CN109596829A - A kind of liver cancer marker protein and its detection method - Google Patents
A kind of liver cancer marker protein and its detection method Download PDFInfo
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Abstract
The present invention provides a kind of liver cancer marker protein and its detection methods, and the 14-3-3 η albumen that specifically the present inventor is surprised to find that in the course of the research in liver cancer patient blood serum significantly increases, therefore can be used as the significant albumen of liver cancer.The blood plasma detection of 14-3-3 η has important value as potential liver cancer early detection, tumor prognosis prediction.The present invention also provides the methods of 14-3-3 η in detection blood plasma can be detected to the 14-3-3 η in blood plasma using method of the invention, significant for studying in laboratory research and clinic.
Description
Technical field
The invention belongs to fields of biomedicine, specifically, the present invention relates to a kind of liver cancer marker protein and its detection sides
Method.
Background technique
Tumour (tumor, neoplasm) refers to that cell loses the normal regulation grown to it under the effect of tumorigenesis factor, leads
The disease for causing paraplasm and generating.Tumour can be divided into benign and malignant tumour two major classes.The former slow growth, with surrounding tissue
Boundary clear does not shift, little to human health damage.The latter's growth rapidly, can be transferred to other body parts, can also
Harmful substance is generated, normal organ structure is destroyed, so that body function is lacked of proper care, life-threatening.
It is to endanger a kind of disease of human health most serious at present that malignant tumour, which is also cancer (cancer),.In the U.S., dislike
The death rate of property tumour is only second to cardiovascular disease and occupies second.Liver cancer (liver cancer) refers to the evil for betiding liver
Property tumour, the death rate are higher.The early symptom and sign of primary carcinoma of liver are unobvious or lack characteristic, and whens some onsets shows liver
Area's distending pain, the performance colic also having, or showed with transfer stove symptom to be earliest.Because its grade of malignancy is high, disease progression is fast, patient
What discomfort typically no in early days, goes to a doctor once there is symptom, has often belonged to middle and advanced stage.Therefore, the early diagnosis of liver cancer is in liver
There is huge meaning in the treatment of cancer.In addition, directly influencing subsequent clinic to the judgement of prognosis in the treatment of liver cancer
Therapeutic scheme, but be still difficult to judge the prognosis of liver cancer in the art.
Summary of the invention
The purpose of the present invention is to provide a kind of liver cancer marker protein and its detection methods.
In the first aspect of the present invention, a kind of purposes of the detection reagent of 14-3-3 η is provided, detection reagent is used to prepare
Box, the detection kit are used to detect the content of 14-3-3 η in body fluid sample.
In another preferred example, the body fluid sample is blood sample, hydrothorax sample, CSF sample, ascites sample.
In another preferred example, the kit is also used to diagnosing liver cancer or judges the prognosis situation of liver cancer treatment.
In another preferred example, the kit is also used to judge the Sorafenib resistance of liver cancer.
In another preferred example, the detection reagent includes the antibody of anti-14-3-3 η.
The second aspect of the present invention provides a kind of method for detecting 14-3-3 η, the method includes the steps:
(1) sample to be detected is provided, contains 14-3-3 η in the sample, and the samples sources are in body fluid；
(2) level of 14-3-3 η in the sample to be detected is detected.
In another preferred example, the body fluid is blood sample, hydrothorax, cerebrospinal fluid, ascites.
In another preferred example, the sample to be detected is plasma sample or serum sample.
In another preferred example, in the step (2) comprising steps of
(2.1) high-abundance proteins in sample are removed, low-abundance protein sample is obtained；
(2.2) level of 14-3-3 η in the low-abundance protein sample is detected.
In another preferred example, the high-abundance proteins include albumin and IgG.
In another preferred example, the high-abundance proteins further include antitrypsin, IgA, transferrins, haptoglobin,
Fibrinogen, alpha2-macroglobulin, α 1- acidoglycoprotein, IgM, apolipoprotein AI, apolipoprotein aii, Complement C_3, He Jiazhuan
Parathyrine transporter.
In another preferred example, the method is non-diagnostic purpose.
It in another preferred example, further include before low-abundance protein sample volume is concentrated into processing in the step (2.1)
The 5%-20% of volume；Preferably 10%.
In another preferred example, detection method is Western blot (Western blotting) in the step (2.2).
In another preferred example, the horizontal method of 14-3-3 η in the low-abundance protein is detected in the step (2.2)
For Western blot (Western blotting), using 15% separation gel, separation gel component is as follows: every aqueous 2.3ml of 10ml,
30% acrylamide 5.0ml, 1.5mol/lTris buffer 2.5ml, 10%SDS0.1ml, urea 6.4g, TEMED4 μ l, 10%
(w/v)AP100μl；Preferably, the pvdf membrane in the aperture 0.2um is selected when transferring film；It is highly preferred that the transferring film time is 25min-
30min。
In another preferred example, in the step (2.2) comprising steps of
1) encapsulating
1. distilled water cleans encapsulating glass plate, dry vertically；
2. preparing 15% separation gel 10ml, separation gel component is as follows: 10ml aqueous 2.3ml, 30% acrylamide 5.0ml,
1.5MTris buffer 2.5ml, 10%SDS0.1ml, urea 6.4g, TEMED4 μ l, 10% (w/v) AP100 μ l；It is stood after mixing
That is encapsulating is closed liquid level with isopropanol, is stored at room temperature；
3. after glue to be separated polymerize completely, the deionized water inclined at the top of it is blotted water with filter paper；
4. preparing 5% concentration glue 6ml, concentration glue component is as follows: water 4ml, 30% acrylamide 1ml, 1.0M Tris buffering
Liquid 1ml, 10%SDS80ul, 10%AP60ul, TEMED8ul；Encapsulating immediately is mixed, is filled to top, and be inserted perpendicularly into Teflon
Stripping fork is stored at room temperature；
5. extracting comb after glue polymerize completely, gel is placed in electrophoresis tank, electrophoretic buffer is added, uses running buffer
Liquid rinses loading hole to remove bubble removing；
2) electrophoresis
1. taking low-abundance protein sample, sample solution is prepared；
2. sample solution is boiled 5min in 100 DEG C of boiling water, it is quenched on ice, 3000 turns/min is centrifuged 1min；
3. every hole adds sample liquid 10ul；Electrophoretic buffer is filled it up with, slot cover is covered, is powered on, 70v constant pressure electrophoresis is first used
30min uses 90v constant pressure electrophoresis instead after indicator bromine Finland enters separation gel；
3) albumen and immune detection are transferred
1. pvdf membrane is impregnated 15s in methyl alcohol in advance, then uses ddH before electrophoresis closes to an end2O rinses 2min, leaching
It steeps and starts subsequent operation after 5min in transfering buffering liquid；
2. prizing glue in water, repairs glue to be soaked in transfering buffering liquid after glue and balance 15min；
3. preparing transferring film " sandwich " in transfering buffering liquid；
4. connecting positive and negative anodes, transfer box is put into electroporation, transferring film buffer is added；
5. electroporation is placed in ice water, 200mA constant current transferring film 70min；
6. after transferring film, taking out pvdf membrane, it is put into 5%BSA room temperature closing 2h；
7. film is taken out, in washing film 5min with TBST on shaking table, totally 3 times；
8. the diluted anti-14-3-3 η antibody of TBST is added in incubation bags, 4 DEG C of overnight incubations；
9. TBST washes film 5min, totally 3 times, the secondary antibody that horseradish peroxidase (HRP) label is added is incubated at room temperature 2h；
10. TBST washes film 10min, and totally 3 times；Film reacts 2min with chemiluminescence detection reagent, takes out film, it is extra to get rid of
Liquid wraps pvdf membrane with preservative film, uses X light reaching the film, development, fixing in darkroom.
The third aspect of the present invention provides a kind of method for early diagnosing liver cancer or predicting prognosis in hcc, and the method for stating includes
Step:
(1) sample to be detected is provided, contains 14-3-3 η in the sample, and the samples sources are in body fluid；
(2) level of 14-3-3 η in the sample to be detected is detected；
(3) level of the 14-3-3 η in the sample to be detected in the horizontal and normal sample of 14-3-3 η.If
The level of 14-3-3 η is significantly higher than the level of the 14-3-3 η in the normal sample in the sample to be detected, then explanation should be to
It is poor from the prognosis of liver cancer patient or the liver cancer patient to detect sample.
In another preferred example, described to be diagnosed as complementary diagnosis.
In another preferred example, the level of 14-3-3 η in the sample to be detected is detected in the step of the method (2)
Method is a kind of method for detection 14-3-3 η that the second aspect of the present invention provides.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows the electrophoresis result of WB detection 14-3-3 η.
Fig. 2 shows the electrophoresis result of WB detection 14-3-3 η in control experiment.
Specific embodiment
The 14-3-3 η albumen that the present inventor is surprised to find that in the course of the research in liver cancer patient blood serum significantly increases, because
This can be used as the significant albumen of liver cancer.The blood plasma detection of 14-3-3 η is pre- as potential liver cancer early detection, tumor prognosis
Measuring tool has important value.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this
Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and
And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention
The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated
" about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method
And material, place enumerates preferred method and material herein.
14-3-3 is a kind of phosphoserine/threonine binding protein family, including α/β, γ, ε, σ, ξ, θ/τ and η seven
Hypotype.Existing research shows: 5 kinds of 14-3-3 protein subunits (α/β, γ, ε, σ and ξ) take part in liver cancer process.The present inventor's research
It was found that: compared to hypotype has been reported, 14-3-3 η exists in the tumour cell and vascular endothelial cell in liver cancer tissue to be divided
Cloth, and its expression gradually rises with liver cancer progress.Be further discovered that: 14-3-3 η is equal in liver cancer and vascular endothelial cell
It can be by forming regenerative feedback loop with p-ERK1/2, and then promote the process of liver cancer, and the high table of liver cancer patient tissue 14-3-3 η
Up to Sorafenib resist and prognosis mala it is related.
The amino acid sequence of 14-3-3 η is as follows:
But the 14-3-3 η detection in liver cancer patient tumor tissues is extremely inconvenient, the present inventor attempts to examine in serum/slurry
The level of 14-3-3 η is surveyed, but since 14-3-3 η molecular weight is smaller, about 28KD, close to the light chain size of albumen, therefore
The difference of 14-3-3 η level in Serum of Cancer Patients/slurry sample and normal human serum/slurry sample can not be observed in testing result
It is different.
The present inventor further removes high-abundance proteins in plasma/serum (such as IgG, IgM, IgA, albumin, α 1- acid
Property glycoprotein etc.) after, 14-3-3 η is detected in serum/slurry sample by the immunoblotting analysis detection method success of improvement, and
And have been surprisingly found that 14-3-3 η level has significant difference in Serum of Cancer Patients/slurry sample and normal human serum/slurry sample.Into
One step, it is compared and analyzed by large sample hepatocarcinoma patient plasma sample and human normal plasma's sample, finds 14-3- in blood plasma
3 η can be used as hepatocarcinoma mark object, can be used for potential liver cancer early detection, tumor prognosis prediction etc..
Although can be relatively easy to detect 14-3-3 η in liver cancer tissue, can not be immunized always in this field
Imprinting method detects 14-3-3 η in serum, therefore before making the present invention it is generally acknowledged that in tumor patient and normal human serum
14-3-3 η level is without significant difference.The present inventor has found under study for action, in routine serum sample containing a large amount of IGg, IGm,
IGa etc., 14-3-3- η content are few and cover in the background of foreign protein, can not be detected and be obtained by immunoblotting analysis.
It tests by deep analysis and repeatedly, the present inventor carries out sample processing method and Western-blotting method
It improves, is finally obtained the technical solution that can detecte 14-3-3 η in blood plasma.It, can be in blood plasma using method of the invention
14-3-3 η detected, in laboratory research 14-3-3 η and clinic study 14-3-3 η it is significant.Than
It such as, can be using the plasma content of method detection 14-3-3 η of the invention in the drug development using 14-3-3 η as target spot.
The present inventor has found under study for action, only removes the high-abundance proteins in serum sample, then carries out at concentration again
Reason, in conjunction with the improved immunoblotting analysis detection method of the present inventor, just it is observed that purpose band.It is needed in immunoblotting analysis detection
Resolving gel concentration can be mentioned 15% using high-crosslinking-degree SDS- urea gel system using the lesser separation gel in aperture；Electrophoresis
When pay close attention to 28Kd similar in albumen Marker position, electrophoretic band arrive separation gel middle position when stopping electrophoresis；
The pvdf membrane that the aperture 0.2um is selected when transferring film, reduces the probability for penetrating film；Shorten the transferring film time, controls in 25min-30min.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002)
Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
Embodiment 1
The Gao Feng in MARSHuman-14 kit (being purchased from agilent company) removal blood sample is used in the present embodiment
Albumen is spent, low-abundance protein is obtained, the specific steps are as follows:
1, plasma/serum high-abundance proteins are removed
1) fresh collection EDTA anticoagulated whole blood, 3000g room temperature are centrifuged 5min, collect supernatant；200ul blood plasma is taken, is added
Buffer A carries out 4 times of dilutions；
2) Filter column of 0.22um is added in the blood plasma after diluting, and 16000g is centrifuged 1min, removes precipitated impurities；
3) purification column is assembled, buffer A is according to 1ml/min speed, pre-activate purification column 4min at room temperature；
4) blood plasma after 800ul dilution is added in purification column, carries out column purification according to the flow velocity of 0.5ml/min；
5) liquid for collecting 10.5-14.5min outflow obtains low-abundance protein, and -20 DEG C save immediately, avoid freezing repeatedly
Melt.
6) buffer B washs purification column 7.5min according to 3ml/min speed；
7) buffer A 3ml/min speed washs purification column 10min, balances purification column, and 2-8 DEG C of preservation purification column pays attention to
Buffer A cannot be killed.
8) albumen after purification is carried out being concentrated into 20ul using freeze drier (Thermo company, MicroModulyo)
Left and right, -80 DEG C of preservations.
2, WB (Western blotting) detects 14-3-3 η
1) encapsulating
1. distilled water cleans encapsulating glass plate, dry vertically.
2. preparing 15% separation gel 10ml according to the above method: water 2.3ml, 30% acrylamide 5.0ml, 1.5MTris buffering
Encapsulating immediately after liquid 2.5ml, 10%SDS0.1ml, urea 6.4g, TEMED4 μ l, 10% (w/v) AP100 μ l are mixed, fills to tooth
It combs 2~3mm of lower edge (marking in advance), with isopropanol closing liquid level to remove bubble removing and completely cut off air, is stored at room temperature 45 points
Clock waits for that glue polymerize completely.
3. after glue to be separated polymerize completely, the deionized water inclined at the top of it is blotted water with filter paper.
4. 5% concentration glue 6ml: water 4ml is prepared, 30% acrylamide 1ml, 1.0M Tris buffer 1ml, 10%
SDS80ul, 10%AP60ul, TEMED8ul mix encapsulating immediately, fill to top, and be inserted perpendicularly into Teflon stripping fork, room temperature is quiet
It sets 20 minutes and polymerize to glue.
5. extracting comb after glue polymerize completely, gel is placed in electrophoresis tank, electrophoretic buffer is added, uses running buffer
Liquid rinses loading hole to remove bubble removing.
2) electrophoresis
1. taking each processing histone extracting solution, protein concentration and the mixing of isometric 5 × sample-loading buffer are adjusted, as
Sample solution.
2. sample solution is boiled 5min in 100 DEG C of boiling water, makes albuminous degeneration, be quenched on ice, 3000 turns/min centrifugation
1min。
3. every hole adds sample liquid 10ul, a hole is stayed to add the Marker of 10 μ l pre-dyed.Electrophoretic buffer is filled it up with, slot cover is covered,
Powering on, first uses 70v constant pressure electrophoresis, about 30min uses 90v constant pressure electrophoresis instead after indicator bromine Finland enters separation gel, when
Stop electrophoresis when its electrophoretic band of 25Kd Marker is to separation gel middle position, close power supply, takes out offset plate.
3) albumen and immune detection are transferred
1. pvdf membrane is impregnated 15s in methyl alcohol in advance, then uses ddH before electrophoresis closes to an end2O rinses 2min, leaching
It steeps and starts subsequent operation after 5min in transfering buffering liquid.
2. prizing glue in water, repairs glue to be soaked in transfering buffering liquid after glue and balance 15min.
3. pressing black flour (cathode) → sponge → filter paper → glue → pvdf membrane (0.2um) → filter paper → sponge → red face (anode)
Sequence prepare transferring film " sandwich ", every layer complete after first drive bubble away and repave another layer.Sanming City is prepared in transfering buffering liquid
Control the generation of avoidable bubble.
4. connecting positive and negative anodes, transfer box is put into electroporation to the direction of anode by film, transferring film buffer is added.
5. electroporation is placed in ice water, 200mA constant current transferring film 70min.
6. after transferring film, quickly removing pvdf membrane, it is put into 5%BSA room temperature closing 2h.
7. film is taken out, in washing film 5min × 3 time with TBST on shaking table.
8. the diluted anti-14-3-3 η antibody (CST company, article No. 9640) of TBST is added in incubation bags, 4 DEG C of overnight incubations.
9. TBST washes film 5min × 3 time, the goat-anti rabbit secondary antibody of horseradish peroxidase (HRP) label (Jackson1:
2000) it is incubated at room temperature 2h.
10. TBST washes film 10min × 3 time.Film reacts 2min in chemiluminescence detection reagent (reagent A: reagent B=1:1),
Film is taken out, extra liquid is got rid of, pvdf membrane is wrapped with preservative film, uses X light reaching the film, development, fixing in darkroom.
For experimental result as shown in Figure 1, in Fig. 1, swimming lane 1,3,5,7 is that In Sera of Patients With Hepatocarcinoma sample removal high-abundance proteins are pure
Immunoblot results after changing concentration have obvious purpose band to occur at about 28kd；Swimming lane 2,4,6,8 is normal human serum
Sample removes the immunoblot results after high-abundance proteins purifying concentration, and purpose band can also be observed at 28kd；On albumen
Sample amount is 50ug.
Fig. 2 is the testing result of control experiment, wherein swimming lane 1,2,5,6 is the hepatocarcinoma patient for not removing high-abundance proteins
The testing result of plasma sample can not observe the purpose band at 28kd；Swimming lane 3,7 is not to be concentrated after removing high-abundance proteins
In Sera of Patients With Hepatocarcinoma sample testing result, can not equally observe at 28kd see purpose band；Swimming lane 4,8 is liver cancer disease
The check sample of human liver cancer tissue, it can be observed that apparent 28kd purpose band.Arrow pointed location is purpose band place
Position.Immunoblotting analysis experimental procedure is same as above, protein content 50ug.
3, result statisticallys analyze
After film progress background adjustment will be scanned, purpose band is carried out by gray value using Gel-Pro analyzer software
It counts, obtains the gray value of each sample purpose band.Using excel T-test function, double tails, double sample Singular variance is analyzed,
Experimental group and control group expression quantity have differences significant (P=0.017310345), and statistic analysis result is as shown in table 1.
Table 1
Statistic analysis result surface, the 14-3-3 η albumen in liver cancer patient blood serum significantly increases, therefore can be used as liver cancer
Significant albumen.The blood plasma detection of 14-3-3 η has important valence as potential liver cancer early detection, tumor prognosis prediction
Value.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
- The purposes of the detection reagent of 1.14-3-3 η, which is characterized in that be used to prepare detection kit, the detection kit is used The content of 14-3-3 η in detection body fluid sample.
- 2. purposes as described in claim 1, which is characterized in that the body fluid sample is blood sample, hydrothorax sample, cerebrospinal fluid Sample, ascites sample.
- 3. purposes as described in claim 1, which is characterized in that the kit is also used to diagnosing liver cancer or judges that liver cancer is controlled The prognosis situation for the treatment of.
- 4. a kind of method for detecting 14-3-3 η, which is characterized in that the method includes the steps:(1) sample to be detected is provided, contains 14-3-3 η in the sample, and the samples sources are in body fluid；(2) level of 14-3-3 η in the sample to be detected is detected.
- 5. method as claimed in claim 4, which is characterized in that the body fluid is blood sample, hydrothorax, cerebrospinal fluid, ascites.
- 6. method as claimed in claim 4, which is characterized in that in the step (2) comprising steps of(2.1) high-abundance proteins in sample are removed, low-abundance protein sample is obtained；(2.2) level of 14-3-3 η in the low-abundance protein sample is detected.
- 7. method as claimed in claim 6, which is characterized in that the high-abundance proteins include albumin and IgG.
- 8. method as claimed in claim 6, which is characterized in that the high-abundance proteins further include antitrypsin, IgA, turn Ferritin, haptoglobin, fibrinogen, alpha2-macroglobulin, α 1- acidoglycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, Complement C_3 and transthyretin.
- 9. method as claimed in claim 4, which is characterized in that further include by low-abundance protein sample in the step (2.1) Volume concentration extremely handles the 5%-20% of front volume；Preferably 10%.
- 10. a kind of early diagnosis liver cancer or the method for predicting prognosis in hcc, which is characterized in that the method includes the steps:(1) sample to be detected is provided, contains 14-3-3 η in the sample, and the samples sources are in body fluid；(2) level of 14-3-3 η in the sample to be detected is detected；(3) level of the 14-3-3 η in the sample to be detected in the horizontal and normal sample of 14-3-3 η.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101334410A (en) * | 2007-06-26 | 2008-12-31 | 沙桂华 | Method and reagent kit for detecting endometriosis |
| CN101952313A (en) * | 2007-11-27 | 2011-01-19 | 不列颠哥伦比亚大学 | 14-3-3 eta antibodies and uses thereof for the diagnosis and treatment of arthritis |
| CN102183661A (en) * | 2011-03-16 | 2011-09-14 | 天津宝瑞生物技术有限公司 | Application of protein and proteome in preparation of liver cirrhosis diagnostic reagent |
| WO2012112156A2 (en) * | 2011-02-15 | 2012-08-23 | The Board Of Regents Of The University Of Oklahoma | Compositions useful for cancer detection and treatment, a cancer stem cell model, and methods of production and use thereof |
| WO2013190075A2 (en) * | 2012-06-20 | 2013-12-27 | Meyer Helmut E | Specific biomarkers for hepatocellular carcinoma (hcc) |
| US20160320395A1 (en) * | 2013-11-13 | 2016-11-03 | Electrophoretics Limited | Materials and methods for diagnosis and prognosis of liver cancer |
-
2017
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101334410A (en) * | 2007-06-26 | 2008-12-31 | 沙桂华 | Method and reagent kit for detecting endometriosis |
| CN101952313A (en) * | 2007-11-27 | 2011-01-19 | 不列颠哥伦比亚大学 | 14-3-3 eta antibodies and uses thereof for the diagnosis and treatment of arthritis |
| WO2012112156A2 (en) * | 2011-02-15 | 2012-08-23 | The Board Of Regents Of The University Of Oklahoma | Compositions useful for cancer detection and treatment, a cancer stem cell model, and methods of production and use thereof |
| CN102183661A (en) * | 2011-03-16 | 2011-09-14 | 天津宝瑞生物技术有限公司 | Application of protein and proteome in preparation of liver cirrhosis diagnostic reagent |
| WO2013190075A2 (en) * | 2012-06-20 | 2013-12-27 | Meyer Helmut E | Specific biomarkers for hepatocellular carcinoma (hcc) |
| US20160320395A1 (en) * | 2013-11-13 | 2016-11-03 | Electrophoretics Limited | Materials and methods for diagnosis and prognosis of liver cancer |
Non-Patent Citations (6)
| Title |
|---|
| SHEN J等: "14-3-3η is a novel growth-promoting and angiogenic factor in hepatocellular carcinoma", 《JOURNAL OF HEPATOLOGY》 * |
| 刘娟等: "肝癌组织中14-3-3基因差异表达的意义", 《世界华人消化杂志》 * |
| 刘桂芬: "14-3-3蛋白亚型在人肝癌组织中的表达及意义", 《中国优秀硕士学位论文全文数据库医药科技卫生辑》 * |
| 汪谦: "《现代医学实验方法》", 31 August 1997, 人民卫生出版社 * |
| 肖忠华: "肝细胞癌患者血浆中低丰度差异表达蛋白质组分析", 《中国优秀硕士学位论文全文数据库医药科技卫生辑》 * |
| 黄大鹏等: "《医用生物化学实验》", 31 October 1992, 中国医药科技出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114705794A (en) * | 2022-04-15 | 2022-07-05 | 西湖大学 | Proteomics analysis method for biological sample |
| CN114705794B (en) * | 2022-04-15 | 2022-12-02 | 西湖大学 | Proteomics analysis method for biological sample |
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