CN107966566A - A kind of kit for being used to detect Xinjiang region patients with esophageal squamous cell carcinoma outcome - Google Patents
A kind of kit for being used to detect Xinjiang region patients with esophageal squamous cell carcinoma outcome Download PDFInfo
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Abstract
The present invention provides a kind of kit for being used to detect Xinjiang region patients with esophageal squamous cell carcinoma outcome, including 1 albumen of phosphatidase ε, IKB α albumen and mutant P 53 protein.Further include 0.01M citrate buffer solutions, the H that 10mM ethylenediamine tetra-acetic acids repair liquid, mass percent concentration is 3%2O2Solution, 0.01M phosphate buffers, BSA confining liquids, immunohistochemistry Envision kits and the DAB colour reagents that mass percent concentration is 5%.The present invention is the expression of joint PLCE1, IKB α, P53 protein antibodies detection patient with esophageal carcinoma paraffin embedding specimens from pri and then predicts the kit of patient's prognosis.The kit of the present invention has the prediction effect of higher than single Indexs measure, is approached with the predictive ability of TNM stage, and the immunohistochemistry two step method that is based on of the present invention be a kind of mature and reliable, can be in the widely used method of basic hospital.
Description
Technical field
The invention belongs to field of biology, is related to a kind of detection kit, specifically being a kind of to be used to detect Xinjiang region
The kit of patients with esophageal squamous cell carcinoma outcome.
Background technology
The cancer of the esophagus occupies the 9th in ten common big malignant tumour rankings of the mankind, global cancer epidemiology system in 2012
Count the global cancer of the esophagus year new cases 323008 of (GLOBOCAN2012) data display, death 281217.China is
The hotspot of world's cancer of the esophagus, especially based on male patient, organization type is mainly squamous cell carcinoma（Abbreviation esophageal squamous cell carcinoma）.
Publish within 2012《China's tumour registration annual report》Latest data show that China's esophageal squamous cell carcinoma occupies Incidence the 5th
It is position, 4th dead, serious threat is constituted to the quality of life of citizen even life security.
The prognosis of Accurate Prediction patient with esophageal carcinoma is of great significance for clinical further treatment and follow-up.However,
At present except international TNM by stages in addition to patient with esophageal carcinoma can also be more accurately predicted without a kind of simple and effective method
Prognosis.The prognosis of TNM stage prediction patient is comparatively laborious, it is necessary to evaluate the invasive depth of tumour, metastatic lymph node exactly
Number, tumour whether there is DISTANT METASTASES IN etc..
Phosphatidase ε 1（Phospholipase C, epsilon 1, PLCEl）It it is recent years headed by Wang Lidong is taught
Researcher find the cancer of the esophagus tumor susceptibility gene.It is phospholipase C by the PLCE1 albumen of PLCE1 gene codes（PLC）Family
New a member, it is except the domain having comprising PLC families（PH, XY, C2 and EF domain）Outside, also comprising it alone
Distinctive two domains（One is to bind domain RAl and RA2 positioned at the Ras of C terminals, and one is N areas RasGEF domains）.Separately
There is article report outside, hormone, cell factor, neurotransmitter and growth factor equimolecular can activate PLCE1, hydrolyzed cellular film
On PIP2（4,5 diphosphonic acid of phosphatidylinositols）Produce IP3（InsP3, inositol trisphosphate) and DAG
（Diacylglycerol, diacylglycerol) two kinds of important Second messenger in cell, influence the increment and differentiation of cell.
IKB α albumen（NF-kappa-B inhibitor α) it is the NF- κ B letter related with the starting of inflammation tumour
The inhibitor of number path, is a kind of transcription factor of pleiotropism, can be occurred with the KB sites of a variety of base promoter sites special
Property combination so as to promoting its transcriptional expression.Studies have reported that IKB α high expression in B cell lymphoma tissue, is B cell leaching
The independent prognostic factor of bar knurl.
P53 is the tumor suppressor gene that the mankind are found earliest, and in most of malignant tumours, P53 undergos mutation, and is a kind of thin
The related gene of born of the same parents' apoptosis, and the invasive ability of tumour cell is added under the assistance of other genes, ultimately result in patient's
Life cycle reduces, so the prognosis to a certain extent to patient is intentional.P53 genes include wild type and saltant type.Saltant type
P53 albumen relative to wild-type P53 protein long half time, so the albumen that ImmunohistochemistryMethods Methods observe is mostly saltant type
P53 albumen.
Although above-mentioned every kind of albumen is respectively provided with the ability of prediction patient with esophageal carcinoma prognosis, 3 kinds of albumen are joined together pre-
The prognosis for surveying patient with esophageal carcinoma then has stronger judgement efficiency, is only second to the predictive abilities of internationally recognized TNM by stages.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of for detecting Xinjiang region patients with esophageal squamous cell carcinoma prognosis
The kit of effect, this kit will solve to detect Xinjiang region patients with esophageal squamous cell carcinoma outcome in the prior art
The cumbersome technical problem of method.
The present invention provides a kind of kit for being used to detect Xinjiang region patients with esophageal squamous cell carcinoma outcome, including phosphatide
1 albumen of enzyme ε, IKB α albumen and mutant P 53 protein.
Further, 0.01M citrate buffer solutions are further included, 10mM ethylenediamine tetra-acetic acids repair liquid, quality percentage
Specific concentration is 3% H2O2Solution, 0.01M phosphate buffers, the BSA confining liquids that mass percent concentration is 5%, immunohistochemistry
Envision kits and DAB colour reagents.
Wherein, above-mentioned immunohistochemical method is mainly the mark for detecting neutral formalin fixation and paraffin embedding
This.
Detection method used in the present invention is immunohistochemistry Envision two step methods.
Further, predict that the method for patient with esophageal carcinoma prognosis includes the following steps：Sample is sliced, dewaxes, after aquation
With the expression of Envision two step method testing goal albumen.
Further, predict that the method for patient with esophageal carcinoma prognosis is specific as follows：Section is through dewaxing to water, antigen retrieval, going out
After endogenous peroxydase and 5%BSA confining liquids closing living, it is built in respectively with primary antibody PLCE1, IKB α, P53 in moisture preservation box
4 DEG C of overnight incubations；Then with redying nucleus through DAB colour developings and haematoxylin after the 37 DEG C of incubations of Envision reagent As liquid；Most
Neutral gum mounting is used afterwards by dehydration of alcohol and dimethylbenzene are transparent, and is scored every immunohistochemical staining section.
Further, the cut-off values of every kind of protein expression height are determined by SPSS softwares, wherein the cut- of PLCE1
Off values are 4 points, and the cut-off values of IKB α are 4 points, and the cut-off values of saltant type P53 are 3 points.
Further, for every sample after being detected respectively using 3 kinds of antibody, the calculating of its 3 kinds of albumen Combined expressions is public
Formula is Y=α × A+ β × B+ γ × C, and wherein α, β, γ is coefficient, can be returned and drawn by Cox, therefore the formula is converted to Y=
0.541×A+0.463×B+0.379×C；A, B, C are respectively the expression shape of PLCE1 in this sample, IKB α, P53 albumen
State, 1 represents low expression, and 2 represent high expression；Arranged after the Y value of every sample is calculated according to order from small to large, wherein
Y value corresponding to centrally located sample is cut-off values, i.e., 2.3154, then the case less than or equal to the value is
Low danger group, the case higher than the value are high-risk group.
Further, only need to be the immune group of 3 albumen of patient when evaluating the information of individual patient with esophageal carcinoma prognosis
Change score corresponding to expression status be inserted in formula, it is possible to draw Y value, then with we determined that cut-off values
2.3154 compare, and the patient less than or equal to the value belongs to low danger group, its 1 year survival rate is 96.3%, and survival rate is within 2 years
87.5%, 3 years survival rates are 77.8%, and the patient more than the value belongs to high-risk group, its 1 year survival rate is 90.3%, 2
Year, survival rate was 86.2%, and 3 years survival rates are 81.1%.
The present invention is had found by studying：Its overall survival of the patient of the high expression of the albumen of PLCE1 is notable in the cancer of the esophagus
Higher than the patient of low expression, and it is that patient with esophageal carcinoma prognosis mala stands alone as hazards.Mutation P53 gene protein exists
High expression and closely related with the prognosis mala of patient in esophageal squamous cell carcinoma tissue.IKB α albumen high table in esophageal squamous cell carcinoma tissue
Reach and closely related with the poor prognosis of patient.
The present invention is the table of joint PLCE1, IKB α, P53 protein antibodies detection patient with esophageal carcinoma paraffin embedding specimens from pri
Up to situation and then the kit of prediction patient's prognosis.PLCE1 of the present invention, IKB α, P53 albumen associated prediction patients
The kit of prognosis has the prediction effect of higher than single Indexs measure, is approached with the predictive ability of TNM stage, and this hair
The bright immunohistochemistry Envision two step methods being based on are a kind of mature and reliables, can be in the widely used side of basic hospital
Method.
The present invention is compared with prior art, its technological progress is significant.The present invention passes through joint-detection amynologic index
PLCE1, IKB a, the prognostic evaluation of saltant type P53 positive expressions amount auxiliary patient with esophageal carcinoma, to solve patient with esophageal carcinoma prognosis
The present situation that evaluation means lack provides a kind of accurately and rapidly Forecasting Methodology.
Brief description of the drawings
Kaplan-Meier survivorship curve figure of the PLCE1 albumen in the esophageal squamous cell carcinoma crowd of different expression is shown in Fig. 1
And dangerous function figure（X2=5.213, P=0.022）
Kaplan-Meier survivorship curve figure and danger of the IKB α albumen in the esophageal squamous cell carcinoma crowd of different expression is shown in Fig. 2
Dangerous functional arrangement（X2=6.784, P=0.009）
Fig. 3 shows Kaplan-Meier survivorship curve figure and dangerous function figure of the mutant P 53 protein in different expression crowds
（X2=5.113, P=0.024）
The Kaplan-Meier survivorship curves that the crowd is judged after 3 kinds of albumen are combined are shown in Fig. 4（X2=11.403, P
=0.001）
ROC curve (note is shown in Fig. 5：The area under the curve formed after 3 kinds of albumen joints is maximum, for 0.590).
Embodiment
The present invention is expanded on further below by specific embodiment and with reference to attached drawing, but is not intended to limit the present invention.
It is attached both to be from medical college of Shihezi Univ first for the wax stone of 168 human esophageal carcinomas used in the embodiment of the present invention
Belong to hospital's cardiothoracic surgery.This experiment has passed through Ethics Committee of First Affiliated Hospital, Shihezi University College of Medicine, and every patient is all
Informed consent form is signed.
Immunohistochemical method, with reference to Wang Guanghui Identification of MXRA5 as a novel in 2013
tissue biomarker for colorectal cancer progression。
Mapping software used is SPSS 20.0.
Used reagent is shown in Table 1 in the embodiment of the present invention, and major experimental instrument is shown in Table 2 with consumptive material, matches somebody with somebody used in experiment
Reagent processed is shown in Table 3.
1 experiment reagent of table
| Title | Company | The place of production |
| Dimethylbenzene | Tianjin Fu Yu Fine Chemical Co., Ltd | China |
| 75% alcohol | Xinjiang Jie Pule bio tech ltd | China |
| 95% alcohol | Xinjiang Jie Pule bio tech ltd | China |
| Absolute ethyl alcohol | Tianjin Fu Yu Fine Chemical Co., Ltd | China |
| Citrate buffer | Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge | China |
| PBS phosphate buffers | Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge | China |
| 30% hydrogenperoxide steam generator | Chengdu Ke Long chemical reagents factory | China |
| PLCE1 primary antibodies | Sigma | The U.S. |
| IKB a primary antibodies | Abcam | Britain |
| P53 primary antibodies | Gene science | China |
| 5%BSA confining liquids | Beijing Suo Laibao Science and Technology Ltd | China |
| Primary antibody dilution | Leica | Germany |
| Envision kits | Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge | China |
| DAB color developing agents | Dako | Germany |
| Haematoxylin | Shanghai Bo Gu bio tech ltd | China |
| Neutral gum | Shanghai Yi Yang Instrument Ltd. | China |
2 laboratory apparatus of table and consumptive material
| Title | Company | The place of production |
| Organization chip makes instrument | ALPHELYS | Italy |
| Slicer | Leica | Germany |
| Light microscope | Olympus | Japan |
| 4 DEG C of refrigerators | Haier | China |
| - 20 DEG C of refrigerators | Haier | China |
| Insulating box | Bright Medical Instruments factory forever of Beijing | China |
| Electric drying oven with forced convection | Bright Medical Instruments factory forever of Beijing | China |
| Micro sample adding appliance | Eppendorf | Germany |
| Micro-wave oven | Beautiful | China |
| 1.5ml EP are managed | Axygen | The U.S. |
| Tip heads | Axygen | The U.S. |
| Glass slide | Citotest Labware Manufacturing Co., Ltd. | China |
| Coverslip | Citotest Labware Manufacturing Co., Ltd. | China |
| Wet box | Safe experiment equipment factory of Haimen City | China |
| Staining rack | Safe experiment equipment factory of Haimen City | China |
3 preparation of reagents of table
It is a kind of to aid in patient with esophageal carcinoma prognosis to be commented by cancer of the esophagus amynologic index PLCE1, IKB α, P53 positive expressions amount
The method of valency, includes the following steps：
（1）168 human esophageal carcinoma samples of surgery excision are subjected to paraffin embedding, the wax stone of 168 human esophageal carcinomas is made；
（2）Dyed according to HE and determine organization type, chosen tumor tissues and be fabricated to organization chip；
（3）The wax stone of histotomy is made to 4 μm of serial section, the histotomy of gained is placed on metallochromy frame,
It is positioned in 65 DEG C of baking ovens that to bake piece 30 minutes paraffin melting to surface；
（4）Histotomy routine dewaxing aquation：
Organization chip embathes 3 times × 5 minutes by dimethylbenzene successively；Absolute ethyl alcohol embathes 1 time, 5 minutes；90% alcohol embathes 1
It is secondary, 5 minutes,；80% alcohol embathes 1 time, 5 minutes；70% alcohol embathes 1 time, 5 minutes, takes off the paraffin on histotomy surface
And abundant aquation, slide embathe 3 times into the water；
（5）High pressure antigen hot repair is answered；Histotomy is dipped in 1 × citrate buffer（PLCE1 albumen and P53 albumen reparations
It is applicable in）, 1 × edta buffer liquid（IKB α albumen reparations are applicable in）In, high fire screen heats 3 minutes in micro-wave oven；Taking-up is put into height
Pot is pressed, 1300W is heated to jet, and 800W is reheated 8 minutes；Cooled down at room temperature after taking-up, section is embathed 3 times into the water；
（6）Deactivating endogenous peroxydase：Histotomy is immersed in 3% hydrogenperoxide steam generator 15 minutes, then organizes to cut
Piece embathes 3 times into the water；
（7）Histotomy is placed in vibration washing 3 times × 5 minutes in 1 × PBS buffer, and blotting paper wipes unnecessary PBS buffer；
（8）100 μ l 5%BSA confining liquids are added dropwise in every section, and 37 DEG C are incubated 30 minutes, get rid of surplus liquid；
（9）Primary antibody is added dropwise：PLCE1 primary antibodies（HPA015598, Sigma）By 1:500 dilutions；It is placed in 4 DEG C of overnight incubations in wet box；
Negative control replaces primary antibody with PBS buffer；
（10）Take out wet box, rewarming 30 minutes；PBS vibration washings 3 times × 5 minutes；Blotting paper wipes unnecessary PBS buffer；
（11）Secondary antibody is added dropwise：The universal immunologic combined detection reagent kit of anti-rabbit/mouse (Envision kits)（PV-6000）, with
Lower abbreviation secondary antibody kit, every histotomy are added dropwise Envision reagent A liquid, and 37 DEG C of incubations 30 minutes, PBS embathes 3
It is secondary × 5 minutes；
（12）DAB develops the color：PBS embathes 3 times × 5 minutes, secondary antibody kit be in stoste and buffer solution according to stoste:Dilution
For 1:100 proportional arrangement adds the nitrite ion of 100 μ l into nitrite ion, every histotomy, under the microscope during control colour developing
Between, it was observed that occur positive findings again without obvious background coloration when stopped reaction, with distilled water flushing 5 minutes；
（13）Haematoxylin is redyed 3 minutes；Histotomy embathes 3 times into the water；Sour alcohol breaks up 0.5s, and histotomy is put into water
In embathe 3 times；Tap water oil blackeite 5 minutes；
（14）It is dehydrated and transparent：Histotomy is embathed 1 time, 5 minutes by 70% alcohol successively；80% alcohol embathes 1 time, 5 points
Clock；Absolute ethyl alcohol embathes 1 time, 5 minutes；Dimethylbenzene embathes 3 times, every time 5 minutes；
（15）Mounting：Placed 5 minutes in fume hood, the dimethylbenzene on histotomy is volatilized, then with neutral gum mounting, envelope
When placement 1 is small in baking oven after piece, natural gum is set to solidify as early as possible；
（16）With Olympus light microscopes at a 200-fold magnification, using double scoring semiquantitative method scorings：<5% cell
The positive is 0 point；6%-25% cell positives are 1 point；26%-50% cell positives are 2 points；51%-75% cell positives are 3 points；
>75% cell positive is 4 points.Tinctorial strength presses the presence or absence of cell colour developing and depth scoring：Cell is 0 point without colour developing；It is light yellow
For 1 point；Brown color is 2 points；Sepia is 3 points.It is multiplied and is classified then in conjunction with positive cell percentage and tinctorial strength, scope is
0~12.Obatained score is 0-1 timesharing, is classified as（-）；2-4 points are（+）；5-8 points are（++）；9-12 points are（+++）.Its
In-it is feminine gender；+ it is weakly positive；++/+++ is strong positive.In order to facilitate statistics, according to step（15）Immune scoring, we
All cases are divided into low expression and high 2 groups of expression, and 1 represents low expression group, and 2 represent high expression group, its cut-off value according to
SPSS20.0 softwares determine.PLCE1：0~3 point is low expression group, and 4~12 points are high expression group；IKB Α：0~3 point is low table
Up to group, 4~12 points are high expression group；Saltant type P53：0~2 point is low expression group, and 3~12 points are high expression group.
Table 4
（17）3 indexs join together to judge that the equation of patient with esophageal carcinoma prognosis is as follows.
（18）Y=α × A+ β × B+ γ × C, wherein α, β, γ are coefficient, and numerical value can be drawn by being returned by Cox, therefore side
Formula is changed into Y=0.541 × A+0.463 × B+0.379 × C.
A, B, C are the expression status of 3 albumen in this sample, and 1 represents low expression, and 2 represent high expression.Meter
Arranged after calculating the Y values of every sample according to order from small to large, wherein the corresponding Y value of centrally located case is
Cut-off values, i.e., 2.3154.Case less than or equal to the value is low danger group, and the case higher than the value is high-risk group, is so divided
Survival analysis is carried out again after into 2 groups.In addition it is also possible to gone by drawing ROC curves under the curve that each albumen of comparison is formed
The size of area (AUC).
（19）The relation of the expression of each albumen and the life span of patient uses Kaplan-Meier survival analysises；Prediction
The independent hazard factor for influencing patient's prognosis uses Cox proportional hazards regression modelses；Every kind of albumen prediction patient with esophageal carcinoma is pre-
Efficiency afterwards uses ROC curves；P ＜ 0.05 are considered having significance,statistical.
The results show：
From figure 1 it appears that the prognosis of the low patient of PLCE1 positive expression amounts is high significantly better than PLCE1 positive expression amounts
Patient, i.e., relatively low PLCE1 positive expressions amount and the preferable prognosis of patient are closely related.
From figure 2 it can be seen that the prognosis of the low patient of IKB α positive expression amounts is high significantly better than IKB α positive expressions amount
Patient, i.e., relatively low IKB α positive expressions amounts and the preferable prognosis of patient are closely related.
From figure 3, it can be seen that the prognosis of the low patient of P53 positive expression amounts is high significantly better than P53 positive expression amounts
Patient, i.e., relatively low P53 positive expressions amount and the preferable prognosis of patient are closely related.
Figure 4, it is seen that the prognosis of the patient of low danger group is significantly better than high-risk group of patient after 3 kinds of albumen joints,
I.e. it is closely related with preferable prognosis to be less than 2.3154 for 3 kinds of albumen Combining diagnosis Y values.
As can be seen from Table 5, Cox risks regression model detects the Risk values after each albumen is expressed alone or in combination
And whether be patient's prognosis independent hazard factor it is as follows.
The expression status of PLCE1 and the life span of patient are closely related, and still independent prognostic factor (Risk
=2.382,95% CI=1.094-5.185, P=0.029).
The expression status of IKB and the life span of patient are closely related, and still independent prognostic factor (Risk=
2.197,95% CI=1.189-4.062, P=0.012).
The expression status of P53 and the life span of patient are closely related, and still independent prognostic factor (Risk=
1.659,95% CI=1.059-2.598, P=0.027).
3 albumen joints have stronger cancer of the esophagus prognosis prediction ability, while are also independent prognostic factor (Risk
=1.553,95% CI=1.166-2.067, P=0.003), but inferior to TNM by stages (Risk=2.291,
95% CI=1.692-3.102, P=8.2 × 10-8) and lymph node status (Risk=2.049,95% CI
=1.345-3.099, P=0.001) predictive ability.
From figure 5 it can be seen that the ROC curve area formed after 3 albumen joints is only second to TNM stage, than independent
Each gene expression predictive value it is all high.
In conclusion it is hazards (the P of independent evaluation patient with esophageal carcinoma prognosis after PLCE1, IKB α, P53 joint
=0.003) corresponding P values (being respectively 0.029,0.012,0.027) during patient, are individually evaluated less than them.
What the Kaplan-Meier survivorship curves evaluation more independent than each index drawn after 3 index joints was drawn
Kaplan-Meier curve difference becomes apparent from, and X2 values are the largest (X2=11.403), and P values are minimum (P=
0.001)。
The ROC area under the curve obtained after 3 indexs joint is 0.590, when being all higher than every kind of index and individually evaluating
The ROC area under the curve formed, is only second to the area under the curve (0.653) that TNM stage is formed.Therefore say PLCE1,
The prognosis of patient with esophageal carcinoma is evaluated after IKB α, saltant type P53 joint has the predictive value of higher, and than TNM by stages more
Good operation and applicability are stronger.
, only need to be obtained 3 protein expressions of the patient when evaluating the clinically prognosis information of individual patient with esophageal carcinoma
The corresponding expression status numerical value of immunohistochemistry fraction (1 represents low expression, and 2 represent high expression) be inserted in formula Y=
In 0.345 × A+0.416 × B+0.287 × C, it is possible to draw Y values, then with we determined that cut-off values
2.3154 compare, and the patient less than the value belongs to low danger group, its 1 year survival rate is 86.8%, and survival rate is within 2 years
65.9%, 3 years survival rates are 28%；And the patient more than the value belongs to high-risk group, its 1 year survival rate is 77.4%, 2
Year, survival rate was 57.4%, and 3 years survival rates are 19.1%.
Claims (2)
- A kind of 1. kit for being used to detect Xinjiang region patients with esophageal squamous cell carcinoma outcome, it is characterised in that：Including phosphatidase ε 1 albumen, IKB α albumen and mutant P 53 protein.
- 2. a kind of kit for being used to detect Xinjiang region patients with esophageal squamous cell carcinoma outcome according to claim 1, its It is characterized in that：Further include 0.01M citrate buffer solutions, 10mM ethylenediamine tetra-acetic acids repair liquid, mass percent concentration is 3% H2O2Solution, 0.01M phosphate buffers, the BSA confining liquids that mass percent concentration is 5%, immunohistochemistry Envision Kit and DAB colour reagents.
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| CN110870879A (en) * | 2018-09-03 | 2020-03-10 | 石河子大学 | Use of PLCE1 inhibitors and recombinant p53 adenoviruses |
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| 李昀等: "PLCE1 调控p53 诱导食管癌细胞凋亡的实验研究", 《中国药理学通报》 * |
| 阿卜拉江•阿卜杜热合曼等: "新疆多民族食管癌患者癌组织中p53 蛋白表达及其临床意义", 《新疆医科大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110870879A (en) * | 2018-09-03 | 2020-03-10 | 石河子大学 | Use of PLCE1 inhibitors and recombinant p53 adenoviruses |
| CN109142755A (en) * | 2018-10-09 | 2019-01-04 | 福建省立医院 | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application |
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