CN101469352B - In situ hybridization detection kit for early uterocarcinoma - Google Patents
In situ hybridization detection kit for early uterocarcinoma Download PDFInfo
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- CN101469352B CN101469352B CN2008100422629A CN200810042262A CN101469352B CN 101469352 B CN101469352 B CN 101469352B CN 2008100422629 A CN2008100422629 A CN 2008100422629A CN 200810042262 A CN200810042262 A CN 200810042262A CN 101469352 B CN101469352 B CN 101469352B
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Images
Abstract
The invention relates to an in-situ hybridization detection reagent kit for early uterine cancer, which comprises a hybridization probe and a marker, wherein a sequence of the hybridization probe is shown in SEQ ID NO.1. The invention also provides an in-situ hybridization detection method for a P2RX7 gene. In addition, the invention also provides application of the reagent kit to preparation of a medicine for detecting a uterine cancer disease. The reagent kit has the advantages that the reagent kit has the characteristics of high sensitivity and strong specificity. The detection method has convenient and simple operation, and can be universally used and promoted in district hospitals and above.
Description
[technical field]
The present invention relates to a kind of test kit, specifically about a kind of early stage uterus carcinoma hybridization in situ detection kit and detection method and application.
[background technology]
The data that provides according to domestic and international authoritative institution, the newly-increased number 1,600,000 of the annual cancer of China, death toll nearly 1,600,000, the patient 6,000,000, the annual newly-increased cancer patients 8,000,000 in the whole world, death toll is near 8,000,000, and the patient has 8,400 ten thousand people approximately, to double to the above number of the year two thousand twenty, this is one group of fearful numeral.Uterus carcinoma is a kind of malignant tumour of serious harm WomanHealth, and sickness rate is stable in recent years rises and the rejuvenation that becomes.Uterus tumor is one of common gynecologic malignant tumor, and sickness rate occupies second in female tumor, according to the world wide statistics, and the tumor of cervix morbidity example of annual estimation 46.6 ten thousand.Wherein 80% case occurs in developing country.China's annual new discovery uterus carninomatosis 13.5 ten thousand accounts for 1/3 of whole world morbidity number, is first of China's gynecologic malignant tumor.World's gynecology recent studies on shows: the uterus carcinoma morbidity all comes from human papilloma virus infection (HPV).Uterus carcinoma is one of common gynecologic malignant tumor, and sickness rate occupies second in women's malignant tumour, be only second to mammary cancer.China has just actively developed the preventing and controlling of uterus carcinoma from the latter stage fifties, some other adhere to that the area of carrying out examination all obtained significant effect as Shanghai textile department, Jingan County, Jiangxi and.The mortality ratio (Chinese population age regulation) of whole nation cervical cancer by dropping to 3.25/10 ten thousand of the nineties, has descended 69% 10.28/10 ten thousand of seventies.Compare with world other countries, also the high level by the seventies drops to medium level.But because China region is wide, populous, still have every year new cases about 100,000, account for 1/5th of world's uterus carcinoma new cases sum.In recent years, according to some countries and regions, the morbidity of uterus carcinoma and mortality ratio were roughly stable, but the part has the year mild case of the trend, particularly cervical cancer of growth to increase to some extent.In recent years, also there is the report of uterus carcinoma patient rejuvenation trend China some areas.Although in the past 20 in the period of, the mortality ratio of China's uterus carcinoma descends significantly, in the midwestern some areas of China, the morbidity of uterus carcinoma and mortality ratio are but high all the time in decades.As counties such as Wudu, Gansu, Yangcheng, Shanxi, the cervical cancer mortality ratio surpasses 10 times of national uterus carcinoma mortality ratio, far above world average level up to 36.00/10 ten thousand.Uterus carcinoma changes to the formation cancer before cancer, how demand eight accomplishes that to ten years early detection and diagnosis are the keys that reduces M ﹠ M.Whether conventional plate coating checking can only be diagnosed is cancer cells, that is to say that finding cancer cells has been late period, and often patient's lifetime is not long.How it has very important clinical meaning in more early discovery (canceration early stage).The all main health tissues of the U.S. think that all the early detection to cancer is to save life, reduces the key of treatment of late stage expense.
Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as cancer genomics, so far, we might do more accurate early diagnosis on gene level, just can accomplish the early prediction diagnosis in canceration early stage or cancer cells formation (during mono-clonal).
American Studies personnel doctor Gorodeski finds that in experiment the P2RX7 gene can be distinguished cancer, pre-cancer and healthy tissues, and the P2RX7 gene becomes the first biomarker that can detect the cell carcinogenesis potentiality.They just observe in breadboard cell cultures at first, uterus carcinoma cell, uterine epithelial cell proliferative cell and normal uterus epithelial cell, the P2RX7 gene is all different with the P2RX7 protein expression, uterine cancers is expressed minimum, the uterine epithelial cell proliferative cell is expressed medium degree, and the normal uterus epithelial cell is expressed 90-100%.With they have analyzed clinical case and normal people with quadrat method, come to the same thing.The P2RX7 gene extensively is present in body epithelium and the epithelial cell, and it is the unique now marker that can organize before the differentiation canceration before tumour takes place.The data that the detection technique (gene diagnosis kit of combination) of P2RX7 gene is used for screening clinical gynaecology uterus carcinoma is not appeared in the newspapers.
Hybridization in situ technique (in situhybridization) is that molecular biology and cytochemistry technology are combined, and is probe with the nucleic acid molecule of mark, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark, under optimum conditions with histocyte in the complementary nucleic acid strand be that target nucleic acid is hybridized, with radioautograph or immunocytochemistry label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.
Chinese patent literature CN1556221 discloses " IC53 gene and associated products diagnosis thereof and the purposes of treatment colorectal carcinoma and a kind of test kit of diagnosing colon cancer ".Chinese patent literature CN1769485 discloses " rickettsial in situ hybridization detection method of Pinna(Atrina)pectinate pathogeny and test kit thereof ".Chinese patent literature CN1556410 discloses " a kind of detection kit of fish virus and detection method thereof ".Chinese patent literature CN1680597 discloses " a kind of PCR kit for fluorescence quantitative that is used for detection by quantitative hepatitis C virus (HCV) ".Chinese patent literature CN2918435 discloses " a kind of oligonucleotide chip and test kit that detects obesity-related gene SNP ".But, do not appear in the newspapers about the hybridization in situ detection kit and the detection technique thereof of P2RX7 gene.
[summary of the invention]
Technical problem to be solved by this invention is that a kind of early stage uterus carcinoma hybridization in situ detection kit and detection method and application are provided.Find uterus carcinoma early, reduce the M ﹠ M of uterus carcinoma.
In order to address the above problem, the invention provides the application of a kind of early stage uterus carcinoma hybridization in situ detection kit in preparation detection uterus carcinoma disease medicament, described test kit comprises hybridization probe, marker, and wherein said hybridization probe sequence is shown in SEQ ID NO.1.The P2RX7 gene order is seen SEQ IDNO.1, and the nucleotide sequence length of P2RX7 gene is 3155bp, and CDS is 97..1884, is positioned at karyomit(e) 12q24 " on the site.
As optional technical scheme, the RNA sequence of described hybridization probe sequence shown in SEQ ID NO.1.
As optional technical scheme, described marker is selected from radionuclide or non-radioactive marker.
As optional technical scheme, described radionuclide is selected from
3H,
35S,
125I or
32A kind of among the P.
As optional technical scheme, described non-radioactive marker is selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.
As optional technical scheme, described non-radioactive marker is preferably from digoxin.
As optional technical scheme, also comprise synergistic agent, described synergistic agent is the alkaline phosphatase enzyme antibody.
The present invention also provides a kind of early stage uterus carcinoma hybridization in situ detection kit, comprises hybridization probe, marker, and described hybridization probe sequence is shown in SEQ ID NO.1, and described marker is selected from radionuclide or non-radioactive marker
The present invention also provides a kind of in situ hybridization detection method of P2RX7 gene, and this method may further comprise the steps:
A, the hybridization probe in the test kit is contacted with RNA to be measured in the substrate, form hybridization complex;
The hybridization complex that b, detection a step obtain.
As optional technical scheme, the condition that forms hybridization complex in the described a step is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hours, and described substrate is selected blood cell sample or histocyte sample for use.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, with the P2RX7 gene is detected object, synthesising probing needle is the DNA or the RNA sequence of P2RX7 gene, and the substrate of detection is the expression amount of the RNA of blood of human body sample white corpuscle or uterine cancer cell cell.The display packing of hybridization in situ technique can provide the sxemiquantitative or the quantitative expression deciding degree of P2RX7 gene.Judge above expression of gene amount according to the immunohistochemical methods colour developing of hybridization back, normal uterine tissue P2RX7 gene is not expressed, promptly do not develop the color, uterine cancer cell hyperplasia is not expressed, and does not develop the color, and the P2RX7 gene is expressed the uterine cancers patient, i.e. colour developing, the unique of this gene expresses when cervical cancer, and be very special, and clinical meaning is very great.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein Za Jiao concrete steps comprise:
Instrumentation:
1). sample to be measured is put into reactive tank;
2). instrument discards liquid automatically, adds Digestive system automatically;
3). instrument discards liquid automatically, and the back is fixing automatically;
4). instrument discards liquid automatically, automatically prehybridization (42 ℃);
5). instrument discards liquid automatically, cleans automatically;
6). instrument discards liquid automatically, automatically hybridization (42 ℃);
7). instrument discards liquid automatically, cleans automatically;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, cleans colour developing automatically;
10). take out the mounting microscopy.
The invention has the advantages that:
1, test kit provided by the invention has characteristics highly sensitive, high specificity.
2, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
3, clinical meaning of the present invention is that more early stage tracking detects cervical cancer generation dynamic process, and the detection and the screening implement that are used for the cervical cancer preventive medicine.Diagnostic kit of the present invention is with other detects the oncofetal protein mark clinically, and the medical imaging inspection has remarkable difference.The present invention can (uterine cervix canceration early stage or cancer cell multiplication) detect the P2RX7 gene unconventionality expression on gene level, before medical imaging inspection and examinations of uterus are not found occupancy cervical cancer focus, before the cancer biochemical indicator does not produce unusually, also do not form before the lump, can accomplish the information acquisition of above abnormal gene expression early, give real early diagnosis of clinical lung cervical cancer sufferer.So just might implement early diagnosis, early prevention, the early treatment of cervical cancer, might be from thorough root cervical cancer foul disease on the source.
[description of drawings]
Fig. 1 is a uterus cancer patient P2RX7 genetic expression picture in the embodiment of the invention.
Fig. 2 is a healthy tissues P2RX7 genetic expression picture in the embodiment of the invention.
Fig. 3 is a uterine cancer cell hyperplasia P2RX7 genetic expression picture.
[embodiment]
Below in conjunction with figure the embodiment of a kind of early stage uterus carcinoma hybridization in situ detection kit provided by the invention and detection method and application is elaborated.
Embodiment 1
A kind of early stage uterus carcinoma hybridization in situ detection kit comprises hybridization probe, marker, synergistic agent, and wherein, described hybridization probe sequence is shown in SEQ ID NO.1.The hybridization probe digoxigenin labeled.Other liquid and sample in the test kit are composed as follows:
Digestive system 100 μ l/ manage 1 pipe/box colourless transparent liquid
Protection liquid 100 μ l/ pipe 1 pipe/box colourless transparent liquid
Prehybridization solution 1300 μ l/ manage 2 pipe/box colourless transparent liquids
Justice hybridization solution 10 μ l/ pipe 1 pipe/box colourless transparent liquid
Antisense hybridization solution 10 μ l/ manage 1 pipe/box colourless transparent liquid
Confining liquid 1000 μ l/ manage 1 pipe/box colourless transparent liquid
Alkaline phosphatase enzyme antibody 1 μ l/ manages 1 pipe/box colourless transparent liquid
Developer A 175 μ l/ manage 1 pipe/box yellow liquid
Developer B 320 μ l/ manage 1 pipe/box colourless transparent liquid
Light yellow or the colourless transparent liquid of damping fluid I 10x 90ml/ bottle 1 bottle/box
Light yellow or the colourless transparent liquid of damping fluid II 10x 80ml/ bottle 1 bottle/box
Light yellow or the colourless transparent liquid of damping fluid III10x 20m/ bottle 3 bottle/boxes
Light yellow or the colourless transparent liquid of damping fluid IV 10x 90ml/ bottle 1 bottle/box
Stationary liquid 90ml/ bottle 1 bottle/box colourless transparent liquid
6/box of positive control sample
Mentioned reagent composition explanation: (all reagent are available from SIGMA)
1, Digestive system: the 20mg/ml Proteinase K, the 100mg Proteinase K adds DEPC-H
2O5ml;
2, the glycine of protection liquid: 0.2g adds 1 * damping fluid I of 1ml;
3, prehybridization solution: 1 * damping fluid II7.5ml
50×D3ml
10mg/ml?yestt-RNA750ul
11mg/ml?SALMON?TESTES?DNA?682ul
0.04M?EDTA?3ml
50％formamide?15ml
4, the bloking of confining liquid: 0.03g (buying from Roche Holding Ag) adds 1ml1 * damping fluid III;
5,10x damping fluid I:(PH7.1-7.4)
NaCl?80g
Na
2HPO
4.12H
2O?360g
KCl?2g
KH
2PO
4?2g
Add tri-distilled water to 1l, and autoclaving;
6,10x damping fluid II:(PH7.0)
NaCl?175.3g
Trisodium Citrate 88.2g
Several of HCl
Add tri-distilled water to 1l, and autoclaving;
7, damping fluid III:(PH7.9)
Tris 121.1g
NaCl 87.66g
About HCl 60ml
Add tri-distilled water to 1l, and autoclaving;
8, damping fluid IV:
1M Tris-HCl (PH9.5): Tirs121.1g adds about HCl 3ml, adds water 900ml, transfers PH to 9.5, adds water to 1l, and autoclaving;
1M NaCl:NaCl 58.44 adds water to 1l, and autoclaving;
0.5M MgCl
2: 101.65g MgCl
2.6H
2O adds water to 1l, and autoclaving;
9, stationary liquid: Paraformaldehyde 96 40g adds 1 * damping fluid I to 1l, and heat (about 50-60 degree) is stirred to dissolving a little;
10, developer A:NBT1g adds 70%DMF11.44ml;
11, developer B:BCIP1g adds 100%DMF30ml.
Test kit of the present invention can many person-portions use or person-portion use.
Embodiment 2
A kind of in situ hybridization detection method of P2RX7 gene and test kit are used
One, sample disposal
1, with the centrifuge tube of 10ml, dress 4.5ml lymphocyte separation medium, again the 3ml anticoagulation is slowly added contain lymphocyte separation medium (blood: in the centrifuge tube of lymphocyte separation medium=1:1.5), the centrifugal 10min of 2000r/min;
2, draw the middle layer white corpuscle to another centrifuge tube, in this pipe, add 1 * damping fluid I of about twice again, mixing, the centrifugal 10min of 1500g/min;
3, abandon supernatant. precipitation adds 1 * damping fluid I of about twice, mixing, the centrifugal 10min of 1500g/min;
4, abandon supernatant, and test tube mouth excess liquid is gone with the tissue suction.Again precipitation is made suspension, drop in push jack on the slide, seasoning.(hospital with good conditionsi can use the pelleter film-making.) 3ml blood, can do 4 slice, thin pieces;
5, use the 40ml4% stationary liquid, in glass jar, fixedly 30min uses 1 * damping fluid I to wash 5min again.Every cylinder can be put 16;
6, sample can be kept at-20 ℃, or continues to do experiment.
Two, reagent in the test kit is mixed with working concentration
1,10 * damping fluid I is diluted to 1 * damping fluid I with tri-distilled water by 1:10;
2,20 * damping fluid II is diluted to 2 * damping fluid II with tri-distilled water by 1:10;
Be diluted to 0.2 * damping fluid II by 1:100; Be diluted to 0.1 * damping fluid II by 1:200;
3,10 * damping fluid III is diluted to 1 * damping fluid III with tri-distilled water by 1:10;
4,10 * damping fluid IV with tri-distilled water by 1:10 be diluted to * damping fluid IV (get 1#, 2#, each 10ml of 3#, add water to 100ml both can).
Three, experimental procedure:
1, gets two of every person's samples to be checked, two of (other two give over to check with) and positive control samples (test at every turn and do a pair of positive control);
2, in glass jar, add Digestive system (Digestive system 100ul adds 1 * damping fluid 199.9ml, is working concentration) 20ml.37 ℃ of water-bath preheatings 10 minutes.Put 16 slides into, handle 12min, use 1 * damping fluid I to wash 5min again for 37 ℃;
3, wash 10min with 0.2% protection liquid (protection liquid 1ml adds 1 * damping fluid I99ml and is working concentration), tri-distilled water is washed 5min, and above process is all carried out at glass jar.The slide seasoning;
4, slide is put into the box of preserving moisture, add prehybridization solution 20ul/ sheet. covered, the lid box of tightly preserving moisture is placed in 42 ℃ of constant water bath box more than the 3h;
5, take out slide, discard cover glass, slide is put into glass jar, with 70%, 90%, 95% ethanol is respectively washed 2min, seasoning;
6, slide is put into the box of preserving moisture, two of every patient specimens, one adds just hybridization solution 20ul/ sheet, and another adds antisense hybridization solution 20ul/ sheet. and covered, the lid box of tightly preserving moisture is placed on 16-24h in 42 ℃ of constant water bath box;
7, take out slide, discard cover glass, slide is put into glass jar
In 42 ℃ of constant water bath box, wash twice, each 15min with 2 * damping fluid II
In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II
In 42 ℃ of constant water bath box, wash twice, each 15min with 0.1 * damping fluid II;
8, wash 30s with 1 * damping fluid III, take out slide, seasoning;
9, slide is put into the box of preserving moisture, add 0.5%l confining liquid (the 1ml confining liquid adds 5ml1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture, at room temperature act on 30min;
10, take out slide, III washes 30s with 1 * damping fluid, seasoning;
11, slide is put into the box of preserving moisture, add alkaline phosphatase enzyme antibody (adding 1.8ml1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture and at room temperature act on 30min;
12, take out slide, wash 3 times, each 15min with 1 * damping fluid III;
13,1 * damping fluid IV washes 2min, adds developer (developer A73.3ul, developer B157.5ul are added among 30ml1 * damping fluid IV, mixing), more than the room temperature lucifuge 12h;
14, wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.
Four, the result judges
100-300 cell of counting calculates the per-cent of catching the purple cell under light microscopic.
What the positive control sample added the antisense hybridization solution should catch purple more than 80%.
All add the negative internal reference of just hybridization solution should be colourless.
Uterus cancer patient P2RX7 genetic expression picture is seen Fig. 1 in the embodiment of the invention.Healthy tissues P2RX7 genetic expression picture is seen Fig. 2 in the embodiment of the invention.Uterine cancer cell hyperplasia P2RX7 genetic expression picture is seen Fig. 3.
The cDNA of digoxigenin labeled, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also having overcome biotin labeled probe is organized the endogenous vitamin H to do shortcomings such as sorrow in the crossover process in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized, method with immunohistochemical methods develops the color again, under light microscopic, observe existence and the location of mRNA,, judge the expression amount of goal gene according to painted cell count.
The inventive method is a nucleic acid hybridization in situ technology commonly used at present, and this method is used for determining by detecting the P2RX7 gene expression amount in the substrate cell whether cervical cancer takes place.Clinical study shows that the P2RX7 gene is the specific gene of cervical cancer, because the P2RX7 gene is not expressed in normal people and hyperplasia of cervix uteri, express in cancer only, illustrate that cervical cancer takes place, be used for determining whether uterine cancers takes place, and hyperplasia or normal.Thereby obtain the diagnostic message of cervical cancer.
Embodiment 3
Detect the uterus carcinoma disease and detect contrast experiment between the uterus carcinoma disease with the P2RX7 kit gene with the HCCR kit gene.
Specificity, susceptibility, accuracy for each comfortable uterus carcinoma disease of scientific evaluation said gene.We use the method for parallel test, detect the mRNA of said gene simultaneously, detection technique adopts the nucleic acid hybridization in situ technology, with same routine uterus carcinoma disease peripheral blood of patients, detect the mRNA (carrying out same procedure and step and reagent that the hybridization in situ technique of embodiment 1 and embodiment 2 is all adopted in nucleic acid hybridization in situ, immunohistochemical staining, mirror numeration down, report as a result etc.) of P2RX7 gene and HCCR gene simultaneously.Find the P2RX7 gene in uterus carcinoma disease patient expression amount than the expression amount height of HCCR gene same disease patient.The result shows that specificity, susceptibility, the accuracy of the medical diagnosis on disease of P2RX7 gene pairs uterus carcinoma are better than HCCR gene, and in situ hybridization genetic expression figure shows that P2RX7 expression of gene amount is 70%, and HCCR expression of gene amount is 50%.The index that test kit of the present invention is done in the uterus carcinoma medical diagnosis on disease has very important clinical meaning.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Rui bends biotechnology (Shanghai) Co., Ltd.
＜120〉a kind of early stage uterus carcinoma hybridization in situ detection kit and detection method and application
<130>/
<160>1
<170>PatentIn?version3.1
<210>1
<211>3155
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
Claims (1)
1. early stage uterus carcinoma hybridization in situ detection kit, comprise hybridization probe, marker, it is characterized in that described hybridization probe sequence is shown in SEQ ID NO.1, described marker is selected from radionuclide or non-radioactive marker, and described radionuclide is selected from
3H,
35S,
125I or
32A kind of among the P, described non-radioactive marker are selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1625565A (en) * | 2001-09-03 | 2005-06-08 | 因特里特有限公司 | Antibodies to non-functional P2X7 receptor, diagnosis and treatment of cancers and other conditions |
| CN101068927A (en) * | 2004-08-31 | 2007-11-07 | 西伦蒂斯私人股份公司 | Methods and compositions to inhibit p2x7 receptor expression |
-
2008
- 2008-08-29 CN CN2008100422629A patent/CN101469352B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1625565A (en) * | 2001-09-03 | 2005-06-08 | 因特里特有限公司 | Antibodies to non-functional P2X7 receptor, diagnosis and treatment of cancers and other conditions |
| CN101068927A (en) * | 2004-08-31 | 2007-11-07 | 西伦蒂斯私人股份公司 | Methods and compositions to inhibit p2x7 receptor expression |
Non-Patent Citations (2)
| Title |
|---|
| Kobayashi K etc..《Differential expression patterns of mRNAs for P2X recptor subunits in neurochemically characterized dorsal root ganglion neurons in the rat》.《J Comp Neurol》.2005,第481卷(第4期),377-390. * |
| 孔虹 等.《P2z/P2x7受体检测的研究进展》.《四川生理科学杂志》.2002,第24卷(第1期),12-15. * |
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