CN101481732A - Prediction reagent kit for early stage primary liver cancer patient postoperative metastasis and recurrence based on immune molecule - Google Patents
Prediction reagent kit for early stage primary liver cancer patient postoperative metastasis and recurrence based on immune molecule Download PDFInfo
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- CN101481732A CN101481732A CNA2008102043627A CN200810204362A CN101481732A CN 101481732 A CN101481732 A CN 101481732A CN A2008102043627 A CNA2008102043627 A CN A2008102043627A CN 200810204362 A CN200810204362 A CN 200810204362A CN 101481732 A CN101481732 A CN 101481732A
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Abstract
The invention provides a prediction kit for the recurrence and metastasis after resection of an early primary carcinoma patient based on immune molecules. The prediction kit is characterized in that the prediction kit comprises interleukin 2 amplification detection reagent, reference gene amplification detection reagent, a reference substance, a positive reference substance and non-RNA enzyme water. The interleukin 2 gene messenger ribonucleic acid expression amount of the invention is taken as a marker which is obviously superior to clinical indexes that are currently used for predicting the postoperative recurrence and survival of the patients with liver cancer and as a prognosis judgment biomarker of the early patients with liver cancer.
Description
Technical field
The present invention relates to a kind of prediction test kit that recurs based on the early primary hepatocarcinoma patient postoperative metastasis of immune molecule, belong to biological technical field.
Background technology
Primary hepatocellular carcinoma (abbreviation liver cancer) is second cancer cause of the death of China, operation is the main treatment means of present early hepatocarcinoma, yet the high transfer and relapse rate of postoperative (5 years recurrence rate reach～70%) 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 important social benefit and economic benefit, explore novel simple biomarker and will greatly prolong liver cancer patient postoperative survival time with diagnostic value.
All microenvironment albumen of cancer or genetic expression become the focus of tumor research to the effect in tumour generation and the progress.Many researchs show that also the imbalance of expression of cancer week immune factor is relevant with the kinds of tumors progress, have found that so far the several genes expression can be used as the tumor prognosis prediction index.Yet, liver cancer 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 are though coating such as has or not also very low at relevant its predictablity rate on the whole with transfer and relapse.Application such as leaf Qinghai contain the cell DNA chip of 9180 genes, the gene expression profile of the primary hepatocarcinoma primary tumor that shifts in the liver is accompanied or do not accompanied to research 40 examples, the molecule predictive model that has or not 153 genes that shift in the liver can be accurately distinguished in foundation, preliminary accuracy 90%.This model is more owing to relating to index at present, more difficult clinical application, and further exploring 17 gene involved in immunity cancers week expression levels on this basis can the predicting liver cancer prognosis.At present, how optimizing existing prediction index instructs liver cancer treatment still to face huge challenge.Real time fluorescent quantitative polymerase chain reaction (PCR) technology has mature and stable, and the characteristics that detection accuracy is high have become the conventional means of molecule and RESEARCH ON CELL-BIOLOGY, and important use is worth having very aspect hepatoma Metastasis and the recurrence prediction.The operation of liver cancer patient belongs in early days mostly, and the difficult point that prognosis is judged also is this type of patient, and it is very wide that exploration and exploitation have prognosis biomarker application prospect.
Summary of the invention
The purpose of this invention is to provide a kind of prediction test kit that recurs based on the early primary hepatocarcinoma patient postoperative metastasis of immune molecule.
In order to achieve the above object, technical scheme of the present invention provides a kind of prediction test kit that recurs based on the early primary hepatocarcinoma patient postoperative metastasis of immune molecule, it is characterized in that, comprise interleukin-22 augmentation detection reagent, internal control gene augmentation detection reagent, normal liver tissue reference substance, negative control product, positive reference substance, no RNA enzyme water;
Wherein, described interleukin-22 augmentation detection reagent is by 5-15mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.0-3.0mM magnesium chloride, 0.3-0.4pmol/ul triphosphate deoxyribose nucleotide, 0.05-0.15U/Taq enzyme, 0.3-0.4pmol fluorescent mark interleukin-22 self cancellation probe and primer, 10-30u/ulRNA enzyme inhibitors and water are formed;
Internal control gene augmentation detection reagent is by 5-15mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.0-3.0mM magnesium chloride, 0.3-0.4pmol/ul triphosphate deoxyribose nucleotide, 0.05-0.15U/Taq enzyme, 0.3-0.4pmol fluorescent mark glyceraldehyde 3-phosphate dehydro-genase gene self cancellation probe and primer, 10-30u/ulRNA enzyme inhibitors and water are formed;
The normal liver tissue reference substance is normal no cancer hepatic tissue cDNA;
The negative control product are no mRNA water;
Positive reference substance is normal people's interleukin II mRNA sample;
No RNA enzyme water is prepared by following method: deionized water was handled 10-12 hour through 0.1vol% coke diethyl phthalate, 110-130 degree centigrade autoclaving 20-35 minute.
The present invention detects the expression of cancer week liver organization interleukin II messenger RNA(mRNA) (hereinafter to be referred as the interleukin-messenger RNA(mRNA)) by the probe method polymerase chain reaction,PCR, find the Th1 cytokines, particularly the interleukin II messenger RNA(mRNA) is as a kind of biomarker of predicting liver cancer transfer relapse of primary liver cancer patient after operation, can become the screening agent of early discovery hepatoma Metastasis recurrence, be used to develop into the diagnostic kit that comprises this cytokine.
The mentioned reagent box can be used to predict the hepatoma Metastasis recurrence of early primary hepatocarcinoma patient operation back, especially at the early hepatocarcinoma patient, and single tumour when wherein being defined as operation in early days, cancer is fastened under no naked eyes and the mirror, no distant metastasis.Its concrete steps are: collect early hepatocarcinoma patient cancer Zhou Zhengchang tissue (pathological examination confirmation acrotism comedocarcinoma is fastened) and the no liver cancer normal liver tissues of 8 examples in contrast ,-80 degree Celsius, cryopreservation.Applied molecular biology ordinary method extracting 25mg organizes total Yeast Nucleic Acid, in liquid nitrogen, the 25mg tissue is milled into powder, taking advantage of liquid nitrogen does not volatilize the light time as yet, with powder transfer in the 1.5ml centrifuge tube, the Trizol reagent (available from American I nvitrogen (hero) life technology company limited) that adds 1.0ml, fully mixing was placed 15 minutes, the back adds the 200ul trichloromethane, 4 ℃, 12000 rev/mins centrifugal 15 minutes, draw supernatant, add the 500ul isopropanol precipitating after, obtain total RNA according to description of commodity, the spectrophotometer detectable level.
The total RNA of 2ug obtains cDNA by conventional reverse transcription.Healthy tissues is by obtaining corresponding cDNA with quadrat method, 8 routine samples are mixed in the back, forms the normal control product.All conventional reagent all have commercialization reagent.Use real-time fluorescence quantitative PCR instrument (ABI7900HT) and detect the interleukin II expression level, GAPDH is as the sample internal reference.
All probe primers all select for use u.s. national library of medicine (NCBI) gene database (Genbank) human IL-12 and GADPH messenger RNA(mRNA) sequence to design for masterplate.
With normal control product gene expression level as criterion, hepatocarcinoma patient interleukin II messenger RNA(mRNA) expression amount is divided by this standard, thereby determine the expression ratio of hepatocarcinoma patient interleukin II messenger RNA(mRNA) with respect to the normal people, wherein more than or equal to 1 for increasing, less than 1 for reducing, the interleukin II messenger RNA(mRNA) is expressed the hepatocarcinoma patient postoperative that increases and is very easily occurred shifting or recurrence and dead in 2 years.
The each detection of this test kit all need be provided with the positive and negative control.
But this test kit is avoided multigelation in subzero 20 degree stably stored 1 year.
Test kit prediction effect: existence or death that the interleukin II messenger RNA(mRNA) transfer and relapse of predicting liver cancer patient postoperative more exactly in 66 routine patients' cancer week tissue, interleukin II messenger RNA(mRNA) can also predicting liver cancer patient postoperatives.As depicted in figs. 1 and 2, cancer beside organism's interleukin-messenger RNA(mRNA), the different patient of expression have significantly different prognosis (p＜0.05), wherein, curve 1 expression interleukin-high expression level among Fig. 1, the low expression of curve 2 expression interleukin-, curve 1 expression interleukin-high expression level among Fig. 2, the low expression of curve 2 expression interleukin-.
The present invention has the following advantages:
1. the present invention proposes the interleukin II messenger RNA(mRNA) first can become the leading indicator that prognosis in hcc is diagnosed separately or with other clinical indices combination.
2. interleukin II messenger RNA(mRNA) level obviously is better than being used at present the clinical indices of predicting liver cancer patient postoperative recurrence and existence as prediction index, as alpha-fetoprotein, tumour size, coating has or not etc., and it is simple reliable to measure this gene expression dose.
Description of drawings
Fig. 1 is the no knurl survival curve figure of interleukin II messenger RNA(mRNA);
Fig. 2 is total survival curve figure of interleukin II messenger RNA(mRNA).
Embodiment
Specify the present invention below in conjunction with embodiment.
A kind of prediction test kit that recurs based on the early primary hepatocarcinoma patient postoperative metastasis of immune molecule comprises interleukin-22 augmentation detection reagent, internal control gene augmentation detection reagent, normal liver tissue reference substance, negative control product, positive reference substance, no RNA enzyme water;
Wherein, described interleukin-22 augmentation detection reagent is by 10mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.5mM magnesium chloride, 0.35pmol/ul triphosphate deoxyribose nucleotide, 0.10U/Taq enzyme, 0.35pmol fluorescent mark interleukin-22 self cancellation probe and primer, 20u/ulRNA enzyme inhibitors and water are formed;
Internal control gene augmentation detection reagent is by 10mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.5mM magnesium chloride, 0.35pmol/ul triphosphate deoxyribose nucleotide, 0.10U/Taq enzyme, 0.35pmol fluorescent mark glyceraldehyde 3-phosphate dehydro-genase gene self cancellation probe and primer, 20u/ulRNA enzyme inhibitors and water are formed;
The normal liver tissue reference substance is normal no cancer hepatic tissue cDNA;
The negative control product are no mRNA water;
Positive reference substance is normal people's interleukin II mRNA sample;
No RNA enzyme water is prepared by following method: deionized water was handled 11 hours 120 degrees centigrade of autoclavings 30 minutes through 0.1% (v/v) coke diethyl phthalate.
The early hepatocarcinoma patient leaves and takes the other normal liver tissue of cancer when performing the operation first ,-80 degree Celsius are preserved.With reference to the molecular biology ordinary method, illustrate according to commercialization RNA extraction agent and reverse transcription reagent: the total RNA of the other normal liver tissue of extracting 25mg cancer, reverse transcription generates the cDNA product.
Test kit uses step:
1. add interleukin-22 augmentation detection reagent in this test kit of 10ul after drawing the above-mentioned reverse transcription product of 2ul, the back provides deoxyribonuclease water to mend to 25ul with test kit; Standby, this mixture is better than use in 12 hours most.
2. draw the identical reverse transcription product adding of 2ul test kit simultaneously internal control gene augmentation detection reagent 10ul is provided, the back provides deoxyribonuclease water to mend to 25ul with test kit, and is standby; In the test kit positive reference substance and negative control product draw respectively 2ul respectively with 10ul interleukin-22 augmentation detection reagent mix, then provide deoxyribonuclease water to mend to 25ul with test kit.Above-mentioned mixed solution all by the same steps as operation, adds in 96 orifice plates, and every part of liver organization sample is provided with three times and repeats contrast.96 orifice plates of first-class sample are used the amplification of the U.S. ABI7900 of application system biotech firm real time fluorescent quantitative instrument, condition be 95 ℃ 10 minutes; 95 ℃ 15 seconds, 60 ℃ totally 40 circulations in 1 minute.Normal liver tissue reference substance interleukin II expression amount in the same operation condition detection kit.Calculating cancer aside cytokine 2 messenger RNA(mRNA) according to cycle number relative quantification method expresses, with normal control product expression ratio, determine that said gene expresses variation tendency, express to reduce and then point out patient's prognosis relatively poor, postoperative need be strengthened monitoring, suitably assisting therapy in addition; It is better that prompting patient prognosis is increased in expression, and postoperative was difficult for recurrence in 2 years.
A kind of prediction test kit that recurs based on the early primary hepatocarcinoma patient postoperative metastasis of immune molecule comprises interleukin-22 augmentation detection reagent, internal control gene augmentation detection reagent, normal liver tissue reference substance, negative control product, positive reference substance, no RNA enzyme water;
Wherein, described interleukin-22 augmentation detection reagent is by 5mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.0mM magnesium chloride, 0.3pmol/ul triphosphate deoxyribose nucleotide, 0.05U/Taq enzyme, 0.3pmol fluorescent mark interleukin-22 self cancellation probe and primer, 10u/ulRNA enzyme inhibitors and water are formed;
Internal control gene augmentation detection reagent is by 5mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.0mM magnesium chloride, 0.3pmol/ul triphosphate deoxyribose nucleotide, 0.05U/Taq enzyme, 0.3pmol fluorescent mark glyceraldehyde 3-phosphate dehydro-genase gene self cancellation probe and primer, 10u/ulRNA enzyme inhibitors and water are formed;
The normal liver tissue reference substance is normal no cancer hepatic tissue cDNA;
The negative control product are no mRNA water;
Positive reference substance is normal people's interleukin II mRNA sample;
No RNA enzyme water is prepared by following method: deionized water was handled 10 hours 110 degrees centigrade of autoclavings 20 minutes through 0.1vol% coke diethyl phthalate.
Operation steps is with embodiment 1.
Embodiment 3
A kind of prediction test kit that recurs based on the early primary hepatocarcinoma patient postoperative metastasis of immune molecule comprises interleukin-22 augmentation detection reagent, internal control gene augmentation detection reagent, normal liver tissue reference substance, negative control product, positive reference substance, no RNA enzyme water;
Wherein, described interleukin-22 augmentation detection reagent is by 15mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 3.0mM magnesium chloride, 0.4pmol/ul triphosphate deoxyribose nucleotide, 0.15U/Taq enzyme, 0.4pmol fluorescent mark interleukin-22 self cancellation probe and primer, 30u/ulRNA enzyme inhibitors and water are formed;
Internal control gene augmentation detection reagent is by 15mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 3.0mM magnesium chloride, 0.4pmol/ul triphosphate deoxyribose nucleotide, 0.15U/Taq enzyme, 0.4pmol fluorescent mark glyceraldehyde 3-phosphate dehydro-genase gene self cancellation probe and primer, 30u/ulRNA enzyme inhibitors and water are formed;
The normal liver tissue reference substance is normal no cancer hepatic tissue cDNA;
The negative control product are no mRNA water;
Positive reference substance is normal people's interleukin II mRNA sample;
No RNA enzyme water is prepared by following method: deionized water was handled 12 hours 130 degrees centigrade of autoclavings 35 minutes through 0.1vol% coke diethyl phthalate.
Operation steps is with embodiment 1.
Claims (1)
1. prediction test kit based on the recurrence of the early primary hepatocarcinoma patient postoperative metastasis of immune molecule, it is characterized in that, comprise interleukin-22 augmentation detection reagent, internal control gene augmentation detection reagent, normal liver tissue reference substance, negative control product, positive reference substance, no RNA enzyme water;
Wherein, described interleukin-22 augmentation detection reagent is by 5-15mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.0-3.0mM magnesium chloride, 0.3-0.4pmol/ul triphosphate deoxyribose nucleotide, 0.05-0.15U/Taq enzyme, 0.3-0.4pmol fluorescent mark interleukin-22 self cancellation probe and primer, 10-30u/ulRNA enzyme inhibitors and water are formed;
Internal control gene augmentation detection reagent is by 5-15mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, 2.0-3.0mM magnesium chloride, 0.3-0.4pmol/ul triphosphate deoxyribose nucleotide, 0.05-0.15U/Taq enzyme, 0.3-0.4pmol fluorescent mark glyceraldehyde 3-phosphate dehydro-genase gene self cancellation probe and primer, 10-30u/ulRNA enzyme inhibitors and water are formed;
The normal liver tissue reference substance is normal no cancer hepatic tissue cDNA;
The negative control product are no mRNA water;
Positive reference substance is normal people's interleukin II mRNA sample;
No RNA enzyme water is prepared by following method: deionized water was handled 10-12 hour through 0.1vol% coke diethyl phthalate, 110-130 degree centigrade autoclaving 20-35 minute.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175843A (en) * | 2011-01-20 | 2011-09-07 | 复旦大学附属中山医院 | Molecular markers of well-differentiated early liver cancer and use thereof |
| CN102206710A (en) * | 2011-04-12 | 2011-10-05 | 复旦大学附属中山医院 | Real time polymerase chain reaction (PCR) microarray kit for predicting postoperative recurrence and metastasis of liver cancer after operation |
| CN102445539A (en) * | 2011-10-08 | 2012-05-09 | 复旦大学附属中山医院 | Antibody chip for adopting in early screening and diagnosis of liver cancer, and kit thereof |
| CN103687963A (en) * | 2011-07-12 | 2014-03-26 | 小利兰·斯坦福大学理事会 | A method of determining the prognosis of hepatocellular carcinomas using a multigene signature associated with metastasis |
| WO2021126100A1 (en) | 2019-12-18 | 2021-06-24 | Sabanci Universitesi | Interleukin 11 (il-11) as a biomarker and drug target in fatal liver cancer |
-
2008
- 2008-12-10 CN CNA2008102043627A patent/CN101481732A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175843A (en) * | 2011-01-20 | 2011-09-07 | 复旦大学附属中山医院 | Molecular markers of well-differentiated early liver cancer and use thereof |
| CN102206710A (en) * | 2011-04-12 | 2011-10-05 | 复旦大学附属中山医院 | Real time polymerase chain reaction (PCR) microarray kit for predicting postoperative recurrence and metastasis of liver cancer after operation |
| CN102206710B (en) * | 2011-04-12 | 2013-01-23 | 复旦大学附属中山医院 | Real time polymerase chain reaction (PCR) microarray kit for predicting postoperative recurrence and metastasis of liver cancer after operation |
| CN103687963A (en) * | 2011-07-12 | 2014-03-26 | 小利兰·斯坦福大学理事会 | A method of determining the prognosis of hepatocellular carcinomas using a multigene signature associated with metastasis |
| CN102445539A (en) * | 2011-10-08 | 2012-05-09 | 复旦大学附属中山医院 | Antibody chip for adopting in early screening and diagnosis of liver cancer, and kit thereof |
| CN102445539B (en) * | 2011-10-08 | 2015-01-07 | 复旦大学附属中山医院 | Antibody chip for adopting in early screening and diagnosis of liver cancer, and kit thereof |
| WO2021126100A1 (en) | 2019-12-18 | 2021-06-24 | Sabanci Universitesi | Interleukin 11 (il-11) as a biomarker and drug target in fatal liver cancer |
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Open date: 20090715 |