CN108929909A - A screening kit for metastatic screening of papillary thyroid microcarcinoma - Google Patents
A screening kit for metastatic screening of papillary thyroid microcarcinoma Download PDFInfo
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- CN108929909A CN108929909A CN201810837364.3A CN201810837364A CN108929909A CN 108929909 A CN108929909 A CN 108929909A CN 201810837364 A CN201810837364 A CN 201810837364A CN 108929909 A CN108929909 A CN 108929909A
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Abstract
The invention discloses a kit for screening the metastasis of papillary carcinoma of thyroid and application thereof. The invention effectively determines whether the detected object has metastatic papillary thyroid cancer, the positive person suggests early operation, and the negative person can continue observation, thus avoiding over-treatment and insufficient treatment of the thyroid cancer and having good clinical application prospect.
Description
Technical field
The present invention relates to the detection methods of the small papillary carcinoma of thyroid gland.
Background technique
Thyroid cancer incidence rapid growth in the world, is the most fast solid malignant of annual growth, it is contemplated that
By 2019, thyroid cancer can become the women common cancer being number three.Most of new cases are all first among these
The small papillary carcinoma of shape gland (PTMC), i.e. diameter of tumor≤1cm thyroid papillary carcinoma.According to the different countries, PTMC exists
Ratio in new hair thyroid cancer is floated between 40%-50%.Whether the small papillary carcinoma patient of thyroid gland deposits both at home and abroad at present
Excessive diagnosing and treating be Thyroid Surgery most it is popular be also it is most disputable if one of inscribe: PTMC patient should select on earth
Select to perform the operation as early as possible and be also to continue with observation? but at present there is no both at home and abroad method accurately judge before surgery PTMC invasion and in advance
Afterwards, it can not also prejudge which PTMC can cause biggish harm that need to treat in time to patient in advance.
Transcription group (RNA-seq) is for transcript regulation, the alternative splicing, gene in comprehensive and accurate identification tumour
Fusion etc. parses important function of Gene Fusion during tumorigenesis that take place frequently.Base is based on by the way that transcription group is this
Because of the molecular label of express spectra, it can not only distinguish the phenotype ownership of cell, can be also used for the diagnosis of disease.Up to now,
It is related to the small papillary carcinoma of thyroid gland (PTMC) (diameter of tumor≤1cm) not yet thyroid papillary carcinoma (PTC) through retrieval
Transcription group research and document report.Transcription group is relatively low to sample size requirements, and a small amount of tissue can be detected, especially suitable
Close the research of PTMC.
Therefore, high for metastatic if the index for accurately prejudging PTMC high metastatic and poorer prognosis in advance can be found
The timely operative treatment of PTMC is to reach best prognosis, for more inert PTMC, selection observation.It thus can be to avoid
The over-treatment of PTMC can both mitigate the psychological burden of patient, can also mitigate the financial burden of national society, can also be more
Reasonable utilization valuableness and limited medical resource.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of determining test object (the small papillary carcinoma of thyroid gland) whether
Method in abnormality (high metastatic or poorer prognosis).
Present invention finds a kind of for detecting the small papillary carcinoma marker development process of thyroid gland and Index for diagnosis
Biomarker.
The present invention provides a kind of metastatic kit for screening of the small papillary carcinoma of thyroid gland, it includes optional is used for
Detect the reagent of the expression of TESC in the small papillary carcinoma tissue of thyroid gland.
TESC:tescalcin [Homo sapiens (human)],
Ensembl:ENSG00000088992MIM:611585；Vega:OTTHUMG00000144168.
Preferably, the reagent of TESC expression is that PCR detection is used in the detection small papillary carcinoma tissue of thyroid gland
Reagent.
Preferably, PCR detection reagent includes primer pair shown in NO.1~2 SEQ ID.
Preferably, the reagent of TESC expression is Western- in the detection small papillary carcinoma tissue of thyroid gland
Blot detection method reagent.
The present invention also provides the reagents of the expression of TESC in the detection small papillary carcinoma tissue of thyroid gland to prepare
Purposes in the metastatic screening agent of the small papillary carcinoma of thyroid gland.
Preferably, the reagent of TESC expression is that PCR detection is used in the detection small papillary carcinoma tissue of thyroid gland
Reagent.
Preferably, PCR detection reagent includes primer pair shown in NO.1~2 SEQ ID.
Preferably, the reagent of TESC expression is Western- in the detection small papillary carcinoma tissue of thyroid gland
Blot detection method reagent.
The expression water that kit of the present invention passes through TESC in the detection small papillary carcinoma specimens of thyroid gland to be checked
It is flat, high metastatic patient screening is come out, for the timely operative treatment of the high PTMC of metastatic to reach best prognosis, for compared with
For inert PTMC, selection observation.The psychology that thus to avoid the over-treatment of PTMC, can both mitigate patient is negative
Load, can also mitigate the financial burden of national society, can also more reasonably utilize expensive and limited medical resource.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 TESC expression testing result figure, a: fluorescence real-time quantitative PCR detects TESC rna expression water in the tissue
Flat detection；B: the protein expression water of TESC in 2 high transfer tissues and 3 negative for metastatic neoplasm tissues is detected with Western method
It is flat；C: the high transfer group of protein expression level and nothing of TESC turns in the high transfer tissue of immunohistochemistry detection and negative for metastatic neoplasm tissue
The gene of differential expression in shifting group.
Specific embodiment
The expression of 1 TESC of embodiment and the relationship of the small papillary carcinoma state of thyroid gland
1. pathology is collected
Collection carries out the PTMC case of first operation, art in May, 2018 from Huaxi Hospital Attached to Sichuan Univ in December, 2016
It is preceding to sign sample reception informed consent form, meet the pathological tissue specimen of the equal Regular operation excision of above-mentioned condition, art
Gland leaf where middle thyroid cancer stove, which is cut, takes cancer stove and normal tissue to be in charge of dress liquid nitrogen, and registration patient money in detail within 15 minutes
Material makes a record, and pathological examination is carried out after some months, and determination is that lymph node height shifts or without transfer, strictly selects and be included in nothing turn
It moves and the expression of the tumor tissues of high transfer group progress TESC detects, no transfer 35, height transfer 26.
The standard that sample is included in is as follows:
1. (because isthmus is relatively thin, therefore TMC is easy to invade out envelope, therefore isthmus for left lobe of thyroid gland or the small papillary carcinoma of lobus dexter
Cancer excludes), color ultrasound prompts tubercle maximum diameter between 5-10mm；
2. non-pregnancy or lactating female (because women disease incidence is apparently higher than male, in addition for avoid sex hormone etc. because
The interference of element, therefore transcript profile sequencing only incorporates female patients, male patient is used for later period functional verification)；
3. case is first operation.
The expression of 2.TESC detects
Rna expression is horizontal in the tissue for 2.1 fluorescence real-time quantitative PCR testing goal genes
The tissue saved in liquid nitrogen is taken out, place 5 minutes makes its softening on ice, is crushed with tissue homogenizer, weighing, often
1ml TRIZOL is added in 100mg tissue.The chloroform of 0.2ml is added in the sample of the TRIZOL reagent cracking of every 1ml, covers tightly pipe
Lid.Manually acutely after oscillation tube body 15 seconds, 15 to 30 DEG C are incubated for 2 to 3 minutes.12000rpm is centrifuged 15 minutes at 4 DEG C.After centrifugation
Mixing liquid is classified into the red phenol chloroform phase of lower layer, middle layer and colourless aqueous phase upper layer.RNA is all distributed in water phase
In.The volume of aqueous top layer is about the 60% of the TRIZOL reagent being added when being homogenized.Aqueous top layer is transferred to a clean nothing
In the centrifuge tube of RNA enzyme.Add isometric isopropanol mixing to precipitate RNA therein, be incubated for after ten minutes for 15 to 30 DEG C after mixing,
12000rpm is centrifuged 10 minutes at 4 DEG C.Sightless RNA precipitate will form glue in bottom of the tube and side wall before being centrifuged at this time
Precipitate block.Supernatant is removed, 75% ethyl alcohol (the 75% ethyl alcohol use of at least 1ml is added in the sample of every 1mlTRIZOL reagent cracking
DEPCH2O is prepared), clean RNA precipitate.After mixing, 7000rpm is centrifuged 5 minutes at 4 DEG C.Most of ethanol solution is carefully sucked,
Keep RNA precipitate 5-10 minutes dry in air at room temperature.When dissolving RNA, the 40 μ l of water that no RNA enzyme is first added is blown and beaten repeatedly with rifle
Several times, it makes it completely dissolved, the RNA solution of acquisition is stored in -80 DEG C for use.Use Agilent 2100Bioanalyzer
The concentration of (6000 Nano Kit of Agilent RNA) detection total RNA.
Sample cDNA synthesis: using article No. to synthesize for the kit of Femantas K1631,4 μ l of reverse transcription buffer, with
0.2 μ l of power traction object, 0.5 μ l, DEPC water of reverse transcriptase 13.3 μ l, 2 μ l of RNA template, 20 μ l of total volume.Tube bottom is flicked to mix solution
It closes, the of short duration centrifugation of 6000rpm.First 70 DEG C dry bath 3 minutes of mixed liquor, then 37 DEG C water-bath 60 minutes.After taking-up immediately 95 DEG C it is dry
Bath 3 minutes, obtaining reverse transcription end solution is cDNA solution, is stored in -80 DEG C for use.
Fluorescence real-time quantitative PCR: reaction system: 2 × PCR enzyme Mix10ul, upstream primer F 0.5ul, downstream primer R
0.5ul, add water to total volume 20ul.Reaction step: 45 PCR cycles (94 DEG C 20 seconds；60 DEG C 5 seconds).It is recorded after completing reaction
Ct value, calculation expression are horizontal.The kit used is bio-rad ssofast evagreen, article No. 172-5204AP.
Primer sequence is as follows:
TESC:Forward:TCAACCCCATCCGATCCAAA
Reverse:ATCTCAGCTTCTCCTTCCGG
2.2Western method (biorad 1620177, tesc antibody are available from Novus, article No. nbp2-13426) inspection
Survey the protein expression level of TESC in 2 high transfer tissues and 3 negative for metastatic neoplasm tissues.
The expression of 3 TESC and the relationship of the small papillary carcinoma state of thyroid gland
Rna expression is horizontal in the tissue for 3.1 fluorescence real-time quantitative PCR testing goal genes
Experimental result is as shown in Fig. 1 a figure and table 1:
The correlation of TESC and the small papillary carcinoma state of thyroid gland in the tissue of table 1
| The expression average level of TESC | |
| The non-diverting sample of the small papillary carcinoma of thyroid gland | 1.83 copy/ng cDNA |
| The small papillary carcinoma height of thyroid gland shifts sample | 8.55 copy/ng cDNA |
As shown in table 1 and Fig. 1 a figure, in the small papillary carcinoma patient of thyroid gland, the expression of the TESC of non-diverting patient
It is low, and the expression of high transfer patient is significantly high, the two significant difference.
In actually detected, if the TESC expression of discovery sample to be examined is greater than 8.55 copy/ng cDNA, it is recommended that
It performs the operation, if the 1.83 copy/ng cDNA of copy/ng cDNA~8.55, then suggests close follow-up check, monitor TESC
Expression is performed the operation when necessary, if less than 1.83 copy/ng cDNA, then suggests observing.
2.2 Western methods detect the protein expression water of TESC in 2 high transfer tissues and 3 negative for metastatic neoplasm tissues
It is flat.
As shown in Fig. 1 b, c figure, in the small papillary carcinoma patient of thyroid gland, the protein expression of the TESC of non-diverting patient is flat
It is horizontal low, and the protein expression level of high transfer patient TESC is significantly high, the two significant difference.
As can be seen from the above results, the non-diverting patient of the small papillary carcinoma of thyroid gland compares, the small mamillary of thyroid gland
The rna expression level and protein expression level of the TESC of cancer height transfer patient significantly increases, and illustrates the small mamillary of thyroid gland
The expression of TESC is positively correlated in the metastatic and tumor tissues of cancer, and it is small that the high expression of TESC can significantly improve thyroid gland
The high metastatic possibility of papillary carcinoma.
Therefore, can by detecting the expression of TESC in small papillary carcinoma specimens of thyroid gland to be checked,
High metastatic patient screening is come out.
The composition and its application method of the kit of 2 present invention detection TESC of embodiment
One, PCR detection kit
1, the composition of kit
Detection kit (50 person-portion):
| Component | Volume |
| Upstream primer | 0.5ul(10μM) |
| Downstream primer | 0.5ul(10μM) |
| 2 × PCR enzyme Mix | 10μL |
| dd water | To 20 μ L of total volume |
Primer sequence is as follows:
TESC:Forward:TCAACCCCATCCGATCCAAA
Reverse:ATCTCAGCTTCTCCTTCCGG
2, the application method of kit
The tumor tissues of sample to be examined are taken out, place 5 minutes makes its softening on ice, is crushed with tissue homogenizer, weighing, often
1ml TRIZOL is added in 100mg tissue.The chloroform of 0.2ml is added in the sample of the TRIZOL reagent cracking of every 1ml, covers tightly pipe
Lid.Manually acutely after oscillation tube body 15 seconds, 15 to 30 DEG C are incubated for 2 to 3 minutes.12000rpm is centrifuged 15 minutes at 4 DEG C.After centrifugation
Mixing liquid is classified into the red phenol chloroform phase of lower layer, middle layer and colourless aqueous phase upper layer.RNA is all distributed in water phase
In.The volume of aqueous top layer is about the 60% of the TRIZOL reagent being added when being homogenized.Aqueous top layer is transferred to a clean nothing
In the centrifuge tube of RNA enzyme.Add isometric isopropanol mixing to precipitate RNA therein, be incubated for after ten minutes for 15 to 30 DEG C after mixing,
12000rpm is centrifuged 10 minutes at 4 DEG C.Sightless RNA precipitate will form glue in bottom of the tube and side wall before being centrifuged at this time
Precipitate block.Supernatant is removed, 75% ethyl alcohol (the 75% ethyl alcohol use of at least 1ml is added in the sample of every 1mlTRIZOL reagent cracking
DEPCH2O is prepared), clean RNA precipitate.After mixing, 7000rpm is centrifuged 5 minutes at 4 DEG C.Most of ethanol solution is carefully sucked,
Keep RNA precipitate 5-10 minutes dry in air at room temperature.When dissolving RNA, the 40 μ l of water that no RNA enzyme is first added is blown and beaten repeatedly with rifle
Several times, it makes it completely dissolved, the RNA solution of acquisition is stored in -80 DEG C for use.Use Agilent 2100Bioanalyzer
The concentration of (6000 Nano Kit of Agilent RNA) detection total RNA.
Sample cDNA synthesis: using article No. to synthesize for the kit of Femantas K1631,4 μ l of reverse transcription buffer, with
0.2 μ l of power traction object, 0.5 μ l, DEPC water of reverse transcriptase 13.3 μ l, 2 μ l of RNA template, 20 μ l of total volume.Tube bottom is flicked to mix solution
It closes, the of short duration centrifugation of 6000rpm.First 70 DEG C dry bath 3 minutes of mixed liquor, then 37 DEG C water-bath 60 minutes.After taking-up immediately 95 DEG C it is dry
Bath 3 minutes, obtaining reverse transcription end solution is cDNA solution, is stored in -80 DEG C for use.
Fluorescence real-time quantitative PCR: reaction system: 2 × PCR enzyme Mix10ul, upstream primer F 0.5ul, downstream primer R
0.5ul adds water to total volume 20ul.Reaction step: 45 PCR cycles (94 DEG C 20 seconds；60 DEG C 5 seconds).It is recorded after completing reaction
Ct value, calculation expression are horizontal.
To sum up, the table that kit of the present invention passes through TESC in the detection small papillary carcinoma specimens of thyroid gland to be checked
Up to level, high metastatic patient screening is come out, can be used for the auxiliary diagnosis of the small papillary carcinoma of clinical thyroid, adopted for patient
Relevant remedy measures or decision are taken to provide effective foundation, potential applicability in clinical practice is good.
Sequence table
<110>Huaxi Hospital Attached to Sichuan Univ
<120>kit for screening of the metastatic screening of the small papillary carcinoma of a kind of thyroid gland
<130> GY026-18P1353
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>TESC-F(Artificial Sequence)
<400> 1
tcaaccccat ccgatccaaa 20
<210> 2
<211> 20
<212> DNA
<213>TESC-R(Artificial Sequence)
<400> 2
atctcagctt ctccttccgg 20
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593771A (en) * | 2018-07-27 | 2019-04-09 | 四川大学华西医院 | A kind of the 1100th bit base mutated gene of mankind MAP2K5 and its detection kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103874768A (en) * | 2011-05-05 | 2014-06-18 | 临床基因组学股份有限公司 | A method of diagnosing neoplasms |
-
2018
- 2018-07-26 CN CN201810837364.3A patent/CN108929909A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103874768A (en) * | 2011-05-05 | 2014-06-18 | 临床基因组学股份有限公司 | A method of diagnosing neoplasms |
Non-Patent Citations (3)
| Title |
|---|
| KSENIA G KOLOBYNINA ET AL.: "Emerging roles of the single EF-hand Ca2+ sensor tescalcin in the regulation of gene expression, cell growth and differentiation", 《J CELL SCI》 * |
| LEIGHTON STEIN ET AL.: "Copy Number and Gene Expression Alterations in Radiation-Induced Papillary Thyroid Carcinoma from Chernobyl Pediatric Patients", 《THYROID 》 * |
| YUN HEE KANG ET AL.: "The EF-hand calcium-binding protein tescalcin is a potential oncotarget in colorectal cancer", 《ONCOTARGET》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593771A (en) * | 2018-07-27 | 2019-04-09 | 四川大学华西医院 | A kind of the 1100th bit base mutated gene of mankind MAP2K5 and its detection kit |
| CN109593771B (en) * | 2018-07-27 | 2022-03-29 | 四川大学华西医院 | 1100 th base mutant gene of human MAP2K5 and detection kit thereof |
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