CN106501517B - Detect application of the material of SPARC protein in serum in examination hepatocellular carcinoma kit is prepared - Google Patents
Detect application of the material of SPARC protein in serum in examination hepatocellular carcinoma kit is prepared Download PDFInfo
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Abstract
The invention discloses a kind of new application for the material for being used to detect SPARC protein concentration.New application provided by the present invention is the application for detecting the material of SPARC protein concentration in preparing examination or aiding in examination hepatocellular carcinoma product.It is demonstrated experimentally that SPARC protein can be as the tumor markers of hepatocellular carcinoma；Examination is carried out in the case of with normal artificial examination object, the sensitivity for being determined as HCC patient is 82%, specificity 93%, AUC 0.89；Examination is carried out in the case of using LC patient as examination object, the sensitivity for being determined as HCC patient is 62%, specificity 86%, AUC 0.73；SPARC protein can be used for diagnosis AFP negative liver cancer；SPARC protein can also combine with AFP for examination or auxiliary diagnosis HCC in normal person or LC patient.
Description
Technical field
Material the present invention relates to SPARC protein in biological technical field, more particularly to detection serum is preparing examination liver
Application in cell cancer kit.
Background technology
Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of common malignant tumour, there is higher hair
Sick rate and the death rate, about 650 000 people die of HCC every year, and its incidence has the trend increased.The disease is mainly in Southeast Asia,
It wherein there are about more than 50% and betide China.A situation arises is a multifactor, multistage process for hepatocellular carcinoma, B-mode liver
Scorching virus (hepatitis B virus, HBV) chronic infection is important high risk factor, the hepatic sclerosis for having hepatitis B background
(liver cirrhosis, LC) patient is the people at highest risk that possible develop into liver cancer.5 years survival rates of hepatocellular carcinoma are poor at present,
Examination early liver cancer patient, takes treatment to arrange in time in the early screening of hepatocellular carcinoma, especially liver cirrhosis patient people at highest risk
Apply, the survival rate secondary to hepatitis b virus infected patients with hepatocellular carcinoma can be improved.
HCC clinical diagnosises at present and state of illness monitoring etc. still depend on the imageological examinations such as B ultrasound and combine blood serum designated object
The detection of alpha-fetoprotein (alpha-fetoprotein, AFP) level.And AFP is as current HCC diagnosis and state of illness monitoring one
Most important biomarker, its sensitivity are still undesirable.So sensitive serum biomarkers in hepatocellular carcinoma thing is found, especially
It can be applied to the liver cancer patient of AFP negative, extremely important for diagnosing hepatocellular carcinoma.
SPARC protein is a kind of glycoprotein, by adjusting cell growth with extracellular matrix and cell factor interaction.
The occurrence and development of SPARC protein and tumour are in close relations, and some researches show that SPARC protein is overexpressed (Le in HCC tissues
Bail,B.et al.Osteonectin/SPARC is overexpressed in human hepatocellular
carcinoma.J Pathol.1999).But the relation of every kind of albumen expression in peripheral blood and tissue is extremely complex,
Simply its expression in peripheral blood, or even sometimes both expression can not be derived from the expression in tissue
Level can be completely opposite.Such as studies have reported that (CES1) albumen of liver carboxylesterase 1 in liver cancer patient tissue
In significantly lower, and the albumen significantly raises (Na, K., et al., Human liver in liver cancer patient blood serum
carboxylesterase 1outperforms alpha-fetoprotein as biomarker to discriminate
hepatocellular carcinoma from other liver diseases in Korean patients.Int J
Cancer,2013)。
The content of the invention
It is an object of the present invention to provide the purposes of the material of SPARC protein concentration in detection serum or blood plasma.
The material of SPARC protein concentration is preparing examination or auxiliary diagnosis liver in detection serum or blood plasma provided by the invention
Application in cell cancer product.
The present invention also provides detection serum or blood plasma in the material of SPARC protein concentration preparing examination or auxiliary diagnosis
Application in α-fetoprotein-negative hepatocellular carcinoma product.
In one embodiment of the invention, the hepatocellular carcinoma is specially to have hepatic sclerosis background secondary to HBV infection
Hepatocellular carcinoma.
The SPARC protein behaviour SPARC protein.
In above application, the material of SPARC protein concentration includes detection serum or blood plasma in the detection serum or blood plasma
Reagent and instrument needed for middle SPARC protein concentration.
In above application, the reagent in the detection serum or blood plasma needed for SPARC protein concentration resists including SPARC protein
Body.
Second purpose of the invention is to provide the material of SPARC concentration and detection serum or blood plasma in detection serum or blood plasma
The purposes of the material of middle alpha-fetoprotein concentration.
The present invention provides alpha-fetoprotein in the material and detection serum or blood plasma of SPARC concentration in detection serum or blood plasma
Application of the material of concentration in examination or auxiliary diagnosis of hepatoma product is prepared.
In above application, the material of SPARC protein concentration includes detection serum or blood plasma in the detection serum or blood plasma
Reagent and instrument needed for middle SPARC protein concentration；
Or, the material of alpha-fetoprotein concentration includes alpha-fetoprotein in detection serum or blood plasma in the detection serum or blood plasma
Reagent and instrument needed for concentration.
In one embodiment of the invention, the reagent detected in serum or blood plasma needed for alpha-fetoprotein concentration is AFPization
Luminescence method kit is learned, instrument is Roche fully automatic electric chemical illumination immunity analysis instrument.
In above application, the reagent in the detection serum or blood plasma needed for SPARC protein concentration resists including SPARC protein
Body.
3rd purpose of the invention is to provide a kind of examination or auxiliary diagnosis of hepatoma product.
Product provided by the invention, it includes the material of SPARC protein concentration in above-mentioned detection serum or blood plasma.
In one embodiment of the invention, the material for being used to detect SPARC protein concentration resists for SPARC protein
Body, is specially SPARC antibody used in ELISA (R＆D Systems companies, article No.：DSP00).It is of course also possible to it is other types
Antibody or it is other can be used for detection SPARC protein concentration material.
In the present invention, the examination of the product or diagnosis object are hepatic sclerosis (LC) patient or Healthy People.
In one embodiment of the invention, hepatic sclerosis (LC) patient is specially the liver cirrhosis patient of HBV infection.
The detection SPARC protein concentration concretely detects the SPARC protein concentration in human serum or blood plasma.
4th purpose of the invention is to provide a kind of examination or auxiliary diagnosis of hepatoma product.
Product provided by the invention, it includes in above-mentioned detection serum or blood plasma the material of SPARC protein concentration and above-mentioned
Detect the material of alpha-fetoprotein concentration in serum or blood plasma.
In the said goods, the product is kit.
In order to find serum biomarkers in hepatocellular carcinoma thing, the tumour of pairing of the inventor to AFP negative liver cancer patient with
Cancer beside organism's interstitial fluid is identified, it is found that SPARC protein significantly raises in liver cancer patient tumor tissues interstitial fluid.Because group
It is the interface for connecting intracellular fluid and instrument for circulation of body fluid to knit interstitial fluid, and result prompting SPARC protein is very likely to from liver cancer group
Knit and be secreted into by interstitial fluid in blood.And then expression of the albumen in clinical serum sample is have detected,
It was found that its serum-concentration significantly increases in liver cancer patient, propose that SPARC protein can be examined as the serum of AFP negative liver cancer patient
Disconnected marker, and can be with AFP Combining diagnosis liver cancer.
It is demonstrated experimentally that serum SPARC protein can be as the tumor markers of hepatocellular carcinoma (HCC).When with it is asymptomatic just
When ordinary person is as examination object, Receiver operating curve (receiver operating characteristic
Curve, ROC) analysis result shows that area under the curve (Area Under roc Curve, AUC) reaches 0.89, sensitivity is
82%th, specificity 93%；In the case of using LC patient as examination object, AUC=0.73, sensitivity 62%, specificity
For 86%.When SPARC protein is used to diagnose AFP negative liver cancer patient, with normal artificial examination object, AUC=0.88, sensitivity
For 78%, specificity 93%；Using LC patient as examination object, AUC=0.72, sensitivity 61%, specificity 84%.
SPARC protein can also combine with AFP for examination or auxiliary diagnosis HCC in LC patient.
Brief description of the drawings
Fig. 1 is normal group (N), SPARC protein is horizontal scattered in liver cirrhosis patient group (LC) and liver cancer patient group (HCC) serum
Point diagram.
Fig. 2 is SPARC protein level in normal group (N), liver cirrhosis patient group (LC) and liver cancer patient group (HCC) serum
ROC curve analysis result.
Fig. 3 is normal group (N), in AFP negative liver cirrhosis patient group (LC) and AFP negative liver cancer patient group (HCC) serum
The ROC curve analysis result of SAPRC protein levels.
Fig. 4 is that liver cirrhosis patient group (LC) and liver cancer patient group (HCC) SPARC and the ROC curve of AFP Combining diagnosis are analyzed
As a result.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Involved explanation of nouns in following embodiments：
Receiver Operating Characteristics (receiver (relative) operating characteristic, ROC) curve is anti-
The balance between sensitivity and specificity is reflected, area is important experimental accuracy index under ROC curve.Each examination is calculated respectively
Area (AUC) under the ROC curve tested is compared, the big person of area, and the diagnostic value of experiment is big.
Sensitivity (True Positive Rate)：Reality is ill and is correctly judged as ill percentage by testing standard, sensitivity
It is the bigger the better, ideal sensitivity 100%.
Specificity (true negative rate)：Reality is disease-free and is correctly judged as disease-free percentage by testing standard, specificity
It is the bigger the better, preferable specificity 100%.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
SPARC protein and the relation of patient HCC in embodiment 1, serum
First, experiment material
Serum sample comes from：
Normal group：Normal person (N) 30；
LC patient's group：There is the liver cirrhosis patient (being not converted into hepatocellular carcinoma, LC) 35 of HBV chronic infection backgrounds；
HCC patient's group：Having the patients with hepatocellular carcinoma (HCC) 50 of HBV chronic infection backgrounds, (wherein 23 cloudy for APF
Property).
Wherein, normal human serum comes from physical examination of healthy population；Liver cirrhosis patient and patients with hepatocellular carcinoma are clinical definite disease
The serum sample of people.
2nd, SPARC protein and the relation of patient HCC in serum
It is horizontal to the SPARC protein in above-mentioned one serum sample to carry out ELISA detections, used kit R＆D
Systems companies SPARC ELISA detection kit (article No.s：DSP00), specific method is as follows：
1st, with dilution (dilution buffer are equipped with kit) 1：40 dilute serum samples, standard items dilution ladder
Spend 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.13ng/ml, 1.56ng/ml, 0ng/ml, serum sample and mark
Quasi- each 100ul of product is added in 96 orifice plates (kit outfit) for being surrounded by primary antibody, when incubation at room temperature 3 is small.
2nd, washed 6 times with washing lotion (wash buffer are equipped with kit), per 350 microlitres of hole, 1 minute every time.
3rd, 200 microlitres of SPARC secondary antibodies (being carried in kit) are added per hole, when 4 degree of incubations 1 are small.
4th, washed 6 times with washing lotion, per 350 microlitres of hole, 1 minute every time.
5th, 200 microlitres of display substrates (substrate solution are equipped with kit) are added per hole, room temperature lucifuge is incubated
Educate 30 minutes.
6th, 50 microlitres of terminate liquids (stop solution are equipped with kit) are added per hole.
7th, 450nm wavelength readings.
8th, standard curve is drawn by SPARC standard proteins concentration OD values corresponding with its.Pass through each hole serum sample institute
The OD values measured, are fitted the standard curve of SPARC using Excel softwares, calculate SPARC protein in each serum sample
Content.
9th, with 16.0 softwares of SPSS respectively to healthy normal person (N) and patients with hepatocellular carcinoma (HCC), liver cirrhosis patient
(LC) and in patients with hepatocellular carcinoma (HCC) serum SPARC protein is horizontal carries out ROC curve analysis, and to liver cirrhosis patient (LC)
AFP is carried out with patients with hepatocellular carcinoma (HCC) to analyze with SPARC Combining diagnosis ROC curve.
ELISA of the SPARC protein expression in the serum sample that normal group, LC patient's group, HCC patient organize detects knot
Fruit：Mean concentration ± the standard deviation of normal group SPARC protein be 72.77 ± 51.79ng/ml, LC patient's group is 165.54 ±
88.47ng/ml, HCC patient group are 319.00 ± 192.66ng/ml.Statistics is carried out to SPARC protein content in each group sample
Analyze (Mann-Whitney test), find serum SPARC protein in normal group and HCC patient's group (p<And LC 0.0001)
Patient organizes and HCC patient's group (p=0.0002) has significant difference (Fig. 1).
Using normal group as control group, HCC patient's group is disease group, horizontal to SPARC protein in serum to carry out ROC curve point
Analysis, area under the curve AUC=0.89 (as shown in A in Fig. 2), sensitivity 82%, specificity 93%；It is pair with LC patient's group
According to group, HCC patient's group is disease group, AUC=0.73 (as shown in B in Fig. 2), sensitivity 62%, specificity 86%.
From the above results, in serum SPARC protein can be used as normal person and HCC patient and LC and HCC patient into
The potential marker of row Distinguishing diagnosis.
When the Distinguishing diagnosis of progress normal person and HCC patient, if SPARC protein concentration is more than or equal in human serum to be measured
130.72ng/ml, then the artificial or candidate to be measured is HCC patient.
When the Distinguishing diagnosis of progress LC patient and HCC patient, if SPARC protein concentration is more than or equal in human serum to be measured
261.80ng/ml, then the artificial or candidate to be measured is HCC patient.
The concentration threshold of above-mentioned two Distinguishing diagnosis is the corresponding threshold value of maximum youden index of ROC curve, with normal person
Exemplified by the Distinguishing diagnosis of HCC patient, youden index and corresponding threshold value refer to table 1 (maximum youden index and corresponding threshold value runic
Mark).
Table 1 is using normal group as the threshold value of control group, sensitivity, specificity and youden index
3rd, application of the SPARC protein in the HCC patient of detection AFP negative in serum
By the normal person in above-mentioned one, the hepatic sclerosis (LC) for 17 AFP negatives for having HBV chronic infection backgrounds and 23
Hepatocellular carcinoma (HCC) the patients serum sample of AFP negative is detected according to above-mentioned two method.
As a result normally the serum-concentration mean+SD of group SPARC protein is 72.77 ± 51.79ng/ml, 17
LC patient's group of AFP negative be 152.25 ± 94.30ng/ml, HCC patient's groups of 23 AFP negatives is 308.62 ±
196.04ng/ml。
With AFP negative (AFP<HCC patient's group 20ng/ml) is disease group, and normal group is control group, in serum
The horizontal progress ROC curve analysis of SPARC protein, area under the curve AUC=0.88 (Fig. 3 A), sensitivity 78%, specificity are
93%.Organized with the HCC patient of AFP negative as disease group, AFP negative liver cirrhosis patient group is control group, to SPARC eggs in serum
White level carries out ROC curve analysis, area under the curve AUC=0.72 (Fig. 3 B), sensitivity 61%, specificity 84%.Say
Bright SPARC protein can be used for the liver cancer patient of Distinguishing diagnosis AFP negative.
When the Distinguishing diagnosis of progress normal person and the HCC patient of AFP negative, if SPARC protein concentration in human serum to be measured
More than or equal to 136.18ng/ml, then the artificial or candidate to be measured is HCC patient.
When the Distinguishing diagnosis of the LC patient of progress AFP negative and the HCC patient of AFP negative, if SPARC in human serum to be measured
Protein concentration is more than or equal to 247.36ng/ml, then the artificial or candidate to be measured is HCC patient.
The concentration threshold of above-mentioned two Distinguishing diagnosis is the corresponding threshold value of maximum youden index of ROC curve.
4th, the application of SPARC protein and AFP albumen in HCC patient is detected
AFP serum protein concentrations detection methods：Using Roche fully automatic electric chemical illumination immunity analysis instrument, AFPization is utilized
The detection of luminescence method kit is learned, detailed step is with reference to instrument specification.
The serum-concentration of normal group AFP albumen is respectively less than 20ng/ml, and LC patient's group is 2260.6 ± 12582.0ng/ml,
HCC patient's group is 13626.6 ± 47251.9ng/ml.The blood that SPARC protein concentration is organized in normal group, LC patient's group, HCC patient
ELISA testing results in final proof sheet：Mean concentration ± the standard deviation of normal group SPARC protein is 72.77 ± 51.79ng/
Ml, LC patient group are 165.54 ± 88.47ng/ml, and HCC patient's group is 319.00 ± 192.66ng/ml.
Statistical analysis (Mann-Whitney test) is carried out to SPARC protein in each group sample and AFP protein concentrations,
It was found that serum SPARC protein and AFP protein concentrations are in normal group and HCC patient's group (p<And LC patient's group and HCC 0.0001)
Patient's group (p=0.0002) has significant difference.
The common standard for judging SPARC protein and AFP albumen returns result of calculation according to binary Logistic, distinguishes HCC
It is with normal group specific formula for calculation：P=1/ (1+e-(-2.415+0.018*SPARC+0.002*AFP)), when p is more than or equal to 0.643, sentence
Break as HCC；Distinguish HCC and LC calculation formula be：P=1/ (1+e-(-1.498+0.008*SPARC+0.000016*AFP)), when p is more than or equal to
When 0.633, it is judged as HCC.
With 16.0 softwares of SPSS respectively to normal person (N) and patients with hepatocellular carcinoma (HCC), hepatic sclerosis (LC) and liver cell
Cancer patient (HCC) carries out the ROC curve analysis that diagnosis HCC is used in combination in AFP and SPARC.
Using normal group as control group, HCC patient's group is disease group, and differentiation is used in combination with AFP albumen in serum SPARC protein
Diagnose the area under the curve AUC=0.93 (as shown in A in Fig. 4) of HCC patient, sensitivity 86%, specificity 97%；With LC
Patient's group is control group, and HCC patient's group is disease group, and AUC=0.78 (such as Fig. 4 of Distinguishing diagnosis HCC patient is used in combination in both
Shown in middle B), sensitivity 67%, specificity 84%.
From the above results, in serum SPARC protein and AFP albumen can be used as normal person and HCC patient and LC with
HCC patient carries out the potential marker of Distinguishing diagnosis.
The above results are summarized in table 2 and table 3, illustrate SPARC protein, AFP albumen and its use in conjunction and are used for liver cell
Cancer patient and normal person or the effect of LC patient's antidiastole.
2 SPARC protein of table and AFP albumen Distinguishing diagnosis normal persons and patients with hepatocellular carcinoma effect compare (SPARC or
SPARC and AFP is used in combination effect and is better than AFP)
3 SPARC protein of table and AFP albumen Distinguishing diagnosis hepatic sclerosis and patients with hepatocellular carcinoma effect compare (SPARC or
SPARC and AFP is used in combination effect and is better than AFP)
From the above results, in serum SPARC protein can be used as normal person and HCC patient and LC and HCC patient into
The potential marker of row Distinguishing diagnosis.
Sequence table
<110>Beijing Proteome Research Center
<120>Detect application of the material of SPARC protein in serum in examination hepatocellular carcinoma kit is prepared
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 303
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> 1
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Ala Pro Gln Gln Glu Ala Leu Pro Asp Glu Thr Glu Val Val Glu
20 25 30
Glu Thr Val Ala Glu Val Thr Glu Val Ser Val Gly Ala Asn Pro Val
35 40 45
Gln Val Glu Val Gly Glu Phe Asp Asp Gly Ala Glu Glu Thr Glu Glu
50 55 60
Glu Val Val Ala Glu Asn Pro Cys Gln Asn His His Cys Lys His Gly
65 70 75 80
Lys Val Cys Glu Leu Asp Glu Asn Asn Thr Pro Met Cys Val Cys Gln
85 90 95
Asp Pro Thr Ser Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val Cys
100 105 110
Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe Phe Ala Thr
115 120 125
Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His Lys Leu His Leu Asp
130 135 140
Tyr Ile Gly Pro Cys Lys Tyr Ile Pro Pro Cys Leu Asp Ser Glu Leu
145 150 155 160
Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys Asn Val Leu Val
165 170 175
Thr Leu Tyr Glu Arg Asp Glu Asp Asn Asn Leu Leu Thr Glu Lys Gln
180 185 190
Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys Arg Leu Glu Ala
195 200 205
Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe Glu Lys Asn Tyr
210 215 220
Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly Gln Leu Asp Gln
225 230 235 240
His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu Ala Pro Leu Arg
245 250 255
Ala Pro Leu Ile Pro Met Glu His Cys Thr Thr Arg Phe Phe Glu Thr
260 265 270
Cys Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Asp Glu Trp Ala Gly
275 280 285
Cys Phe Gly Ile Lys Gln Lys Asp Ile Asp Lys Asp Leu Val Ile
290 295 300
Claims (3)
1. the material for detecting SPARC protein concentration in serum or blood plasma is thin in preparation examination or auxiliary diagnosis α-fetoprotein-negative liver
Application in born of the same parents' cancer product.
2. application according to claim 1, it is characterised in that：SPARC protein concentration in the detection serum or blood plasma
Material includes the reagent and instrument needed for SPARC protein concentration in detection serum or blood plasma.
3. application according to claim 2, it is characterised in that：SPARC protein concentration institute in the detection serum or blood plasma
The reagent needed includes SPARC protein antibody.
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| CN107102147B (en) * | 2017-04-05 | 2019-09-13 | 北京蛋白质组研究中心 | Application of the THBS2 Protein Detection object in preparation prognosis of HCC assessment kit |
| CN107037210A (en) * | 2017-04-05 | 2017-08-11 | 北京蛋白质组研究中心 | Application of the THBS2 Protein Detections thing in diagnosis of hepatoma kit is prepared |
| GB201721308D0 (en) * | 2017-12-19 | 2018-01-31 | Nordic Bioscience As | SPARC assay |
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