CN103123355B - Kit for diagnosis of prostate cancer bone metastases and use method thereof - Google Patents
Kit for diagnosis of prostate cancer bone metastases and use method thereof Download PDFInfo
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- CN103123355B CN103123355B CN201210579863.XA CN201210579863A CN103123355B CN 103123355 B CN103123355 B CN 103123355B CN 201210579863 A CN201210579863 A CN 201210579863A CN 103123355 B CN103123355 B CN 103123355B
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Abstract
The invention relates to a kit for diagnosis of prostate cancer bone metastases and a use method of the kit. The kit is used for determining the antibacterial protein Hepcidin content in patient serum, and can provide reference data for the diagnose of the prostate cancer bone metastasis and a technology platform for exploring treatment of the human body prostate cancer bone metastasis. Simultaneously, an effective monitoring basis can be further provided for the early diagnosis and a prognosis therapeutic effect evaluation of the prostate cancer bone metastasis.
Description
Technical field
The present invention relates to a kind of kit for prostate cancer with osseous metastasis diagnosis and using method thereof, belong to field of medicaments.
Background technology
Prostate cancer is global the sixth-largest malignant tumour.In the U.S., prostate-cancer incidence occupies male malignancy first, and mortality ratio is only second to lung cancer, and its incidence of disease is raise trend year by year in China.Prostate cancer can diffuse to the arbitrary tissue of whole body or organ by Blood route metastasis, modal DISTANT METASTASES IN position is bone, so prostate cancer is the malignant tumour that Bone tumour the most easily occurs, can there is Bone tumour in the patients with prostate cancer more than 80%, and dying from 85%-100% merging Bone tumour in patients with prostate cancer.
Koeneman etc. propose the Mechanism Hypothesis of prostate cancer osteogenic transfer on the basis of summing up forefathers' research, point three phases: 1. cancer metastasis is to bone, ramp under growth factor bFGF abundant in bone matrix, the effect of TGF β and IGFs.2. cancer cell is bred in a large number, starts to secrete PSA, TSP2 1 (thrombospondin21), and the latter is a kind of stromatin.By suppressing host immune system, promote growth of tumour cell.TGF2 β regulates bone matrix protein deposition and remodeling type.3. in progressive stage, bFGF, EGF, keratinocyte growth factor (keratinocyte growth factor, KGF) and ET21 etc. are in high level, thus occur osteogenic response.The coefficient results such as the rise of osteoblast differentiation, bone matrix deposition, alkaline phosphatase, calcium concentration rising, cause fiber bon e formation.
The initial stage of prostate cancer bone transfer does not often accompany symptom, the pain that can occur together when being in a bad way, weak and dysfunction.The common site of Bone tumour comprises backbone, hipbone, rib and shoulder blade, and the patients with terminal generation ostalgia of about 60%, is common in waist, sacrum portion, buttocks, hip pelvis.Ostalgia has the different forms of expression, and some patient can show as rest pain, and some patient then shows as intermittent pain.Ostalgia can be confined to a certain privileged site of health, also can show as the migrans pain of health different parts; May change in intraday different time ostalgia, also different with movable reaction to having a rest.If because tumor invading makes sclerotin obviously become fragile, there is pathologic fracture possibly.Some position is arthritic common site, as knee joint and shoulder joint, the pain occurred at these positions might not be caused by prostate cancer transfer, need whether inspection is clear and definite further exists prostate cancer with osseous metastasis, and early diagnosis has decisive significance to prostate cancer therapy, Bone tumour whether is had mainly through x-ray photo and radionuclide bone scan video picture at present for detecting prostate cancer, but shortage specificity, not only the skeletal diseases such as old fracture can not be differentiated, and be easily subject to the impact of medicine, as diphosphate.
Along with the continuous understanding to prostate cancer with osseous metastasis mechanism, the bone matrix turnover label relevant to prostate cancer with osseous metastasis also progressively comes into one's own, but still there is no sensitivity at present, special label, so be badly in need of wanting we the world of medicine experts and scholars jointly go to study a kind of prostate cancer with osseous metastasis responsive easily to be surveyed, method that specificity is high, for we prostate cancer with osseous metastasis patient provides definite clinical diagnosis foundation, we patient can be obtained the effective treatment in the very first time.The detection method that preferably this sensitivity is easy can at any time for prostate cancer with osseous metastasis anaphase and prognosis situation provide trace detection, and being convenient to us can take therapeutic scheme targetedly according to the progress of the state of an illness in time.
Summary of the invention
The present inventor by prostate cancer from cercinoma prophase pathologic change, further investigation to serial procedures such as transfers in late period, test has found that one is applicable to prostate cancer with osseous metastasis and detects, and the scheme that result is responsive, specificity is high, the present invention provides this diagnostic kit for diagnosing prostate cancer Bone tumour at this.And this diagnostic kit is the kit for measuring antibacterial protein Hepcidin content in serum, by measuring the antibacterial protein Hepcidin in patients serum, can effectively prostate cancer with osseous metastasis and the bone disease such as old fracture, primary bone tumor be differentiated.
The present invention provides a kind of monitoring reagent box for the follow-up evaluation to prostate cancer with osseous metastasis treatment situation on the other hand, by the monitoring of kit, be convenient to doctor and the testing result different according to patient can understand its rehabilitation situation, and formulate targeted treatment schemes, treat timely and effectively for patient provides.
The diagnostic kit of the prostate cancer with osseous metastasis described in the present invention and prostate cancer with osseous metastasis Prognosis scoveillance kit are by measuring the antibacterial protein Hepcidin in patients serum, the content measuring antibacterial protein in serum is diagnosed or is monitored, Hepcidin be derive from liver body in regulator of iron homeostasis, small intestine can be suppressed the absorption of iron and utilization, and the iron release of reticuloendothelial system macrophage, play center adjustment effect.Found Hepcidin and infection, tumour and chronic inflammation closely related.The present inventor is through experimental studies have found that, the expression of serum Hepcidin, BMP6, IL-6, sTfR and the index of routine blood test of prostate cancer with osseous metastasis patient is measured by ELISA method, find that serum Hepcidin expresses in prostate cancer with osseous metastasis group to increase without Bone tumour group, hyperplasia of prostate control group bright all showing compared with prostate cancer, gap has statistical significance (P < 0.05).Illustrate that the high expressed of serum Hepcidin in prostate cancer with osseous metastasis patient has value of clinical studies, patients with prostate cancer generation Bone tumour and Hepcidin in close relations.By quantitatively detecting the important evidence that can be used for as prostate cancer with osseous metastasis clinical diagnosis to serum of patients with prostate cancer Hepcidin, and the evaluation index of prostate cancer with osseous metastasis prognosis.
Hepcidin diagnosis, monitoring reagent box and Hepcidin enzyme linked immunological kit, using method:
A. the dilution of standard items: prepare small test tube 6, finish number successively, first adds standard dilutions 100ul in each small test tube, then gets original content standard items 100ul and adds in the test tube of the number of finishing, fully mix; In this test tube, get 100ul again add in second test tube, fully mix; In this test tube, get 100ul again add in the 3rd test tube, fully mix; In this test tube, get 100ul again add in the 4th test tube, fully mix; In this test tube, get 100ul again add in the 5th test tube, fully mix; Then in this test tube, get 100ul, discard.6th test tube is as No. 0 standard items.After dilution, each pipe concentration respectively: 280ng/ml, 140ng/ml, 70 ng/ml, 35ng/ml, 17.5ng/ml, 0 ng/ml.At enzyme mark bag by the accurate sample wells of bidding on plate, add the standard items 50ul of variable concentrations successively.
B. sample: patient's empty stomach in early morning venous blood samples 3ml is placed in EDTA anti-coagulants vacuum tube, and 4 DEG C of centrifugal l0min of 1000xg, isolate serum, detect as early as possible; Or put-80 DEG C of Refrigerator stores, before use must equilibrate at room temperature.
C. application of sample: establish blank well respectively, blank control wells does not add sample, enzyme marking reagent and biotin labeled anti-Hepcidin antibody, and respectively step operation is identical for all the other, testing sample hole.In enzyme mark bag is by testing sample hole on plate, first adds sample 40 μ l, and then adds biotin labeled Hepcidin antibody 10 μ l.Sample is added on bottom ELISA Plate hole by application of sample, does not touch hole wall as far as possible, rocks mixing gently.
D. incubation: use the rearmounted 37 DEG C of incubations of shrouding film shrouding 30 minutes.
E. dosing: by for subsequent use after 30 times of concentrated cleaning solution distilled water 30 times dilution.
F. wash: carefully take shrouding film off, discard liquid, dry, cleansing solution is filled it up with in every hole, leaves standstill and discards after 30 seconds, so repeats 5 times, pats dry.
G. enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.
H. incubation: operate same D.
I. wash: operate same F.
J. develop the color: every hole first adds developer A50 μ l, then adds developer B50 μ l, and shake mixing gently, 37 DEG C of lucifuges develop the color 15 minutes.
K. stop: every hole adds stop buffer 50 μ l, cessation reaction (now blue standing turns yellow).
L. measure: measure within 15 minutes after adding stop buffer, sequentially measure the absorbance (OD value) in each hole with blank air-conditioning zero, 450nm wavelength.
Embodiment
The following examples can help those skilled in the art more fully to understand the present invention.But do not limit the present invention in any way.
embodiment 1. prostate cancer with osseous metastasis is diagnosed
One, case is chosen
Choose prostate cancer with osseous metastasis patient 30 example, prostate cancer without Bone tumour patient 30 example and Patients with Prostatic Hyperplasia 30 example.50 ~ 70 years old age, average 65 years old [exclusion standard: acute inflammatory reaction: c reactive protein (C-Reactive Protein CRP) >6mg/L), dysfunction of liver (prothrombin time and glutamic-pyruvic transaminase are extremely), has heart and lung diseases medical history.] 。Prostate cancer with osseous metastasis group PSA baseline is greater than 20 μ g/L, and scope 20.0 ~ 1500 μ g/L, median 138.0 μ g/L, all the pathological diagnosis of row aspiration biopsy of prostatic gland is prostate cancer, and the inspection of whole body nuclide bone scan is diagnosed as Bone tumour.Without Bone tumour group PSA 3.5 ~ 28.2 μ g/L, median 10.2 μ g/L, the pathological diagnosis of row aspiration biopsy of prostatic gland is prostate cancer, and the inspection of whole body nuclide bone scan has no Bone tumour stove.Patients with Prostatic Hyperplasia group PSA0.3 ~ 14.2 μ g/L, median 3.7 μ g/L, row TURP Pathological diagnoses is Patients with Prostatic Hyperplasia.Have prostatic tubercle, 5 routine PSA > 10 μ g/L, 7 routine PSA 4 ~ 10 μ g/L but FPSA/TPSA < 0.16 row aspiration biopsy of prostatic gland for 2 examples, pathological diagnosis is Patients with Prostatic Hyperplasia.
Two, serum Hepcidin, BMP6, IL-6, sTfR measurement result
Early morning, venous blood samples 3ml was placed in EDTA anti-coagulants vacuum tube on an empty stomach, and 4 DEG C of centrifugal l0min, isolate serum, put-80 DEG C of Refrigerator stores.Adopt competitive homophase enzyme-linked immunosorbent assay (ELISA) quantitative measurement sample serum Hepcidin, IL-6, sTfR, BMP6 respectively.
Each group of serum Hepcidin, IL-6, sTf, BMP6 expression and Hb value are in table 1.Hepcidin prostate cancer with osseous metastasis group express apparently higher than without Bone tumour group and with Patients with Prostatic Hyperplasia group, difference has statistical significance (P<0.05), compare with Hb, no significant difference (P>0.05).Prostate cancer is without Bone tumour group and Patients with Prostatic Hyperplasia group comparing difference not statistically significant (P>0.05).
Table 13 groups of patients serums Hepcidin, IL-6, sTfR, BMP6 express and Hb value compares
| Project | Prostate cancer with osseous metastasis group | Prostate cancer is without Bone tumour group | Hyperplasia of prostate control group |
| Hepcidin(μg/L) | 70.5±13.3a | 39.2±14.3 | 33.9±12.6 |
| IL-6(μg/L) | 23.8±6.4a | 12.3±7.1 | 11.5±5.9 |
| sTfR(μg/L) | 5.3±2.3 a | 7.5±1.9 | 7.9±2.7 |
| BMP6(μg/L) | 430.1±56.2 a | 334.8±47.3 | 289.9±40.6 |
| Hb（g/L） | 127.8±10.7 | 138.1±11.7 | 142.3±10.4 |
Note: a compares P<0.05 with prostate cancer without Bone tumour group, Patients with Prostatic Hyperplasia group.
Three, conclusion
The main pathological characteristics of prostate cancer with osseous metastasis shows as prostate cancer tissue companion's osteoblast active proliferation of transfer and a large amount of new bone formation, also can show as osteolytic or Combination.The diagnosis of prostate cancer with osseous metastasis is mainly through nuclide bone scan inspection, but and insensitive.
The present inventor finds after deliberation, serum Hepcidin expresses in prostate cancer with osseous metastasis group and comparatively increases without Bone tumour group, Patients with Prostatic Hyperplasia group bright all showing, illustrate that the high expressed of serum Hepcidin in prostate cancer with osseous metastasis patient has value of clinical studies, patients with prostate cancer generation Bone tumour and Hepcidin in close relations, Bone tumour progress and body Micro-inflammation state, iron metabolism have close relationship.
This research finds that the BMP6 of prostate cancer with osseous metastasis patient expresses and comparatively obviously increases without Bone tumour group and Patients with Prostatic Hyperplasia group, and the expression of BMP6 and being proportionate property of Hepcidin, what show that prostate cancer with osseous metastasis patients serum Hepcidin expresses increases, and is likely caused by BMP6/SAMD signal path.The high expressed of prostate cancer with osseous metastasis patient B MP6 have stimulated the expression of the Molecular regulator Hepcidin of iron metabolism, causes the change of inflammatory conditions in body iron metabolism and body, thus causes Bone tumour.This research simultaneously also finds that the IL-6 of prostate cancer with osseous metastasis group expresses and obviously increases, and serum Hepcidin positive correlation.Microinflammation stimulates the expression of Hepcidin, and IL-6 is the main mediated factor that inflammatory reaction regulates.IL-6 regulates the expression of serum Hepcidin, its mechanism of action may be the expression by stimulating JAK/STAT signal path to raise phosphorylated signal transduction and activating transcription factor 3 (phosphor-rylated signal transduction and activators of transcription-3 p-STAT3), p-STAT3 suppresses the generation of antibacterial peptide (Hepcidin antimicrobial peptide HAMP), thus the synthesis of serum Hepcidin is increased.IL-6 stimulates the mediation of JAK/STAT signal path, causes Hepcidin to change in the expression of prostate cancer with osseous metastasis.
So the expression of serum Hepcidn, can as the sensitive indicator of diagnosing prostate cancer Bone tumour.The content simultaneously detecting Hepcidn in patients serum may be used for the auxiliary diagnosis means of prostate cancer with osseous metastasis nuclide bone scan inspection.
embodiment 2. prostate cancer with osseous metastasis prognosis condition monitoring
One, case is chosen
Choose prostate cancer with osseous metastasis patient 50 example, 55 ~ 75 years old age, average 66 years old, [exclusion standard: acute inflammatory reaction: c reactive protein (C-Reactive Protein CRP) >6mg/L), dysfunction of liver (prothrombin time and glutamic-pyruvic transaminase are extremely), has heart and lung diseases medical history.] 。Prostate cancer with osseous metastasis group PSA baseline is greater than 20 μ g/L, and scope 20.0 ~ 1500 μ g/L, median 138.0 μ g/L, all the pathological diagnosis of row aspiration biopsy of prostatic gland is prostate cancer, and the inspection of whole body nuclide bone scan is diagnosed as Bone tumour.
Two, serum Hepcidin measurement result
Hepcidin enzyme linked immunological kit assay method: the dilution of A. standard items: prepare small test tube 6; finish number successively; first in each small test tube, add standard dilutions 100ul, then get original content standard items 100ul and add in the test tube of the number of finishing, fully mix; In this test tube, get 100ul again add in second test tube, fully mix; In this test tube, get 100ul again add in the 3rd test tube, fully mix; In this test tube, get 100ul again add in the 4th test tube, fully mix; In this test tube, get 100ul again add in the 5th test tube, fully mix; Then in this test tube, get 100ul, discard.6th test tube is as No. 0 standard items.After dilution, each pipe concentration respectively: 280ng/ml, 140ng/ml, 70 ng/ml, 35ng/ml, 17.5ng/ml, 0 ng/ml.At enzyme mark bag by the accurate sample wells of bidding on plate, add the standard items 50ul of variable concentrations successively.B. sample: patient's empty stomach in early morning venous blood samples 3ml is placed in EDTA anti-coagulants vacuum tube, and 4 DEG C of centrifugal l0min of 1000xg, isolate serum, detect as early as possible, or puts-80 DEG C of Refrigerator stores necessary equilibrate at room temperature before use.C. application of sample: establish blank well respectively, blank control wells does not add sample, enzyme marking reagent and biotin labeled anti-Hepcidin antibody, and respectively step operation is identical for all the other, testing sample hole.In enzyme mark bag is by testing sample hole on plate, first adds sample 40 μ l, and then adds biotin labeled Hepcidin antibody 10 μ l.Sample is added on bottom ELISA Plate hole by application of sample, does not touch hole wall as far as possible, rocks mixing gently.D. incubation: use the rearmounted 37 DEG C of incubations of shrouding film shrouding 30 minutes.E. dosing: by for subsequent use after 30 times of concentrated cleaning solution distilled water 30 times dilution.F. wash: carefully take shrouding film off, discard liquid, dry, cleansing solution is filled it up with in every hole, leaves standstill and discards after 30 seconds, so repeats 5 times, pats dry.G. enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.H. incubation: operate same D.I. wash: operate same F.J. develop the color: every hole first adds developer A50 μ l, then adds developer B50 μ l, and shake mixing gently, 37 DEG C of lucifuges develop the color 15 minutes.K. stop: every hole adds stop buffer 50 μ l, cessation reaction (now blue standing turns yellow).L. measure: measure within 15 minutes after adding stop buffer, sequentially measure the absorbance (OD value) in each hole with blank air-conditioning zero, 450nm wavelength.
Result calculates record: with the concentration of reference material for horizontal ordinate, OD value is ordinate, and typical curve drawn by coordinate paper, and OD value per sample finds corresponding concentration by typical curve, record, and record bone scanning testing result, respectively after two weeks, serum Hepcidin mensuration is carried out to treatment, bone scanning detects, and record measurement result, with two weeks before result compare, the results are shown in 2.Divide into groups by the difference of Hepcidin testing result change, wherein, raising in 20 μ g/L-30 μ g/L intervals is A group, raising in 10 μ g/L-20 μ g/L intervals is B group, raising in 0 μ g/L-10 μ g/L interval is C group, being reduced in 0 μ g/L-10 μ g/L interval is D group, and being reduced in 10 μ g/L-20 μ g/L intervals is E group.Measure serum Hepcidin again to these 5 groups after surrounding and carry out the detection of whole body nuclide bone scan, and recording result, result contrasts after two weeks respectively at the former treatment of surrounding, and comparing result is in table 3.
Hepcidin detection after two weeks treated by table 2, bone scanning detects and compares with testing result before two weeks
| Group | Hepcidin testing result compares | Case load | Bone scanning testing result compares |
| A | Raise 20 μ g/L ~ 30 μ g/L | 7 examples | Scanning result display 2 example obviously worsens, and 3 routine slight deterioration, 2 routine contrast differences are not obvious |
| B | Raise 10 μ g/L ~ 20 μ g/L | 19 examples | Bone scanning testing result display 1 example obviously worsens, and 5 routine slight deterioration, 13 routine contrast differences are not obvious |
| C | Raise 0 μ g/L ~ 10 μ g/L | 17 examples | 1 example display slight deterioration, 16 routine contrast differences are not obvious |
| D | Reduce by 0 μ g/L ~ 10 μ g/L | 5 examples | Contrast difference is all not obvious |
| E | Reduce by 10 μ g/L ~ 20 μ g/L | 2 examples | Contrast difference is all not obvious |
After surrounding treated by table 3, Hepcidin detects, bone scanning detects and compare with testing result before two weeks and before surrounding
| group | case load | hepcidin testing result compares change with before two weeks | bone scanning testing result compares with before two weeks | hepcidin testing result compares change with before surrounding | bone scanning testing result compares with before surrounding |
| a | 7 examples | 12 μ g/L ~ 34 μ g/L | 1 example obviously worsens, and 3 routine slight deterioration, 3 routine contrast differences are not obvious | 36 μ g/L ~ 55 μ g/L | comparing result display all obviously worsens |
| b | 19 examples | 4 μ g/L ~ 21 μ g/L | 2 examples obviously worsen, and 5 routine slight deterioration, 12 routine contrast differences are not obvious | 23 μ g/L ~ 39 μ g/L | comparing result display 17 obviously worsens, 2 routine slight deterioration. |
| c | 17 examples | -2 μ g/L ~ 27 μ g/L | 1 example obviously worsens, and 3 example display slight deterioration, 13 routine contrast differences are not obvious | 7 μ g/L ~ 32 μ g/L | comparing result display 14 obviously worsens, and 1 routine slight deterioration, 1 routine contrast difference is not obvious |
| d | 5 examples | -8 μ g/L ~ 22 μ g/L | contrast difference is all not obvious | -4 μ g/L ~ 25 μ g/L | comparing result shows, and 2 routine slight deterioration, 3 routine contrast differences are not obvious |
| e | 2 examples | -7 μ g/L ~ 12 μ g/L | contrast difference is all not obvious | -19 μ g/L ~ 1 μ g/L | contrast difference is all not obvious |
Comprehensive above-mentioned test findings is known, wherein A group Hepcidin after treatment two weeks raise the highest, but it can determine by bone scanning to be just defined as only 2 examples that obviously worsen being all deterioration by bone scanning after surrounding.After B group treatment two weeks its by bone scanning can determine obviously to worsen less than 10%.B, C two groups of bone scanning result differences are unconspicuous all more than 65%, and testing result deterioration rate after surrounding lacks and reaches more than 80%.Known by Hepcidin Monitoring Data to E, D two groups in addition, these two groups treatments all have certain effect, but bone scanning Comparative result difference is not obvious, and testing result is clinical in directive significance.In sum, Hepcidin testing result, as the sensitive indicator of diagnosing prostate cancer Bone tumour, can follow up a case by regular visits to index as prognosis simultaneously, judge treatment curative effect.To serum Hepcidin periodic detection, to tracking in time prognosis situation can be played in prostate cancer with osseous metastasis, for clinical treatment provides better guidance.
Claims (2)
1. measure the application of ELISA reagent in the kit for the preparation of prostate cancer with osseous metastasis Prognosis scoveillance of antibacterial protein Hepcidin content in patients serum.
2., according to the application described in claim 1, it is characterized in that the ELISA reagent measuring antibacterial protein Hepcidin content in patients serum is Hepcidin ELISA reagent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011071368A1 (en) * | 2009-12-11 | 2011-06-16 | Umc St. Radboud | Method for measuring hepcidin |
| CN102809599A (en) * | 2011-06-03 | 2012-12-05 | 国家纳米科学中心 | Method and kit for detecting hepcidin |
| CN202631537U (en) * | 2012-03-03 | 2012-12-26 | 北京勤邦生物技术有限公司 | Enzyme-linked immuno sorbent assay (ELISA) kit used for detecting malachite green |
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| US8017737B2 (en) * | 2002-11-19 | 2011-09-13 | Hasan Kulaksiz | Diagnostic methods for diseases by screening for hepcidin in human or animal tissues, blood or body fluids; monoclonal antibodies specific to human hepcidin and associated uses therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011071368A1 (en) * | 2009-12-11 | 2011-06-16 | Umc St. Radboud | Method for measuring hepcidin |
| CN102809599A (en) * | 2011-06-03 | 2012-12-05 | 国家纳米科学中心 | Method and kit for detecting hepcidin |
| CN202631537U (en) * | 2012-03-03 | 2012-12-26 | 北京勤邦生物技术有限公司 | Enzyme-linked immuno sorbent assay (ELISA) kit used for detecting malachite green |
Non-Patent Citations (1)
| Title |
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| HEPCIDIN, ANAEMIA, AND PROSTATE CANCER;Toshihiko Tanno et al.;《Bjui Letters》;20110228;第107卷(第4期);678-679 * |
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