CN102346188A - Novel purpose of THBS-1 - Google Patents
Novel purpose of THBS-1 Download PDFInfo
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- CN102346188A CN102346188A CN2010102444441A CN201010244444A CN102346188A CN 102346188 A CN102346188 A CN 102346188A CN 2010102444441 A CN2010102444441 A CN 2010102444441A CN 201010244444 A CN201010244444 A CN 201010244444A CN 102346188 A CN102346188 A CN 102346188A
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Abstract
The invention discloses a novel purpose of THBS-1, which is an application of thrombospondin 1 (THBS-1) in designing and/or preparing kits used for diagnosing prostate cancer. THBS-1 concentration is tested by using THBS-1 monoclonal antibody or a kit used for testing THBS-1, and then prostate cancer is diagnosed. As a result, a positive diagnostic efficiency of prostate cancer is 100%. Moreover, with the method, PSA negative (wherein a serum PSA level is lower than 10ng/ml) prostate cancer patients are diagnosed as positive. Therefore, the precision of the method is substantially higher than that of the existing PSA diagnostic method.
Description
Technical field
The present invention relates to the new purposes of THBS-1.
Background technology
(Prostate Cancer PCa) is one of modal malignant tumour among the north america male sex to prostate cancer, shows according to American Cancer Society's statistical figure in 2009, and the patient who newly is diagnosed as prostate cancer accounts for this year and newly is diagnosed as 25% of malignant tumor patient
[1], account for first of male tumor; Death toll accounts for 9% of total male sex's malignant tumour death toll, is only second to lung cancer (33%).In China, along with the progressively change of aging and living environment of population structure, the incidence of disease of prostate cancer increases year by year, is in Rapid development stage.
The secretory protein group is meant and can be secreted into extracellular all protein summation, the implication of its broad sense in several ways, can " discharge " to extracellular all protein.Part in the tumor cell secretion albumen can be released into blood, becomes potential blood serum designated object.Mass-spectrometric technique is that the discovery of tumor markers provides a huge protein sequence database.Directly the secretory protein of malignant cell is analyzed, to identifying that the malignant tumour label in the serum is a more high-efficiency method.
Prostate is an androgen-dependent organ; The androgen withdrawal treatment is effective in early days for prostate cancer; But the patient tends to after such treatment about two years and develops into androgen independence; The androgen withdrawal treatment has just lost effect, and tumour tends to obtain stronger aggressive and transfer ability.LNCaP (androgen-dependent AR is positive) is representative prostate cancer cell line, and C4-2 cell (androgen independence and AR are positive) is to be developed and next clone by LNCaP.LNCaP is inoculated in subcutaneous 8 weeks of nude mice and with the nude mice castration, it is subcutaneous in nude mice that tumor inoculations are taken out in 4 week backs, once more with the nude mice castration.So just obtained the C4-2 cell, they are identical with LNCaP cytogenetics background, but have obtained the energy for growth of the non-dependence of androgen, and have stronger one-tenth knurl property.LNCaP and C4-2 cell have been simulated patient's PD process clinically well, are the prostate cancer research cell models of admitting in the world.PC3 is the cell model that the prostate cancer bone shifts, and androgen receptor is negative; C4-2B, this clone also is to be developed by LNCaP, the clone for bone shifts equally also has the character of the non-dependence of androgen; DU145 is the clone that the prostate cancer brain shifts, and androgen receptor is negative.
(thrombospondin-1 THBS-1) is the principal ingredient of the α granule of platelet to the responsive albumen 1 of blood coagulation, and the migration and the tube chamber that can combine with cell surface CD36 to suppress endothelial cell form.The amino acid sequence of THBS-1 is shown in SEQID NO:1.
The amino acid sequence of PSA (PSA) is shown in SEQ ID NO:2.
Summary of the invention
An object of the present invention is to provide the responsive albumen 1 of blood coagulation new purposes, the responsive albumen 1 of anticoagulation antibody new purposes and detect the new purposes of the kit of the responsive albumen 1 of blood coagulation.
The new purposes of the antibody of the responsive albumen 1 of anticoagulation provided by the present invention is the polyclonal antibody of monoclonal antibody and/or the responsive albumen 1 of anticoagulation of the responsive albumen 1 of anticoagulation is used for the kit of auxiliary diagnosis cancer in preparation application.
Above-mentioned antibody all is the antibody that obtains from commercial sources, or cultivates the antibody that obtains through cells in vitro, specifically can be available from R&D company.
The new purposes of the kit of the responsive albumen 1 of detection blood coagulation provided by the present invention is the kit of the responsive albumen 1 of detection blood coagulation is used for the kit of auxiliary diagnosis cancer in preparation application.
The new purposes of the new purposes of the responsive albumen 1 of blood coagulation provided by the present invention is the responsive albumen 1 of blood coagulation is used for the kit of auxiliary diagnosis cancer in design and/or preparation application.
In above-mentioned arbitrary said application, the monoclonal antibody of the responsive albumen 1 of said anticoagulation is responsive albumen 1 mouse monoclonal antibody of anticoagulation.
In above-mentioned arbitrary said application, said cancer is prostate cancer, carcinoma of urinary bladder or clear cell carcinoma of kidney.
In above-mentioned arbitrary said application, said prostate cancer is the negative prostate cancer of PSA (PSA).
In above-mentioned arbitrary said application, said PSA feminine gender be in the serum PSA concentration smaller or equal to 10ng/ml.
In above-mentioned arbitrary said application, the kit that detects the responsive albumen 1 of blood coagulation specifically can be available from R&D company, and catalog number is DTSP-10.
In above-mentioned arbitrary said application, the amino acid sequence of the responsive albumen 1 of said blood coagulation is shown in SEQ ID NO:1; The amino acid sequence of PSA (PSA) is shown in SEQ ID NO:2.
The experiment proof; Detect the THBS-1 concentration value with the monoclonal antibody of THBS-1 or the kit of detection THBS-1; And then diagnosing prostate cancer, be 100% to the positive diagnosing rate of prostate cancer, more outstanding is; This method can be diagnosed as the positive with the patients with prostate cancer of PSA negative (Serum PSA level is less than 10ng/ml), and the degree of accuracy that this method is described is apparently higher than existing P SA diagnostic method.
Description of drawings
Fig. 1 is the typical curve that logarithm and the serum double fastener heart ELISA of THBS-1 concentration detects the logarithm match of 450nm light absorption (OD450Correct) after the correction that obtains.
Fig. 2 is THBS-1 in patients with prostate cancer and other male urinary system tumor patient expression of serum apparently higher than normal the elderly.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The composition of embodiment 1, kit
One, kit is formed
Kit (Human Thrombospondin-1 quantification kit) is available from R&D company, and catalog number is DTSP-10, the concrete composition as follows:
1, Thrombospondin-1Micorplate: the 96 hole polyphenyl propylene microplates (8 holes * 12) that encapsulated anti-Thrombospondin-1 mouse monoclonal antibody;
2, the Thrombospondin-1Conjugate:21ml coupling the anti-Thrombospondin-1 polyclonal antibody of horseradish peroxidase;
3, Thrombospondin-1Standard:1000ng Thrombospondin-1 recombinant protein freeze-dried powder;
4, Assay Diluent RD1-56:17ml buffering protein solution;
5, Calibrator Diluent RD5-33Concentrate:2 pipe (21ml/ pipe) buffering protein alkali;
6, Wash Buffer Concentrate:21ml/ pipe 25 * padded surface activating agent concentrated solution (containing antiseptic);
7, Color Reagent A:12.5ml stable state superoxol;
8, Color Reagent B:12.5ml stable state chromogen matter (tetramethyl benzidine);
9, Stop Solution:6ml 2mol/L aqueous sulfuric acid;
10, Plate Covers: 4 of microplate sealing strips.
Embodiment 2, kit diagnosing prostate cancer
One, the method that detects with kit
(1) preparation of working fluid and standard items, dilution of sample
Wash Buffer working fluid: 20ml Wash Buffer Concentrate adds deionized water and is settled to 500ml.
Calibrator Diluent RD5-33 working fluid: 20ml Calibrator Diluent RD5-33Concentrate and the abundant mixing of 20ml deionized water.
Thrombospondin-1 standard items dilution: 1000ng Thrombospondin-1 recombinant protein freeze-dried powder adds the 1ml deionized water; Stir gently; Leave standstill then more than 15 minutes it is fully dissolved, promptly get 1000ng/mlThrombospondin-1 standard items stock solution.With Calibrator Diluent RD5-33 working fluid Thrombospondin-1 standard items stock solution is carried out 7 doubling dilution, has obtained concentration and be respectively 500,250,125,62.5,31.3,3.1,15.6 with the standard items dilution of 7.8ng/ml.Use the Calibrator DiluentRD5-33 working fluid that does not add standard items as blank (0ng/ml);
Be used for sample of blood final proof article: 10 μ l serum add 990 μ l Calibrator Diluent RD5-33 working fluids, fully mixing.
(2) operation steps:
2.1 the preparation of typical curve
(1) take out two Thrombospondin-1Micorplate, remainder is used the tinfoil paper good seal again;
(2) every hole adds 100 μ l Assay Diluent RD1-56;
(3) to add 50 μ l Thrombospondin-1 concentration respectively be 500,250,125,62.5 in every hole, 31.3,3.1,15.6 with standard items dilution and the blank of 7.8ng/ml, each concentration is done two repetitions.With each hole of Plate Covers sealing, normal temperature was hatched two hours at miniature shaking table (rotating speed is 500rpm);
(4) thoroughly discard sample in every hole; Wash Buffer working fluid (about 400 μ l) is filled it up with in every hole; Normal temperature is washed in the ELISA special use and was washed on the plate machine 3 minutes, and thoroughly reject Wash Buffer working fluid repeats this cleaning 4 times; Behind last the end, Thrombospondin-1Micorplate is tipped upside down on the paper handkerchief of cleaning.
(5) every hole adds 200 μ l Thrombospondin-1Conjugate, and with each hole of Plate Covers sealing, normal temperature was hatched two hours at miniature shaking table (rotating speed is 500rpm);
(6) thoroughly discard Thrombospondin-1Conjugate in every hole; Wash Buffer working fluid (about 400 μ l) is filled it up with in every hole; Normal temperature is washed in the ELISA special use and was washed on the plate machine 3 minutes, and thoroughly reject Wash Buffer working fluid repeats this cleaning 4 times; Behind last the end, Thrombospondin-1Micorplate is tipped upside down on the paper handkerchief of cleaning.
(7) Color Reagent A mixes with Color Reagent B equal-volume, mixes in back 15 minutes and must use;
(8) every hole adds 200 μ l (7) mixed liquors, and the normal temperature lucifuge was hatched 30 minutes;
(9) every hole adds 50 μ l Stop Solution, and color should become yellow by blueness, if irregular colour one is patted microplate its content is fully mixed;
In (10) 30 minutes with microplate (Microplate Reader; Model 680; Biorad) read the 450nm and the 570nm light absorption reading in each hole; The latter deducts the OD450* reading that the former obtains every hole, calculates two mean values that repeat the OD450* reading of each concentration, and this value deducts two in blank hole and repeats the OD450Correct reading that the mean value of OD450* reading is each Thrombospondin-1 concentration correspondence. and each Thrombospondin-1 concentration is taken the logarithm respectively with corresponding OD450Correct; Use Microsoft Office Excel 2003 softwares and draw scatter diagram; And the match linear dependence, obtain typical curve (for example like table 1, Fig. 1).The functional expression of typical curve is y=0.6664X-1.325.R
2＝0.9762。
Table 1Thrombospondin-1 double fastener heart ELISA detects
2.2 the detection of serum T hrombospondin-1 concentration.
(1) take out some Thrombospondin-1 Micorplate, remainder is used the tinfoil paper good seal again
(2) every hole adds 100 μ l Assay Diluent RD1-56
(3) every hole adds 50 μ l respectively and is used for sample of blood final proof article (seeing dilution of sample), adds to do a blank hole step (3) in the method same 2.1 again.With each hole of Plate Covers sealing, normal temperature was hatched two hours at miniature shaking table (rotating speed is 500rpm);
(4) thoroughly discard sample in every hole; Wash Buffer working fluid (about 400 μ l) is filled it up with in every hole; Normal temperature is washed in the ELISA special use and was washed on the plate machine 3 minutes, and thoroughly reject Wash Buffer working fluid repeats this cleaning 4 times; Behind last the end, Thrombospondin-1Micorplate is tipped upside down on the paper handkerchief of cleaning.
(5) every hole adds 200 μ l Thrombospondin-1Conjugate, and with each hole of Plate Covers sealing, normal temperature was hatched two hours at miniature shaking table (rotating speed is 500rpm);
(6) thoroughly discard Thrombospondin-1Conjugate in every hole; Wash Buffer working fluid (about 400 μ l) is filled it up with in every hole; Normal temperature is washed in the ELISA special use and was washed on the plate machine 3 minutes, and thoroughly reject Wash Buffer working fluid repeats this cleaning 4 times; Behind last the end, Thrombospondin-1Micorplate is tipped upside down on the paper handkerchief of cleaning.
(7) Color Reagent A mixes with Color Reagent B equal-volume, mixes in back 15 minutes and must use
(8) every hole adds 200 μ l (7) mixed liquors, and the normal temperature lucifuge was hatched 30 minutes
(9) every hole adds 50 μ l Stop Solution, and color should become yellow by blueness, if irregular colour one is patted microplate its content is fully mixed
In (10) 30 minutes with microplate (Microplate Reader; Model 680; Biorad) read the 450nm and the 570nm light absorption reading in each hole, the latter deducts the OD450* reading that the former obtains every hole, calculates two mean values that repeat the OD450* reading of each concentration; This value deducts blank hole OD450* reading and is the corresponding OD450Correct reading of each blood serum sample. and this reading is taken the logarithm ask x, 100 * lg as y substitution typical curve
(1)X is serum T hrombospondin-1 content.
PSA (PSA) concentration detection method: Applied Electrochemistry luminescence method; Use Roche Elecsys2010 fully automatic electric chemical illumination immunity analysis instrument and supporting PSA reagent thereof (the German Luo Shi diagnosis company limited by forming a complete production network with instrument provides), all use in the phase in effect.The preparation of typical curve and serum detect and carry out in strict accordance with operating process.The content value of PSA is with M (Q
R) expression, M representes the median of THBS-1 content in the sample; Q
RThe interquartile range of THBS-1 content, i.e. Q in the expression sample
RThe absolute value of the difference of=upper quartile value and lower quartile numerical value.
(3) detect patient's serum
According to method described in the experiment (two) the THBS-1 concentration in following experimenter's the serum is detected: the normal elderly men of 10 examples; 105 routine patients with prostate cancer; Gerontal patient's (carcinoma of urinary bladder 10 examples, clear cell carcinoma of kidney 13 examples) of 23 other male urinary system tumours of example.Experimenter's agreement is all passed through in the collection of serum.
Result such as table 2 are with shown in Figure 2.Blood-serum P SA concentration is with M (Q
R) expression, serum T HBS-1 concentration is with (mean+SD) expression.
Show that THBS-1 is extremely remarkable normal the elderly and patients with prostate cancer expression of serum amount difference, T verifies difference has statistical significance (* * *: p＜0.001).Proof can be come diagnosing prostate cancer through the serum-concentration that detects THBS-1.
Further the serum T HBS-1 concentration with PSA≤10ng/ml patients with prostate cancer in THBS-1 concentration in the normal elderly men serum and the serum compares, and the blood-serum P SA concentration to them also compares simultaneously.The THBS-1 level is all apparently higher than normal elderly men in the patients with prostate cancer serum of the patients with prostate cancer of result: PSA≤10ng/ml (*) and PSA＞10ng/ml (*).Use the T check and confirm that it has statistical significance (p equals 0.0010 and 0.0004 respectively).And in the patient of PSA≤10ng/ml, PSA level and normal the elderly and no significant difference (p=0.0937), but THBS-1 concentration value and normal elderly men significant difference.Explain: THBS-1 helps the diagnosis of Serum PSA level smaller or equal to the prostate patient of 10ng/ml, and the Serum PSA level of these patients with prostate cancer and normal elderly men and no significant difference (table 2).
Further with THBS-1 concentration in the normal elderly men serum and carcinoma of urinary bladder 10 examples, clear cell carcinoma of kidney 13 routine gerontal patients' serum T HBS-1 concentration compares, and serum T HBS-1 concentration is with (mean+SD) expression).The result: the gerontal patient's of carcinoma of urinary bladder and clear cell carcinoma of kidney serum T HBS-1 level is all apparently higher than normal the elderly.Use the T check and confirm that it has statistical significance (p equals 0.0022 and 0.0014 respectively).Explain that THBS-1 helps the diagnosis of elderly men carcinoma of urinary bladder and clear cell carcinoma of kidney.
Show, detect the THBS-1 concentration value, can be used for diagnosing prostate cancer with this kit.
Claims (7)
1. the polyclonal antibody of the monoclonal antibody of the responsive albumen 1 of anticoagulation and/or the responsive albumen 1 of anticoagulation is used for the application of the kit of auxiliary diagnosis cancer in preparation.
2. the kit that detects the responsive albumen 1 of blood coagulation is used for the application of the kit of auxiliary diagnosis cancer in preparation.
3. the responsive albumen 1 of blood coagulation is used for the application of the kit of auxiliary diagnosis cancer in design and/or preparation.
4. according to arbitrary described application among the claim 1-3, it is characterized in that: said cancer is prostate cancer, carcinoma of urinary bladder or clear cell carcinoma of kidney;
The monoclonal antibody of the responsive albumen 1 of said anticoagulation is responsive albumen 1 mouse monoclonal antibody of anticoagulation.
5. application according to claim 4 is characterized in that: said prostate cancer is the negative prostate cancer of PSA.
6. according to arbitrary described application among the claim 1-5, it is characterized in that: said PSA negative for PSA concentration in the serum smaller or equal to 10ng/ml.
7. according to arbitrary described application among the claim 1-6, it is characterized in that: the amino acid sequence of the responsive albumen 1 of said blood coagulation is shown in SEQ ID NO:1; The amino acid sequence of said PSA is shown in SEQ IDNO:2.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6303324B1 (en) * | 1997-03-19 | 2001-10-16 | Oncotech, Inc. | Methods for cancer prognosis and diagnosis |
| CN1867679A (en) * | 2003-07-17 | 2006-11-22 | 环太平洋生物技术有限公司 | Markers for detection of gastric cancer |
| CN1977049A (en) * | 2004-04-26 | 2007-06-06 | 儿童医疗中心有限公司 | Platelet biomarkers for the detection of disease |
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2010
- 2010-08-03 CN CN201010244444.1A patent/CN102346188B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6303324B1 (en) * | 1997-03-19 | 2001-10-16 | Oncotech, Inc. | Methods for cancer prognosis and diagnosis |
| CN1867679A (en) * | 2003-07-17 | 2006-11-22 | 环太平洋生物技术有限公司 | Markers for detection of gastric cancer |
| CN1977049A (en) * | 2004-04-26 | 2007-06-06 | 儿童医疗中心有限公司 | Platelet biomarkers for the detection of disease |
Non-Patent Citations (10)
| Title |
|---|
| 《Eur.J.Biochem.》 20030228 Nathalie Heuzé -Vourćh et al. Complex alternative splicing of the hKLK3 gene coding for the tumor marker PSA (prostate-specific-antigen 706-714 1-7 , 第270期 * |
| 《THE JOURNAL OF UROLOGY》 20091130 Dragomir P. Zubac, et al. The Expression of Thrombospondin-1 and p53 in Clear Cell Renal Cell Carcinoma: Its Relationship to Angiogenesis, Cell Proliferation and Cancer Specific Survival 第2144-2149页 1-5 第182卷, * |
| DRAGOMIR P. ZUBAC, ET AL.: "The Expression of Thrombospondin-1 and p53 in Clear Cell Renal Cell Carcinoma: Its Relationship to Angiogenesis, Cell Proliferation and Cancer Specific Survival", 《THE JOURNAL OF UROLOGY》, vol. 182, 30 November 2009 (2009-11-30), pages 2144 - 2149, XP026677709, DOI: doi:10.1016/j.juro.2009.07.015 * |
| GARY D. GROSSFELD ET AL.: "Thrombospondin-1 Expression in Bladder Cancer: Association With p53 Alterations, Tumor Angiogenesis, and Tumor Progression", 《JOURNAL OF THE NATIONAL CANCER INSTITUTE》, vol. 89, no. 3, 5 February 1997 (1997-02-05), pages 219 - 227 * |
| JONATHAN CHARLES GODDARD ET AL.: "Reduced Thrombospndin-1 at Presentation Predicts Disease Progression in Superficial Bladder Cancer", 《EUROPEAN UROLOGY》, vol. 42, no. 5, 30 November 2002 (2002-11-30), pages 464 - 468 * |
| KEMIN TAN ET AL.: "The Structures of the Thrombospondin-1 N-Terminal Domain and Its Complex with a Synthetic Pentameric Heparin", 《STRUCTURE》, vol. 14, no. 1, 2 July 2010 (2010-07-02), pages 33 - 42 * |
| MICHAEL W. SHAFER ET AL.: "Antibody Array Profiling Reveals Serum TSP-1as a Marker to Distinguish Benign From Malignant Prostatic Disease", 《THE PROSTATE》, no. 67, 27 December 2006 (2006-12-27), pages 255 - 267 * |
| NATHALIE HEUZÉ –VOURćH ET AL.: "Complex alternative splicing of the hKLK3 gene coding for the tumor marker PSA (prostate-specific-antigen", 《EUR.J.BIOCHEM.》, no. 270, 28 February 2003 (2003-02-28), pages 706 - 714, XP002316593, DOI: doi:10.1046/j.1432-1033.2003.03425.x * |
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