CN106319057A - Method for early monitoring radioactive pneumonia fibrosis caused by tumor radiotherapy - Google Patents
Method for early monitoring radioactive pneumonia fibrosis caused by tumor radiotherapy Download PDFInfo
- Publication number
- CN106319057A CN106319057A CN201610781509.3A CN201610781509A CN106319057A CN 106319057 A CN106319057 A CN 106319057A CN 201610781509 A CN201610781509 A CN 201610781509A CN 106319057 A CN106319057 A CN 106319057A
- Authority
- CN
- China
- Prior art keywords
- pneumonia
- radiation
- radiotherapy
- early
- radiation pneumonia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for early monitoring radioactive pneumonia fibrosis caused by tumor radiotherapy, in particular to a method for early diagnosis and prognosis evaluation of radioactive pneumonia by using a molecular marker. By the design of primers S100A8 and S100A9 and antibodies of a human, generation of the radioactive pneumonia and the pulmonary fibrosis can be monitored in the early stage, and the prognosis of diseases can be estimated. The method has the advantages of simple operation, high sensitivity and high specificity.
Description
Technical field
The present invention relates to a kind of Fibrotic method of radiation pneumonia caused for early monitoring tumor radiotherapy, specifically relate to
And a kind of molecular marked compound is for the method for radiation pneumonia early diagnosis with prognosis evaluation.
Background technology
Radiation pneumonia (radiation pneumonitis, RP), be Several Kinds of Malignancy (include pulmonary carcinoma, breast carcinoma and
The esophageal carcinoma) after radiotherapy, there is abnormal pathological manifestations in lung tissue, belong to common complication, acute radiation can be divided into
Inflammation changes and chronic fibrosis pathological changes.The lighter's non-evident sympton, severe one can cause the extensive fibrosis of lung, respiratory dysfunction,
Even respiratory failure, is also the main cause of death of induced lung injury.At present for the countermeasure mainly morning of radiation pneumonia
Phase prevention and early discovery, but occur the monitoring index developed the fewest, the most clinically to radiation for detection of radioactive pneumonia
Property pneumonia does not still have gratifying remedy measures, limits the dosage of tumor radiotherapy simultaneously, and pulmonary status is directly connected to
The therapeutic effect of tumor patient and quality of life.Therefore, it was predicted that and reduce radiation pneumonia be chest cancer radiation control
Treat the important component part of strategy.
At present, the pathogenesis of radiation pneumonia is not yet fully apparent from, although have been found that some indexs and lung radiation injury
Inflammation has dependency, but its significant diagnostic threshold does not determines.So, in order to reduce the incidence rate of radiation pneumonia with serious
Degree, needs to further investigate the Clinical criteria that its molecular mechanism occurred, screening specificity and sensitivity are the most suitable, enters one
Step improves radiation therapy technology, and according to conditions of patients, it is considered to various possible factors, treat with a certain discrimination, work out rational therapy meter
Draw, reduce the generation of radiation pneumonia as far as possible.Radiation pneumonia once occurs, the most irreversible, therefore the meaning of its prevention
More even more important than treatment.
If patient one Nian Nei pulmonary accepted radiotherapy, engender pulmonary's diseases such as continuing more than 2 weeks coughs, dyspnea
Shape, lamellar or streak shade that lung image display simultaneously is consistent with irradiating the visual field will consider concurrent radiation pneumonia
May, despite noninfective, also to differentiate with infectious pneumonia, and usually merge after radiation pneumonia generation
Infect.Both discriminating main points mainly from radiotherapy history, iconography and Infectious marker, (such as routine blood test, train by expectorant clinically
Support) start with.
S100A8 and S100A9 is calbindin family member, and both are not only presented in monomer, and how with
The structure of heterodimer plays a role, and is the most usually selected by people to compare research simultaneously.During they are mainly expressed in
Property granulocyte, mononuclear phagocyte and some keratinocyte, have powerful antibacterial characteristics, be therefore referred to as " calprotectin ".
Research shows, under internal stress state, and the neutrophilic granulocyte of activation, macrophage energy stably excreting S100A8 and S100A9, and
Both can recruit and activate again more inflammatory cell, thus forms positive feedback effect.Macrophage is the important cells of lungs,
Chronic inflammatory disease environment can be recruited by multiple adhesion molecule and chemotactic factor and assemble and discharge inflammatory mediator, with lung radiation injury
Scorching generation is closely related.Have research display In vitro culture cell line can induce under the irradiation of x-ray S100A8 and
The expression of S100A9, with ionizing radiation simulation inflammatory factor effect, its mechanism may promote that macrophage activation is relevant.At some
In the pathological process that inflammation participates in, S100A8 and S100A9 is effector or the amplifier of putative pro-inflammatory.Therefore,
Both are used as monitoring inflammatory conditions development and evaluating the biomarker of therapeutic effect.
In recent years, increasing research finds that S100A8 with S100A9 is relevant to many diseases of planting, such as both difference
In high expressed in tumor and inflammatory tissue, play an important role in chronic inflammatory disease acute, the multiple disease of research is occurred,
Development, treatment and prognosis all have directive significance, be expected to become detection relevant disease New Set.Early-stage Study basis finds,
In the inflammatory model that mice eye is set up, it has been found that the expression of S100A8 and S100A9 is the most obvious at gene and protein level
Rising.There is no its report being applied to radiation pneumonia early diagnosis and prognosis at present.
Summary of the invention
The purpose of the present invention be design a kind of can detection of radioactive pneumonia is sensitive in whole blood warning index, relate to
The method that two kinds of inflammatory factor S100A8 and S100A9 are used for the radiation pneumonia that early monitoring tumor radiotherapy causes, key point is
The design of S100A8 and the S100A9 primer of people.Specify that in peripheral blood, S100A8 and S100A9 (includes in different malignant tumor
Pulmonary carcinoma, breast carcinoma and the esophageal carcinoma) after radiotherapy, the patient of concurrent radiation pneumonia expresses and substantially increase, and without complication patient
There is notable significant difference.Height that wherein S100A8 expresses and radiation pneumonia patient's prognosis, including pneumonia reaction, fine
The change of dimensionization and pulmonary function has dependency, and radiation pneumonia patient's prognosis of prompting high expressed S100A8 is poor, has early
The generation of phase early warning radiation pneumonia and assess the using value of its prognosis.There is the generation of early monitoring radiation pneumonia, and
And the feature of disease prognosis can be assessed, its operation has simple, susceptiveness and the high advantage of specificity.
For achieving the above object, the radiation pneumonia that the present invention uses a kind of early monitoring tumor radiotherapy to cause is Fibrotic
Method, sending out by S100A8 and S100A9 primer and the design of antibody of people, early monitoring radiation pneumonia and pulmonary fibrosis
Raw, and assess disease prognosis.
Comprise the following steps:
(1) determine target detection object, get rid of IV phase extensive transferrer, row pulmonary surgery person, have asthma, severe chronic to prop up
Tracheitis, emphysema, pulmonary heart disease, Severe Pulmonary Infection person or have other serious disease persons；
(2) genome of peripheral blood in patients is extracted, with real time quantitative PCR method detection S100A8 and S100A9 at gene water
Flat excursion, leave and take sample time point be radiotherapy respectively before, radiotherapy 1 week, 2 weeks, 3 weeks, after radiotherapy 1 month, 2 months, 3
Month；
(3) the expression difference of analysis purpose gene；
(4) check and evaluation monitoring radiation pneumonia and pulmonary fibrosis in early days are got rid of according to result and infectious pneumonia.
Further, in described step (1), target detection object is malignant tumor and accepts 3 dimensional conformal radiation therapy
Patient.
Further, the detecting step of described step (2) is:
Venous blood collection 5ml was in routine blood test pipe on an empty stomach, through 0.9%NaCl solution two-fold dilution after splitting erythrocyte early morning
After, it being added slowly in the centrifuge tube equipped with equal-volume Ficoll 100 solution, 2500rpm rotating speed is centrifuged 10 minutes, in absorption
Between mononuclearcell be placed in 1.5ml EP pipe, 5000rpm is centrifuged 5 minutes, abandons supernatant, is precipitated as peripheral blood single core thin
Born of the same parents；
In every part of PERIPHERAL BLOOD MONONUCLEAR CELL, add 1ml Trizol solution, vibration mixing, add 200 chloroforms, reverse
For several times, 13000rpm is centrifuged 15 minutes, and upper water is transferred in another EP pipe mutually in mixing, adds equal-volume aqueous isopropanol, room
Gentle and quiet putting 5 minutes, 13000rpm is centrifuged 15 minutes, abandons supernatant, adds 500 μ l 70% washing with alcohol twice, vacuum drying, heavy
Shallow lake is dissolved in 20 μ l TE solution；
1ug RNA reverse transcription in the system of 20ul of each sample is become cDNA, with cDNA as template, expands purpose base
Because of S100A8 and S100A9, with GAPDH gene as internal reference, reaction system is 25ul, response procedures: denaturation 95 DEG C,
10min；Circulate 95 DEG C, 15sec, 60 DEG C, 1min, totally 40 circulations.
Further, described people's S100A8cDNA sequence:
ATGTTGACCGAGCTGGAGAAAGCCTTGAACTCTATCATCGACGTCTACCACAAGTACTCC
CTGATAAAGGGGAATTTCCATGCCGTCTACAGGGATGACCTGAAGAAATTGCTAGAGACC
GAGTGTCCTCAGTATATCAGGAAAAAGGGTGCAGACGTCTGGTTCAAAGAGTTGGATATC
AACACTGATGGTGCAGTTAACTTCCAGGAGTTCCTCATTCTGGTGATAAAGATGGGCGTG
GCAGCCCACAAAAAAAGCCATGAAGAAAGCCACAAAGAGTAG
Primer sequence:
Product length=235
Forward primer:CGAGCTGGAGAAAGCCTTGA 20
Reverse primer:TGCCACGCCCATCTTTATCA 20
People S100A9:
CDNA sequence:
ATGACTTGCAAAATGTCGCAGCTGGAACGCAACATAGAGACCATCATCAACACCTTCCAC
CAATACTCTGTGAAGCTGGGGCACCCAGACACCCTGAACCAGGGGGAATTCAAAGAGCTG
GTGCGAAAAGATCTGCAAAATTTTCTCAAGAAGGAGAATAAGAATGAAAAGGTCATAGAA
CACATCATGGAGGACCTGGACACAAATGCAGACAAGCAGCTGAGCTTCGAGGAGTTCATC
ATGCTGATGGCGAGGCTAACCTGGGCCTCCCACGAGAAGATGCACGAGGGTGACGAGGGC
CCTGGCCACCACCATAAGCCAGGCCTCGGGGAGGGCACCCCCTAA
Primer sequence:
Product length=178
Forward primer:GCTGGTGCGAAAAGATCTGC 20
Reverse primer:GTCACCCTCGTGCATCTTCT 20
People GAPDH:
CDNA sequence:
ATGGGGAAGGTGAAGGTCGGAGTCAACGGATTTGGTCGTATTGGGCGCCTGGTCACCAGG
GCTGCTTTTAACTCTGGTAAAGTGGATATTGTTGCCATCAATGACCCCTTCATTGACCTC
AACTACATGGTTTACATGTTCCAATATGATTCCACCCATGGCAAATTCCATGGCACCGTC
AAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATCACCATCTTCCAGGAGCGA
GATCCCTCCAAAATCAAGTGGGGCGATGCTGGCGCTGAGTACGTCGTGGAGTCCACTGGC
GTCTTCACCACCATGGAGAAGGCTGGGGCTCATTTGCAGGGGGGAGCCAAAAGGGTCATC
ATCTCTGCCCCCTCTGCTGATGCCCCCATGTTCGTCATGGGTGTGAACCATGAGAAGTAT
GACAACAGCCTCAAGATCATCAGCAATGCCTCCTGCACCACCAACTGCTTAGCACCCCTG
GCCAAGGTCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGACCACAGTCCATGCC
ATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGC
GGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTC
ATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTG
TCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAG
GTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAG
GTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGC
ATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGTATGACAACGAATTTGGCTAC
AGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAGTAA
Primer sequence:
Forward primer:CGGAGTCAACGGATTTGGTCGTAT 24
Reverse primer:AGCCTTCTCCATGGTGGTGAAGAC 24.
Further, described step (3) gene relative expression quantity computing formula is 2-△ △ ct, the table of analysis purpose gene
The amount of reaching difference.
Further, described infectious pneumonia gets rid of the one being detected as blood routine examination, Sputum culturing or imaging examination
Or several combination；
Wherein, what routine blood test detected specifically comprises the following steps that patient's venous blood collection about 5mL, is examined by blood cell analyzer
Survey various types of cells and the change of composition.Neutrophilic granulocyte and lymphocyte count and ratio in selective analysis result.If it is white
Total cellular score is more than 10 × 109Individual/L, neutrophilic granulocyte percentage ratio more than 70%, then prompts for bacterial pneumonia；If lymph
Cell counting or ratio raise, then be thought of as viral pneumonia more.The leukocyte of simple radiation pneumonia patient or lymphocyte
Ratio and counting often less than normal value.
Typically being preferred with expectorant in morning when specifically comprising the following steps that specimen taken of Sputum culturing detection, direct smear, light Microscopic observation
Cell quantity, every low power field squamous cell<10, leukocyte>25, or squamous cell/leukocyte<1:2.5,
Inoculated and cultured can be proceeded as " qualified " specimen.If continuous quadratic is separated to identical antibacterial, concentration 105-106CFU/ml,
It is regarded as the pathogenic bacterium of pneumonia.The Sputum culturing of simple radiation pneumonia patient is often negative.
Imaging examination specifically comprise the following steps that Thoracic CT scan, particularly high-resolution tomography can find more hidden
The focus hidden and characteristic change, and the diagnosis to infection and the discriminating of noninfectious disease, special infection all has the highest reality
By value.Selective analysis consolidation shadow, tuberosity shadow, cavity shadow, Diffuse alveolar is needed to ooze out the Differential Diagnosis of shadow in the application.Consolidation
Mostly shadow is the sign of antibacterial or fungal infection.If patient one Nian Nei pulmonary accepted radiotherapy, lung image display with irradiate
Lamellar that the visual field is consistent or streak shade will consider the possibility of concurrent radiation pneumonia.
The beneficial effects of the present invention is:
(1) research finds that S100A8 and S100A9 can apply to the early diagnosis of radiation pneumonia and pulmonary fibrosis with pre-
After；
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described.
Fig. 1 is MCP-1 cell expression of S100A8mRNA after lonizing radiation irradiate；
Fig. 2 is MCP-1 cell expression of S100A9mRNA after lonizing radiation irradiate；
Detailed description of the invention
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making
The every other embodiment obtained under creative work premise, broadly falls into the scope of protection of the invention.
Embodiment 1:
A kind of Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy causes, by the S100A8 of people and
S100A9 primer and the design of antibody, early monitoring radiation pneumonia and the generation of pulmonary fibrosis, and assess disease prognosis.
Comprise the following steps:
(1) determine target detection object, get rid of IV phase extensive transferrer, row pulmonary surgery person, have asthma, severe chronic to prop up
Tracheitis, emphysema, pulmonary heart disease, Severe Pulmonary Infection person or have other serious disease persons；
(2) genome of peripheral blood in patients is extracted, with real time quantitative PCR method detection S100A8 and S100A9 at gene water
Flat excursion, leave and take sample time point be radiotherapy respectively before, radiotherapy 1 week, 2 weeks, 3 weeks, after radiotherapy 1 month, 2 months, 3
Month；
(3) the expression difference of analysis purpose gene；
(4) check and evaluation monitoring radiation pneumonia and pulmonary fibrosis in early days are got rid of according to result and infectious pneumonia.
Infectious pneumonia: refer to the pneumonia caused by pathogenic microorganism such as antibacterial, virus, mycoplasmas.Generally with anti-
Raw element, antiviral therapy are main.
Non-infectious pneumonia: be the pneumonia caused by lonizing radiation, imbedibility foreign body or mechanical injuries.For the cause of disease
Symptomatic treatment.Radiation pneumonia belongs to Non-infectious pneumonia.
This research is to carry out detecting in early days for specific crowd (carrying out the tumor patient of radiotherapy to the chest), and prevention is often
See the generation of complication radiation pneumonia, instruct clinician to adjust therapeutic scheme and dosage in time, it is to avoid unfavorable treatment
Bring the damage of patient.
It is proposed that in addition to clear and definite radiotherapy history, the following detection means of best incorporated differentiate radiation pneumonia with
Infectious pneumonia, makes reasonably diagnosis.
1. blood routine examination
Patient's venous blood collection about 5mL, by blood cell analyzer detection various types of cells and the change of composition.Selective analysis
Neutrophilic granulocyte and lymphocyte count and ratio in result.If total white blood cells is more than 10 × 109Individual/L, neutral grain is thin
Born of the same parents' percentage ratio more than 70%, then prompts for bacterial pneumonia；If lymphocyte count or ratio raise, then it is thought of as virus more
Property pneumonia.The leukocyte of simple radiation pneumonia patient or percentage of lymphocyte and counting are often less than normal value.
2. Sputum culturing
Typically it is preferred with expectorant in morning during specimen taken, direct smear, observation of cell quantity under light microscopic, in every low power field squamous
Chrotoplast<10, leukocyte>25, or squamous cell/leukocyte<1:2.5 can proceed as " qualified " specimen
Inoculated and cultured.If continuous quadratic is separated to identical antibacterial, concentration 105-106CFU/ml, it is believed that be causing a disease of pneumonia
Bacterium.The Sputum culturing of simple radiation pneumonia patient is often negative.
3. imaging examination
Thoracic CT scan, particularly high-resolution tomography can find that the focus more hidden and characteristic change, to sense
Dye and the discriminating of noninfectious disease, the diagnosis of special infection all have the highest practical value.Need selective analysis in the application
Consolidation shadow, tuberosity shadow, cavity shadow, Diffuse alveolar ooze out the Differential Diagnosis of shadow.Mostly consolidation shadow is levying of antibacterial or fungal infection
As.If patient one Nian Nei pulmonary accepted radiotherapy, lamellar that lung image display is consistent with the irradiation visual field or streak the moon
Shadow will consider the possibility of concurrent radiation pneumonia.
Experiment show:
(1) infrastest:
Human macrophage MCP-1 cell lies in containing 10% hyclone (Fetal bovine serum, FBS)
RPMI1640 culture fluid, cultivates under the conditions of 5%CO2 saturation by 37 DEG C.24h before logarithmic proliferation phase cell irradiation, changes fresh cultured
Base, 60Co gamma-rays irradiates, absorbed dose 10Gy, and 24h after irradiation adds lipopolysaccharide LPS to final concentration 5ug/ml, continues to cultivate
24h。
With RT-PCR method detection human macrophage MCP-1 cell line by intracellular S100A8 after ionizing radiation and
The change of S100A9mRNA expression, it is seen that MCP-1 cell is merely by 48h or 5ug/mL after the irradiation of 10Gy 60Co gamma-rays
LPS cultivates 24h, S100A8mRNA and expresses enhancing；But 24h adds 5ug/mL LPS continuation training after 10Gy 60Co gamma-rays irradiates
Foster 24h, S100A8 and S100A9mRNA express and are remarkably reinforced (see Fig. 1, Fig. 2), illustrate that gamma-rays and LPS are collaborative and strengthen huge biting carefully
The expression of S100A8 and S100A9mRNA in born of the same parents MCP-1.
(2) clinical experiment:
1, the inclusive criteria of object of study: turn out to be malignant tumor in my institute through pathology and accept three-dimensional conformal radiation and control
Treat, patient's totally 67 example of the concurrent radiation pneumonia of clinical diagnosis, wherein pulmonary carcinoma 34 example, 22 cases of breast cancer, the esophageal carcinoma 11 example, it is contemplated that
Surviving >=6 months, KPS marks >=80 points, signs Informed Consent Form.
Exclusion standard: IV phase extensive transferrer, row pulmonary surgery person, have asthma, severe chronic bronchitis, emphysema,
Pulmonary heart disease, Severe Pulmonary Infection person or have other serious diseases.Year at age (59 ± 13)；Female 39 example, male 28 examples.Wherein there are 23 examples
There occurs radiation pneumonia, for pulmonary carcinoma 13 example, breast carcinoma 7 example, the esophageal carcinoma 3 example, individual month of morbidity 2-5 after radiotherapy, average out to
(3.2 ± 1.2) individual month, all meet the diagnostic criteria of radiation pneumonia.
2, diagnostic criteria: and late period radiation pulmonary injuries grade scale acute by U.S. tumour radiotherapy cooperative groups (RTOG)
Evaluating, the diffusivity lamellar density consistent with coverage field raises shadow to have symptoms of pneumonia and sign, particularly breast CT to show, examines
Break as radiation pneumonia.Because >=2 grades of radiation pneumonias i.e. occur persistent cough and (or) dyspnea, need to carry out at clinic
Reason, therefore selecting to observe terminal is >=2 grades of radiation pneumonias.According to whether occur radiation pneumonia that 60 example patients are divided into radioactivity
Pneumonia group (23 example) and "dead" pneumonia group (44 example).
3, therapeutic scheme:
Using IMRT, chest and abdomen body film is fixed, CT analog scanning, scanning slice spacing 5mm；Count at Varian Eclipse DX
Draw system drawing target outline, according to ICRU50, No. 62 reports, the vitals such as target area and lung, esophagus, heart, spinal cord are hooked
Draw.Tumor target (GTV) is visual tumors on CT (including lung inner disease foci and mediastinal lymph nodes)；Clinical target area (CTV) is outside GTV
Expanding 6-8mm, bronchus puts 12mm outside direction, including part lymphatic drainage district；Plan field is that corresponding CTV extends out 5-10mm
(considering the factors such as respiratory movement amplitude and Set-up errors to determine)；Launched field direction is utilized to see (beam eye view) function
Designing 4-6 irradiation field, application DVH and isodose curve, in conjunction with Pulmonary Function state with jeopardize organ by comprehensive quality evaluation
Determine treatment plan；Radiotherapy dosimetry is 2Gy/ time, 5 times/week, 60-70Gy；Collect the mean dose of GTV, maximal dose, minimum
Dosage, lungs V5, V10, V20 mean dose, Normal Tissue Complication probability (NTCP), esophagus V45, the average radioactive dose of heart
With spinal cord maximum radioactive dose totally 11 physical parameter data.
4, detecting step: extract the genome of peripheral blood in patients, detects S100A8 with real time quantitative PCR method (RT-PCR)
With S100A9 in the excursion of gene level, leave and take sample time point be radiotherapy respectively before, radiotherapy 1 week, 2 weeks, 3 weeks, radiotherapy
Latter 1 month, 2 months, 3 months.Early morning, venous blood collection 5ml was in routine blood test pipe on an empty stomach, molten through 0.9%NaCl after splitting erythrocyte
After liquid two-fold dilution, being added slowly in the centrifuge tube equipped with equal-volume Ficoll 100 solution, 2500rpm rotating speed is centrifuged 10 points
Clock, draws middle mononuclearcell and is placed in 1.5ml EP pipe, and 5000rpm is centrifuged 5 minutes, abandons supernatant, is precipitated as peripheral blood
Mononuclearcell.In every part of PERIPHERAL BLOOD MONONUCLEAR CELL, add 1ml Trizol solution, vibration mixing, add 200 chloroforms, top
Falling to mix for several times, 13000rpm is centrifuged 15 minutes, and upper water is transferred in another EP pipe mutually, adds equal-volume aqueous isopropanol,
Room temperature stands 5 minutes, and 13000rpm is centrifuged 15 minutes, abandons supernatant, adds 500 μ l 70% washing with alcohol twice, vacuum drying,
Resolution of precipitate is in 20 μ l TE solution.1ug RNA reverse transcription in the system of 20ul of each sample is become cDNA, with cDNA
For template, amplifying target genes S100A8 and S100A9, with GAPDH gene as internal reference, reaction system is 25ul, reaction interval
Sequence: denaturation 95 DEG C, 10min；Circulating 95 DEG C, 15sec, 60 DEG C, 1min, totally 40 circulations, gene relative expression quantity calculates public affairs
Formula: 2-△ △ ct, the expression difference of analysis purpose gene.
1 liang of table group patient's S100A8mRNA level determination results contrast before, during and after radiotherapy
2 liang of table group patient's S100A9mRNA level determination results contrast before, during and after radiotherapy
Before radiotherapy, radiation pneumonia group and S100A8 and S100A9mRNA level error in "dead" pneumonia group peripheral blood
Different not statistically significant；In Patients During Radiotherapy, radiation pneumonia group S100A8 and S100A9mRNA level gradually rise, "dead"
It is inconspicuous that pneumonia group S100A8 and S100A9mRNA level raise trend；Radiation pneumonia group patient before, during and after radiotherapy each
In time point blood, S100A8 and S100A9mRNA level is above "dead" pneumonia group (P < 0.05), is shown in Table 1, table 2.
The full group the shortest follow up time of patient is 3 months, the longest asks to be 12.5 months when following up a case by regular visits to, and the median follow-up time time is 6
Month, without case lost to follow-up.Follow up a case by regular visits to the period total incidence rate of acute RP 34% (23/67), 2 grades and more than 2 grades induced lung injuries generations
Rate is 30.4% (7/23).Statistics Application software SPSS 22.0, according to S100A8 and S100A9 expression and lung fibroblast
The incidence rate changed, by Pearson check analysis, finds that the expression of S100A8 and the generation of lung fibrosis are in the most just
Relevant (P ＜ 0.05), the expression of S100A9 is inconspicuous with the generation dependency of lung fibrosis.
Claims (8)
1. the Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy causes, it is characterised in that: by the S100A8 of people
With the design of S100A9 primer, early monitoring radiation pneumonia and the generation of pulmonary fibrosis, and assess disease prognosis.
2. the Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy as claimed in claim 1 causes, its feature exists
In comprising the following steps:
(1) determine target detection object, get rid of IV phase extensive transferrer, row pulmonary surgery person, have asthma, severe chronic bronchus
Inflammation, emphysema, pulmonary heart disease, Severe Pulmonary Infection person or have other serious disease persons；
(2) genome of peripheral blood in patients is extracted, with real time quantitative PCR method detection S100A8 and S100A9 at gene level
Excursion, leave and take sample time point be radiotherapy respectively before, radiotherapy 1 week, 2 weeks, 3 weeks, after radiotherapy 1 month, 2 months, 3 months；
(3) the expression difference of analysis purpose gene；
(4) check and evaluation monitoring radiation pneumonia and pulmonary fibrosis in early days are got rid of according to result and infectious pneumonia.
3. the Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy as claimed in claim 1 or 2 causes, its feature
It is: in described step (1), target detection object is malignant tumor the patient accepting 3 dimensional conformal radiation therapy.
4. the Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy as claimed in claim 1 or 2 causes, its feature
It is: the detecting step of described step (2) is:
Early morning, venous blood collection 5ml was in routine blood test pipe on an empty stomach, after splitting erythrocyte after 0.9%NaCl solution two-fold dilution, slow
Delaying joins in the centrifuge tube equipped with equal-volume Ficoll 100 solution, and 2500rpm rotating speed is centrifuged 10 minutes, draws middle single
Nucleus is placed in 1.5ml EP pipe, and 5000rpm is centrifuged 5 minutes, abandons supernatant, is precipitated as PERIPHERAL BLOOD MONONUCLEAR CELL；
In every part of PERIPHERAL BLOOD MONONUCLEAR CELL, add 1ml Trizol solution, vibration mixing, add 200 chloroforms, reverse mixing
For several times, 13000rpm is centrifuged 15 minutes, and upper water is transferred in another EP pipe mutually, adds equal-volume aqueous isopropanol, and room temperature is quiet
Putting 5 minutes, 13000rpm is centrifuged 15 minutes, abandons supernatant, adds 500 μ l 70% washing with alcohol twice, vacuum drying, precipitates molten
Solution is in 20 μ l TE solution；
1ug RNA reverse transcription in the system of 20ul of each sample is become cDNA, with cDNA as template, amplifying target genes
S100A8 and S100A9, with GAPDH gene as internal reference, reaction system is 25ul, response procedures: denaturation 95 DEG C,
10min；Circulate 95 DEG C, 15sec, 60 DEG C, 1min, totally 40 circulations.
5. the Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy as claimed in claim 1 or 2 causes, its feature
It is:
Described people's S100A8cDNA sequence:
ATGTTGACCGAGCTGGAGAAAGCCTTGAACTCTATCATCGACGTCTACCACAAGTACTCC
CTGATAAAGGGGAATTTCCATGCCGTCTACAGGGATGACCTGAAGAAATTGCTAGAGACC
GAGTGTCCTCAGTATATCAGGAAAAAGGGTGCAGACGTCTGGTTCAAAGAGTTGGATATC
AACACTGATGGTGCAGTTAACTTCCAGGAGTTCCTCATTCTGGTGATAAAGATGGGCGTG
GCAGCCCACAAAAAAAGCCATGAAGAAAGCCACAAAGAGTAG
Primer sequence:
Product length=235
Forward primer:CGAGCTGGAGAAAGCCTTGA 20
Reverse primer:TGCCACGCCCATCTTTATCA 20
People S100A9:
CDNA sequence:
ATGACTTGCAAAATGTCGCAGCTGGAACGCAACATAGAGACCATCATCAACACCTTCCAC
CAATACTCTGTGAAGCTGGGGCACCCAGACACCCTGAACCAGGGGGAATTCAAAGAGCTG
GTGCGAAAAGATCTGCAAAATTTTCTCAAGAAGGAGAATAAGAATGAAAAGGTCATAGAA
CACATCATGGAGGACCTGGACACAAATGCAGACAAGCAGCTGAGCTTCGAGGAGTTCATC
ATGCTGATGGCGAGGCTAACCTGGGCCTCCCACGAGAAGATGCACGAGGGTGACGAGGGC
CCTGGCCACCACCATAAGCCAGGCCTCGGGGAGGGCACCCCCTAA
Primer sequence:
Product length=178
Forward primer:GCTGGTGCGAAAAGATCTGC 20
Reverse primer:GTCACCCTCGTGCATCTTCT 20
People GAPDH:
CDNA sequence:
ATGGGGAAGGTGAAGGTCGGAGTCAACGGATTTGGTCGTATTGGGCGCCTGGTCACCAGG
GCTGCTTTTAACTCTGGTAAAGTGGATATTGTTGCCATCAATGACCCCTTCATTGACCTC
AACTACATGGTTTACATGTTCCAATATGATTCCACCCATGGCAAATTCCATGGCACCGTC
AAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATCACCATCTTCCAGGAGCGA
GATCCCTCCAAAATCAAGTGGGGCGATGCTGGCGCTGAGTACGTCGTGGAGTCCACTGGC
GTCTTCACCACCATGGAGAAGGCTGGGGCTCATTTGCAGGGGGGAGCCAAAAGGGTCATC
ATCTCTGCCCCCTCTGCTGATGCCCCCATGTTCGTCATGGGTGTGAACCATGAGAAGTAT
GACAACAGCCTCAAGATCATCAGCAATGCCTCCTGCACCACCAACTGCTTAGCACCCCTG
GCCAAGGTCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGACCACAGTCCATGCC
ATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGC
GGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTC
ATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTG
TCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAG
GTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAG
GTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGC
ATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGTATGACAACGAATTTGGCTAC
AGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAGTAA
Primer sequence:
Forward primer:CGGAGTCAACGGATTTGGTCGTAT 24
Reverse primer:AGCCTTCTCCATGGTGGTGAAGAC 24.
6. the Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy as claimed in claim 1 or 2 causes, its feature
It is: described step (3) gene relative expression quantity computing formula is 2-△ △ ct, the expression difference of analysis purpose gene.
7. the Fibrotic method of radiation pneumonia that early monitoring tumor radiotherapy as claimed in claim 1 or 2 causes, its feature
It is: described infectious pneumonia gets rid of one or more combinations being detected as blood routine examination, Sputum culturing or imaging examination.
8. the purposes of a S100A8 and S100A9 marker gene, it is characterised in that: for radiation pneumonia and pulmonary fibrosis
Early diagnosis and prognosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610781509.3A CN106319057A (en) | 2016-08-31 | 2016-08-31 | Method for early monitoring radioactive pneumonia fibrosis caused by tumor radiotherapy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610781509.3A CN106319057A (en) | 2016-08-31 | 2016-08-31 | Method for early monitoring radioactive pneumonia fibrosis caused by tumor radiotherapy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106319057A true CN106319057A (en) | 2017-01-11 |
Family
ID=57788405
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610781509.3A Pending CN106319057A (en) | 2016-08-31 | 2016-08-31 | Method for early monitoring radioactive pneumonia fibrosis caused by tumor radiotherapy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106319057A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660206A (en) * | 2018-08-14 | 2018-10-16 | 安徽未名天佳基因科技有限公司 | The genetic biomarker object and its application of prediction or auxiliary prediction lung radiation future trouble radiation pneumonitis risk |
| CN108893533A (en) * | 2018-08-14 | 2018-11-27 | 安徽未名天佳基因科技有限公司 | For predicting or assisting the kit of prediction lung radiation future trouble radiation pneumonitis risk |
| CN109628571A (en) * | 2018-12-10 | 2019-04-16 | 浙江省肿瘤医院 | Radiation pneumonitis incubation period sensitive gene quantitative detecting method |
| CN109825568A (en) * | 2019-01-09 | 2019-05-31 | 中国人民解放军军事科学院军事医学研究院 | Radiosensitive gene marker and the application in the low LET ray radiation of identification |
| CN110211695A (en) * | 2019-06-14 | 2019-09-06 | 上海市肺科医院 | A kind of pulmonary fibrosis severity appraisal procedure |
| CN111912973A (en) * | 2020-06-15 | 2020-11-10 | 上海焕一生物科技有限公司 | Kit for predicting lung injury clinical progress of breast tumor patient after radiotherapy |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550440A (en) * | 2008-04-02 | 2009-10-07 | 中国医学科学院肿瘤研究所 | Radiation pneumonitis related genes ATM and P53 as well as polymorphic locus and application thereof |
| CN104805197A (en) * | 2015-04-09 | 2015-07-29 | 嘉兴市第二医院 | Method for taking molecular marker in diagnosis and prognosis evaluation of breast cancer |
-
2016
- 2016-08-31 CN CN201610781509.3A patent/CN106319057A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550440A (en) * | 2008-04-02 | 2009-10-07 | 中国医学科学院肿瘤研究所 | Radiation pneumonitis related genes ATM and P53 as well as polymorphic locus and application thereof |
| CN104805197A (en) * | 2015-04-09 | 2015-07-29 | 嘉兴市第二医院 | Method for taking molecular marker in diagnosis and prognosis evaluation of breast cancer |
Non-Patent Citations (7)
| Title |
|---|
| CHUNGUANG HAN: "Time-dependent expression of leukotriene B4 receptors in rat collagen-induced arthritis", 《PROSTAGLANDINS & OTHER LIPID MEDIATORS》 * |
| 沈敬华: "《医学免疫学与病原生物学实验指导》", 30 June 2016, 北京大学医学出版社 * |
| 王剑青: "S100A8基因在肺纤维化大鼠模型肺组织中的表达", 《贵州医药》 * |
| 饶亚岚: "钙结合蛋白S100A8在放射性肺纤维化早期的表达及意义", 《辐射研究与辐射工艺学报》 * |
| 马青山: "放射性肺损伤PI预测模型的建立和应用研究", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》 * |
| 高明奇: "《临床医学概论》", 31 January 2016, 中国医药科技出版社 * |
| 魏晓丽: "钙结合蛋白S100A8及其临床应用前景", 《实用医院临床杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660206A (en) * | 2018-08-14 | 2018-10-16 | 安徽未名天佳基因科技有限公司 | The genetic biomarker object and its application of prediction or auxiliary prediction lung radiation future trouble radiation pneumonitis risk |
| CN108893533A (en) * | 2018-08-14 | 2018-11-27 | 安徽未名天佳基因科技有限公司 | For predicting or assisting the kit of prediction lung radiation future trouble radiation pneumonitis risk |
| CN108660206B (en) * | 2018-08-14 | 2022-11-22 | 天佳吉瑞基因科技有限公司 | Genetic biomarker for predicting or assisting in predicting risk of radiation pneumonitis after lung radiation and application of genetic biomarker |
| CN109628571A (en) * | 2018-12-10 | 2019-04-16 | 浙江省肿瘤医院 | Radiation pneumonitis incubation period sensitive gene quantitative detecting method |
| CN109825568A (en) * | 2019-01-09 | 2019-05-31 | 中国人民解放军军事科学院军事医学研究院 | Radiosensitive gene marker and the application in the low LET ray radiation of identification |
| CN109825568B (en) * | 2019-01-09 | 2022-07-19 | 中国人民解放军军事科学院军事医学研究院 | Radiation sensitive gene marker and application thereof in identifying low LET (low-LET-rate) ray radiation |
| CN110211695A (en) * | 2019-06-14 | 2019-09-06 | 上海市肺科医院 | A kind of pulmonary fibrosis severity appraisal procedure |
| CN110211695B (en) * | 2019-06-14 | 2021-08-06 | 上海市肺科医院 | Pulmonary fibrosis severity evaluation method |
| CN111912973A (en) * | 2020-06-15 | 2020-11-10 | 上海焕一生物科技有限公司 | Kit for predicting lung injury clinical progress of breast tumor patient after radiotherapy |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106319057A (en) | Method for early monitoring radioactive pneumonia fibrosis caused by tumor radiotherapy | |
| US9708669B2 (en) | Method for detecting genes sensitive to low-level ionizing radiation | |
| Liu et al. | Correlations between the expression of C-erB-2, CD34 and ER in breast cancer patients and the signs of conventional ultrasonography and ultrasound elastography. | |
| CN108254557A (en) | The system that the related primary carcinoma of liver prognosis of HBV is determined with PD-1 in the T cell of the CD8 positives and TIGIT | |
| CN107012207A (en) | LRP5 is used as clinical diagnosis and the application of suppression digestive system tumor | |
| RU2808194C1 (en) | Method of predicting development of radiation skin damage after radiation therapy in patients with breast cancer | |
| CN105200114A (en) | Method for predicting chemicotherapy sensitivity by using cervical carcinoma side population cell | |
| CN107217055B (en) | A kind of cancer diagnosis chip and its kit | |
| CN104713874A (en) | Breast cancer urine detection reagent, detection kit, as well as preparation and detection methods thereof | |
| Zhong et al. | PATH01. 03 Mutational Landscape and Prognosis Prediction for Immune Checkpoint Blockades of DNA Damage Response Pathways in Non-Small Cell Lung Cancer | |
| Herold-Mende et al. | MODL-04. ESTABLISHMENT OF A RELIABLE GLIOBLASTOMA TUMOR-ORGANOID PLATFORM ENABLES AUTOMATED HIGH-THROUGHPUT DRUG SCREENING ADVANCING PERSONALIZED MEDICINE | |
| Machida et al. | EP077/# 342 Exploratory study for precision medicine in cervical adenocarcinoma | |
| Tallberg | Anatomical location of metastatic lymph nodes in anal carcinoma | |
| Van De Voort et al. | 77P The cost effectiveness and cosmetic outcome of vacuum assisted excision compared to surgical excision for the treatment of benign and high-risk lesions | |
| Bruno et al. | JS06. 4. A Intracranial ependymomas of the adult: outcome and response to treatments across molecular subtypes | |
| Deantonio et al. | Hypofractionated volumetric modulated arc therapy (VMAT) in the treatment of advanced oesophageal cancer | |
| CN107674918A (en) | Applications of the 5p of has mir 195 as biomarker in pulmonary cancer diagnosis kit is prepared | |
| Sun et al. | Adiponectin-AdipoR1 axis in renal cell carcinoma plays a pivotal role in tumor progression and drug resistance | |
| Liu et al. | Carbon ion irradiation combined with PD-1 inhibitor trigger abscopal effect in Lewis lung cancer via a threshold dose | |
| Imai et al. | P3. 01-43 Predictive Value of Computed Tomography Characteristics for Nivolumab Response in Pretreated Non-Small Cell Lung Cancer | |
| Ahmad et al. | Clinical Outcomes Of Patients With Pathologic Residual Nodal Disease After Neo-Adjuvant Chemoradiation For Esophageal Carcinoma | |
| Nenakhova et al. | Dynamics of circulating tumor cells during neoadjuvant chemotherapy in patients with locally-advanced breast cancer | |
| Uchiyama et al. | The Relationship with Radiation Pneumonitis Followed By Stereotactic Body Radiotherapy and Emphysema Evaluated By CT | |
| Yu et al. | CT Diagnosis of COVID-19 Based on the Imaging Signs of Secondary Pulmonary Lobular | |
| Mody et al. | Rare Case of Primary Papillary Adenocarcinoma of the Lung |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170111 |