CN107144695A - Application of the Arl13b albumen in cancer diagnosis - Google Patents
Application of the Arl13b albumen in cancer diagnosis Download PDFInfo
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- CN107144695A CN107144695A CN201710255066.9A CN201710255066A CN107144695A CN 107144695 A CN107144695 A CN 107144695A CN 201710255066 A CN201710255066 A CN 201710255066A CN 107144695 A CN107144695 A CN 107144695A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Abstract
Application of the Arl13b albumen in cancer diagnosis, Arl13b albumen or its nucleotide sequence can be used for the reagent or kit for preparing diagnosis cancer.Anti- Arl13b protein specific antibodies or Arl13b protein-specifics nucleic acid probe are reacted as cancer diagnosing agent and cell sample, by detecting that the detectable group with probe or antibody coupling obtains the binding capacity of antibody or probe, it is compared with the binding capacity of normal cell, it is convenient to which tentative diagnosis is made to conditions of patients.Arl13b is as the mark of tumour in the present invention, condition-inference and Index for diagnosis available for tumour；Arl13b is alternatively arranged as the potential therapy target of tumour.
Description
Technical field
The invention belongs to technical field of molecular biology, more particularly to gene diagnosis.
Background technology
With the change of human habitat, living standard and life style, malignant tumour turns into increasingly common and serious
Threaten one of principal disease of human life and quality of life.Global annual new cancer more than 1,400 ten thousand, wherein relatively conventional
Be lung cancer, breast cancer；The annual whole world has 8,200,000 people to die from cancer, and the most common cause of the death is lung cancer.Pathogenesis of cancer map shows
Show, although China's cancer morbidity is 2.8% for global medium level, but the death rate is in medium level on the upper side, is
27%.The incidence of China's cancer has certain features：Onset of liver cancer is 50.5%, and the death rate is 51.4%, accounts for the whole world
Onset of liver cancer, dead half；The cancer of the esophagus, stomach cancer equally account for whole world morbidity, dead nearly 50%；Lung cancer ratio is higher, accounts for
Global new cases 1/3.Globocan is predicted to global pathogenesis of cancer, death, to the year two thousand twenty, it is contemplated that about 17,140,000 people will
Cancer is suffered from, 10,050,000 people will die from cancer.
Although cancer patient is continuously increased in recent years, the five year survival rate of cancer is significantly improved.With nineteen ninety-five -1999
Year is compared, and 5 years survival rates of -2009 years 2005 major cancers of China have obtained obvious raising, such as：Stomach cancer by
15.3% increases to 31.3%, and colon cancer increases to 54.6% by 33.5%, and the carcinoma of the rectum increases to 53.2%, liver cancer by 28.9%
12.5% is increased to by 2.4%, lung cancer increases to 17.5% by 7.5%, and breast cancer increases to 80.9%, cervical carcinoma by 53.8%
59.9% is increased to by 40.1%.The raising for the treatment of technology progress such as surgical technic level；Advanced chemotherapeutics and radiotherapy skill
The application of art and increasing efficiently discovery of targeted drug etc. are the major reasons that cancer cure rate is improved.But people
More given the credit to cancer focus it is early find, early diagnosis and early treatment.
Arl13b is a small molecule GTP enzyme, and it specifically assembles in the primary cilium of a variety of organs.Arl13b pairs
The formation of primary cilium and the performance of function play very crucial effect, participate in vesicle transport, cell differentiation, cell movement and
The processes such as cytoskeleton formation.In the case of Arl13b protein delations, cilium is short and small and structural damage.In the mankind,
The mutation of Arl13b genes can cause Joubert syndromes.The mouse of Arl13b gene delections have ciliary structures impaired and
The phenomenons such as Hedgehog signal paths activity exception, and show the symptom similar to Joubert syndrome patients.In the recent period, have
Document report Arl13b adjusts the growth of tumour cell.
Therefore, the accurate and specific diagnostic reagent or kit that research is invented for cancer have urgent demand.
The content of the invention
It is an object of the invention to provide index, diagnostic reagent or kit, the Arl13b albumen for being capable of Accurate Diagnosis cancer
Diagnostic kit purposes, and vitro detection its expression quantity method.
It is an object of the present invention to provide a kind of index of cancer diagnosis.
Second object of the present invention has been to provide a kind of cancer diagnosing kit.In a preference of this aspect,
Anti- Arl13b antibody couplings have detectable group, can detect group and are selected from chromophore, chemiluminescent groups, fluorogen or same position
Element.
Third object of the present invention has been to provide a kind of cancer diagnosing kit.In a preference of this aspect,
Nucleic acid probe coupling containing Arl13b protein-specifics has detectable group, can detect group and is selected from chromophore, chemiluminescence
Group, fluorogen or isotope.
Fourth object of the present invention is that Arl13b albumen or its nucleotide sequence are preparing the use of diagnostic reagent or kit
On the way.In a preference of this aspect, cancer diagnosing agent is that anti-Arl13b protein specific antibodies or Arl13b albumen are special
Specific nuclease probe.
The 5th purpose of the present invention has been to provide a kind of method of vitro detection specificity Arl13b protein expressions, wraps
Include：Reacted with anti-Arl13b protein specific antibodies or Arl13b specific dna probes with cell sample, using normal cell as
Control；Compare the binding capacity of antibody or probe, wherein the amount higher than control shows that the cell is cancer cell, less than or equal to control
Amount show the cell be normal cell.In a preference of this aspect, binding capacity is by detection and probe or antibody
What the detectable group of coupling was measured.
The present invention has advantages below：(1) Arl13b as tumour mark, available for the condition-inference of tumour and pre-
After judge；(2) Arl13b can as tumour potential therapy target.
Brief description of the drawings
Fig. 1 is influences of the silence Arl13b to tumor cell proliferation ability.
Influences of the Fig. 2 for overexpression Arl13b to tumor cell proliferation ability.
Fig. 3 is influences of the silence Arl13b to tumor cell invasion ability.
Influences of the Fig. 4 for overexpression Arl13b to tumor cell invasion ability.
Fig. 5 is influences of the silence Arl13b to tumor cell migration ability.
Influences of the Fig. 6 for overexpression Arl13b to tumor cell migration ability.
Fig. 7 is that Arl13b silences suppress growth of the stomach cancer cell in nude mouse.
Fig. 8 is that Arl13b is overexpressed the growth for promoting stomach cancer cell in nude mouse.
Fig. 9 is the expression quantity that Western Bolt methods detect stomach organization and Arl13b in cancer beside organism.
The expression that Figure 10 is the different degrees of Arl13b of Kaplan-Meier survival analysises is survived with Overall survival and without disease
The relation of phase.
In above figure, N-Shh：N-Shh stimulates liquid；Control：Control medium；Vector：Compare stomach cancer cell；
Arl13b-KD：Stable Gastric cancer cell MKN45；GAPDH：Glyceraldehyde-3-phosphate dehydrogenase.
Embodiment
Embodiment is provided below embodiment of the present invention is described in detail, the following example is only used for illustrating
The bright present invention, and be not construed as limiting the scope of the present invention.Unreceipted actual conditions person, enters according to normal condition in embodiment
OK.
Embodiment 1：Plate clone method detects influences of the Arl13b to tumor cell proliferation ability.
Take the logarithm growth period Arl13b silences stable Gastric cancer cell MKN45 (Arl13b-KD) and control stomach cancer cell
(Vector) or be overexpressed Arl13b stable stomach cancer cell MKN28 (Arl13b) and control stomach cancer cell (Vector), removal
Culture medium, plus pancreatin fully digest and added after 2-3min in DMEM high glucose mediums of the 500 μ l containing 10% serum and pancreatin, piping and druming
Cell is mixed, makes cell separation into unicellular.1000 cells/wells are taken to be inoculated into 6 orifice plates, plus N-Shh stimulates liquid (N-Shh)
Or control medium (Control), and make cell dispersed.It is positioned over 37 DEG C, 5%CO2Cultivated 14-20 days in incubator.
When observing macroscopic clone, culture is terminated.Remove culture medium, plus the fixed 10min of 37 DEG C of 4% paraformaldehyde solution.
Remove after fixer, after being washed 3 times through PBS, add 0.2% violet staining 20-30min.PBS washes away dyeing liquor extremely many times
It is placed in and is air-dried after PBS clarifications.6 orifice plates are inverted in into professional scanner to be scanned.Background is removed using ImageJ softwares
And to unit area (1cm2) violet staining clone number calculated, * * represent p<0.01.As a result show, Arl13b promotes
The propagation (Fig. 1, Fig. 2) of tumour cell.
Embodiment 2：Influences of the Transwell experiment detections Arl13b to tumor cell invasion ability.
Tumour cell is through Nature enemy 12h.With 50mg/LMatrigel 1：8 dilutions are coated with Transwell cells bottom
The upper chamber face of film, 4 DEG C air-dry.Residual liquid in culture plate is suctioned out, serum-free mediums of the 50ul containing 10g/LBSA is added per hole,
37 DEG C, 30min.Transwell cells are put into 24 orifice plates, the serum free medium of 300 μ l pre-temperatures, room temperature are added in upper chamber
Lower standing 15-30min, makes matrigel rehydration.Remaining culture liq is sucked again.Digest the stable stomach cancer cell of Arl13b silences
MKN45 (Arl13b-KD) and control stomach cancer cell (Vector) or the stable stomach cancer cell MKN28 for being overexpressed Arl13b
(Arl13b) and control stomach cancer cell (Vector), terminate digestion after centrifugation discard nutrient solution, washed with PBS 1-2 times, with containing BSA
Serum free medium be resuspended, adjustment cell density is to 1-10 × 105.The μ l of cell suspension 200 are taken to add Transwell cells,
Room adds 500 culture mediums of the μ l containing 20%FBS under 24 orifice plates, plus N-Shh stimulates liquid (N-Shh) or control medium
(Control) 24h, is cultivated.Cell is transferred in new culture hole, is washed to appropriate color, small interior is gone to cotton swab
Portion does not pass through the cell of upper chamber film surface.Toward the small indoor μ l PBS of addition 100, the cell that inverted microscope observation is gone through, 200 ×
The invasion cell number in 10 visuals field is counted under light microscopic, its average value is taken, tumour cell is represented with the relative number of invasion cell
Invasive ability, * * represent p<0.01.As a result show, Arl13b promotes the invasion and attack (Fig. 3, Fig. 4) of tumour cell.
Embodiment 3：Influences of the Migration experiment detections Arl13b to tumor cell migration ability.
Compared with ruler in 6 orifice plates behind with marker, uniformly draw horizontal line, it is, horizontal per every about 0.5~1cm together
Through hole.Per hole at least across 5 lines.About 5 × 10 are added in hole5The stable Gastric cancer cell MKN45 of individual Arl13b silences
(Arl13b-KD) and control stomach cancer cell (Vector) or be overexpressed Arl13b stable stomach cancer cell MKN28 (Arl13b) and
Stomach cancer cell (Vector) is compareed, inoculation principle reaches 100% for rear fusion rate overnight.Compare ruler with pipette tips within second day, to the greatest extent
The horizontal line cut perpendicular to behind is measured, pipette tips are vertical, it is impossible to tilt and (same pipette tips are preferred between different holes).PBS
Wash cell 3 times, remove the cell under drawing, adding the N-Shh of serum-free stimulates liquid (N-Shh) or serum-free control medium
(Control).37 DEG C of 5%CO2 incubators are put into, are cultivated.Can be by 0,12,24h point in time sampling, taking pictures, (the specific time is according to reality
Test depending on needs).Opened using Image J softwares after picture, random draw takes 6 to 8 horizontal lines, calculates the equal of iuntercellular distance
Value, * * represent p<0.01.As a result show, Arl13b promotes the migration (Fig. 5, Fig. 6) of tumour cell.
Embodiment 4：Arl13b promotes the growth of stomach cancer cell in vivo.
Buy 24 BALB/c-nu Female nude mices, 4-6 week old, body weight 18-22g；By table of random number by 24 nude mices
It is divided into two groups：1. slow virus silence control group (Vector)+Arl13b silences group (Arl13b-KD)；2. slow virus be overexpressed pair
According to group (Vector)+Arl13b overexpressions group (Arl13b)；And weigh the body weight of every nude mice.Use 0.25% Trypsin Induced
The stable Gastric cancer cell MKN45 (Arl13b-KD) and control stomach cancer cell (Vector) of Arl13b silences are overexpressed Arl13b
Stable stomach cancer cell MKN28 (Arl13b) and control stomach cancer cell (Vector), 1000r/min centrifugation, PBS wash 2 times, use
PBS is with restriction I × 108Cell/ml.0.2ml, which is extracted, with the syringe of No. 6 syringe needles of band contains 2 × 107The cell suspension of individual living cells,
It is inoculated in the medial thigh skin of every nude mice (left and right is respectively control group, interference or overexpression group).After injection at the 7th day,
Come into effect when control group and roughly equal treatment group knurl body in reference water and add Doxycycline (2mg/ml) induced expression, every 2
It changes water once, is administered 3-4 weeks:The every three days volumes (V) with transplantable tumor of vernier caliper measurement：V=a × b2/ 2 (a for length
Footpath, b is minor axis), and observe the change of the physical signs such as nude mice spirit, active situation.After 3-4 weeks, euthanasia nude mice is anaesthetized,
Transplantable tumor is cut, and weighs its weight.It is stable to lower Arl13b groups (Arl13b-KD) mouse compared with control group (Vector)
Knurl body it is smaller, knurl weight is lighter (Fig. 7).However, compared with control group (Vector), Arl13b groups are overexpressed stable
(Arl13b) in testing, it is observed that being overexpressed, Arl13b posterior tuberosity bodies are bigger, and knurl weight is heavier (Fig. 8).
Embodiment 5：Detect the expression quantity of stomach organization and Arl13b in cancer beside organism.
Trash ice is got out, 5ml centrifuge tubes are weighed and carry out mark, the precooling on ice together with PBS.Take 8 gastric cancers at random
The cancerous tissue of people and its effective thermos cup transfer equipped with liquid nitrogen of corresponding cancer beside organism's tissue freezing, clip portion of tissue are put
Enter in the centrifuge tube of precooling, remaining tissue is put back in cryopreservation tube, is replaced；Weigh tissue, record；Washed with the PBS of precooling
Tissue is washed, 2-3 times, the blood of adhesion is washed away；Tissue is shredded, addition contains 1：100 protease inhibitors and 1mM PMSF's
RIPA buffer, additional proportion tissue：Buffer=1:10(g/ml)；The homogenised tissue on historrhexis's instrument, 13000g,
10s, it is more broken to organizing；Tissue will be maintained on ice at the moment；On gyroscope, 4 DEG C of rotation 30min make tissue fully crack；Cracking
Liquid is transferred to EP pipes, centrifugation, 4 DEG C, 12000g, 15min；Supernatant is taken, histone lysate needed for producing；100 μ l albumen are taken to determine
Western Blot are measured and are, if remaining is temporarily without putting -80 DEG C of preservations.BCA protein quantifications, it is quantitative according to the numerical value of gained
Matched curve, obtains the protein concentration after linear equation and dilution, calculates actual protein concentration.Take appropriate albumen
Western sample detections normal structure and the protein content of Arl13b in cancerous tissue.As a result Arl13b tables in cancerous tissue are shown
Up to amount (Fig. 9) higher than normal structure.
Embodiment 6：Arl13b expression quantity is related to the state of an illness of Patients with Gastric Cancer and prognosis.
Pathology preserves FFPE sample in 154 gastric cancer cases chosen, and organization type is through 1 experienced pathology
Section doctor is determined to the histotomy HE dyeing for being selected in wax stone source.To 154 stomach cancer samples, (including 154 corresponding
Cancer beside organism) carry out immunohistochemical analysis.SABC scoring is scored according to " German sxemiquantitative points-scoring system ", according to 0
~12 points of standards of grading, it is negative and low expression to delimit 0~6 point,>6 points are middle high expression.Will be each according to Arl13b points of height
Index scoring for two groups and with clinical pathologic characteristic progressive correlation analysis.Analysis result is shown in Table 1, as a result points out：Height expression
Arl13b and tumor size, invasive depth are relevant.
Influence of the high expression to patient's prognosis in stomach organization for clear and definite Arl13b, we enter to the patient for including research
Row follow-up.Observing time is set as Overall survival (overall surviva), and without disease life cycle (Disease-free
survival).Single factor test survival analysis discovery is carried out with Kaplan-Meier analyses：Arl13b height expression can significantly affect trouble
Person OS and DFS, the higher patient survival that scores are shorter (Figure 10).Show that Arl13b and clinical prognosis have significant correlation.
Relation between table 1Arl13b and clinical pathologic characteristic
Summary specific embodiment measurement result, Arl13b is expressed in tumor tissues height, the propagation of promotion tumour cell,
Migration and invasion and attack.
Therefore, Arl13b albumen or its nucleotide sequence can be used for the reagent or kit for preparing diagnosis cancer.Will be anti-
Arl13b protein specific antibodies or Arl13b protein-specifics nucleic acid probe react as cancer diagnosing agent and cell sample,
By detecting that the detectable group with probe or antibody coupling obtains the binding capacity of antibody or probe, the binding capacity with normal cell
It is compared, the diagnosis available for cancer.
Claims (3)
- Application of the 1.Arl13b albumen in cancer diagnosis.
- Application of the 2.Arl13b albumen in cancer diagnosing kit is prepared.
- Application of the 3.Arl13b specific dna probes in cancer diagnosing kit is prepared.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111269982A (en) * | 2020-02-20 | 2020-06-12 | 南昌大学 | Application of SNEP1 protein in diagnosis of colorectal cancer |
| CN112294947A (en) * | 2020-11-10 | 2021-02-02 | 南昌大学 | Application of polypeptide in preparing medicine for preventing and/or treating tumor |
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| CN107144695B (en) | 2019-02-26 |
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