CN101441213B - Liver cancer patient postoperative transferring recrudescence polymolecular forecasting reagent kit based on inflammation factor - Google Patents
Liver cancer patient postoperative transferring recrudescence polymolecular forecasting reagent kit based on inflammation factor Download PDFInfo
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- CN101441213B CN101441213B CN2008102081506A CN200810208150A CN101441213B CN 101441213 B CN101441213 B CN 101441213B CN 2008102081506 A CN2008102081506 A CN 2008102081506A CN 200810208150 A CN200810208150 A CN 200810208150A CN 101441213 B CN101441213 B CN 101441213B
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Abstract
The invention provides a multimolecular prediction kit for postoperative recurrence and transfer for the patients with liver cancer based on inflammatory factor, characterized by comprising a coating agent, a washing liquid, a horseradish peroxidase-labeled antibody, a substrate solution, a stop solution, and prediction reference values, wherein, the coating agent is composed of a carbonate coating buffer solution of PH9.6, and an interleukin 2 antibody or an interleukin 15 antibody or an interleukin 5 antibody; based on the 4mug/mul of protein concentration of extract from liver peri-cancer tissue, the prediction reference values of interleukin 2, interleukin 15, and interleukin 5 are respectively 20-30pg/ml, 32-43pg/ml, and 18-28pg/ml, and the prediction reference values vary proportionably along with the sample concentration. In the invention, by using the interleukin 2, interleukin 15, and interleukin 5 in liver peri-cancer tissue, the postoperative recurrence and transfer for the patients with liver cancer can be accurately predicted.
Description
Technical field
The present invention relates to a kind of liver cancer patient postoperative metastasis recurrence polymolecular prediction kit, belong to biological technical field based on inflammatory factor.
Background technology
Liver cancer is second cancer cause of the death of China, and operation is present main treatment means.Yet the high transfer and relapse rate of postoperative (recurrence rate reached 70% in 5 years, and small liver cancer also reaches 50%) has become the bottleneck of further raising late result, also is the important key of capturing liver cancer.If can carry out the prediction of early stage transfer and relapse to the specific crowd behind the liver cancer radical correction, in time give therapeutic intervention, accomplish early to find early treatment, can further improve radical-ability postoperative recurrence patient's survival rate, also have very big social benefit and economic benefit.
The effect of cancer week microenvironment in tumour takes place and makes progress in recent years becomes the focus of tumor research.Many researchs also show the unbalance relevant with kinds of tumors of Th1/Th2 immunity.It is closely related that the inflammation immune response state that the research work in we has confirmed cancer week microenvironment in early stage and Patients with Primary postoperative metastasis recur, and can more accurately predict transfer and relapse.And present clinical still do not have transfer and relapse and existence situation enough responsive and that special prediction index is come the predicting liver cancer postoperative patient; Alpha-fetoprotein, tumour size; Though coating has or not etc. relevant with transfer and relapse but its predictablity rate is also very low on the whole, and is especially more unpredictable to the postoperative recurrence of early liver cancer.
Summary of the invention
The purpose of this invention is to provide a kind of liver cancer patient postoperative metastasis recurrence polymolecular prediction kit based on inflammatory factor.
In order to achieve the above object; Technical scheme of the present invention provides the liver cancer patient postoperative metastasis recurrence polymolecular prediction kit based on inflammatory factor; It is characterized in that, form by the antibody that encapsulates reagent, washing lotion, horseradish peroxidase-labeled, substrate solution, stop buffer and prediction reference;
Encapsulate wherein that reagent encapsulates damping fluid by pH9.6 carbonate and interleukin 2 antibody, interleukin 15 antibody or t cell growth factor antibody are formed;
Washing lotion is the pH7.4 phosphate buffer;
The antibody of horseradish peroxidase-labeled is made up of the interleukin 2 antibody of horseradish peroxidase-labeled, the interleukin 15 antibody of horseradish peroxidase-labeled and the t cell growth factor antibody of horseradish peroxidase-labeled;
Substrate solution is tetramethyl benzidine substrate solution or ABTS substrate solution;
Stop buffer is a 1.8-2.1M sulfuric acid;
Prediction reference is that 4ug/ul is as the criterion with all tissue extracts of liver cancer protein concentration: interleukin 2 is that 20-30pg/ml, interleukin 15 are 32-43pg/ml; T cell growth factor is 18-28pg/ml; Different application of sample concentration, the proportional variation of prediction reference.
It is sodium carbonate 1.59g and soda mint 2.93g to be dissolved in the 1000ml distilled water process that said carbonate encapsulates damping fluid.
Described phosphate buffer is the potassium dihydrogen phosphate with 0.2g 0.15M, the 2.9g disodium hydrogen phosphate dodecahydrate, and 8.0g sodium chloride, 0.2g potassium chloride, the 0.5ml Tween-20 is added in the 1000ml distilled water and processes.
The present invention has the following advantages:
1. interleukin 2, interleukin 15 and the t cell growth factor transfer and relapse of predicting liver cancer patient postoperative more exactly in cancer week hepatic tissue; Accuracy rate is respectively 69.2%, 61.7%, 61.7% in 133 routine early liver cancer patients; The existence or the death of all right predicting liver cancer patient postoperative of interleukin 2, accuracy rate is 72.9%.
2. interleukin 2, interleukin 15 and t cell growth factor obviously are superior to being used at present the clinical indices of predicting liver cancer patient postoperative recurrence and existence; Like alpha-fetoprotein, tumour size; Coating has or not etc., and especially the prediction to the recurrence of early liver cancer postoperative metastasis has tangible value and significance.And it is simple and to patient's non-invasive (use be the hepatic tissue that excises in the liver cancer patient art) to measure these interleukins.
Description of drawings
Fig. 1 is the XPS synoptic diagram;
Fig. 2 is interleukin 2, interleukin 15 and the t cell growth factor prediction case figure to the transfer and relapse of liver cancer patient postoperative;
Fig. 3 is the no knurl survivorship curve of interleukin 2;
Fig. 4 is total survivorship curve of interleukin 2;
Fig. 5 is the no knurl survivorship curve of interleukin 15;
Fig. 6 is the no knurl survivorship curve of t cell growth factor.
Embodiment
Below in conjunction with specific embodiment the present invention is described.
Embodiment 1
A kind of kit of predicting liver cancer transfer relapse of primary liver cancer patient after operation is made up of the antibody that encapsulates reagent, washing lotion, horseradish peroxidase-labeled, substrate solution, stop buffer and prediction reference;
Encapsulate wherein that reagent encapsulates damping fluid by pH9.6 carbonate and interleukin 2 antibody, interleukin 15 antibody or t cell growth factor antibody are formed;
Washing lotion is the pH7.4 phosphate buffer;
The antibody of horseradish peroxidase-labeled is made up of the interleukin 2 antibody of horseradish peroxidase-labeled, the interleukin 15 antibody of horseradish peroxidase-labeled and the t cell growth factor antibody of horseradish peroxidase-labeled;
Substrate solution is the tetramethyl benzidine substrate solution;
Stop buffer is a 1.8M sulfuric acid;
Prediction reference is that 4ug/ul is as the criterion with all tissue extracts of liver cancer protein concentration: interleukin 2 is that 20-30pg/ml, interleukin 15 are 32-43pg/ml; T cell growth factor is 18-28pg/ml; Different application of sample concentration, the proportional variation of prediction reference.
It is sodium carbonate 1.59g and soda mint 2.93g to be dissolved in the 1000ml distilled water process that said carbonate encapsulates damping fluid.
Described phosphate buffer is the potassium dihydrogen phosphate with 0.2g 0.15M, the 2.9g disodium hydrogen phosphate dodecahydrate, and 8.0g sodium chloride, 0.2g potassium chloride, the 0.5ml Tween-20 is added in the 1000ml distilled water and processes.
Use this kit to predict that the concrete steps of primary hepatocarcinoma patient operation back hepatoma Metastasis recurrence are:
The first step: encapsulate: using carbonate to encapsulate damping fluid is 5 μ g/ml with interleukin 2, interleukin 15 and t cell growth factor antibody dilution to protein content respectively; Add interleukin 2, interleukin 15 antibody, t cell growth factor antibody diluent respectively in the 1st, 2, the 3 row reacting holes in XPS as shown in Figure 1; Add the interleukin 2 antibody diluent at the 4th, 5,6 row; Add the interleukin 15 antibody diluent at the 7th, 8,9 row; Add the t cell growth factor antibody diluent at the 10th, 11,12 row, add 0.1ml in each reacting hole, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 3 minutes with phosphate buffer.
Second step: application of sample: the cancer week hepatic tissue extract 0.1ml to be checked that adds 4ug/ul puts 37 ℃ and hatched 1 hour in the above-mentioned reacting hole that has encapsulated.Wash 2 times with phosphate buffer then.Simultaneously the 1st, 2,3 row reacting holes are done the serial dilution standard items hole of interleukin 2, interleukin 15, t cell growth factor respectively.
The 3rd step: the antibody that adds horseradish peroxidase-labeled: in each reacting hole, add horseradish peroxidase-labeled antibody (dilutability after the titration) 0.1ml of corresponding fresh dilution.Hatched 1 hour the same method washing for 37 ℃.
The 4th step: add substrate solution colour developing: in each reacting hole, add tetramethyl benzidine (TMB) substrate solution 0.1ml, 37 ℃ 20 minutes.
The 5th step: cessation reaction: in each reacting hole, add 2M sulfuric acid 0.05ml.
The 6th step: survey each hole absorbance: on the ELISA detector; In 450nm (if develop the color with ABTS; 410nm then) locates; Survey each hole absorbance with zeroing back, blank hole,, calculate sample interleukin 2 to be checked, interleukin 15 and t cell growth factor concentration according to standard items testing result drawing standard curve.
The 7th step: be applied to prediction: the concentration of the detected interleukin 2 of kit, interleukin 15 and t cell growth factor; Be used for the existence or the death of the transfer and relapse and the liver cancer patient postoperative of predicting liver cancer patient postoperative; When interleukin 2 concentration is lower than 25.76pg/ml (when application of sample concentration is 4ug/ul); Interleukin 15 concentration is lower than 37.16pg/ml (when application of sample concentration is 4ug/ul), or t cell growth factor concentration is lower than 23.173pg/ml (when application of sample concentration is 4ug/ul), the easy transfer and relapse of judgement liver cancer patient postoperative; When interleukin 2 concentration is lower than 25.76pg/ml (when application of sample concentration is 4ug/ul), judge that the liver cancer patient postoperative is prone to death.
Embodiment 2
A kind of kit of predicting liver cancer transfer relapse of primary liver cancer patient after operation is made up of the antibody that encapsulates reagent, washing lotion, horseradish peroxidase-labeled, substrate solution, stop buffer and prediction reference;
Encapsulate wherein that reagent encapsulates damping fluid by pH9.6 carbonate and interleukin 2 antibody, interleukin 15 antibody or t cell growth factor antibody are formed;
Washing lotion is the pH7.4 phosphate buffer;
The antibody of horseradish peroxidase-labeled is made up of the interleukin 2 antibody of horseradish peroxidase-labeled, the interleukin 15 antibody of horseradish peroxidase-labeled and the t cell growth factor antibody of horseradish peroxidase-labeled;
Substrate solution is the ABTS substrate solution;
Stop buffer is a 2.1M sulfuric acid;
Prediction reference is that 4ug/ul is as the criterion with all tissue extracts of liver cancer protein concentration: interleukin 2 is that 20-30pg/ml, interleukin 15 are that 32-43pg/ml is a criteria for classification; T cell growth factor is 18-28pg/ml; Different application of sample concentration, the proportional variation of prediction reference.
It is sodium carbonate 1.59g and soda mint 2.93g to be dissolved in the 1000ml distilled water process that said carbonate encapsulates damping fluid.
Described phosphate buffer is the potassium dihydrogen phosphate with 0.2g 0.15M, the 2.9g disodium hydrogen phosphate dodecahydrate, and 8.0g sodium chloride, 0.2g potassium chloride, the 0.5ml Tween-20 is added in the 1000ml distilled water and processes.
Use this kit to predict that the concrete steps of primary hepatocarcinoma patient operation back hepatoma Metastasis recurrence are:
The first step: encapsulate: using carbonate to encapsulate damping fluid is 5 μ g/ml with interleukin 2, interleukin 15 and t cell growth factor antibody dilution to protein content respectively; Add interleukin 2, interleukin 15 antibody, t cell growth factor antibody diluent respectively in the 1st, 2, the 3 row reacting holes in XPS as shown in Figure 1; Add the interleukin 2 antibody diluent at the 4th, 5,6 row; Add the interleukin 15 antibody diluent at the 7th, 8,9 row; Add the t cell growth factor antibody diluent at the 10th, 11,12 row, add 0.1ml in each reacting hole, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 3 minutes with phosphate buffer.
Second step: application of sample: the cancer week hepatic tissue extract 0.1ml to be checked that adds 4ug/ul puts 37 ℃ and hatched 1 hour in the above-mentioned reacting hole that has encapsulated.Wash 2 times with phosphate buffer then.Simultaneously the 1st, 2,3 row reacting holes are done the serial dilution standard items hole of interleukin 2, interleukin 15, t cell growth factor respectively.The 3rd step: the antibody that adds horseradish peroxidase-labeled: in each reacting hole, add horseradish peroxidase-labeled antibody (dilutability after the titration) 0.1ml of corresponding fresh dilution.Hatched 1 hour the same method washing for 37 ℃.
The 4th step: add substrate solution colour developing: in each reacting hole, add or ABTS substrate solution 0.1ml, 37 ℃ 20 minutes.
The 5th step: cessation reaction: in each reacting hole, add 2M sulfuric acid 0.05ml.
The 6th step: survey each hole absorbance: on the ELISA detector; In 450nm (if develop the color with ABTS; 410nm then) locates; Survey each hole absorbance with zeroing back, blank hole,, calculate sample interleukin 2 to be checked, interleukin 15 and t cell growth factor concentration according to standard items testing result drawing standard curve.
The 7th step: be applied to prediction: the concentration of the detected interleukin 2 of kit, interleukin 15 and t cell growth factor; Be used for the existence or the death of the transfer and relapse and the liver cancer patient postoperative of predicting liver cancer patient postoperative; When interleukin 2 concentration is lower than 25.76pg/ml (when application of sample concentration is 4ug/ul); Interleukin 15 concentration is lower than 37.16pg/ml (when application of sample concentration is 4ug/ul), or t cell growth factor concentration is lower than 23.173pg/ml (when application of sample concentration is 4ug/ul), the easy transfer and relapse of judgement liver cancer patient postoperative; When interleukin 2 concentration is lower than 25.76pg/ml (when application of sample concentration is 4ug/ul), judge that the liver cancer patient postoperative is prone to death.
Show with the testing result of kit among the embodiment 1-2 in 133 routine early primary hepatocarcinoma patients:
1. as shown in Figure 2; Wherein a represents interleukin 2; B represents interleukin 15, and c represents t cell growth factor, and experimenter's performance curve shows the transfer and relapse that interleukin 2, interleukin 15 and t cell growth factor can predicting liver cancer patient postoperatives.Their areas under a curve are respectively 0.627,0.633,0.602, and the P value is respectively 0.013,0.009,0.046.
2.Kaplan-Meier existence and recurrence analyze to show: when application of sample concentration (being liver cancer week tissue extract's protein concentration) during for 4ug/ul, IL-2 is criteria for classification with 25.76pg/ml, and IL-15 is criteria for classification with 37.16pg/ml; IL-5 with 23.173pg/ml is criteria for classification, and is as shown in Figure 3, wherein; A represents the low group of interleukin 2, and b represent that interleukin 2 is high to be organized, low-level IL-2 than high-level patient's IL-2 recurrence rate obviously raise (p=3.48E-8), as shown in Figure 4; Wherein, A represents the low group of interleukin 2, and b represents the high group of interleukin 2, life cycle obviously shorter (p=0.02); As shown in Figure 5, wherein, a represents the low group of interleukin 15, and b represents the high group of interleukin 15, and low-level IL-15 is than the obvious recurrence rate rising of high-level patient IL-15 (p=0.004); As shown in Figure 6, wherein, a represents the low group of t cell growth factor, and b represents the high group of t cell growth factor, and low-level IL-5 is than the obvious recurrence rate rising of high-level patient IL-5 (p=0.02).
3. when application of sample concentration (be liver cancer week tissue extract protein concentration) during for 4ug/ul; IL-2 is reference value with 25.76pg/ml; Be lower than 25.76pg/ml and be predicted as transfer and relapse, be higher than 25.76pg/ml and be predicted as not transfer and relapse, IL-2 prediction transfer and relapse accuracy rate 69.2%. is reference value with 37.16pg/ml with same reference value IL-2 predicting survival accuracy rate 72.9%.IL-15; Be lower than 37.16pg/ml and be predicted as transfer and relapse; Be higher than 37.16pg/ml and be predicted as not transfer and relapse, IL-15 prediction transfer and relapse accuracy rate 61.7%.IL-5, is lower than 23.173pg/ml and is predicted as transfer and relapse for being reference value with 23.173pg/ml; Be higher than 23.173pg/ml and be predicted as not transfer and relapse, IL-5 prediction transfer and relapse accuracy rate also is 61.7%.
Claims (3)
1. a kit that is used to predict the recurrence of Patients with Primary postoperative metastasis is characterized in that, is made up of the antibody that encapsulates reagent, washing lotion, first antibody, horseradish peroxidase-labeled, substrate solution, stop buffer and prediction reference;
Encapsulate wherein that reagent encapsulates damping fluid by pH9.6 carbonate and interleukin 2 antibody, interleukin 15 antibody or t cell growth factor antibody are formed;
Washing lotion is the pH7.4 phosphate buffer;
The antibody of horseradish peroxidase-labeled is made up of the interleukin 2 antibody of horseradish peroxidase-labeled, the interleukin 15 antibody of horseradish peroxidase-labeled and the t cell growth factor antibody of horseradish peroxidase-labeled;
Substrate solution is tetramethyl benzidine substrate solution or ABTS substrate solution;
Stop buffer is a 1.8-2.1M sulfuric acid.
2. a kind of kit that is used to predict the recurrence of Patients with Primary postoperative metastasis as claimed in claim 1 is characterized in that, it is sodium carbonate 1.59g and soda mint 2.93g to be dissolved in the 1000ml distilled water process that described carbonate encapsulates damping fluid.
3. a kind of kit that is used to predict the recurrence of Patients with Primary postoperative metastasis as claimed in claim 1; It is characterized in that; Described phosphate buffer is the potassium dihydrogen phosphate with 0.2g 0.15M, 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride; 0.2g potassium chloride, the 0.5ml Tween-20 is added in the 1000ml distilled water and processes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7101542B1 (en) * | 1999-05-26 | 2006-09-05 | Vallera Daniel A | Cell-mediated targeting of toxins to pathogenic cells |
| CN101039951A (en) * | 2003-11-03 | 2007-09-19 | 基因信息公司 | Liver cancer biomarkers |
| CN101221178A (en) * | 2006-11-28 | 2008-07-16 | 创盛科技股份有限公司 | Method for diagnosing primary malignancy hepatic tumor hepatocellular carcinoma |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7101542B1 (en) * | 1999-05-26 | 2006-09-05 | Vallera Daniel A | Cell-mediated targeting of toxins to pathogenic cells |
| CN101039951A (en) * | 2003-11-03 | 2007-09-19 | 基因信息公司 | Liver cancer biomarkers |
| CN101221178A (en) * | 2006-11-28 | 2008-07-16 | 创盛科技股份有限公司 | Method for diagnosing primary malignancy hepatic tumor hepatocellular carcinoma |
Non-Patent Citations (1)
| Title |
|---|
| 赵彩彦等.肝细胞癌患者血清IL-6、IL-2***的变化及相互关系.《中国肿瘤临床》.1999,第26卷(第11期), * |
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