CN107907686A - A kind of immunohistochemistry detects NAP2 expressions in liver cancer and cancer beside organism - Google Patents

Abstract

The invention discloses a kind of immunohistochemistry to detect NAP2 expressions in liver cancer and cancer beside organism, and immunohistochemistry detection NAP2 is expressed in liver cancer and cancer beside organism：The results are shown in Figure 1, NAP2 high expression in liver cancer tissue, substantially very low without expression or expression quantity in control tissue by cancer.The present invention finds effective tumor markers and is of great significance for Carcinogenesis Mechanism research, the early diagnosis of disease and Index for diagnosis.By bioinformatic analysis, effect of these key proteins in system signal network is parsed；Verified again with MRM absolute quantitations, assess these liver cancer key protein Polypeptide Diagnostic liver cancer value and analysis and clinical correlation, propose their feasibilities in terms of the clinical practices such as the prevention, early diagnosis and Index for diagnosis of liver cancer.

Description

A kind of immunohistochemistry detects NAP2 expressions in liver cancer and cancer beside organism

Technical field

The present invention relates to genetic transcription group and proteomics field, especially a kind of immunohistochemistry detects NAP2 in liver The elemental method expressed in cancer and cancer beside organism.

Background technology

Due to primary carcinoma of liver early stage generally without any symptom, once there is clinical manifestation, the state of an illness have been enter into mostly in, evening Phase, and grade malignancy height, the fast, poor prognosis that is in progress, invasion are strong, therefore, find effective tumor markers and are used for canceration machine Managing research, the design of drug targets, the early diagnosis of disease and the examination of people at highest risk becomes working as Guangxi province liver cancer research Business is anxious.

At present, liver cancer discovery and diagnosis be by Serum AFP detection combine imageological examination.It is wherein clinical more to generally acknowledge Diagnosing cancer of liver standard be：Serum concentration of AFP>400 μ g/L, for 4 weeks, iconography finds that liver has Solid lesion and can arrange Except diseases such as hepatitis activity, reproduction embryonal tumors.But only rely on serum concentration of AFP and carry out diagnosing liver cancer, sensitivity is 39% ~ 64% Between, specificity is 76% ~ 91%, and for positive predictive value only 9%~32%, this allows for many liver cancer patient missing inspections, or false positive And false negative, directly affect the early diagnosis and therapy of liver cancer patient.

With the fast development of biotechnology and the completion of the Human Genome Project (HGP) genome sequencing, Ren Leijin The genome times afterwards comprehensively are entered, the research of wherein proteomics has been to be concerned by more and more people.With proteomic techniques come Detection, analysis and definite marker protein and target protein, the change for illustrating oncoprotein expression are different from tumor development The correlation and its rule in stage, are the current best approach for solving early diagnosis of tumor problem；Meanwhile and tumour And its newest and most strong means in drug screening research.

Mass-spectrometric technique is that the core of specific tumour marker is screened, identified in proteomic techniques, and common is surface Strengthen laser desorption/ionization time of flight mass mass-spectrometric technique（surface-enhanced laserdesorption/ Inionation-time of flightmass spectrometry, SELDI-TOF-MS）With substance assistant laser desorpted electricity From flight time mass spectrum（Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS）.This seminar once utilizes SELDI-TOF-MS combination LC-MS HPLC/ MALDI-TOF-MS technologies analyze the blood serum sample of the relevant liver cancer of hepatitis B, chronic benign hepatopathy, normal person, obtain people The specific serum marker NAP2 of class hepatitis B associated hepatocellular carcinoma（71）And NAP2（73）.There is similar detection using to SELDI Principle but the preferable CLINPROT systems of repeatability, it is obvious in two groups of blood serum samples of liver cancer and normal person to screen two differences Protein and peptide, one is accredited as from fibrinogen（Fibrinogen）A secretory piece, fibrinopeptide A （Fibrinopeptide A）, this is completely the same with the result of Eduard etc.；Another peptide identification is kininogen （Kininogen）.And the iTRAQ of new development over the past two years（isobaric tags for relative and absolute Quantification, iTRAQ）Mark combines MALDI-TOF MS/MSF technologies, establishes in iTRAQ isotope labeling reagents On the basis of, an including electrically charged reporter group, in addition an also peptide fragment reactive group and a balance group, wherein flat Weigh group balance molecule amount, and it is all 145 that guarantee, which is combined the total relative molecular weight of rear iTRAQ reagents with each reporter gene, peptide Section reactive group can measure E amino groups with the N-terminal and lysine of peptide fragment and react, and can so mark biological sample All peptide fragments of middle generation, strengthen the peptide fragment coverage of any given albumen, while after remaining the translation of such as some albumen The important feature information of modification, provides quantitative information so that seminar is new in liver cancer for the discovery and identification of disease marker It is more further strengthened in terms of the screening of marker.

From 2005, liver cancer-specific marker screened through above-mentioned mass-spectrometric technique in this seminar, and a series of of acquirement grind Study carefully achievement, the discovery and identification of these functional proteins and potential disease protein markers have benefited from Different Proteomics and grind The development studied carefully, but the gene expression abundance of these protein how is further detected, to illustrate its function and the meaning in disease research Justice, has become more and more important.At present, the research mode of marker discovery-verification-clinic confirmation has obtained researchers' It is widely recognized as.Found in discovery phase from disease organism sample and the discrepant protein of normal sample；To Qualify Phase The label screened is quantified, assesses its accuracy, specificity, scope etc., and the marker being verified is special with clinic Sign, result are associated；The most possible candidate molecules for becoming biomarker that Qualify Phase is found out, into clinic in next step Confirmation.This research mode makes the discovery of biomarker in body fluid and verification is more effective, science and rationally.

In above-mentioned research mode, the quantitative technique of target protein is the bridge that marker leads to verification from discovery.Matter The high sensitivity of spectrum can help people to excavate into biological sample low-abundance protein, polypeptide letter in the discovery phase of marker Breath, but in the quantitative verification stage, since complex component be total to the interference of effluent and the wide dynamic range up to more than 9 orders of magnitude, It greatly affected quantitative detection of the mass spectrum to low abundance proteins, polypeptide in biological fluid.Although researchers attempt to use A variety of methods, including chromatographic isolation as sufficient as possible, remove high-abundance proteins matter etc., but the preci-sion and accuracy of its result Still far from the requirement for meeting analysis, until mass spectrum multiple-reaction monitoring (multiple reaction monitoring, MRM) The appearance of technology.M R M technologies apply the advantages of above presenting its method in quantitative proteomics.

M R M technologies are based on Given information or assume information setting Mass Spectrometer Method rule, to legal ion into Row signal records, and removal is not largely inconsistent the interference of normally ion signal, so as to obtain a kind of data recipient of Information in Mass Spectra Formula.Specifically, it is according to polypeptide parent ion mass number and fragment ion masses number, selects parent ion-daughter ion pair, permits The parent ion for being permitted to meet setting enters collision cell, and after the completion of collision, only record sets daughter ion signal.By parent ion and son from The selection twice of son, removes interfering ion, reduces Chemical Background, improves sensitivity.When carrying out MRM analyses, Modern Mass Spectrometry instrument Large batch of sample can be monitored by MRM scan patterns, check whether mother-daughter ion of this arrangement to occurring.If letter Number occur, it was demonstrated that this molecule exists in the sample, and instrument will automatically switch to MS/MS scan patterns, determines the broken of this parent ion Piece ion information, and identify again and confirm the structure of the molecule.If in MRM scanning works, expected mother/daughter is not found Ion pair, just illustrates that the molecule is not present in this sample.Since MRM scan patterns are efficiently quick, and there is high sensitivity, Therefore it is very suitable for high throughput verification and validation biomarker.In addition, the high-throughout feature of MRM technologies, is also more hatching eggs Condition is quantitatively provided while white matter.M R M technologies are right by being combined with isotope-dilution analysis or m T R A Q technologies True peptide fragment in internal standard peptide fragment and sample that known quantity adds is marked respectively, selects different mother-daughter ions to obtaining not Same chromatography detecting signal, compares both signals, so as to produce absolute quantitation result^[15].At present, by the blood of MRM quantitative verifications Clear marker is the best way for avoiding false positive marker.

Therefore, this research is intended sieving seminar with SELDI-TOF-MS protein chips, iTRAQ isotope labelling techniques in advance The liver cancer-specific protein marker of choosing, it is clear and definite using real-time fluorescence quantitative RT-PCR and immunoblotting analysis Western blot technologies The expression and positioning of the new protein marker of liver cancer, and goal in research is further selected；Transfected using eucaryon, SiRNA Perturbation technique observes influence of the new marker of liver cancer to hepatoma cell strain biological characteristics, while using in nude mice lotus knurl model Its effect in invasion in HCC transfer of experimental observation；By bioinformatic analysis, parse these key proteins and believe in system Effect in number network；The absolute quantitation that large sample is finally carried out with MRM technologies is verified, assesses these liver cancer key protein polypeptides Diagnosing liver cancer is worth and analysis and clinical correlation, proposes that they face in prevention, early diagnosis and Index for diagnosis of liver cancer etc. The feasibility of bed application aspect, this research will be that finally filtering out liver cancer key protein/polypeptide establishes solid foundation.

The content of the invention

The object of the present invention is to provide a kind of immunohistochemistry to detect NAP2 expressions in liver cancer and cancer beside organism.

This immunohistochemistry detects NAP2 expressions in liver cancer and cancer beside organism,

Comprise the steps of：

Immunohistochemistry detection NAP2 is expressed in liver cancer and cancer beside organism：The results are shown in Figure 1, NAP2 high tables in liver cancer tissue Reach, it is substantially very low without expression or expression quantity in control tissue by cancer.

Invention beneficial effect：

The present invention finds effective tumor markers and is used for Carcinogenesis Mechanism research, the early diagnosis of disease and Index for diagnosis with weight Want meaning.To the new protein marker of liver cancer filtered out, by gene knock-in, knock out that the inside and outside such as to test with transplanted tumor in nude mice real Test, observe candidate gene expression be blocked or raise after influence to tumor biological behavior, orient influence human liver cancer The key protein of occurrence and development；By bioinformatic analysis, effect of these key proteins in system signal network is parsed； Verified again with MRM absolute quantitations, assess these liver cancer key protein Polypeptide Diagnostic liver cancer value and analysis and clinical correlation, It is proposed their feasibilities in terms of the clinical practices such as the prevention, early diagnosis and Index for diagnosis of liver cancer.

Brief description of the drawings

Fig. 1 is the schematic diagram of the present invention.

NAP2 immunohistochemical experiment result A liver cancer tissue B cancer beside organisms.

Embodiment

Embodiment：

Comprise the steps of：

Immunohistochemistry detection NAP2 is expressed in liver cancer and cancer beside organism：The results are shown in Figure 1, NAP2 high tables in liver cancer tissue Reach, it is substantially very low without expression or expression quantity in control tissue by cancer.

The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto, Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.

Claims (1)

1. a kind of immunohistochemistry detects NAP2 expressions in liver cancer and cancer beside organism, it is characterised in that：By following steps group Into：

Immunohistochemistry detection NAP2 is expressed in liver cancer and cancer beside organism：The results are shown in Figure 1, NAP2 high tables in liver cancer tissue Reach, it is substantially very low without expression or expression quantity in control tissue by cancer.