CN106405084A - Application of nucleolin to preparation of diagnostic reagent and therapeutic for intestinal cancer - Google Patents
Application of nucleolin to preparation of diagnostic reagent and therapeutic for intestinal cancer Download PDFInfo
- Publication number
- CN106405084A CN106405084A CN201510467697.8A CN201510467697A CN106405084A CN 106405084 A CN106405084 A CN 106405084A CN 201510467697 A CN201510467697 A CN 201510467697A CN 106405084 A CN106405084 A CN 106405084A
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- China
- Prior art keywords
- intestinal cancer
- paranuclein
- nucleolin
- application
- cancer
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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
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
Abstract
The invention relates to multi-functional ribosomal protein nucleolin and application thereof, specifically to application of nucleolin as a molecular marker for the intestinal cancer of human beings and application of nucleolin to preparation of a preparation or kit used for detecting and diagnosing the intestinal cancer of human beings and a therapeutic for the intestinal cancer, belonging to the field of biotechnology. According to the invention, ELISA detection is carried out on both the blood plasma sample of a patient with the intestinal cancer and a normal control blood plasma sample; detection results show that the level of nucleolin in the blood plasma of the patient with the intestinal cancer is substantially higher than the level of nucleolin in the normal control blood plasma sample and that nucleolin is highly expressed in the intestinal cancer and secreted to blood; thus, nucleoli can be used as a noninvasive marker for diagnosis of the intestinal cancer. The invention further provides application of nucleolin to preparation of a diagnostic reagent and kit for the intestinal cancer and a drug with nucleolin as a target and for treatment of the intestinal cancer. The invention provides a novel assistant determination means for diagnosis of a colorectal cancer.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of multi-functional non-ribosomal protein paranuclein and purposes and in particular to application paranuclein as people's intestinal cancer molecular marker and its is preparing the purposes in the preparation detecting or diagnosing people's intestinal cancer or test kit and medicine.
Background technology：
Raising recently as our people's standard of living and the change of dietary habit, the M ＆ M of colorectal cancer is also in the trend increasing year by year.Research display, the prognosis of colorectal cancer is by stages closely related with disease, and early diagnosiss and early treatment can significantly improve prognosis, extend life cycle.In clinical practice at present, relatively current, conventional inspection is serum blood inspection, the conventional blood serum tumor markers related to intestinal, as all relatively low to the specificity of diagnosis of colorectal carcinoma and sensitivity in S-CEA (CEA), intestinal cancer related antigen (CCA), CA19-9 (CA19-9) etc.；Simultaneously because colorectal cancer early stage can no significantly clinical symptoms, these all cause certain difficulty to the early discovery of colorectal cancer, early diagnosiss；Therefore, find the promising tumor marker of colorectal cancer, accurately judge this illness clinical stagess and prognosis become the focus of attention of this area researcher.
Prior art discloses paranuclein is a kind of multi-functional non-ribosomal protein, and it is mainly distributed in nucleus, has and is distributed on a small quantity on caryoplasm, Cytoplasm and cell membrane；It participates in the DNA metabolism of many aspects, also widely participates in the regulation process of RNA, such as processing of transcription, the holding of ribosome assembling, the translation of mRNA and its stability and microRNA etc.；Research report, paranuclein said target mrna s after regulatory transcription, promote cell proliferation and anti-apoptotic, thus ring the progress of disease in cellular layer face；Particularly in tumor and virus infection, the function by rna regulation for the paranuclein, the nearly pathological evolution process of involved in diseases.
Studies have reported that, in Cytoplasm, paranuclein is combined with Bcl-2 mRNA, AKT1 mRNA, CCNI mRNA, strengthens its stability, thus increasing the expression of Bcl-2, AKT1 and cyclin I, playing anti-apoptotic and promoting tumorigenic effect；It can be combined with the noncoding region 5 ' end of TP53mRNA, suppresses its translation, thus reducing the expression of tumor-inhibiting factor P53.
Research is had to disclose gastrin, as a kind of albumen of expression high in gastrointestinal cancer, also regulated and controled by paranuclein, paranuclein, by being combined with Gastrin mRNA, strengthens its stability, increase the expression of gastrin, research display, in colorectal cancer cells, gastrin can raise the expression of COX-2, increase the expression of PGE2, promote the propagation of cancerous cell；Paranuclein can also be held with MMP9 mRNA noncoding region 3 ' and combine, and strengthen its stability, increase it and be translated as the function of matrix metalloproteinase；Matrix metalloproteinase can degradation of cell epimatrix composition, it is essential with the survival processes of blood vessel in the healing of wound, plays particularly important effect in the transfer of tumor and the pathophysiological process of progress；In addition, paranuclein can also play the tumorigenic effect of rush, such as miR-15a and miR-16 etc. by the miRNA that regulation and control promote tumor generation.
Also studies have found that malignant tumor tissue surface of cell membrane has the high expression of paranuclein, path is adjusted by mediate calcium, lipopolysaccharide is expressed, P selectin induces path etc. to adjust transfer and the progress of tumor cell；The paranuclein of surface of cell membrane can as the receptor of carcinogen Tipa, in combination after carcinogen be transported to intracellular play carcinogenesis.
Present Research based on prior art, the abnormal diagnosis for colorectal cancer that applicant of the present invention intends by research, detection paranuclein in PATIENTS WITH LARGE BOWEL body there may be expression provides new auxiliary judgment means, especially provides purposes in preparation intestinal cancer diagnostic reagent and test kit and medicine for the paranuclein.
Content of the invention：
Present invention aims to lacking the present situation of effective diagnosis of colorectal carcinoma serological index in prior art, provide purposes in preparation intestinal cancer diagnostic reagent and test kit and medicine for the paranuclein.Intestinal cancer diagnostic reagent especially with paranuclein as target and test kit and medicine.
The present invention passes through the exception that detection paranuclein in PATIENTS WITH LARGE BOWEL body has expression, and paranuclein provides new auxiliary judgment means as the serologic marker thing index of diagnosis of colorectal carcinoma, the diagnosis for colorectal cancer.
Further, purposes in preparation intestinal cancer diagnostic reagent and test kit for the paranuclein is provided.
Further, the medicine of the treatment intestinal cancer with paranuclein as target is provided.
For achieving the above object, the present invention carries out the ELISA detection of paranuclein to the plasma sample of patients with bowel cancer and normal control：Including：
1, reagent：ELISA kit is purchased from Uscn Life Science company；
2, patient and sample：
1) randomly select the preoperative blood specimen of the patient being diagnosed as intestinal cancer, using health examination patient's blood as comparison；
2) adopt kernel cellulose content in double antibody ELISA method quantitative determination human plasma, corresponding operating is carried out according to kit specification：
3) map (seven point diagrams) after each standard substance and sample O.D. value deduction blank well O.D. value, with the concentration of standard substance as vertical coordinate (or logarithmic coordinates), O.D. it is worth for abscissa (or logarithmic coordinates), draw standard curve, preferably press the R2 value of regression equation calculation in embodiments of the invention, more leveled off to R2 value and 1 be preferred, according to sample O.D. value, corresponding concentration is found by standard curve, is multiplied by extension rate；Or the concentration with reference material calculates the regression equation of standard curve with O.D. value, the O.D. value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, obtain the actual concentrations of sample.
Experimental result shows：In intestinal cancer patient's blood plasma, paranuclein level is significantly higher than normal control, P=0.0005 (as shown in Figure 1)；Show that described paranuclein height in intestinal cancer is expressed and is secreted in blood, therefore, paranuclein can be used as the noinvasive mark of intestinal cancer diagnosis.
The present invention passes through the exception that detection paranuclein in PATIENTS WITH LARGE BOWEL body has expression, shows that described paranuclein can be used as the serologic marker thing index of diagnosis of colorectal carcinoma, the diagnosis for colorectal cancer provides new auxiliary judgment means.
Paranuclein of the present invention can be used for preparing intestinal cancer diagnostic reagent and test kit.
Test kit of the present invention includes the antibody for kernel fibroin；Described detection kit contains paranuclein standard substance.
Further, paranuclein of the present invention can be additionally used in preparing the target of the medicine for the treatment of intestinal cancer.
On the other hand, present invention also offers a kind of detection method of people's intestinal cancer mark paranuclein, it includes step：
(1) genomic DNA in separation and Extraction sample or albumen；
And/or
(2) expression of the genomic DNA of detection (1) gained or paranuclein in albumen.
The present invention passes through the exception of detection paranuclein expression in PATIENTS WITH LARGE BOWEL body, and in intestinal cancer patient's blood plasma, paranuclein level is significantly higher than normal control, there is provided paranuclein is as the serologic marker thing index of diagnosis of colorectal carcinoma.
Figure of description
Fig. 1 is intestinal cancer patient and paranuclein horizontal expression in the blood plasma of normal control, which show paranuclein horizontal expression in intestinal cancer patient's blood plasma and is significantly higher than normal control.
Specific embodiment
Embodiment 1 carries out the ELISA detection of paranuclein to the plasma sample of patients with bowel cancer and normal control
Reagent：ELISA kit is purchased from Uscn Life Science company.
Patient and sample：
Randomly selecting -2015 years 2 months in January, 2013 is newly diagnosed as the preoperative blood specimen of 30 patients of intestinal cancer, chooses 28 health examination patient's blood as comparison simultaneously.All blood specimen collection schemes are all ratified through Huashan Hospital Ethical Committee.
Kernel cellulose content in double antibody ELISA method quantitative determination human plasma：
Corresponding operating is carried out according to kit specification；
1, collection of specimens and holding
Patient blood specimen 1000 × g is centrifuged 20 minutes, takes supernatant (blood plasma) to be placed in -20 DEG C or -80 DEG C preservations；
2nd, operating procedure：
1) before testing, standard substance, reagent and sample prepare；
2) it is loaded：Set gauge orifice, testing sample hole, blank well respectively；If gauge orifice 7 hole, sequentially add the standard substance of 100 μ L variable concentrations, blank well adds 100 μ L, remaining hole adds testing sample 100 μ L, ELISA Plate adds overlay film, 37 DEG C incubate 2 hours；
3) discard liquid, dry, do not wash；
4) every hole adds detection solution A working solution 100 μ L, and ELISA Plate adds overlay film, and 37 DEG C incubate 1 hour；
5) discard in the hole liquid, every hole is washed with the cleaning mixture of 350 μ L, soak 1-2 minute, suck or get rid of the liquid in ELISA Plate, place mat paper is inhaled or flapped and pats dry in the hole liquid, repeat to wash plate 3 times；For the last time, after washing, the cleaning mixture of in the hole is dried completely；
6) every hole adds detection solution B working solution 100 μ L, adds overlay film, and 37 DEG C incubate 30 minutes；
7) discard in the hole liquid, dry, wash plate 5 times, method is with step 4；
8) every hole adds substrate solution 90 μ L, and ELISA Plate adds overlay film, 37 DEG C of lucifuge colour developings, and the response time controls in 15-25 minute, does not exceed 30 minutes.When the front 3-4 hole of gauge orifice has obvious gradient blue, when rear 3-4 gradient pores are inconspicuous, you can terminate；
9) every hole adds stop bath 50 μ L, terminating reaction, and now blueness is vertical turns yellow；The addition sequence of terminate liquid should be identical with the addition sequence of substrate solution, uneven color one such as, rocks ELISA Plate so that solution mix homogeneously；
10) after ELISA Plate bottom no water droplet and in the hole bubble-free, with the optical density (O.D. value) in each hole of 450nm wavelength measurement for the microplate reader.
3rd, calculate
Map (seven point diagrams) after each standard substance and sample O.D. value deduction blank well O.D. value, such as setting multiple holes, then should take its mean value calculation；With the concentration of standard substance as vertical coordinate (or logarithmic coordinates), O.D. value is abscissa (or logarithmic coordinates), draws standard curve, in the present embodiment, according to the R2 value determination of regression equation calculation, is more leveled off to R2 value and 1 is preferred；Make Curve Software (curve expert 1.30) using specialty to be analyzed, according to sample O.D. value, corresponding concentration is found by standard curve, is multiplied by extension rate；Or the concentration with reference material calculates the regression equation of standard curve with O.D. value, the O.D. value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, as the actual concentrations of sample.
Experimental result shows：In intestinal cancer patient's blood plasma, paranuclein level is significantly higher than normal control, P=0.0005 (as shown in Figure 1), shows that paranuclein height in intestinal cancer is expressed and is secreted in blood, and therefore, described paranuclein can be used as the noinvasive mark of intestinal cancer diagnosis.
Claims (7)
1. paranuclein is for preparing the purposes in intestinal cancer diagnostic reagent.
2. paranuclein is for preparing the purposes in intestinal cancer diagnostic kit.
3. the purposes as described in claim 1 or 2 is it is characterised in that described paranuclein is examined as colorectal cancer
Disconnected serologic marker thing index.
4. the purposes as described in claim 1 or 2 is it is characterised in that described diagnostic kit detection exists
The expression of paranuclein in PATIENTS WITH LARGE BOWEL body.
5. a kind of medicine treating intestinal cancer is it is characterised in that wherein, with paranuclein for treatment bowelcancer medicine
Target.
6. a kind of detection kit is it is characterised in that it includes the antibody for kernel fibroin, Yi Jihan
There are paranuclein standard substance.
7. a kind of detection method of people's intestinal cancer mark paranuclein is it is characterised in that it includes step：
(1) genomic DNA in separation and Extraction sample or albumen；
And/or
(2) expression of the genomic DNA of detection (1) gained or paranuclein in albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510467697.8A CN106405084A (en) | 2015-07-31 | 2015-07-31 | Application of nucleolin to preparation of diagnostic reagent and therapeutic for intestinal cancer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510467697.8A CN106405084A (en) | 2015-07-31 | 2015-07-31 | Application of nucleolin to preparation of diagnostic reagent and therapeutic for intestinal cancer |
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| CN106405084A true CN106405084A (en) | 2017-02-15 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110763845A (en) * | 2018-07-27 | 2020-02-07 | 成都市第三人民医院 | Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050053607A1 (en) * | 2002-04-08 | 2005-03-10 | Bates Paula J. | Method for the diagnosis and prognosis of malignant diseases |
| CN102539734A (en) * | 2005-05-12 | 2012-07-04 | 清华大学 | Nucleolin-assistant cancer diagnosis and treatment method |
-
2015
- 2015-07-31 CN CN201510467697.8A patent/CN106405084A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050053607A1 (en) * | 2002-04-08 | 2005-03-10 | Bates Paula J. | Method for the diagnosis and prognosis of malignant diseases |
| CN102539734A (en) * | 2005-05-12 | 2012-07-04 | 清华大学 | Nucleolin-assistant cancer diagnosis and treatment method |
Non-Patent Citations (1)
| Title |
|---|
| ABEER HAMMOUDI ET AL: "Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer(CRC)", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110763845A (en) * | 2018-07-27 | 2020-02-07 | 成都市第三人民医院 | Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit |
| CN110763845B (en) * | 2018-07-27 | 2022-10-28 | 成都市第三人民医院 | Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit |
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Application publication date: 20170215 |