of specificity, the monoclonal antibody and the polyclonal antibody that comprise various anti-thioredoxin protein 2s, because it can be used in the expression that detects thioredoxin protein 2, thereby can be used to detect liver cancer, perhaps be used to prepare the preparation that detects liver cancer or kit etc., this is conspicuous for a person skilled in the art.
Though dynamic biological function of relevant thioredoxin protein 2 and tumour related mechanism are still waiting further research, but be sure as the label that detects liver cancer with it.Thioredoxin protein 2 can be used as the potential sign of hepatocellular carcinoma, and its biological function prompting thioredoxin protein 2 in born of the same parents may be as the prognosis molecule mark of liver cancer and the target molecule of clinical treatment.
Claims (7)
1. a thioredoxin protein 2 is as the application of the protein molecular marker that detects liver cancer.
2. application as claimed in claim 1 is characterized in that, described is to detect the expression of this albumen in the liver cell tissue as the protein molecular marker that detects liver cancer.
3. application as claimed in claim 2 is characterized in that, the expression of this albumen of described detection in the liver cell tissue is to detect this albumen whether to have up-regulated expression in the liver cell tissue.
4. the application of the antibody of an anti-thioredoxin protein 2 is characterized in that, is used to prepare the preparation that detects liver cancer.
5. application as claimed in claim 4 is characterized in that the antibody of described anti-thioredoxin protein 2 comprises monoclonal antibody and polyclonal antibody.
6. the application of the antibody of an anti-thioredoxin protein 2 is characterized in that, is used to prepare the kit that detects liver cancer.
7. application as claimed in claim 6 is characterized in that the antibody of described anti-thioredoxin protein 2 comprises monoclonal antibody and polyclonal antibody.
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| PL394194A1 (en) | 2011-03-14 | 2011-09-12 | Read-Gene Spółka Akcyjna | Selenoprotein genotypes and selenium levels in organisms and the natural diet as the key risk factors for cancers in humans |
| CN102586202A (en) * | 2012-03-13 | 2012-07-18 | 中国科学院海洋研究所 | Thioredoxin and preparation method and application thereof |
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| Title |
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| Sadek CM et.al.Characterization of human thioredoxin-like 2. A novel microtubule-binding thioredoxin expressed predominantly in the cilia of lung airway epithelium and spermatid manchette and axoneme..《The Journal of Biological Chemistry》.2003,第278卷(第15期),13133-13142. * |
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