CN106908609B - Purposes of the tumour-specific trnaplantation antigen albumen TSTA3 in preparing oesophagus squama cancer diagnosis reagent - Google Patents
Purposes of the tumour-specific trnaplantation antigen albumen TSTA3 in preparing oesophagus squama cancer diagnosis reagent Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention belongs to biomedicine technical fields, provide a kind of purposes of tumour-specific trnaplantation antigen albumen TSTA3 in preparing oesophagus squama cancer diagnosis reagent, this purposes solves in the prior art the technical issues of molecular target of patients with esophageal squamous cell carcinoma diagnosis and prognosis information is considerably less, and judgment criteria differs.Immune scoring is carried out by TSTA3 protein expressions situation, the relationship with patient diagnosis and prognosis, the diagnosis for assisting patients with esophageal squamous cell carcinoma and prognostic evaluation are then determined by the expression quantity of TSTA3 albumen.The present invention assists the diagnosis of patients with esophageal squamous cell carcinoma by amynologic index TSTA3 expressing quantities and evaluates prognosis, and the present situation to solve patients with esophageal squamous cell carcinoma diagnosis and prognostic evaluation means shortage provides a kind of accurately and rapidly prediction technique.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of tumour-specific trnaplantation antigen albumen TSTA3 exists
Prepare the purposes in oesophagus squama cancer diagnosis reagent.
Background technology
The cancer of the esophagus is one of malignant tumour common in world wide, and China is that Incidence of esophageal cancer and the death rate are highest
Country, annual new cases 47.8 ten thousand, death 37.5 ten thousand, incidence and the death rate occupy the third of all kinds of malignant tumours respectively
Position and the 4th.China's Incidence of Esophageal Cancer is concentrated mainly on several districts occurred frequently, the Taihang Mountain that wherein Henan, Shanxi, Hebei have a common boundary
Area's incidence highest.Different from western countries, the histological type 90% of China's cancer of the esophagus is squamous cell carcinoma, Most patients
Middle and advanced stage is in when making a definite diagnosis, progression of the disease is fast, poor prognosis, 5 years survival rates only 10% or so.Esophageal carcinoma therapy is still with hand at present
Based on art combination chemicotherapy, but to being unable to the patient of early detection often without radical-ability operative chance, while to relapse and metastasis patient
The general curative effect of complex treatment is still unsatisfactory.Compared with other tumor types, esophageal squamous cell carcinoma (Esophageal squamous
Cell carcinoma, ESCC) early diagnosis marker and the target spot of targeted therapy very limit to, lacking can be for reference
International standard.Therefore, the occurrence and development mechanism for furtheing investigate the cancer of the esophagus, for the molecular marker and system of clinical discovery early diagnosis
Fixed effective clinical intervention measure seems most important.
One universals of malignant tumour are that significant change has occurred in the glycosylation modified of cell.Glycosylation often participates in
Folding, polymerization, maturation and the transport of protein peptide chain play an important role to cell and protein function adjusting aspect.Swollen
In the generation of tumor, evolution, the glycosylation modified of protein can change with disease process, to influence tumour cell
The processes such as adherency, invasion, transfer.The glycoprotein of tumor cell surface exception also exists frequently as the marker of tumour.Therefore,
Glycosylated abnormal change feature is for the early diagnosis of tumour, monitoring the process, prognosis evaluation in searching cancer of the esophagus Carcinogenesis
And the searching of therapy target can provide important information.
Abnormal fucosylation and tumor development are closely related：Fucosylation is glycoprotein, the modification of glycolipid oligosaccharides
In most common modification mode, GDP-L- fucoses are unique donors of fucosylation, synthesis remedy synthesis and from the beginning
Synthesize two kinds of approach（Fig. 1）：The former passes through L-fucose kinases and GDP-L- fucose pyrophosphorylations by free L-fucose
Two steps of enzyme catalyze and synthesize；The latter is that GDP-D mannoses are changed into GDP-L- fucoses by three steps of enzymatic reactions, respectively
Including -3,5 isomerase -4- reductases of GDP mannose -4,6- dehydratases (GMDS) and GDP-4- ketone -6-de-oxy-L-mannose
(TSTA3).Wherein, de novo synthesis has synthesized in human body 90% GDP-L- fucoses, becomes the conjunction of GDP-L- fucose biologies
At main source.Fucosylation also needs to participation and the fucosyltransferase (FUT) of GDP fucose transporters in cell
Catalysis.
Oneself warp of the relationship of abnormal fucosylation and tumour becomes the research hotspot of diagnosis of malignant tumor and treatment.Document report
The abnormal main mechanism close with relation between tumor of the fucosylation in road has：1. fucosido, which may participate in, constitutes certain important adherency
The sugar chain structure of molecule, it is in close relations with metastases.Composition as directly participated in Lewis antigenic determinants, Lewis antigens
It is the binding partner of selectin, the effect in metastases has gained public acceptance；In integrin and E-cadherin molecules
The reduction of core fucosylation and the scarce forfeiture for such as leading to its function, passing through influences cell-ECM and cell-ECM outer room
The interaction of matter causes the variation for invading transfer ability.2. being influenced by EGF-R ELISA (EGFR) fucosylation
The generation of tumour.EGFR has a potential N glycosylation sites, wherein more than half there are fucosylation modification, N glycosylations inhibit
Agent is substantially reduced the combination of EGF and EGFR, the affinity higher of the EGFR and EGF of core fucosylation.3. fucosylation
It is abnormal related to tumor immune escape.Moriwaki etc. has found that GMDS is mutated in colon cancer cell, leads to cell fucosylation
Level declines, to escape natural kill（NK）Tumor necrosin relative death inducing is resisted in cell-mediated immunosurveillance
The Apoptosis of ligand (TRAIL) induction；The presence of surface sLex promotes the killing of NK cells to live in Leukemia K562 cell
Property.There are many abnormal fucosylation indexs to be applied to clinical practice at present, such as：Cell surface fucosylation LeYOligosaccharides
It is a tumor associated antigen, is high expression in the breast cancer of many epithelial origins, oophoroma, liver cancer and intestinal cancer, currently, needle
To the monoclonal antibody of the antigen（IGN-311 and hu3s193）As the potential medicine of epithelial origin immunotherapy of tumors
Object, and enter II clinical trial phases；The alpha-fetoprotein of core fucosylation（AFP-L3）Liver cancer is had more compared to AFP
There is the diagnostic value of specificity；The haptoglobin of fucosylation（HPT）Level is in oophoroma, lung cancer, breast cancer and pancreas
It is increased in the malignant tumours such as cancer, becomes the molecular marker of kinds of tumors.
TSTA3 genes are also known as FX, P35B, and coding albumen is the key that GDP-L- fucose de novo formation rate-limiting enzymes, and
GDP-L- fucoses are unique donors of fucosylation（Fig. 1）.It is less that the gene studies report in tumour.Colorectal cancer,
There are the height expression of TSTA3 genes in the tumours such as hepatocellular carcinoma, breast cancer, compared with colorectal carcinoma cell line sw480 in situ,
High expression TSTA3 in metastatic cancer cell system sw620, the expression with cell SLea is positively correlated, and it is interior to enhance tumour-
Epithelial Cell Adhesion；The missing of fucosylation can lead to intestinal inflammatory in TSTA3 knock out mice bodies, and then cause galandular epithelium
The generation of dysplasia and gland cancer, the process are related to the downward of the inactivation and downstream Hes1 of Notch accesses；TSTA3 is in breast cancer
High expression in tissue is closely related with TNM stage and poor prognosis, can be as independent prognostic factor, in breast cancer
The knockout of TSTA3 can cause the isogenic notable downward of CXCR4, CXCL12, MMP9, related to the decline of cancer cell invasion ability；
And the comparative study in 419 crowds such as Hu finds that TSTA3 is deposited in the peripheral blood and cancerous tissue of early stage patients with lung adenocarcinoma
In increasing for expression quantity, it may be possible to the early diagnosis marker of non-small cell lung cancer.
TSTA3 genes may play critical oncogene effect in esophageal squamous cell carcinoma.Early period pair 14 and 90 ESCC are swollen
Tumor and full-length genome and full sequencing of extron group have been carried out respectively with normal tissue (I phase 51, III phase 53), based on list
It is found in the analysis of nucleotide diversity (SNVs) and small fragment insertion/deletion (Indels), fucose/mannose metabolic pathway exists
The frequency of mutation in I phase case was significantly higher than for III phase, and the early stage that the variation of the access may take part in the cancer of the esophagus is prompted to occur,
The rate-limiting enzyme of the middle TSTA3 gene codes access, the frequency of mutation 2% are missense mutation；Meanwhile based on copy number variation
(CNAs) analysis finds that the regions 8q24 where TSTA3 genes have the copy number significantly and repeated in ESCC in I phase case
Amplification.Part ESCC relevant group sequencing results are published in Cell periodical Am J Hum Genet（It is chose as " Best of
AJHG 2014 to 2015”）And on GigaScience magazines.
On this basis, Primary Study has been carried out to the function of TSTA3 genes.In two plants of oesophaguses of KYSE510 and KYSE150
The proliferation of low TSTA3 gene pairs cancer cell is struck in squamous cell carcinoma without influence, but can significantly inhibit the invasion and migration of cancer cell
Ability；On the contrary, the overexpression of TSTA3 genes can significantly increase the invasion migration of cell while not influencing cancer cell multiplication
Ability (p<0.05)。
To sum up, TSTA3 and esophageal squamous cell carcinoma are closely related：Based on esophageal squamous cell carcinoma and pairing cancer beside organism's full-length genome and outer aobvious
Subgroup sequencing finds the esophageal squamous cell carcinoma related gene that TSTA3 is candidate, and the albumen of its coding is logical for fucose/sweet dew glycometabolism
The rate-limiting enzyme on road, the frequency of mutation 2% are missense mutation；Genomics sequencing result finds that fucose/sweet dew glycometabolism is logical
The frequency of mutation of the road in I phase case was significantly higher than for III phase, prompted the variation of the access that may take part in the early stage hair of the cancer of the esophagus
It is raw, meanwhile, the analysis based on copy number variation (CNAs) is found, the regions 8q24 have the copy number significantly and repeated in ESCC
Amplification, region overlay TSTA3 genes.Prompt TSTA3 genes and cancer of the esophagus occurrence and development closely related.But TSTA3 with
There is not been reported for the relationship of esophageal squamous cell carcinoma occurrence and development, is also had not been reported with the relationship of patients with esophageal squamous cell carcinoma prognosis.The present invention is
It determinesTSTA3The relationship of expression and patients with esophageal squamous cell carcinoma clinical prognosis inquires into it and predicts esophageal squamous cell as molecular marker
The application value of cancer patient's prognosis.
Invention content
The present invention is directed to above-mentioned technical problem in the prior art, provides a kind of tumour-specific trnaplantation antigen albumen
Purposes of the TSTA3 in preparing oesophagus squama cancer diagnosis reagent, this purposes solve esophageal squamous cell carcinoma trouble in the prior art
The technical issues of person diagnoses and the molecular target of prognosis information is considerably less, and judgment criteria differs.
The present invention provides a kind of tumour-specific trnaplantation antigen albumen TSTA3 to prepare diagnosis and/or prognostic evaluation food
Purposes in pipe squamous cell carcinoma patients reagent.
The present invention carries out immune judge by TSTA3 protein expressions situation, is then determined by the expression quantity of TSTA3 albumen
With the relationship of patient diagnosis and prognosis, the diagnosis for assisting patients with esophageal squamous cell carcinoma and prognostic evaluation.
Compared to the prior art, the present invention assists esophageal squamous cell carcinoma to the present invention by amynologic index TSTA3 expressing quantities
The diagnosis of patient and prognosis is evaluated, the present situation to solve patients with esophageal squamous cell carcinoma diagnosis and prognostic evaluation means lack provides
A kind of accurately and rapidly prediction technique.
Description of the drawings
Fig. 1 is the rate-limiting enzyme action diagram of GDP-L- fucoses route of synthesis and TSTA3 albumen；
Fig. 2 is TSTA3 in ESCC tumor tissues and Carcinoma side normal tissue（104 primary carcinoma and 60 pairing cancer beside organisms）
In protein expression level figure, in figure, A:Immuning tissue chip figures of the TSTA3 in ESCC tissue and Carcinoma side normal tissue（×
40,100）；B:TSTA3 is in ESCC tumor tissues and Carcinoma side normal tissue（104 primary carcinoma and 60 cancer beside organisms）In egg
White expression difference scatter plot；C:Protein expressions of the TSTA3 in 60 ESCC tumor tissues and pairing Carcinoma side normal tissue
Level difference scatter plot；
Fig. 3 is that ROC curve screens TSTA3 expression threshold figure in ESCC tumor tissues；
Fig. 4 is to divide TSTA3 high expression and low expression proportion figure in ESCC tumours according to Fig. 3 threshold values；
Fig. 5 is TSTA3 high expression and low expression state in the total crowds of ESCC（A）, without history of drinking history (B), different sexes (C),
To the predicting function figure of prognosis in different location (D) ESCC patient；
Fig. 6 is TSTA3 high expression and low expression state in all ages and classes (A), different lymph node status (B), difference
To the predicting function figure of prognosis in invasive depth (C) ESCC patient.
Specific implementation mode
It is expanded on further below by specific embodiment and in conjunction with attached drawing to the present invention, but is not intended to limit the present invention.
104 esophageal squamous cell carcinomas used in the embodiment of the present invention（ESCC）Tissue is both from Shanxi Province Tumor Hospital（STH
Taiyuan, China）, surgical resection therapy is carried out for the first time, it is preoperative without neoadjuvant, chemotherapy or radiotherapy.104 pieces of primary foods
Pipe cancerous tissue and the normal esophageal epithelial tissue sample of wherein 60 pieces pairings are fabricated to organization chip and are used for subsequently through wax embedding
Immunohistochemical staining analysis.The clinical scale of ESCC is specified with reference to american cancer joint committee and International Union Against Cancer
7th edition TNM grade scale in 2010.This experiment has passed through Mountain Western Medicine S University and participates in hospital.The acquisition of all samples is equal
Sufferers themselves are passed through and family members agree to and endorsed informed consent form.
The expression quantity of TSTA3 albumen in cancerous tissue and cancer beside organism is assessed by Immunohistochemical Staining.Specific mark
This corresponding clinical case data such as tumor size, histological grade, lymphatic metastasis situation, clinical scale are shown in Table 1.
Table 1：ESCC patient clinical pathological datas
Used statistical analysis method is：TSTA3 expressions compare using order in ESCC and Carcinoma side normal tissue
And inspection.Utilize Receiver operating curve（Receiver operating characteristic curve, ROC are bent
Line）Find the best cut off value of the expression of TSTA3 albumen height and low expression.The correlation of the expression of TSTA3 and clinical case factor
Analysis chi-square test of four-fold table.Kaplan-Meier survival analysis and Log-rank check analysis TSTA3 expression status with not
With clinical pathological factors patient's prognosis existence correlation.Cox proportional hazards regression models carry out single factor test and multifactor existence point
Analysis, analysis TSTA3 express the effect in ESCC patient's prognosis prediction.All statistics and corresponding draw use SPSS18.0 softwares
It carries out.Total life span begins to dead day by operation or is finally calculated with visiting Japan.PValue is considered as having statistics meaning less than 0.05
Justice.
Used reagent, major experimental instrument and consumptive material are in the embodiment of the present invention：Resinene, 4% formal
Woods, Tween20：Biotopped Science and Technology Ltd.s；Paraffin：Shanghai Hua Yong paraffin Co., Ltd；Dimethylbenzene：Tianjin richness space
Fine Chemical Co., Ltd；Absolute ethyl alcohol：Tianjin great Mao chemical reagent factories；PH6.0 sodium citrates（Citrate repairs liquid）、
DAB colour reagent boxes：Fuzhou Maixin biotechnology Development Co., Ltd；Fast-type enzyme mark sheep antigen mouse/rabbit igg polymer：Good fortune
State steps neoformation technological development Co., Ltd；3% hydrogen peroxide：Dezhou peace prompt high-tech sterilized articles therefrom Co., Ltd；Haematoxylin dye liquor：
Beijing Suo Laibao Science and Technology Ltd；Organization chip array wax stone preparing instrument：Beijing Chloe Science and Technology Ltd.；Slicer：
Card is come, Germany；Full automatic digital pathology scanning device：The U.S., Aperio companies；Microscope：Card is come, Germany；Refrigerator：Haier,
China；Horizontal shaker：Its woods Bell, China；Micropipettor：Eppendorf, Germany；Electric heating constant-temperature blowing drying box：Shanghai
Zhi Cheng analytical instrument Manufacturing Co., Ltd,；Glass slide, coverslip：Citotest Labware Manufacturing Co., Ltd.；Wet box, staining rack：
Safe experiment equipment factory of Haimen City.
Reagent preparation is used in experiment：PBS phosphate buffers：NaCl（Shanghai Sheng Gong bioengineering Co., Ltd）
1.6g, Na2HPO4 .7H2O（Shanghai Sheng Gong bioengineering Co., Ltd）10.9g Na2HPO4（It is limited that work bioengineering is given birth in Shanghai
Company）5.78g NaH2PO4（Shanghai Sheng Gong bioengineering Co., Ltd）5.29, it is dissolved in 2000ml tri-distilled water mixings.
1xPBST：TWEEN：PBS=1：1000 configurations.
Embodiment 1：One kind is auxiliary by esophageal squamous cell carcinoma amynologic index tumour-specific trnaplantation antigen albumen TSTA3 expression quantity
The method for helping the diagnosis of patients with esophageal squamous cell carcinoma and being evaluated prognosis, immunohistochemical staining step are edited according to Liu Zenghui《Disease
Manage staining technique》（It publishes People's Health Publisher）It carries out.Organization chip is with 1：400 TSTA3 antibody（Anti-rabbit monoclonal is anti-
Body, ab190002, Abcam）It is incubated at 4 DEG C 14 hours, then uses DAB detection kits（Step new, Foochow, China）Processing.Through
Hematoxylin indigo plant shoots × 100 enlarged drawings after contaminating mounting.Using Full automatic digital pathology scanning device (Aperio,
America) and in image analysis software analysis tumor tissues and normal epithelial tissues cell cytosol staining power reflects TSTA3
The expression of albumen.
Specifically comprise the following steps：
（1）104 primary carcinoma of operation excision and 60 pairing cancer beside organism samples are subjected to paraffin embedding, wax is made
Block, the method for specifically preparing wax stone are：The tissue of operation excision is fixed with 10% neutral formalin cup, dehydration embedding, stone
Wax is sliced into 2 μm, is affixed on glass slide, and 65 DEG C make sample be fixed on slide in roasting piece 3-5 hours, cooling preservation；
（2）Dewaxing：Histotomy bakes piece 10min in air dry oven keeps sample paraffin melting, then immerses two respectively
Toluene 3 times, each 10min；Immerse absolute alcohol 2min, absolute alcohol 2min, 80% alcohol 2min, 70% alcohol respectively again
2min；
（3）The elimination of endogenous peroxydase：Slice after dewaxing and aquation is moved to the 3%H for being protected from light processing2O2In
20min reduces unspecific staining to eliminate peroxidase present in histocyte；
（3）It develops a film：Tri-distilled water is developed a film 5min, immerses PBS 3 times, each 3min；
（4）Antigen exposure and antigen retrieval：Histotomy immerses citrate buffer (the 6.46g sodium citrates of PH6.0
It is dissolved in 1800ml tri-distilled waters) hot high pressure 120 DEG C of 2min of reparation, naturally cool to 15-30 DEG C；1xPBST is handled 2min*3 times；
（5）Antibody incubation：Add TSTA3 primary antibodies（Rabbit-anti TSTA3,1：100）, it is desirable that primary antibody working solution all covers tissue,
And cannot be there are bubble, 4 DEG C of wet box are incubated overnight, and take out within second day and place room temperature, 1 hour 1 × PBST of rewarming washes 10min*3
（It is for use that water-bath enters 37 DEG C of casees）；Add secondary antibody（Goat-anti rabbit, 1：500）, tissues are all covered, and cannot be there are bubble, 37 DEG C of incubations
20min；PBST is washed 5min*3 times；
（6）DAB develops the color：1ml DAB substrate solutions are taken to be placed in being protected from light in EP pipes of 1.5ml with pipettor, under the conditions of being protected from light
A drop DAB liquid is added dropwise to manage in EP, mixes well.Draw appropriate colour developing working solution, rapid accurate drop is on histotomy, specific side
The same primary antibody of method, by micro- sem observation staining power, timely control time, after microscopic observation 3-5min cytoplasm becomes faint yellow,
Tap water color development stopping is reacted；
（7）Haematoxylin is redyed：Histotomy is moved on metallochromy frame, is placed in haematoxylin dyestuff liquid cylinder and redyes 1 point
Then histotomy is placed in terminate in tap water and dye by clock.It moves in the liquid cylinder for filling 70% acidic alcohol and carries out after taking-up
Color separation（Differentiation）5 seconds.Finally histotomy taking-up is moved in the liquid cylinder for fill ammonium hydroxide and carries out returning indigo plant（Lan Hua）.Under microscope
Intensity is redyed in observation.If it is low to redye intensity, it is continuing with 70% acidic alcohol and ammonium hydroxide differentiation and orchidization.
（8）Dehydration：Glass slide immerses 70% alcohol 5min, 90% alcohol 5min, absolute alcohol 10min, absolute alcohol respectively
10min, dimethylbenzene 10min, dimethylbenzene 10min, dimethylbenzene 10min；
（9）Resinene mounting：It after taking out histotomy, is put at well-ventilated, the histotomy that fully volatilizees is remaining
Dimethylbenzene.With neutral tree fat drips in organization center, slowly tile coverslip, and tissue is made to be closed completely by resinene, and does not stay
Bubble, slice are completely dried rear microscopic observation and preserve.
（10）Passed through to immunohistochemical staining using the pathology scanning system ScanScope AT of Aperio companies
Cytoplasm in tissue samples afterwards（TSTA3 is distributed in cytoplasm）The scanning recognition of the dyeing depth calculates H values, i.e. generation automatically
The expression of table TSTA3 albumen.
（11）Statistical analysis：TSTA3 expressions compare using rank sum test in ESCC and Carcinoma side normal tissue.It utilizes
Receiver operating curve（Receiver operating characteristic curve, ROC curve）It finds
TSTA3 albumen height is expressed and the best demarcation threshold of low expression, and all samples are expressed H values according to its TSTA3 to be grouped by threshold value,
It is high expression group higher than threshold value, is low expression group less than threshold value.The correlation point of the expression of TSTA3 and clinical case factor
Analysis chi-square test of four-fold table.Kaplan-Meier survival analysis and Log-rank check analyses TSTA3 and different clinical pathologies
Factor patient's prognosis existence correlation.Cox proportional hazards regression models carry out single factor test and multifactor survival analysis, analysis
TSTA3 expresses the effect in ESCC patient's prognosis prediction.All statistics and corresponding draw are carried out using SPSS18.0 softwares.Always
Life span begins to dead day by operation or is finally calculated with visiting Japan.PValue is considered as statistically significant less than 0.05.
Experimental result：
The TSTA3 eggs in 104 primary esophageal squamous cell carcinomas and 60 pairing cancer beside organisms are observed using ImmunohistochemistryMethods Methods
White expression.The results show that TSTA3 expression is in ESCC and the cell cytosol of normal structure（Fig. 2A）, TSTA3 is in cancerous tissue
Middle expression is apparently higher than non-cancer tissue.The H values in cancerous tissue are detected between 66.3158 to 297.3680, median is
92.67.H values in cancer beside organism are between 67 to 251.9230, median 64.88.TSTA3 protein levels are in ESCC cancers
Normal esophageal squamous epithelial tissue (P=0.00014, t=3.900, Fig. 2 B) is apparently higher than in tissue, pairing cancerous tissue and
This difference is same apparent (P=0.0002, t=3.916, Fig. 2 C) in cancer beside organism.
According to the ROC curve relationship (credibility interval AUC=0.703, P=0.00037,95%:0.600-0.818;Fig. 3),
With youden index（Youden index）=（Susceptibility+specificity -1）Corresponding point is excellent diagnostics dividing value when maximum（Boundary
Threshold value）.It chooses H values 195.2735 and is used as demarcation threshold, all cancerous tissue samples are divided into two groups：TSTA3low(H values≤
195.2735) and TSTA3high(H values>195.2735 ).Rank sum test result is shown in cancerous tissue and Carcinoma side normal tissue
The distribution that two groups of middle height there are notable difference, the significantly high expression of TSTA3 in ESCC cancerous tissues (P=0.0018, χ2=9.766；
Fig. 4).
Expression that table 2 is TSTA3 and its with both ESCC patient clinical pathological datas correlation, can from table 2
Go out, TSTA3 protein expression levels and patient age (P=0.017, χ 2=5.983), history of drinking history (P=0.007, χ2=9.817)、
Clinical stages (P=0.010, χ 2=6.996) and status of lymph node metastasis (P=0.043, χ 2=4.876) there is apparent correlation
Property, in older patient, clinical end-stage（III+IV）Patient has in patients with lymph node metastasis, and TSTA3 high expression ratio is big,
Show the malignancy higher of TSTA3 high expression, be easier to shift.TSTA3 protein expression levels with gender, group
The happening part for knitting credit, smoking history and the cancer of the esophagus is unrelated.Although being not statistically significant, TSTA3 protein expression levels
Related with ESCC invasive depth possibility (P=0.055, χ 2=4.131, T1+T2vs.T3；), show the tumour of TSTA3 high expression
Invasion transfer (table 2) more easily occurs.
Table 2：The expression of TSTA3 and its with both ESCC patient clinical pathological datas correlation
It can show that TSTA3 low expression patients overall survival's times are 34 months to 55 months from follow up data, it is 45 average
Month.TSTA3 high expression patients overall survival's times are 26 months to 41 months, 33 months average.The Kaplan-of Fig. 5 and Fig. 6
Meier analysis shows that in all ESCC patients patients overall survival's time of high expression TSTA3 be significantly inferior to low expression patient, in advance
Poor afterwards (P=0.048, Fig. 5 A), this trend in the patient that lesion is located at distal esophagus (P=0.038, Fig. 5 D), nothing is drunk
In the patient of history (P=0.048, Fig. 5 B) it equally exists.In male patient this trend become apparent (P=0.017, figure
5C).In addition, at the age more than in 60 years old patient, the patients overall survival of height expression TSTA3 be inferior to the time low expression patient (P=
0.059, Fig. 6 A), in lymph node positive patient, height expression TSTA3 patients overall survival be inferior to the time low expression patient (P=
0.064, Fig. 6 B), in invasive depth is T3 patient, the patients overall survival of height expression TSTA3 is inferior to high expression patient (P the times
=0.068, Fig. 6 C), although not statistically signigicant, there is apparent trend.Show that high expression TSTA3 may be
One mark of ESCC patient's poor prognosis.
Table 3 is to have evaluated TSTA3 protein expression levels to the survival of patients time using single factor test and multiplicity method
Predictive value.Age, gender, knub position, Pathologic Grading, lymphatic metastasis are analyzed respectively using single factor analysis method
The relationship of the factors and survival of patients time such as state, invasive depth, clinical stages, TSTA3 expressions（Table 3）, there is no send out
The expression of existing life span and TSTA3, which have, obviously to be contacted, and the Proportional hazards of TSTA3 high expression are not statistically significant, but have
There are visible trend (HR=1.967; Cl=0.987-3.923;P=0.055).Further it is the more of Cox proportional hazards regression models
Above-mentioned all Relevant Clinical Factors are included in analysis integrated consideration, when the expression quantity of TSTA3 survives to prognosis by factor analysis simultaneously
Between have specific predictive value, the results are shown in Table 3.The death risk of height expression TSTA3 is 2.755 times (95% of low expression group
Credibility interval=1.241-6.113,P =0.013).Show TSTA3 can as ESCC patient's prognosis an independent prediction because
Son.
Table 3：The multiplicity of Cox proportional hazards regression models
To sum up, the immunohistochemistry results of 104 ESCC are shown, 1. TSTA3 expression quantity is notable in ESCC tumor samples
Higher than with normal tissue (183.9 ± 54.6vs152.8 ± 48.1,P=0.0002)；2. in non-matching ESCC tumor samples
TSTA3 expression quantity is also significantly higher than normal structure (187.0 ± 58.8 vs152.1 ± 48.3,P=0.00014）；③TSTA3
The life span of high expression group patient is considerably shorter than low expression group (33.0 ± 5.5vs45.0 ± 5.4,P=0.048）；④
TSTA3 gene expressions and history of drinking history (P=0.007), invasive depth (P=0.055, T1+T2 VS T3), clinical stages (P=
0.010), lymphatic metastasis (P=0.043) related；5. multiplicity prompt TSTA3 is an independent prediction of ESCC prognosis
Factor.Accordingly, TSTA3 genes may play key effect in esophageal squamous cell carcinoma occurrence and development.
The present invention carries out TSTA3 expressions and ESCC patient clinical pathological parameter relationships by immunohistochemical assay
Research, has evaluated predictive values of the TSTA3 to ESCC prognosis.Observe that the expression of TSTA3 will be apparently higher than in tumor tissues
Normal control tissue.The clinical stages of the high expression level and patient's cancer of TSTA3, whether there is or not the depths of transfer and infiltration for lymph node
It spends related.TSTA3 high expression the survival of patients time is shorter, prognosis is poor.Importantly, TSTA3 high expression can conduct
The independentpredictor of ESCC patient's poorer prognosis.
Since the glycoprotein of most of experience fucosylations are the memebrane proteins of secretory protein or cell surface, and from primary
The cancer cell that tumor locus is fallen off or is discharged into blood is often overexpressed fucosylated glycan on its surface, therefore tumour is thin
The fucose albumen of cellular surface exception exists frequently as the marker of tumour.If cell surface fucosylation Le Y oligosaccharides is one
Tumor associated antigen is high expression in the breast cancer of many epithelial origins, oophoroma, liver cancer and intestinal cancer, currently, for should
The monoclonal antibody (IGN-311 and hu3s193) of the antigen potential drug as epithelial origin immunotherapy of tumors；Core
The alpha-fetoprotein (AFP-L3) of heart fucosylation is compared to AFP for liver cancer with more the diagnostic value of specificity；Fucosido
The haptoglobin (HPT) of change is horizontal to be increased in the malignant tumours such as oophoroma, lung cancer, breast cancer and cancer of pancreas, becomes a variety of
The molecular marker of tumour.TSTA3 is high in ESCC in the present invention expresses and is prompted in ESCC occurrence and development with the relationship of prognosis
In there are significant fucosylation exception, fucose albumen is especially targeted to further investigation of the gene in ESCC and is expected to
The new Carcinogenesis Mechanism of the cancer of the esophagus is disclosed, reason is provided for the new antineoplaston scheme of new identification ESCC early diagnosis marker and design
By basis.
In conclusion the present invention be it is a kind of by amynologic index TSTA3 expressing quantities assist patients with esophageal squamous cell carcinoma
It diagnoses and to the purposes that prognosis is evaluated, i.e. the height according to TSTA3 protein expressions in patients with esophageal squamous cell carcinoma tumor tissues is straight
Connect diagnosis and the prognosis information of determining patient.
The above is only the citing of embodiments of the present invention, it is noted that for the ordinary skill of the art
For personnel, without departing from the technical principles of the invention, several improvement can also be made, these improvement also should be regarded as this
The protection domain of invention.
Claims (2)
1. a kind of purposes of tumour-specific trnaplantation antigen albumen TSTA3 in preparing esophagus cancer diagnosis reagent, it is characterised in that：
The tumour-specific trnaplantation antigen albumen TSTA3 is preparing the purposes in diagnosing patients with esophageal squamous cell carcinoma reagent.
2. a kind of purposes of tumour-specific trnaplantation antigen albumen TSTA3 in preparing prognostic evaluation patients with esophageal squamous cell carcinoma reagent.
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| CN111948394B (en) * | 2020-08-10 | 2023-07-28 | 山西医科大学 | Application of TSTA3 and LAMP2 as targets in esophageal squamous carcinoma cell metastasis detection and drug screening |
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