CN104140999A - MRNA level in-situ hybridization detection kit of AE1 gene in earlier stage of pathologic evolution of stomach cancer, detection method and application - Google Patents
MRNA level in-situ hybridization detection kit of AE1 gene in earlier stage of pathologic evolution of stomach cancer, detection method and application Download PDFInfo
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Abstract
The invention discloses an in situ hybridization detection kit which comprises a hybridization probe and a marker. The invention also discloses a method for in situ hybridization detection of AE1 (ASYMMETRIC LEAVES1/2ENHANCER1) gene's mRNA expression change which is closely related with pathologic evolution of stomach cancer canceration in the earlier stage by the use of the kit. The method comprises the following steps: (1) under the condition that a hybridization probe and a target sequence can form a stable hybrid complex, RNA to be tested in a substrate is contacted with the hybridization probe so as to form a hybrid complex; and (2) the hybrid complex is detected. According to the detection kit and the detection method, expression quantity of AE1 gene can be detected at the RNA level, the index of the detection method is ealier than imaging medicine and present clinical biochemical detection index (protein detection index), and genuine RNA level screening of canceration in the earlier stage can be realized. Meanwhile, the detection method provided by the invention is simple and convenient; cost is low; and the method is convenient for popularization and application in district-level hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to change with cancer of the stomach Pathologic rna expression the correlation detection technology of (Pathologic process).
Background technology
The data providing according to domestic and international authoritative institution, the newly-increased number of the annual cancer of China has reached 3,120,000, death toll nearly 2,600,000, patient exceedes 7,000,000, the annual newly-increased cancer patients 8,000,000 in the whole world, death toll approaches 8,000,000, and patient approximately has more than 8,400 ten thousand people, to double to the above number of the year two thousand twenty, this is one group of fearful numeral.Cancer diagnosis and treatment cost is more and more higher, by cancer patients's year medical expense 200,000, (poor area may be higher, developed regions may exceed 200,000 far away), more than 700 ten thousand patients, annual cost is 1.4 trillion Renminbi, deduction cost 35% approximately 400,000,000,000, approximately has 1,000,000,000,000 Renminbi to consume in vain every year.And cancer patients is most of can be dead soon after treatment.Therefore, existing clinical cancer diagnosis and treatment pattern must change, and innovative point of the present invention is to accomplish in advance preventative examination, then gets involved in time preventative regulation and control and prophylactic treatment, accomplishes preventiveing treatment of disease of gene level cancer.
It is failure that eight units such as U.S. sanitary research institute in 2005, cancer research institute, Disease Control and Prevention Center have done in anticancer Great War, conclusion is that cancer mortality does not reduce, and it lists and causes several factors of anticancer Great War failure to be: 1. tumour cell heterogeneity (polymorphism); 2. tumor cell drug resistance; 3. cancer therapy drug mentality of designing imperfection (animal model designs not science) etc.Meanwhile, in this report, also propose to answer the measure of the existing diagnosis and treatment cancer of re-examine.
The inventor is in the middle discovery that studies for a long period of time, and causing the major reason that cancer mortality is not fallen is to accomplish real early diagnosis.Carry out diagnosing cancer according to existing clinical medicine image (B ultrasonic, CT, Magnetic resonance imaging etc.) and with other biochemistry (cancer antigen, carcinomebryonic antigen, carbohydrate hormone, the cytolemma factor, nuclear factor, cell streaming technology) index, all that tumour forms rear diagnosis, the former will learn in a organized way and change or existing occupying lesion, the latter's major part be tumour form rear secreted, discharge or the marker of tumour.The clinical idea of tradition thinks that occupancy cancer piece is the diagnosis that belongs to early-stage cancer under 2 centimeters, this concept is worth conscientiously discussing, it is rigorous not that 2 centimeters of early stage these of following cancer piece genus define science, analyze from cytology angle, the lump of 1 centimeter approximately has 100,000,000 tumour cells, its three-dimensional cell stack number of the lump of 2 centimeters is far above 200,000,000 tumour cells, produce and form the cancer piece of 2 centimeters to mono-clonal cancer cells early stage from canceration, its Pathologic process is quite long, it may be 5 years or 10 years, or even 10 years above (except special case), what be difficult to confirm is in this Pathologic process, lump is the unique spot of cancer and independent focus, possible cancer cells moves to other tissue or organ growth already.Clinical study confirms already, and once form lump, other cancer cells moves to other position clonal growth by different approaches, once after excision primary tumor, other organ recurrence kitchen range or multiple cancer piece kitchen range successively form.Therefore, whether define in early days with 2 centimeters of following cancerous swelling piece sizes clinically, rigorous not (some case, in the time finding primary lesion, find metastatic lesion simultaneously, not in the content of our statement), be at this moment diagnosis in late period and treatment of late stage in fact, this is the true cause that causes cancer mortality not fallen.
Along with molecular biotechnology is day by day perfect, functional genomics, the deep expansion of the research such as cancer gene group, in order to seek more early stage diagnosing cancer, treatment cancer and preventing cancer, makes great progress.So far, we likely do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level or microRNA) of gene, front in canceration early stage or cancer cells formation (mono-clonal), just can accomplish early prediction and examination.
In large flux examination, find AE1 gene mRNA great expression in patients with gastric cancer, AE1 gene high expression level in stomach cancer cell can play proto-oncogene effect, promotes cancer cell growth and division.Experimental study further finds, the expression that reduces AE1 can make drug-induced Mouse Gastric Cancer incidence obviously reduce.And, AE1 gene mRNA high expression level all in various ethnic groups (white man, Black people, yellow) peripheral blood from patients with gastric cancer white corpuscle.
The present invention selects many group clinical samples (Patients with Gastric Cancer, high risk population, normal control) to adopt nucleic acid hybridization in situ technology and ImmunohistochemistryMethods Methods to detect analysis to the early warning of AE1 gene mRNA and cancer of the stomach.The inventor finds that AE1 gene mRNA has obvious expression amount to change patients with gastric cancer, cancer high risk population's (chronic atrophic gastritis and stomach have Leptospira), normal control people, has very important clinical diagnosis meaning in earlier stage using AE1 gene mRNA as the pathological change of early screening cancer of the stomach under study for action.The Preventive early warning of doing in after cancer of the stomach canceration examination in early stage and curing gastric cancer also has very important clinical meaning.
Contriver is in long-term research, draw a kind of new concept, the clinical diagnosis and treatment pattern of cancer and other clinical major disease must change, can not only stop present treating the disease affected (diagnosis and treatment after morbidity), accomplish preventative diagnosis and treatment, accomplish to preventive treatment of disease, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, contriver, in the rna level kit for screening and medicine of development and production major disease, innovates theoretical and technical.Particularly screen clinical samples (normal population, cancer high risk population, tumour patient), break through the consistency research and development thinking of healthy tissues and tumor tissues comparison, find and develop the rna level becoming before cancer, develop closely related with cancer early gene physiopathology, and the extremely important target of clinical meaning (disease gene), tumour is clinically formed to rear diagnosis and treatment pattern and becomes the preventative diagnosis and treatment of tumour, time and the space of having striven for tumor diagnosis and treatment, reach preventing cancer.
At present, the analytical technologies such as the Northern hybridization for method of AE1 expression profiling, expression chip, real-time fluorescence quantitative PCR and Solexa order-checking, and these methods are used for scientific research aspect, be not suitable with clinical application, and detect RNA, than genetic analysis, more scientific (DNA analysis major part is in the presentation of susceptibility, RNA is functional embodiment), than analysis of protein more reliable (contriver finds on mRNA and albumen transcript and expression space-time in the research of employing Double Labelling Technique, sometimes asynchronous).The detection technique and the test kit that adopt hybridization in situ technique and groupization immunization method to detect AE1 gene mRNA horizontal expression amount according to existing documents and materials have no report.
The inventor is in the requirement for novelty invention, design (patients with gastric cancer, high risk population, normal control) different pieces of information example group, detect with hybridization in situ technique, result shows above Patients with Gastric Cancer AE1 gene mRNA high expression level, high risk population has and expresses in various degree 10-20%, and normal control is all zero expression.Show that AE1 gene mRNA is the important symbol thing of cancer of the stomach canceration examination in early stage.
Hybridization in situ technique (in situ hybridization) is that molecular biology and Cytochemical Technique are combined, taking the nucleic acid molecule of mark as probe, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is the nucleic acid strand (being probe) that makes to contain distinguished sequence, passes through mark, under optimum conditions with histocyte in 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.
The probe of in situ hybridization is the molecule of known array or sequence the unknown but known nucleic acid molecule (though indefinite this molecule full sequence of molecule, but known its for what target molecule), the kind of probe can be divided into again DNA probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe by properties of nucleic acids difference.For the ease of spike, probe must, with certain means mark in addition, be beneficial to later detection.Conventional marker comprises radionuclide and the large class of non-radioactive marker two.Conventional isotopic label has
3h,
35s,
125i and
32p.The advantages such as susceptibility is high although isotopic label has, back end is comparatively clear, because radio isotope all can damage human and environment, have the trend being replaced by heterotope recently.At present the most frequently used in heterotope marker have three kinds of vitamin H, digoxin and fluoresceins.The method that detects these markers is all extremely sensitive.
Can be divided into again DNA-DNA, RNA-DNA, RNA-RNA hybridization according to probe used and the difference that will detect nucleic acid.No matter but the hybridization of any form, all must be through five large processes, i.e. histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the Crossing system of RNA-RNA, synthetic probe (RNA) and the target RNA detecting are the principles that adopts base complementrity (hybridization is complementary), simultaneously through long-time research with observe, start and termination place residue does not affect the result detecting.
In view of the diagnosis of cancer of the stomach clinically (gastroscope, medical imaging and biochemical indicator thing are all the diagnosis after tumour forms) is at present diagnosis in late period, treatment is also treatment of late stage, is also the treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to improve at present the diagnosis and treatment pattern of major disease clinically, become preventative preventiveing treatment of disease from treating the disease affected, reach preventative diagnosis and treatment, current medical imaging means and numerous biochemical marker cannot be detected to becoming rna level before cancer quantizes change technology, do the technological breakthrough of novelty, provide cancer the front rna level examination technology that becomes.Make to have had clinically a front technology that becomes the real early screening of rna level of new cancer, for the diagnosis and treatment of clinical cancer of the stomach are raced against time and space.
In sum, first object of the present invention is to provide a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and marker.Secondly, the present invention shifts the relevant in situ hybridization detection method of early warning after also will providing mentioned reagent box for the examination in early stage of cancer of the stomach Pathologic and treatment.
Summary of the invention
For realizing object of the present invention, technical scheme of the present invention is as follows:
First the present invention provides a kind of hybridization in situ detection kit, it comprises hybridization probe and marker, and wherein, described hybridization probe has respectively sequence number: S68680 shown in sequence table SEQ ID NO.1, nucleotide sequence length is that in 462bp, CDS sequence is 119...462bp, total length 344bp.
A preferred version of test kit of the present invention is that described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
A preferred version of test kit of the present invention is also to comprise hybridization solution.
A preferred version of test kit of the present invention is also to comprise toughener.
A preferred version of test kit of the present invention is also to comprise developer.
Cancer of the stomach Pathologic of the present invention AE1 gene mRNA in early stage kit for screening using value is, to the examination in early stage of cancer of the stomach Pathologic, and early warning occurs for Preventive, diffusion after canceration or after treatment, further coordinates clinical treatment.
The present invention also provides a kind of detection method of AE1 in situ hybridization, comprises the following steps:
(1) described hybridization probe and target sequence can form under the condition of stablizing hybridization complex in the above, RNA to be measured in substrate contacted to formation hybridization complex with hybridization probe; With
(2) detect described hybridization complex.
Detection method of the present invention, wherein preferably, the condition that hybridization complex is stablized in described forming is: the temperature of nucleic acid hybridization is 42 DEG C; The time of nucleic acid hybridization is 16-24 hour.
Detection method of the present invention, wherein preferably, described substrate is selected people's blood leucocyte sample or other organ-tissue cell specimen.More preferably, described blood preparation or other organ-tissue cell specimen are from the high risk population of patients with gastric cancer, cancer of the stomach, healthy normal population.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, taking AE1 gene mRNA as detected object, synthesising probing needle is AE1 gene recombination complementary sequence, and the substrate of detection is the expression amount of blood of human body sample white corpuscle or histiocytic AE1 gene mRNA.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of AE1 gene mRNA.The expression amount of judging above mRNA according to immunohistochemical methods colour developing after hybridization, normal people AE1 zero expresses, and does not develop the color; The low expression of high risk population, a small amount of cell dyeing; Patients with Gastric Cancer high expression level, a large amount of cell dyeings.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, the compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that molecular biology insider all knows, and concrete operation step is under sample disposal, prehybridization, hybridization, immunohistochemical staining, mirror, to carry out quantitative analysis, report the test, and wherein the concrete steps of hybridization comprise:
1). sample to be measured is put into reactive tank;
2). instrument discards liquid automatically, automatically adds Digestive system;
3). instrument discards liquid automatically, automatically rear fixing;
4). instrument discards liquid automatically, automatically prehybridization (42 DEG C);
5). instrument discards liquid automatically, automatically cleans;
6). instrument discards liquid automatically, automatically hybridization (42 DEG C);
7). instrument discards liquid automatically, automatically cleans;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, automatically cleans colour developing;
10). take out mounting microscopy.
The scheme of a preferred embodiment of the present invention is: with the synthetic (cDNA of digoxigenin labeled of digoxigenin labeled for nucleic acid probe of AE1 gene mRNA, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also overcome biotin labeled probe and in crossover process, organized the shortcomings such as the dry sorrow of Endogenous Biotin in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized, develop the color by the method for immunohistochemical methods again, in existence and the location of light Microscopic observation RNA, according to the cell count of dyeing, judge the expression amount of object RNA.
The inventive method is current conventional nucleic acid hybridization in situ technology, the method is by the AE1 gene mRNA expression amount in detection substrate cell, be used for determining the RNA variable quantity in cancer of the stomach Pathologic early stage, early warning cancer of the stomach whether occur and patients with gastric cancer treatment after the whether prediction of Preventive.Because AE1 gene mRNA is zero expression in normal people, AE1 gene has the carcinogenesis of proto-oncogene, if expression amount increases, the risk getting a cancer of the stomach is described, illustrate that cancer of the stomach change occurs, or the postoperative Preventive of Patients with Gastric Cancer, thereby the diagnostic message of acquisition cancer of the stomach.
A test kit can many person-portions use or person-portion use.
The present invention has following beneficial effect:
Clinical meaning of the present invention is to follow the tracks of more in early days the variation that detects AE1 gene mRNA expression amount in cancer of the stomach pathology evolution process, and early warning cancer of the stomach occurs, development trend.Diagnostic kit of the present invention and other detection and cancer markers clinically, and medical imaging inspection has and is showing difference.The present invention can detect at rna level the unconventionality expression of AE1 gene, before the recurrence of occupancy carninomatosis kitchen range is not found in medical imaging inspection, before cancer biochemical indicator does not produce extremely, also before not forming tumour, can accomplish early the information acquisition of AE1 abnormal gene expression, give real early warning of clinical patients with gastric cancer, so just likely implement early screening, early prevention, the early treatment of cancer, likely from source, thoroughly effect a radical cure cancer of the stomach foul disease.
In addition, feature highly sensitive, high specificity that test kit provided by the invention has, meanwhile, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
Brief description of the drawings
Fig. 1 is that illustration (nucleic acid hybridization in situ techniqueflow chart) is implemented in AE1 gene mRNA in situ hybridization of the present invention;
Fig. 2 is Patients with Gastric Cancer AE1 gene mRNA expression picture in the embodiment of the present invention;
Fig. 3 is high risk population AE1 gene mRNA expression picture;
Fig. 4 is normal people AE1 gene mRNA expression picture in the embodiment of the present invention.
Embodiment:
Below in conjunction with embodiment, be described more specifically content of the present invention.Should be appreciated that, the following examples are non-limiting content of the present invention for explanation, and any pro forma change or accommodation will fall into protection scope of the present invention.
Embodiment 1
Prepare the in situ hybridization test kit of the present embodiment according to ordinary method, this test kit comprises hybridization probe, marker, the specification sheets with the design of AE1 gene mRNA, wherein:
The probe mark thing of the present embodiment is selected digoxin.
Test kit hybridization solution composition:
Reagent preparation working concentration
1). by 10 × damping fluid I with tri-distilled water by being diluted to 1 × damping fluid I at 1: 10;
2). by 20 × damping fluid II with tri-distilled water by being diluted to 2 × damping fluid II at 1: 10;
By being diluted to 0.2 × damping fluid II at 1: 100; By being diluted to 0.1 × damping fluid II at 1: 200;
3). by 10 × damping fluid III with tri-distilled water by being diluted to 1 × damping fluid III at 1: 10;
4) .10 × damping fluid IV with tri-distilled water by 1: 10 be diluted to × damping fluid IV (get 1#, 2#, the each 10mL of 3#, add water to 100mL both can).
Embodiment 2
The implementation process of application nucleic acid hybridization in situ detection method to each group of blood preparation AE1 gene mRNA expression amount:
1). get two of samples to be measured;
2). in glass jar, add Digestive system (Digestive system 100 μ L add 1 × damping fluid I99.9ml, are working concentration) 50ml, 37 DEG C of water-bath preheating 10min, put 16 slides into, process 12min, then use 1 × damping fluid I to wash 5min for 37 DEG C;
3). protection liquid with 0.2% (protection liquid 1ml adds 1 × damping fluid I, and 99ml is working concentration) is washed 10min, and tri-distilled water is washed 5min (above process is all carried out at glass jar), takes out slide, allows its seasoning;
4). slide is put into moisture preservation box, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered, covers tightly moisture preservation box, is placed in 42 DEG C of constant water bath box more than 3h;
5). take out slide, discard cover glass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into moisture preservation box, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered, covers tightly moisture preservation box, is placed on 16-24h in 42 DEG C of constant water bath box;
7). take out slide, discard cover glass, slide is put into glass jar:
In 42 DEG C of constant water bath box, wash twice with 2 × damping fluid II, each 15min;
In 42 DEG C of constant water bath box, wash once each 15min with 0.2 × damping fluid II;
In 42 DEG C of constant water bath box, wash twice with 0.1 × damping fluid II, each 15min;
8). with 1 × damping fluid, III washes 30s, takes out slide, seasoning;
9). slide is put into moisture preservation box, add 0.5% confining liquid (1ml confining liquid adds 5ml1 × damping fluid III), 100 μ L/ sheets, cover tightly moisture preservation box, at room temperature act on 30min.(this step does not need to add cover glass);
10). take out slide, with 1 × damping fluid, III washes 30s, seasoning;
11). slide is put into moisture preservation box, add X-AP antibody and (get a pipe alkaline phosphatase enzyme antibody, add wherein 1.8ml1 × damping fluid III) 100 μ L/ sheets, cover tightly moisture preservation box and at room temperature act on 30min, time can not be long, otherwise can produce false positive (this step does not need to add cover glass);
12). take out slide, wash 3 times with 1 × damping fluid III, at every turn 15min;
13). with 1 × damping fluid, IV washes 2min, adds developer (developer A73.3 μ L, developer B157.5 μ L is added in 30mL1 × damping fluid IV, mixes), and room temperature lucifuge 16h is to more than 18h;
14). wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 × damping fluid I mix) mounting microscopy.
Nucleic acid hybridization in situ detection method of the present invention is by goal gene digoxigenin labeled, become RNA nucleic acid probe, the RNA nucleic acid to be measured of probe and human leukocytes is hybridized, develop the color by the method for immunohistochemical methods again, therefore in existence and the location of light Microscopic observation RNA, according to the cell count of dyeing, judge the expression amount of object RNA.
20 of Patients with Gastric Cancer, 20 of high risk population's (chronic atrophic gastritis and stomach have leptospiral infection), 20 of Normal groups.The peripheral blood 3-5 milliliter (separation white corpuscle) of taking out all people to be checked does in situ hybridization.Result represents, all patients with gastric cancer AE1 gene mRNA expression amounts are very high, and cell dyeing is dark; High risk population expresses slightly and reduces, decimal dyeing; Normal group AE1 gene mRNA zero is expressed, and cell does not dye, and concrete outcome is shown in Fig. 2, Fig. 3, Fig. 4.
Claims (10)
1. a hybridization in situ detection kit, comprises hybridization probe and marker, it is characterized in that, described hybridization probe has respectively the CDS fragment in the sequence shown in sequence table SEQ ID NO.S68680, from 119...462bp, and sequence total length 344bp.
2. test kit as claimed in claim 1, is characterized in that, described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
3. test kit as claimed in claim 1, is characterized in that, this test kit also comprises hybridization solution.
4. test kit as claimed in claim 1, is characterized in that, this test kit also comprises synergistic agent.
5. test kit as claimed in claim 1, is characterized in that, this test kit also comprises developer.
6. an AE1 gene hybridization in situ detection method, is characterized in that, the method comprises the following steps:
(1) can form under the condition of stablizing hybridization complex at hybridization probe claimed in claim 1 and target sequence, RNA to be measured in substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect described hybridization complex.
7. detection method as claimed in claim 6, is characterized in that, the condition that hybridization complex is stablized in described forming is: the temperature of nucleic acid hybridization is 42 DEG C; The time of nucleic acid hybridization is 16-24 hour.
8. detection method as claimed in claim 6, is characterized in that, described substrate choosing is human peripheral blood white corpuscle sample.
9. detection method as claimed in claim 6, is characterized in that, described blood preparation (cancer, high risk population, normal people's sample).
10. detection method as claimed in claim 9, is characterized in that, described in comprise high risk population's cancer of the stomach clinically Pathologic process in early stage, and the recurrence of cancer of the stomach after treatment and shift Pathologic process.
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