CN104142320A

CN104142320A - Serum surface enhanced Raman spectrum based parotid tumor diagnosis technology

Info

Publication number: CN104142320A
Application number: CN201310228012.5A
Authority: CN
Inventors: 李龙江; 闫冰
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-06-08
Filing date: 2013-06-08
Publication date: 2014-11-12

Abstract

The invention relates to a serum surface enhanced Raman spectrum based parotid tumor diagnosis technology, which is a novel technology directed at parotid tumor diagnosis by head and neck tumor surgery and oral and maxillofacial surgery. The technology mainly solves the problem that a conventional biopsy technique cannot be employed for definite diagnosis of parotid tumor patients before operation, replaces the parotid tumor local puncture biopsy technology, avoids complications caused thereby, simultaneously alleviates the work intensity of pathologists, and provides the basis for surgeons to adopt different operative treatment modes according to the benign/malignant nature of tumors. According to the technology, nanoscale gold sol particles are added into parotid tumor patient serum to enhance the intensity of a Raman spectrum so as to obtain a characteristic surface enhanced Raman spectrum of the tumor patient serum. Through analysis of a support vector machine on characteristic spectral data, a differential diagnosis model can be established for identifying different parotid tumors and providing the objective basis for preoperative diagnosis, thus establishing a quick and noninvasive new diagnosis technique.

Description

Parotid tumor diagnostic techniques based on serum Surface enhanced raman spectroscopy

Technical field

Parotid tumor is a modal class in Salivary Gland Tumors, accounts for 64% ~ 80% of Salivary Gland Tumors, and the incidence of disease is the trend increasing year by year in recent years.Parotid tumor histopathology complicated classification, tumor type is numerous, according to < < World Health Organization (WHO) staging (version in 2005): the Salivary Gland Tumors histopathologic classification in head and neck neoplasm pathology and science of heredity > >, parotid tumor pathological classification comprises 41 kinds of benign tumour, malignant tumour, soft tissue neoplasm, lymph hematopoietic system cancer and the large classes of secondary tumor five.This brings difficulty at diagnosis and differential diagnosis parotid tumor to clinician and Pathology Doctors ', and because parotid tumor is forbidden conventional preoperative biopsy, more difficult to its preoperative diagnosis.

Along with the development of surgical technic, the concept of functional surgery or keeping quality surgery is more and more advocated, and in treatment disease, preserves as far as possible the function of tissue and recovery organ.With regard to surgical treatment for parotid tumors-A report, for some benign tumours, the outer resection of coating of tumour is advocated by more neck surgeon, compare the shallow leaf of traditional parotid tumor and body of gland or full lobectomy, the outer resection of parotid tumor coating can be preserved parotid gland tissues as much as possible, recover function of parotid, reduce the postoperative complications such as postoperative facial paralysis, gustatory sweating syndrome.But the prerequisite that this operation is carried out must will have clear and definite preoperative diagnosis based on parotid tumor.In addition on the one hand, in clinical position, due to parotid tumor and facial nerve adhesion, cannot clear and definite cancer pathology type or good pernicious matter in the situation that, the excision of facial nerve and reservation be also usually perplexed to neck surgeon.

To sum up, the preoperative diagnosis of parotid tumor is difficulty comparatively, but the differentiation of clear and definite preoperative diagnosis or benign from malignant tumors has positive role to the treatment of parotid tumor again.At present, the technology and the method that are applied to the preoperative diagnosis of parotid tumor have Imaging Method as B ultrasonic, CT, MRI etc., and pathology method is as Cytologic diagnosis technology etc., but have its limitation and shortcoming.Imaging Method is more to provide tumour and surrounding tissue anatomical structure relation, for concrete pathological diagnosis, cannot provide enough data, more relies on image department doctor's subjective clinical experience.And the accuracy rate of diagnosis of Cytologic diagnosis technology usually depends on puncture procedure person's technology and the experience of Pathology Doctors ', can not provide one to stablize objective reference for preoperative diagnosis, puncture technique itself belongs to one simultaneously wound operation, can produce stimulate tumour, cause the complication such as infection of the sending out of tumour, paracentesis area.Therefore, find one without wound, quick, convenient, objectively diagnose new technology, new method all to there is important clinical meaning and value to the preoperative diagnosis of parotid tumor and treatment.

Background technology

Biochemical substances change and metabolism inside and outside the generation of tumour cell, development and cell are in close relations, particularly before genetic morphology variation, there is significant change in biochemical substances structure, composition and the metabolism etc. of tumour cell, mainly be reflected in the change of the protein conformation of cell and body fluid, the abnormal and lipid metabolism variation of nucleic acid content etc., produce the biomarker much with important value, as prostate specific antigen PSA, the AFP of liver cancer etc., it all diagnoses and evaluates important indicator and the reference of prognosis as relevant disease.In parotid tumor, some biomarker structural change or metabolic disorder etc., occurring in cellular morphology not yet there is pathologic change before and is present among the whole process of tumour cell generation, development, this makes as the body fluid components of tumour cell metabolism overall situation and forms corresponding variation also occurs, the molecular marker or the metabolic product that even comprise some malignant tumour, make the researchs such as body fluids such as blood, saliva also become early screening a kind of approach that diagnoses the illness.But at present to the examination technology of tumor disease biomarker if metabonomic technology, protein science technology, genomics technology etc. are because it requires high to sample process, complex operation is consuming time, the factors such as somewhat expensive, have brought Bottleneck Restrictions to the experimental study of large sample amount.

Raman spectroscopy, its principle is based on inelastic scattering, the light scattering frequency shift forming according to molecular vibration carrys out the information such as composition of reaction molecular structure, group, there is high sensitivity and specificity, simultaneously its sample without special processing, applied widely, test the advantages such as quick, be widely used in biomedical sector.Raman spectroscopy has the incomparable advantage of other spectral techniques, as it is high compared with infrared spectrum resolution to compose peak, be not subject to moisture interference, imaging real-time etc., be specially adapted to infrared spectrum to disturb the stronger biological specimen that contains large quantity of moisture research, can provide in a large number about interactional information between molecular composition, molecular structure and molecule in cell, tissue or body fluid, material composition variation, the structural changes etc. such as reflection protein, nucleic acid, lipid, be called as molecular fingerprint technology.But due to Raman signal a little less than, be subject to background fluorescence and disturb, so limit it in the application of the detection of biological samples of complicated component.And the introducing of Surface enhanced raman spectroscopy technology has solved above problem.The principle of Surface enhanced raman spectroscopy technology is simply illustrated as Raman scattering intensity and is directly proportional to molecule local light electromagnetic intensity square of living in, the localized electromagnetic field with metallic substrates generation under incident laser irradiates of nanoscale coarse structure strengthens, make to be adsorbed on the Raman scattering of molecule strength-enhanced in metallic substrates, thereby raising Raman signal intensity that can be exponential (improves 10 ⁶~ 10 ⁸doubly).In Medical Biology field, Surface enhanced raman spectroscopy technology is because it is without wound, the feature such as quick, easy, accurate, sensitivity is high, become a kind of detection and research cell, tissue and body fluid, understand the new technique that it forms structure, metabolism state, molecule variation etc.

Summary of the invention

The summary of the invention application oxide-reduction method of this invention patent, prepares by golden chloric acid and trisodium citrate aqueous solution the nanometer grade gold sol particle that is suitable for the research of parotid tumor patients serum Surface enhanced raman spectroscopy.To parotid tumor blood samples of patients and normal human serum, carry out centrifugal, extract serum, add nano gold sol particle, application Renishaw inVia Raman Microscope carries out Surface enhanced raman spectroscopy research, obtains the Surface enhanced raman spectroscopy of parotid tumor patient and normal human serum.Appliance computer software is analyzed spectroscopic data, removes background noise, carries out data smoothing and obtains characteristic spectrum data, the variation of different molecular composition and structure in analysis spectroscopic data.Spectroscopic data is converted into .xls form, input support vector machine, application support vector machine is analyzed spectroscopic data, adopt " leaving-one method " to set up diagnostic model, distinguish parotid tumor patient and normal human serum, further distinguish different parotid tumor patients serums simultaneously, thereby parotid tumor is carried out to preoperative diagnosis and antidiastole.Its accuracy rate of diagnosis and antidiastole accuracy rate all reach more than 84%.

Embodiment

1. pass through oxide-reduction method, the 100ml HAuCl that is 0.01% by concentration ₄aqueous solution is heated to boiling, and then adding rapidly concentration is 1% sodium citrate aqueous solution 0.7ml, and uniform stirring also keeps the aurosol particle of fluidized state about 0.03mol/L of cooling generation concentration after 30 minutes.

2. extract parotid tumor patient and normal person and play fasting blood 5ml morning, low-speed centrifugal (3400 revs/min) 10 minutes after standing 4 hours, extracts the about 1ml of upper serum under 4 degrees Celsius, standby.

3. draw 4ml aurosol solution to centrifuge tube, high speed centrifugation (6000 revs/min) 10 minutes, removes supernatant, adds 0.4ml serum, and ultrasonic water bath vibration 20 seconds is standing.

4. the serum that adds aurosol is dripped on cover glass, application Renishaw inVia Raman Microscope, excitation wavelength 633nm, object lens X50, power 0.4W, carries out spectral scan 10 seconds sweep times, obtains serum Surface enhanced raman spectroscopy.

5. application WiRE 2.0 softwares convert spectroscopic data to .txt form, and input LabSpec2.0 software carries out smoothly, noise reduction, removal fluorescence background, and data are output as to .xls form.

6. spectroscopic data is inputted to support vector machine, adopt k-fold cross-validation method to select best penalty coefficient C and kernel function related coefficient gamma, set up diagnostic model, tumour and normal serum spectroscopic data are distinguished, again serum spectroscopic data between different tumours is distinguished, adopted accuracy rate, specificity, sensitivity and prediction stability test result.

Claims

1. patent of the present invention is that application surface enhancing Raman spectroscopy is studied parotid tumor patients serum, obtains characteristic spectrum data, is applicable to the preparation method of the nanometer grade gold colloidal sol of parotid gland serum Surface enhanced raman spectroscopy research in the present invention.

2. nano gold sol is added and in serum, obtain this technology of the distinctive Surface enhanced raman spectroscopy of parotid tumor patients serum.

3. in the present invention, apply support vector machine characteristic spectroscopic data analyzed, set up diagnostic model, application support vector machine to characteristic to parotid tumor serum Surface enhanced raman spectroscopy data analysis and set up the method for diagnostic model.