CN109680063A - A kind of kit for early stage of lung cancer diagnosis and First-line chemotherapy reaction assessment early warning - Google Patents
A kind of kit for early stage of lung cancer diagnosis and First-line chemotherapy reaction assessment early warning Download PDFInfo
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Abstract
The present invention relates to one kind to be based on blood platelet mRNAs:MAX、MTURN、HLA‑BThe early stage of lung cancer diagnosis and First-line chemotherapy reaction assessment early warning kit research and development, belong to medicine and hygiene fields.First the invention proposes a kind of marker for early stage of lung cancer diagnosis and First-line chemotherapy reaction assessment early warning, the marker is in plasma platelet mRNAMAX、MTURN、HLA‑BOr its joint；Kit is made in marker simultaneously, for early stage of lung cancer diagnosis and First-line chemotherapy reaction assessment early warning.Compared to health donors, the mRNA in early stage of lung cancer patientMAX、MTURN、HLA‑BExpression is significant to be increased, and has good early diagnosis ability.Combining diagnosis ROC curve analysis shows that, AUC 0.765, sensitivity 75%, specificity be 84.1%, better than individually diagnosis；MRNA simultaneouslyMAX、MTURN、HLA‑BExpression is related with First-line chemotherapy effect, can be used for assessing lung cancer First-line chemotherapy reaction result.
Description
Technical field
The present invention relates to one kind to be based on blood platelet mRNAs:MAX、MTURN、HLA-BThe early stage of lung cancer diagnosis and First-line chemotherapy
The kit research and development of reaction assessment early warning, belong to medicine and hygiene fields.
Background technique
Lung cancer is one of highest malignant tumour of morbidity and mortality in worldwide, and onset concealment is faced in early days
Bed performance lacks specificity, and most of patient has been in Locally Advanced when making a definite diagnosis or DISTANT METASTASES IN occurs, and prognosis is often poor,
5 years survival rates only 2%.Therefore, there is an urgent need for a kind of more sensitive diagnostic markers now, for DISTANT METASTASES IN occurs
Early diagnosis, early treatment and the Index for diagnosis of NSCLC provides objective basis.
The blood platelet (Tumor-educated platelets, TEPs) of tumor microenvironment education has been proved to can be with
Promote cancer progression, " education " of the blood platelet by tumour cell and tumor microenvironment, function may change, thus help
It helps tumour cell to escape immunologic surveillance, promotes growth and metastasis of tumours.These blood platelet (tumor- by tumour education
Educated platelets, TEP) function substantially change, show its RNA spectrum and protein spectrum change, make
Liquid biopsy based on TEP is able to for cancer diagnosis at may.The separation of blood platelet and its use in clinic can be replaced
For invasive surgical to diagnose.In addition, TEPs is easy to obtain in blood sample, and convenient for storage, it can be in blood circulation
It is arrived by stable detection, becomes the ideal biological marker of pulmonary cancer diagnosis.
Blood platelet is initiated by the multi-functional cytode fragment of bone marrow megakaryocyte, its rich content is only in peripheral blood
Inferior to red blood cell.Blood platelet takes part in the development and transfer of tumour, and tumour cell and its microenvironment can directly or indirectly change
Become the content of blood platelet RNA and protein.These are changed by the platelet function of tumour education, can be by a variety of
Mode promotes the survival and transfer of tumour cell.Tumour cell is indirect by different signaling molecules or passes through different receptors
(such as platelet activation receptor palatelet-selectin) directly interacts with blood platelet.Can be discharged after platelet activation a variety of growths because
Son and angiogenic factors, such as platelet derived growth factor, hepatocyte growth factor, vascular endothelial growth factor.Blood platelet
These factors can be discharged in transfer ecological niche, the microenvironment for promoting tumour growth is provided.In addition, blood platelet RNA express spectra has
Dynamic and instantaneity, by tumour cell and/or the blood platelet (Tumor-educated of tumor microenvironment education
Platelets, TEPs) function substantially change, show that the expression of its mRNA changes and have tumour-specific,
There are certain potentiality in terms of lesion detection and diagnosis.
Summary of the invention
Regarding to the issue above and status, the present invention provides a kind of easy to use, quick, specificity it is high for examining in early days
The kit of disconnected lung cancer.
A kind of marker for early stage of lung cancer diagnosis and First-line chemotherapy reaction assessment early warning, the marker is blood plasma blood
In platelet mRNAMAX、MTURN、HLA-BOr its joint.
A kind of kit for early stage of lung cancer diagnosis, comprising: marker, reverse transcription reaction system, quantitative fluorescent PCR are anti-
Answer system and internal reference system.
It include being directed in the quantitative fluorescent PCR reaction system respectivelyMAX、MTURNOrHLA-BForward primer and anti-
To universal primer, SYBR Green mixed liquor, pure water.
It include the forward and reverse primer of internal reference ACTB in the internal reference system.
The reagent of the reverse transcription reaction system includes that poly A polymerase, reverse transcriptase mixed liquor, reverse transcription are slow
Fliud flushing and nuclease free distilled water.
It is a kind of assessment lung cancer First-line chemotherapy reaction assessment early warning kit, comprising: marker, reverse transcription reaction system,
Quantitative fluorescent PCR reaction system and internal reference system.
It include being directed in the quantitative fluorescent PCR reaction system respectivelyMAX、MTURNOrHLA-BForward primer and anti-
To universal primer, SYBR Green mixed liquor, pure water.
It include the forward and reverse primer of internal reference ACTB in the internal reference system.
The reagent of the reverse transcription reaction system includes that poly A polymerase, reverse transcriptase mixed liquor, reverse transcription are slow
Fliud flushing and nuclease free distilled water.
Beneficial effect of the present invention
1, mRNA in Plasma of The Patients With Lung Cancer blood platelet:MAX、MTURN、HLA-BExpression significantly increases
Present invention firstly provides compared to health donors, mRNA in Plasma of The Patients With Lung Cancer blood platelet:MAX、MTURN、HLA-BTable
Increased up to horizontal significant, Combining diagnosis ROC curve analysis shows that, AUC is 0. 74, sensitivity 82.9%, and specificity is
62.4%, better than individually diagnosis.
2, mRNA in plasma platelet in early stage of lung cancer patient:MAX、MTURN、HLA-BExpression is significant to be increased
Compared to health donors, the mRNA in early stage of lung cancer patient:MAX、MTURN、HLA-BExpression is significant to be increased, and is had good
Early diagnosis ability.Combining diagnosis ROC curve analysis shows that, AUC is 0. 765, and sensitivity 75%, specificity is
84.1%, better than individually diagnosis.
3, lung cancer First-line chemotherapy reaction assessment early warning
Using Chi-square Test to the mRNA of First-line chemotherapy Plasma of The Patients With Lung Cancer blood platelet:MAX、MTURN、HLA-BLevel is determined
It measures, as the result is shown mRNA:MAX、MTURN、HLA-BExpression is related with First-line chemotherapy effect, can be used for assessing lung cancer one
Line chemotherapy side effect result.
Detailed description of the invention
Fig. 1: plasma platelet between patients with lung cancer and Healthy PeopleMAXThe differential expression of mRNA；
Fig. 2: plasma platelet between patients with lung cancer and Healthy PeopleMTURNThe differential expression of mRNA；
Fig. 3: plasma platelet between patients with lung cancer and Healthy PeopleHLA-BThe differential expression of mRNA；
Fig. 4: plasma platelet between early stage of lung cancer patient and Healthy PeopleMAX The differential expression of mRNA；
Fig. 5: plasma platelet between early stage of lung cancer patient and Healthy PeopleMTURN The differential expression of mRNA；
Fig. 6: plasma platelet between early stage of lung cancer patient and Healthy PeopleHLA-B The differential expression of mRNA；
Fig. 7:MAX、MTURN、HLA-BDiagnostic of the triple combination between patients with lung cancer and Healthy People；
Fig. 8:MAX、MTURN、HLA-BDiagnostic of the triple combination between early stage of lung cancer patient and Healthy People；
Fig. 9:MAX mRNAPR and Non-PR ratio after low expression and high expression group First-line chemotherapy；
Figure 10:MTURNPR and Non-PR ratio after low expression and high expression group First-line chemotherapy；
Figure 11:HLA-BPR and Non-PR ratio after low expression and high expression group First-line chemotherapy.
Specific embodiment
Implement below for illustrating the present invention, but is not intended to limit the scope of the invention.Unless otherwise specified, case study on implementation
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Case study on implementation 1:mRNAs:MAX、MTURN、HLA-BApplication in the diagnosis early stage of lung cancer
1, experimental design
The experimental design is that acquisition Shandong Tumor Hospital is diagnosed as cure the disease at the beginning of lung cancer people and healthy volunteer's plasma sample, point
From enrichment blood platelet and obtain blood platelet RNA.It is detected by the method for qPCRMAX、MTURN、HLA-BExpression, statisticallys analyze it
The difference between healthy volunteer and lung cancer, the early stage of lung cancer is expressed, and analyzes its diagnostic.
2, patient and sample involved in testing
It is included in 184 normal healthy controls persons that attached Shandong tumour hospital, Shandong University accepts for medical treatment in January, 2016 in October, 2018
And 223 patients with lung cancer, and checked based on postoperative pathological and carry out TNM classification.The clinical data that this research is included in mainly wraps
The age is included, gender, is drunk at AJCC neoplasm staging (the 8th edition), smoking history and the histological type of lung cancer etc..Patients with lung cancer totally 80
Example, the age 27 ~ 89 years old, normal healthy controls person totally 184, the age 29 ~ 74 years old.All patients receive fiberoptic bronchoscopy,
Percutaneous puncture or open surgical biopsy.In chemotherapy, subject's peripheral blood sample about 2ml is acquired before radiotherapy and operative treatment, is placed in and contains
It is mixed in the vacuum tube of EDTA anti-coagulants, and in 120g, room temperature is centrifuged 10 minutes, takes supernatant, repeatedly twice.360g later, 20
Minute.Then platelet sample is collected in centrifuge tube and be stored in -80 DEG C, it is spare.
3, blood platelet in blood plasmaMAX、MTURN、HLA-BHorizontal measurement
Blood platelet in patients with lung cancer and the blood plasma of healthy volunteer is analyzed using qPCR methodMAX、MTURN、HLA-BExpression water
It is flat, the specific steps are as follows:
3.1, the extraction of total serum IgE
0.5 ml TRIzol Reagent is added in the pellet platelets obtained to low-speed centrifugation, blows and beats repeatedly and places 10
Min-20 min keeps platelet lysates abundant；100 μ L chloroforms are added, concussion mixes, and is incubated for 10 min.4 ℃
12000 g are centrifuged 15 min(mixtures and are separated into lower layer's red phenol-chloroform liquid phase, and middle layer is White Flocculus, and upper layer is
Colourless water phase, RNA are distributed in water phase).Upper strata aqueous phase is transferred to newly without (attention in RNase EP pipe with pipettor
Middle layer is not drawn onto).250 μ L isopropanols are added, after being incubated for 10min, 4 DEG C of 12000 g is centrifuged 10 min, occurs white
The visible RNA sediment of color removes supernatant liquid.750 μ L, 75% ethyl alcohol is added, concussion mixes, 4 DEG C of 7500 g centrifugation 5
Min washs RNA, removes supernatant liquid.It is air-dried the RNA pipe min of 5 min~10,15 μ of μ L~20 L are added without RNase
Water dissolves RNA, is blown and beaten repeatedly with pipettor, and 55~60 DEG C of water-baths are incubated for the min of 5 min~10, dissolves RNA sufficiently.By RNA
Sample is placed in -80 DEG C and saves backup.Software detection sample concentration and purity are analyzed with ASP-3700.
3.2, reverse transcription reaction and qPCR
(1) genomic DNA removal reaction: reaction system: 2 μ 5 × gDNA of L Eraser Buffer, 1 μ L gDNA
Eraser,7 μL TotalRNA.Reaction condition: 5 min-30 min of 42 DEG C of 2 min(or room temperature) 4 DEG C keep.
(2) reverse transcription reaction: reaction system: the reaction solution of 10 μ L steps 1,1 μ L PrimeScript RT
Enzyme Mix I, 1 μ L RT Primer Mix, 4 μ L 5 × PrimeScript Buffer 2(for Real Time), 4
μL RNase Free dH2O.Reaction condition: 37 DEG C of 15 min, 85 DEG C of 5 sec, 4 DEG C of holdings.According to sample size, press
Following table reaction system, has configured mixed reaction solution, and concussion mixes.
1 reverse transcription reaction system of table
(3) internal reference system: the forward and reverse primer of internal reference ACTB.
In in Biohazard Safety Equipment, the reaction solution that the above configuration is completed is separately added into the hole of corresponding PCR amplification plate
It is interior, pay attention to avoiding pollution, encloses hyaline membrane later.Notice that each sample standard deviation does 1 secondary orifices, experiment is repeated 3 times.Careful centrifugation,
And it mixes.Operate the computer, detected using 480 quantitative PCR apparatus of LightCycler, the program of according to the form below setting carries out anti-
It answers.
2 PCR reaction condition of table
4, mathematical statistics are analyzed
Statistical analysis is carried out using SPSS 22.0 and 6.0 software of GraphPad Prism.If measurement data is comparing obedience just
State distribution parametric test is simultaneously indicated with average ± standard deviation；If disobeying normal distribution with non-parametric test Mann-
Whitney is examined and with median, and quartile spacing indicates.Comparison between three groups uses DKruskal-Wallis H
test.Comparison between frequency uses Chi-square Test.Diagnosis combination is established using dualistic logistic regression, is worked by subject special
Sign curve (ROC curve) and area under the curve (AUC) determine the mRNAs of differential expression.It is that difference has statistics meaning with p < 0.05
Justice.
5, patients with lung cancer case feature summarize and withMAX、MTURN、HLA-BRelationship between expression
The table 3 interpretation of result Clinical symptoms of patients with lung cancer, including age, gender, Smoking And Drinking situation, histological type and swollen
Tumor size.MAX、HLA-BExpression and the age, gender, smoking state and histological type it is unrelated (P> 0.05), difference without
Statistical significance.MTURNExpression except with gender, smoke, drink it is related in addition to, it is unrelated with other Clinical symptoms.MTURN
In the plasma platelet mRNA in patients with lung cancer women expression be significantly higher than male (P=0.004), difference has statistics
Learn meaning; MTURNIn non-smoking Plasma of The Patients With Lung Cancer blood platelet in mRNA expression be significantly higher than smoker (P=
0.0197), difference is statistically significant; MTURNExpression is significant in non-alcoholic Plasma of The Patients With Lung Cancer blood platelet mRNA
Higher than alcohol user (P=0.0194), difference is statistically significant.
3 cases of lung cancer feature of table summarize and withMAX、MTURN、HLA-BRelationship between expression
6, early stage of lung cancer patient cases feature withMAX、MTURN、HLA-BRelationship between expression
4 interpretation of result of the table Clinical symptoms of early stage of lung cancer patient, including age, gender, Smoking And Drinking situation, histological type
And tumor size.As the result is shownMAX、MTURN、HLA-BExpression and age, gender, Smoking And Drinking situation and tissue
Learn type, tumor size it is unrelated (P>0.05).No significant difference.
4 early stage of lung cancer case feature of table summarize and withMAX、MTURN、HLA-BRelationship between expression
7, plasma platelet mRNAMAX、MTURN、HLA-BIn Healthy People and patients with lung cancer level difference
Using the method for qPCR to the mRNA of the plasma platelet of patients with lung cancer and healthy donorsMAX、MTURN、HLA-BIt is horizontal
It is quantified, to determine mRNAMAX、MTURN、HLA-BWhether the biomarker of diagnosing can be used as.As the result is shown
mRNA MAX、MTURN、HLA-BExpression be remarkably decreased in patients with lung cancer, difference have statistical significance (p <
0.0001, p < 0.0001 p=0.0016 and), see Fig. 1,2,3.Similarly, using the method for qPCR to early stage of lung cancer patient and
The mRNA of the plasma platelet of healthy donorsMAX、MTURN、HLA-BLevel is quantified, to determine mRNAMAX、MTURN、HLA-BWhether the biomarker of the diagnosis early stage of lung cancer can be used as.MRNA as the result is shownMAX、MTURN、HLA-BExpression water
Put down and be remarkably decreased in early stage of lung cancer patient, difference have statistical significance (and of p=0.0101, p < 0.0001 p=
0.007) see Fig. 4,5,6.
8, plasma platelet mRNAMAX、MTURN、HLA-BDiagnostic of the triple combination to lung cancer
The present invention utilizes ROC curve to analyze in turn, detects mRNAMAX、MTURN、HLA-BTo the diagnostic of lung cancer, ROC is bent
Line analysis shows that AUC is 0. 74, sensitivity 82.9%, and specificity is 62.4%, better than individually diagnosis.See Fig. 7.
9, plasma platelet MAX combinesMTURNWithHLA-BTo the diagnostic of the early stage of lung cancer
The present invention utilizes ROC curve to analyze in turn, detects mRNAMAXJointMTURNWithHLA-BTo the diagnosis effect of the early stage of lung cancer
Can, Combining diagnosis ROC curve analysis shows that, AUC is 0. 765, sensitivity 75%, and specificity is 84.1%, better than individually examining
It is disconnected.See Fig. 8.
Case study on implementation 2:mRNAs:MAX、MTURN、HLA-BAssess the reaction of lung cancer front-line chemotherapeutic agents
1, experimental design
The experimental design is that acquisition Shandong Tumor Hospital is diagnosed as cure the disease at the beginning of lung cancer people and healthy volunteer's plasma sample, point
From enrichment blood platelet and obtain blood platelet mRNA.It is detected by the method for qPCRMAX、MTURN、HLA-BExpression, statisticallys analyze it
The expression of plasma platelet mRNA reacted with front-line chemotherapeutic agents between relationship, to predict its chemotherapy side effect.
2, patient and sample involved in testing
It is included in 37 patients with lung cancer that attached Shandong tumour hospital, Shandong University accepts for medical treatment in January, 2016 in October, 2018, year
Age 36 ~ 71 years old, in chemotherapy, subject's peripheral blood sample about 2ml is acquired before radiotherapy and operative treatment, is placed in the anti-coagulants containing EDTA
It is mixed in vacuum tube, and in 120g, room temperature is centrifuged 10 minutes, takes supernatant, repeatedly twice.360g later, 20 minutes.Then it collects
Platelet sample is in centrifuge tube and is stored in -80 DEG C, spare.In addition, 37 patients with lung cancer are based on after First-line chemotherapy according to reality
Body tumor the standard of curative effect evaluation carries out curative effect evaluation, i.e. the sum of target lesion maximum diameter reduces >=30% and (alleviates part) for PR;Target disease
Become the sum of maximum diameter to increase >=20% or new lesion occur into PD(progression of disease);Variation is alleviated in part
SD(stable disease).In MAX gene, having 23 patients is PR, and 13 patients are non-PR (PD+SD);In MTURN gene,
Having 17 patients is PR, and 8 patients are non-PR (PD+SD);In HLA-B gene, having 16 patients is PR, and 7 patients are non-
PR(PD+SD)。
3, blood platelet in blood plasmaMAX、MTURN、HLA-BHorizontal measurement
With case 1
4, statistical analysis
Statistical analysis is carried out using SPSS 22.0 and 6.0 software of GraphPad Prism.If measurement data is comparing obedience just
State distribution parametric test is simultaneously indicated with average ± standard deviation；If disobeying normal distribution with non-parametric test Mann-
Whitney is examined and with median, and quartile spacing indicates.Comparison between frequency uses Chi-square Test.Use binary logic
It returns and establishes diagnosis combination, differential expression is determined by Receiver operating curve's (ROC curve) and area under the curve (AUC)
MRNAs.It is that difference is statistically significant with p < 0.05.
5, plasma platelet mRNAMAXRelationship between being reacted with First-line chemotherapy
Interpretation of result patients with lung cancer mRNAMAXExpression reacted with front-line chemotherapeutic agents between relationship, Chi-square Test result
It showing, the number of cases of PR is lower than Non-PR in patients with lung cancer height expression group, and the number of cases of PR is higher than Non-PR in low expression group,For
0.030(P< 0.05), difference is statistically significant.It, may be pre- to illustrate that MAX mRNA expression is related with chemotherapy side effect
Survey chemotherapy side effect.
5 mRNA of tableMAXExpression reacted with First-line chemotherapy between relationship
6, plasma plateletMTURNRelationship between being reacted with First-line chemotherapy
Interpretation of result patients with lung cancer mRNAMTURNExpression reacted with First-line chemotherapy between relationship, Chi-square Test result is aobvious
To show, the number of cases of PR is lower than Non-PR in patients with lung cancer height expression group, and the number of cases of PR is higher than Non-PR in low expression group,For
0.023(P< 0.05), difference is statistically significant.It, may to illustrate that MTURN mRNA expression is related with chemotherapy side effect
Predict chemotherapy side effect.
6 mRNA MTURN expression of table reacted with First-line chemotherapy between relationship
7、Plasma plateletHLA-BRelationship between being reacted with First-line chemotherapy
Interpretation of result patients with lung cancer mRNA HLA-B expression reacted with First-line chemotherapy between relationship, Chi-square Test result is aobvious
To show, the number of cases of PR is lower than Non-PR in patients with lung cancer height expression group, and the number of cases of PR is higher than Non-PR in low expression group,For
0.011 (P < 0.05), difference is statistically significant.It, may to illustrate that HLA-B mRNA expression is related with chemotherapy side effect
Predict chemotherapy side effect.
7 mRNA of tableHLA-BExpression reacted with First-line chemotherapy between relationship
Claims (9)
1. a kind of marker for early stage of lung cancer diagnosis and First-line chemotherapy reaction assessment early warning, which is characterized in that the mark
Object is in plasma platelet mRNAMAX、MTURN、HLA-BOr its joint.
2. a kind of kit of the early stage of lung cancer diagnosis using marker described in claim 1 preparation, which is characterized in that packet
It includes: marker, reverse transcription reaction system, quantitative fluorescent PCR reaction system and internal reference system.
3. kit according to claim 2, which is characterized in that include point in the quantitative fluorescent PCR reaction system
Safety pin pairMAX、MTURNOrHLA-BForward primer and reverse primer, SYBR Green mixed liquor, pure water.
4. kit according to claim 2, which is characterized in that in the internal reference system including internal reference ACTB forward direction and
Reverse primer.
5. kit according to claim 2, which is characterized in that the reagent of the reverse transcription reaction system includes poly gland
Thuja acid polymerase, reverse transcriptase mixed liquor, reverse transcription buffer and nuclease free distilled water.
6. a kind of kit of the lung cancer First-line chemotherapy reaction assessment early warning using marker described in claim 1 preparation, packet
It includes: marker, reverse transcription reaction system, quantitative fluorescent PCR reaction system and internal reference system.
7. kit according to claim 6, which is characterized in that include point in the quantitative fluorescent PCR reaction system
Safety pin pairMAX、MTURNOrHLA-BForward primer and reversed universal primer, SYBR Green mixed liquor, pure water.
8. kit according to claim 6, which is characterized in that in the internal reference system including internal reference ACTB forward direction and
Reverse primer.
9. kit according to claim 6, which is characterized in that the reagent of the reverse transcription reaction system includes poly gland
Thuja acid polymerase, reverse transcriptase mixed liquor, reverse transcription buffer and nuclease free distilled water.
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| CN110042163A (en) * | 2019-05-17 | 2019-07-23 | 宋现让 | It is a kind of for pulmonary cancer diagnosis and shift early warning kit |
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| CN110042163A (en) * | 2019-05-17 | 2019-07-23 | 宋现让 | It is a kind of for pulmonary cancer diagnosis and shift early warning kit |
| CN110042163B (en) * | 2019-05-17 | 2022-05-17 | 宋现让 | Kit for lung cancer diagnosis and metastasis early warning |
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