CN108504637A - Chinese's clear cell carcinoma of ovary cell line - Google Patents
Chinese's clear cell carcinoma of ovary cell line Download PDFInfo
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- CN108504637A CN108504637A CN201710554757.9A CN201710554757A CN108504637A CN 108504637 A CN108504637 A CN 108504637A CN 201710554757 A CN201710554757 A CN 201710554757A CN 108504637 A CN108504637 A CN 108504637A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
Abstract
The invention belongs to technical field of microbe cell line, it is related to a kind of Chinese's clear cell carcinoma of ovary cell line and its method for building up.The present invention establishes Chinese's clear cell carcinoma of ovary cell line by the original cuiture of clear cell carcinoma of ovary subject ex vivo's sample, is named as FDOV1, preserving number NO.13812, and biological property includes:Monolayer adherence is grown, and loses contact inhibition, and form differs, rounded ellipse or spindle is flat or irregular shape;In vitro culture well-grown, continuous-stable were passaged to for more than 50 generations;There are complicated transposition, the chromosome aberrations such as missing in chromosome karyotype analysis result near diploid cell line;Exon sequencing is now shown is detected simultaneously by ARID1A fs mutation and PIK3CA H1047R mutation in FDOV1 and clinical tumor tissue;Tumor formation is good, can be used for establishing clear cell carcinoma of ovary animal model.The FDOV1 cell lines can be used for occurrence and development and the metastasis of clear cell carcinoma of ovary, new medicament screen, resistance mechanism and preclinical phase research.
Description
Technical field
The invention belongs to microorganism animal cell line technical fields, are related to clear cell carcinoma of ovary cell line, and in particular to one
Kind Chinese clear cell carcinoma of ovary cell line FDOV1 and its method for building up.
Background technology
It is the highest gynecologic malignant tumor of grade malignancy prior art discloses oophoroma.Clear cell carcinoma of ovary
(Ovarian Clear Cell Carcinoma, OCCC), is the second largest common histological subtypes, sees Asia women, mesh especially
The preceding result of study delivered is mostly from Japan.The country has research to deliver series of articles, wherein analyzing China more comprehensively
The characteristics of patient, prompts clear cell carcinoma of ovary to have special clinicopathological characteristics and biological behaviour, e.g., grade malignancy
High, poor prognosis, classic chemotherapy is insensitive, and effective treatment means etc. are lacked after recurrence, therefore, it is saturating further to explore ovary
The biological behaviour and molecular mechanism of clear cell carcinoma are particularly important in clinical research with practice.
Know in the industry, the foundation and identification of human tumor cell line are outstanding to the research of tumor biological behavior and molecular mechanism
It is important.It is reported that only there are 2 plants of people's clear cell carcinoma of ovary cell lines in Unite States Standard biology product collecting center (ATCC) at present
(ES-2, TOV-21G), and China's National Laboratory cellular resources shared platform only has 1 plant (ES-2).The text delivered in 2016
Offer the people's clear cell carcinoma of ovary cell line for disclosing and establishing and identifying so far:14 plants are shared between 1987 to 2016,
Only 1 plant comes from Hong-Kong, and other cell strains are Japanese scholars foundation.
Present situation based on the prior art, present inventor is proposed vertical and identifies that the ovary in China patient source is transparent
Cell carcinoma line, and in particular to a kind of Chinese's clear cell carcinoma of ovary cell line FDOV1 and its method for building up.
Have in the relevant prior art of the present invention and bibliography,
1.Chen W,Zheng R,Baade PD,Zhang S,Zeng H,Bray F,et al.Cancer
statistics in China,2015.CA Cancer J Clin.2016;66:115-32.
2.Sung PL,Chang YH,Chao KC,Chuang CM.Global distribution pattern of
histological subtypes of epithelial ovarian cancer:a database analysis and
systematic review.Gynecol Oncol.2014;133:147-54.
3.Okamoto A,Glasspool RM,Mabuchi S,Matsumura N,Nomura H, Itamochi H,
et al.Gynecologic Cancer InterGroup(GCIG)consensus review for clear cell
carcinoma of the ovary.Int J Gynecol Cancer. 2014;24:S20-5.
4.Ye S,Yang J,You Y,Cao D,Bai H,Lang J,et al.Comparative study of
ovarian clear cell carcinoma with and without endometriosis in People's
Republic of China.Fertil Steril.2014;102:1656-62.
5.Ye S,You Y,Yang J,Cao D,Bai H,Huang H,et al.Comparison of pure and
mixed-type clear cell carcinoma of the ovary:a clinicopathological analysis
of 341chinese patients.Int J Gynecol Cancer. 2014;24:1590-6.
6.Ye S,Yang J,Cao D,Bai H,Huang H,Wu M,et al.Characteristic and
prognostic implication of venous thromboembolism in ovarian clear cell
carcinoma:a 12-year retrospective study.PLoS One.2015;10:e0121818. 7.Ye S,
Yang J,You Y,Cao D,Huang H,Wu M,et al.Comparison of Clinical Characteristic
and Prognosis between Ovarian Clear Cell Carcinoma and Serous Carcinoma:A 10-
Year Cohort Study of Chinese Patients.PLoS One.2015;10:e0133498.
8.Yamada T,Hattori K,Satomi H,Okazaki T,Mori H,Hirose Y.
Establishment and characterization of a cell line(HCH-1)originating from a
human clear cell carcinoma of the ovary.J Ovarian Res. 2016;9:32..
Invention content
The purpose of the present invention is overcoming defect of the existing technology, a kind of clear cell carcinoma of ovary cell line is provided, is had
Body is related to a kind of Chinese's clear cell carcinoma of ovary cell line FDOV1 and its method for building up.
The present invention establishes a kind of clear cell carcinoma of ovary cell line from Chinese patients, which can stablize
Passage, Tumor formation is good, can be suitably used for establishing animal model.The cell line can be used for clear cell carcinoma of ovary occurrence and development and
The research of metastasis, drug resistance principle and new medicament screen etc..
A kind of Chinese's clear cell carcinoma of ovary cell line provided by the invention, name for people's clear cell carcinoma of ovary it is thin
Born of the same parents strain FDOV1, on March 16th, 2017 by China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC) preservation, depositary institution address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number CGMCC
NO.13812。
Chinese's clear cell carcinoma of ovary cell line of the present invention is made by following methods,
(1) it draws materials
In vitro fresh ovarian neoplasm Operated Specimens are obtained, lesion tissue immerses sterile PBS solution cleaning;
(2) original cuiture
Harder tumor tissues are chosen, the tissue several piece of grain of rice size is cut into, are uniformly inoculated in the big wares of 10cm
In, stationary culture, every 4 days half amounts change liquid 1 time, and cell is grown from tissue block after about 1 week;
(3) cell purification
Inoculation passes 1st generation after about 2 weeks, quiet by cell suspension inoculation in culture dish with 0.25% trypsin digestion
Set about 15~it is observed under inverted phase contrast microscope after twenty minutes, see that part cell is adherent, draws cell suspension and enter another culture
Continue to cultivate in bottle;As stated above, it is repeated 2 times passage, keeps tumour cell completely separable with fibroblast;
(4) secondary culture
When cell is covered with bottom of bottle, culture solution, PBS buffer solution rinsing, 0.25% trypsin digestion, by 1 are absorbed:2
Ratio passes on, and reaches for more than 50 generations at present;
Chinese's clear cell carcinoma of ovary cell line is obtained through Identification of Biological Characteristics, is named as FDOV1, preserving number is
NO.13812。
In the present invention:External conventional culture conditions are containing 10%FBS, 1% dual anti-Medium199 cell culture mediums, training
It is 37 DEG C to support temperature, gaseous environment 5%CO2/ 95% air, humidity are saturated humidity.
The cell line FDOV1 of the present invention has following biological characteristics:
1. monolayer adherence is grown, contact inhibition is lost, form differs, and rounded ellipse or spindle are flat, or not
Regular shape;
2. in vitro culture well-grown, passage in every 4 days is primary, can continuous-stable passage, reached for more than 50 generations at present;
3. it is near diploid cell line that chromosome karyotype analysis result, which prompts the cell, there is complicated transposition, missing etc.
Chromosome aberration;
4. fluidic cell cycle detection prompts:G1 phases cell 42.28%;G2 phases cell 36.10%;S phase cells
21.62%.
5. tumor-marker analyte detection:CA125:33.7U/ml;CA199:0.78U/ml; CA153:<1.00U/ml;
CA724:2.96U/ml;AFP:<0.61ng/ml;CEA:0.21ng/ml; HE4:<15nmol/L.
6. two generation sequencing results:FDOV1 and tumor tissues are detected simultaneously by ARID1A fs mutation and PIK3CA H1047R
Mutation.
7.FDOV1 immunocytochemical stains are consistent with clinical tumor Immunohistochemical coloration result, it was demonstrated that FDOV1
From tumor tissues.
8. the 18th generation FDOV1 cell is pressed 1*107The order of magnitude is inoculated in NOD/SKID nude mices, and transplantable tumor is formed after 20 days,
Pathological section prompts transplantable tumor histological characteristic similar to primary tumor.
The present invention provides a kind of Chinese's clear cell carcinoma of ovary cell line FDOV1, which can
To stablize repeatedly passage, it can be used for the research of clear cell carcinoma of ovary biological behaviour;With stronger oncogenicity, can be used for
Clear cell carcinoma of ovary animal model is established, obtained animal model is suitable for point of clear cell carcinoma of ovary occurrence and development
Sub- Mechanism Study can further probe into it to the resistance mechanism of classic chemotherapy drug and screen effective targeted drug, this is thin
Born of the same parents system is the ideal cell line of the basic research of people's clear cell carcinoma of ovary and preclinical phase application.
Description of the drawings
Fig. 1 .FDOV1 morphological observations (inverted phase contrast microscope 100 ×).
Fig. 2 .FDOV1 cell growth curves.
Fig. 3 .FDOV1 chromosome karyotype analysis.
Fig. 4 flow cytometry analysis FDOV1 cell cycles.
Fig. 5 bis- generations sequencing results.A:FDOV1 and tumor tissues single nucleotide variations (SNV) interpretation of result;B:FDOV1
And tumor tissues gene copy number variation (CNV) interpretation of result.
Fig. 6 .FDOV1 cells tumor formation MRI in NOD/SCID Mice Bodies is imaged.
Fig. 7 .FDOV1 cells, clinical tumor tissue and the analysis of transplantable tumor pathological section immunochemistry.
Specific implementation mode
Embodiment 1 prepares Chinese's clear cell carcinoma of ovary cell line FDOV1
(1) it draws materials
Obtaining in vitro fresh clear cell carcinoma of ovary operation Operated Specimens in May, 2016, (Specimen origin is big from Fudan University
Learn attached tumour hospital's clear cell carcinoma of ovary patient, female, 67 years old, pathological diagnosis:Left attachment clear cell carcinoma of ovary), take disease
Stove tissue 1cm3Sterile PBS solution cleaning is immersed, and removes connective tissue and slough;
(2) original cuiture
Tumor tissues are cut into about 1mm3Tissue block is uniformly inoculated in the big wares of 10cm by the fritter of size, stands training
It supports, every 4 days half amounts change liquid 1 time, observe that cell is grown from tissue block after about 1 week;
(3) cell purification
Inoculation passes 1st generation after about 2 weeks, with 0.25% trypsin digestion, is resuspended and is inoculated with serum free medium after centrifugation
In a culture dish, 15~20min is stood, is observed under inverted phase contrast microscope, sees that part cell is adherent, it is careful to draw
Cell suspension, which enters in another culture bottle, to be continued to cultivate.Passage processing 2 times as stated above, can make tumour cell and at fiber finer
Born of the same parents are completely separable;
(4) secondary culture
When cell is covered with bottom of bottle, the old culture solution in former bottle is absorbed, is rinsed 1~2 time with PBS buffer solution, with 0.25%
Trypsin digestion, by 1:2 ratio passage, reached for more than 50 generations at present;Chinese's clear cell carcinoma of ovary cell is made
System names as people clear cell carcinoma of ovary cell strain FDOV1, on March 16th, 2017 by Chinese microorganism strain preservation pipe
The reason committee common micro-organisms center (CGMCC) preservation, depositary institution address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Deposit number is CGMCC NO.13812.
It is containing 10%FBS, 1% dual anti-Medium199 cell culture mediums, culture that this, which tests external conventional culture conditions,
Temperature is 37 DEG C, gaseous environment 5%CO2/ 95% air, humidity are saturated humidity;
(5) identification of biological characteristics
Cellular morphology:It is observed under inverted phase contrast microscope, FDOV1 cell monolayer adherent growths lose contact inhibition, shape
State differs, rounded ellipse or spindle is flat or irregular shape (as shown in Figure 1);
Cell growth curve:The cell of logarithmic growth phase, adjustment cell density are per hole 2*103/ 100ul, is inoculated in
96 orifice plates, if 6 multiple holes;100ul CCK-8 reagents and culture medium 1 is added in the same time within continuous 10 days after inoculation:9 mixing
Liquid reacts 2 hours, and the absorbance (OD) at 450nm is measured with microplate reader, and using the time as horizontal axis, OD mean values are drawn for the longitudinal axis
Cell growth curve (as shown in Figure 2);
Chromosome karyotype analysis:Logarithmic growth phase cell is handled 6 hours, 37 DEG C of mistakes with 0.25 μ g/ml colchicines
Night acquires metaphase cell, fixer (methanol:Glacial acetic acid=3:1) fixed, sample is used after trypsin digestion is handled
Giemsa dye liquors dye, and are counted in microscopically observation, and 45 cells of random counter, wherein 2n=45/46 has 35
(78%), 2n=47 have 3 (7%), and 4n=86-90 has 6 (13%), other numbers 1 (2%), and chromosome exists
Complicated transposition, the chromosome aberrations such as missing, as a result meets the genetics characteristics (as shown in Figure 3) of malignant tumour;
Fluidic cell cycle detection:Collect 1*106Cell is in 15ml centrifuge tubes, after the PBS resuspensions twice of precooling, from
The heart abandons supernatant.Mixing is resuspended with 3ml-20 DEG C of 75% ethyl alcohol in cell precipitation, sets -20 DEG C overnight, and supernatant is abandoned in centrifugation, is added
500ulPI dyeing liquors are incubated 20min, flow cytometry analysis sample, with 488mm exciting lights, the filter of 600nm wavelength for 4 DEG C in the dark
Device detects PI fluorescence;The result shows that:G1 phases cell 42.28%;G2 phases cell 36.10%;21.62% (such as Fig. 4 of S phases cell
It is shown);
Tumor-marker analyte detection prepares 2*10^6 cell (about 2 25T culture bottles, every bottle of about 7ml culture), cultivates 48h
Afterwards, supernatant is collected, changes liquid, after 48h, collects supernatant, inspection CA125 again:33.7U/ml;CA199:0.78U/ml;
CA153:<1.00U/ml; CA724:2.96U/ml;AFP:<0.61ng/ml;CEA:0.21ng/ml;HE4:<15nmol/L;
Gene spectrum analysis was sequenced in two generations:By experimental procedure in Qiagen DNA extraction kits to patient's tumor tissues, blood
And FDOV1DNA is extracted, and DNA concentration is detected through microplate reader, gel electrophoresis checks DNA integrated degrees, will using Covaris
GDNA is interrupted at random, is purified with sample purification beads, the end of product by repairing at flat end,
Then carry out segment screening with magnetic bead, then add A in 3 ' ends, connection PCR reacts connector, carries out PCR amplification, then with make
Hybridized in hybridization buffer with Sure Select capture library, is hybridized using Dynal magnetic captures
Target fragment simultaneously isolates and purifies, then the DNA fragmentation and purified pcr product of PCR amplification capture carry out the quality inspection in capture library,
It is measured including agarose gel electrophoresis quality inspection, Qubit concentration mensurations and 2100Bioanalyzer fragment lengths, by DNA to be measured
Library concentration completes cluster generation on cBot, and then Flowcell is transferred on sequencing system, according to
The normal process of Illumina carries out the sequencing of two generations and CNV, SNV data analysis (sample effective depth>The region 250X, target
Capture rate about 70%), the results show that FDOV1 and tumor tissues are detected simultaneously by ARID1A fs mutation and PIK3CA H1047R
It is mutated (as shown in Figure 5);
FDOV1 cells and tumor tissues Immunochemical Identification:By specimens from pri and monoclonal FDOV1 cells Fu Er
Malin fixes, specimens paraffin embedding slices, carries out HE dyeing (as shown in Fig. 6), chooses HNF1 β, PAX8 to monoclonal FDOV1 cells
Immunocytochemical stain is carried out, is compared (as shown in Figure 7) with tumor tissues immunohistochemical staining, the results showed that, exempt from
Epidemic disease cytochemical staining is consistent with immunohistochemical staining result, it was demonstrated that it remains the phenotypic differentiation of primary tumor cell;
Cell Tumor formation:External extensive amplifying cells, by the 18th generation FDOV1 cell with 1*107The order of magnitude inoculates
3 nude mice whole transplantable tumors can be observed after NOD/SCID mouse 3,20 days to grow, after continued growth 20 days, toy MRI
Imaging shows that transplantable tumor is in nodositas, there is coating (as shown in Figure 6) outside;
The pathology of tumour are identified:Continue culture after a week, (remaining 1 mouse is continuing to train 2 mouse of conventional treatment
Die of pneumonia during supporting), tumor tissues are fixed through formalin, specimens paraffin embedding slices, carry out HE dyeing and immuning tissue
Learn dyeing, the results showed that, transplantable tumor immunohistochemical staining is consistent with former clinical samples immunohistochemical staining result,
Form correspondence (as shown in Figure 7).
The present invention provides a kind of Chinese's clear cell carcinoma of ovary cell line FDOV1, which can
To stablize repeatedly passage, it to be used for the research of clear cell carcinoma of ovary biological behaviour;With stronger oncogenicity, can be used for building
Vertical clear cell carcinoma of ovary animal model, animal model obtained are suitable for the molecule machine of clear cell carcinoma of ovary occurrence and development
System research and drug screening.
Claims (6)
1. a kind of Chinese's clear cell carcinoma of ovary cell line, which is characterized in that the cell line was named as FDOV1, in 2017 3
The moon 16 is by China General Microbiological culture presevation administrative center (CGMCC) preservation, preserving number NO.13812.
2. Chinese's clear cell carcinoma of ovary cell line as described in claim 1, which is characterized in that its biological property is:
Monolayer adherence is grown, and loses contact inhibition, and form differs, rounded ellipse or spindle is flat or irregular shape;
In vitro culture well-grown, passage in every 4 days is primary, being capable of continuous-stable more than 50 generations of passage;Chromosome karyotype analysis result is close
There are complicated transposition, the chromosome aberrations such as missing in diploid cell line;Cycle analysis is G1 phases cell 42.28%;The G2 phases are thin
Born of the same parents 36.10%;S phases cell 21.62%;Tumor markers testing result is CA125:33.7U/ml;CA199:0.78U/ml;
CA153:<1.00U/ml;CA724:2.96U/ml;AFP:<0.61ng/ml;CEA:0.21ng/ml;HE4:<15nmol/L;
FDOV1 is similar to tumor tissues gene profile analysis result height to be detected simultaneously by ARID1A fs mutation and PIK3CA H1047R are prominent
Become;FDOV1 immunocytochemical stains are consistent with primary tumor immunohistochemical staining result;Tumor formation is good, transplantable tumor with
The immunohistochemical staining result of primary tumor is consistent.
3. Chinese's clear cell carcinoma of ovary cell line as described in claim 1, which is characterized in that by following methods system
It is standby, using in vitro tumor tissues secondary culture after processing, Chinese clear cell carcinoma of ovary cell line FDOV1 is built up, is wrapped
Include following steps:
(1) it draws materials:In vitro fresh clear cell carcinoma of ovary tumor resection sample is obtained, lesion tissue immerses sterile PBS solution
Cleaning, and remove connective tissue and slough;
(2) original cuiture:Tumor tissue is inoculated in the big wares of 10cm, stationary culture, cell is grown from tissue block after 1 week;
(3) cell purification:1st generation is passed after being inoculated with 2 weeks, with 0.25% trypsin digestion, by cell suspension inoculation in culture dish
In, it is observed under inverted phase contrast microscope, sees that part cell is adherent, drawn cell suspension and enter in another culture bottle to continue to cultivate;
As stated above, it is repeated 2 times passage, keeps tumour cell completely separable with fibroblast;
(4) secondary culture:When cell is covered with bottom of bottle, culture solution is absorbed, PBS buffer solution rinses, 0.25% trypsin digestion,
By 1:2 ratio passed on for more than 40 generations;
(5) Identification of Biological Characteristics obtains Chinese's clear cell carcinoma of ovary cell line FDOV1, preserving number NO.13812.
4. Chinese's clear cell carcinoma of ovary cell line as described in claim 3, which is characterized in that in the preparation method,
The condition of in vitro culture used for:Containing 10%FBS, 1% dual anti-Medium199 cell culture mediums, cultivation temperature is 37 DEG C, gas
Body environment is 5%CO2/ 95% air, humidity are saturated humidity.
5. Chinese's clear cell carcinoma of ovary cell line described in claim 1 is for establishing clear cell carcinoma of ovary animal mould
Purposes in type.
6. purposes as described in claim 5, which is characterized in that the clear cell carcinoma of ovary animal model of foundation is used to prepare ovum
Nest clear cell carcinoma occurrence and development, metastasis, new medicament screen and resistance mechanism research platform.
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Cited By (2)
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CN109486767A (en) * | 2018-11-20 | 2019-03-19 | 上海市奉贤区中心医院 | A kind of people's epithelial ovarian cancer cell SHOV4 and its application |
CN109666747A (en) * | 2019-02-20 | 2019-04-23 | 天津脉络医学检验有限公司 | A kind of primer combination of probe object and its application |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109486767A (en) * | 2018-11-20 | 2019-03-19 | 上海市奉贤区中心医院 | A kind of people's epithelial ovarian cancer cell SHOV4 and its application |
CN109666747A (en) * | 2019-02-20 | 2019-04-23 | 天津脉络医学检验有限公司 | A kind of primer combination of probe object and its application |
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