CN106596523A - Discovery and applications of sCD40L liver cancer early screening and diagnosis marker - Google Patents
Discovery and applications of sCD40L liver cancer early screening and diagnosis marker Download PDFInfo
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Abstract
The present invention provides discovery and applications of a sCD40L liver cancer early screening and diagnosis marker, and relates to the technical field of diagnostic reagents, more particularly to a new hepatocellular carcinoma (HCC) early screening detection marker and associated and developed hepatocellular carcinoma screening and early diagnosis kits. In order to improve the accuracy and the sensitivity of the hepatocellular carcinoma screening and early diagnosis, the present invention provides a newly discovered marker sCD40L for early diagnosis of hepatocellular carcinoma and hepatocellular carcinoma screening of high risk population, and two monitoring system kits respectively based on a magnetic bead chemiluminescence method and a liquid phase antibody sorting magnetic beads and used for detecting the sCD40L in serum and plasma, wherein the newly discovered marker sCD40L has the higher sensitivity than the existing serum and plasma hepatocellular carcinoma molecular diagnostic markers. According to the present invention, the sCD40L detection system has characteristics of rapid immune reaction, high specificity, good reproducibility, low cost, simple experiment requirement, and the like.
Description
Technical field
Invention is related to a kind of new liver cancer（HCC）Early diagnosis, recurrence early diagnosis and liver cancer population at risk（Liver cirrhosis patient）The detection mark sCD40L and novel liver cancer examination and the getting up early diagnostic test kits of exploitation related to this of liver cancer early screening, the specifically patent are to the new sCD40L liver cancer early screening that we have found that and early diagnosis marker and products thereof（The two class detection technique systems that chemiluminescence detection system based on horseradish enzyme or alkaline phosphatase and the detection architecture for sorting magnetic bead based on Liquid phase antibodies are constituted）Protected.Its core is that we are found that first sCD40L is that the Hepatocarcinoma screening to hepatocarcinoma early diagnosis and people at highest risk has the mark for having more hypersensitivity than existing serum and blood plasma liver cancer molecular diagnostic markers.Its clinical practice will greatly change the serious delayed situation of diagnosing cancer of liver.Therefore there is important clinical value.
Background technology
Hepatocellular carcinoma (hepatoeellular carcinoma, HCC) is one of China's incidence of disease and death rate highest malignant tumour.Data shows that HCC occupies the 3rd in mortality of malignant tumors.China increases every year the people of patient HCC 13.5 ten thousand newly, and dead about 110,000 people account for the 45% of whole world HCC death toll.Most HCC has been middle and advanced stage in diagnosis, and now soon, recurrence after operation rate is high for cancer metastasis, poor to sensitiveness such as conventional chemotherapeutic drug such as 5-Fu and adriamycin, and still difficult at present to obtain gratifying therapeutic effect, patient is rear more poor.Most of patients HCC experienced from chronic hepatitis to cirrhosis, finally develop into the Pathologic course of liver cancer.Hence with hepatocarcinoma early diagnosis mark examination early detection liver cancer, early liver cancer is especially found in people at highest risk, for patient vitals are saved, reduce family and social medical treatment cost is respectively provided with important value.
The existing many of the blood serum designated object of diagnosing cancer of liver in recent years, wherein of greatest concern is 5 serologic marker things that the EDRN groups of U.S. NCI are just advancing phase iii clinical trial.They are AFP/AFP-L3, DCP, GP73, HGF and osteopontin.The fucosylated level of their liver cancer-specific of wherein AFP-L3, DCP, GP73, HGF Main Analysis.Their early-stage Study is displayed in hepatocarcinoma early diagnosis has certain value.But these albumen are all through tumor cell secretion or spill, and these albumen are in close relations with hepatocyte growth, therefore often cirrhosis and the horizontal platoon ratio of liver cancer compared with when significant difference, but when for population at risk's dynamic tracking examination, often there is patient that liver cancer increases in the cirrhosis stage because the presence of hepatic regenerative nodules has just been increased, therefore not can determine that so far with it as liver cancer warning index.
The generation of most liver cancer is inseparable with the long-term chronic pathologic process of liver, often experiences from chronic hepatitis liver cirrhosis again to the pathologic process of liver cancer.We have found that sCD40L as the big advantage of liver cancer the marker of early-warning at least two.First, it is by intra platelet free calcium to blood plasma, and cirrhosis is compared with normal population, platelet levels are remarkably decreased because of hypersplenia, therefore the background level of sCD40L is significantly reduced in liver cirrhosis patient compared with normal population, therefore higher sensitiveness is had when being occurred by cirrhosis to liver cancer.Second, during liver cancer, sCD40L is an enzymatic reaction process from intra platelet free calcium, there is lasting Cascaded amplification effect, therefore these liver cancer secreting type blood plasma molecular markers are compared with AFP, GP73 etc., Sensitivity and Specificity greatly strengthens, and the effective examination of liver cancer for making cirrhosis population at risk is really possibly realized.
The content of the invention
Present invention aim at we have found a kind of blood plasma molecular marker that one kind can be used for liver cancer early screening, liver cancer recurrence early diagnosis and liver cancer routine diagnosis, we carry out patent protection at the use to the mark.The content of protection is included using the mark（sCD40L）Carry out（1）The clinic of liver cancer high risk population and scientific research dynamic examination；（2）Liver cancer patient clinical diagnosis；（3）The recurrence early diagnosis of liver cancer patient liver transfer operation, liver cancer minimally-invasive treatment and Post hepatectomy of liver cancer.
We are used for above-mentioned three class and diagnose two class diagnostic kits that the magnetic bead chemoluminescence method using mark exploitation based on sCD40L and the detection architecture that sorts magnetic bead based on Liquid phase antibodies constitute of working together.
The invention thinking of the present invention is based on following two aspects.First, HCC has accounted at present the second of cancer mortality in China, 80% patient for coming from cirrhosis, the cancer of to be therefore one have catastrophe risk crowd, it is found that special liver cancer serum or blood plasma molecular marker are used for the liver cancer early screening of the special population and are always the technology that whole world associated medical worker most pays close attention to；Second, although and up to now still without a molecular marker that effectively can be used for liver cancer early screening is found, American National tumor research institute is just concentrating carries out phase iii clinical trial to 5 blood plasma molecular markers for having potential value in hepatocarcinoma early diagnosis.
In order to reach above-mentioned purpose of the present invention, we are contrasted to the mark AFP for being clinically used for liver cancer serum diagnosis that the mark and the present whole world are generally acknowledged, the detection of AFP is using the clinical general enzyme-linked chemiluminescence detecting method of Roche AFP：
（1）Verified using cross section sample and find that sCD40L is a clinical liver cancer detection mark for being completely superior to AFP.
Although up to now, existing AFP/AFP-L3, DCP, GP73, the mark such as HGF and osteopontin is used for the diagnosis of liver cancer, but clinically generally or AFP, but its positive rate is less than 60%, and often only have middle and advanced stage just sensitive, consequently found that new mark should be extremely important. we are directed to the sCD40L marks that we have found that in normal population, chronic hepatitis, cirrhosis and the class cross section crowd of liver cancer four (each 50) are analyzed, and as a result see Fig. 1, not only HCC and normal population, chronic hepatitis, there were significant differences between cirrhosis (P<, and the level of liver cirrhosis patient is significantly reduced compared with normal population 0.01)（P<0.05）Although. and the AFP levels of the crowd are in minority HCC late period（20%）Significantly increase, but it is most in low-level, because dispersion is greatly without significant difference（Fig. 2）.
（2）Using before and after liver cancer patient liver transfer operation, liver cancer minimally-invasive treatment and RESECTION OF LIVER CANCER and recurrence sample checking find sCD40L be one be completely superior to AFP clinical liver cancer recurrence early diagnosis detection mark.
First we have carried out the analysis of early stage relapse diagnosis in the patient that 10 radio-frequency ablation of liver cancer minimally-invasive treatments recur, and as a result show, 10 HCC patients have contents of 8 sCD40L before minimally-invasive treatment in blood plasma in high level（First point of Fig. 3）, the 3rd day to the 7th day after minimally-invasive treatment is just preferably minimized level（Third and fourth point of Fig. 3）.Absorbing is early stage recurring for second（Diameter<3CM）SCD40L increases rapidly again（The 5th point of Fig. 3）.The level of sCD40L is again rapid after minimally-invasive treatment declines（The 6th of Fig. 3 and the 7th point）.And the AFP contents of the only AFP of 4 before minimally-invasive treatment in blood plasma in above-mentioned 10 patients, in high level, there is also the decline after minimally-invasive treatment, but the HCC recurrence times point increased in sCD40L can not all detect increasing for AFP levels（Fig. 3）, AFP is pointed out the sensitiveness that liver cancer recurrence diagnose is far below sCD40L. and then our levels using the transplanting Plasma Before And After sCD40L horizontal analysis display sCD40L levels of the patient of 50 liver Transplantation for Hepatocellular Carcinoma after the transfer in the blood plasma of 5 to 7 days are significantly reduced（Fig. 4）.This is further illustrated, and blood plasma sCD40L is closely related with the presence of liver cancer, can be used for the early diagnosis of liver cancer recurrence or transfer and relapse after liver Transplantation for Hepatocellular Carcinoma, Post hepatectomy of liver cancer and liver cancer minimally-invasive treatment.
（3）Find that sCD40L is a detection mark for most holding promise for being diagnosed for the clinical liver cancer early screening of liver cancer population at risk up to now using the dynamic examination checking of cirrhotic population.
Most of patients HCC experienced from chronic hepatitis to cirrhosis, finally develop into the Pathologic course of liver cancer.Hence with hepatocarcinoma early diagnosis mark examination early detection liver cancer, early liver cancer is especially found in people at highest risk, for patient vitals are saved, reduce family and social medical treatment cost is respectively provided with important value.We show for the detection of 135 cirrhosis dynamic examination queues, and for same patient, the level of sCD40L reaches 95% in the testing result display consistency of two time points at continuous more than 6 months intervals（Fig. 5）, and 11 early liver cancers found in this queue, the upper monitoring point of blood plasma sCD40L that liver cancer is made a definite diagnosis a little is high 2 to 5 times（Fig. 6）, and this 11 are shown by the AFP dynamic detection results of cirrhosis to liver cancer, AFP makes a definite diagnosis in liver cancer and be not significantly different between blood plasma level a little and a upper monitoring point（Fig. 7）.While sCD40L can be used for cirrhosis, to the early warning examination of liver cancer patient, AFP can not.
Beneficial effects of the present invention：
（1）Present invention finds a blood plasma molecular marker that can be used for liver cancer early screening, liver cancer recurrence early diagnosis and diagnosing cancer of liver（sCD40L）. liver cancer accounts for the second of tumor mortality rate in China, and the major reason of high mortality is to early diagnose, therefore sCD40L early diagnosis is extremely important for the life for saving liver cancer patient.Consider from performance, the value of life for saving a patient surpasses million yuan, therefore the invention has great social value.
（2）We are for the discovery; develop based on the examination of sCD40L novel liver cancers and getting up early diagnostic test kits; specifically the patent is protected to sCD40L in the diagnostic application of liver cancer; meanwhile, we are developed based on horseradish enzyme or the chemiluminescence detection system of alkaline phosphatase and two class detection technique systems of the detection architecture composition that magnetic bead is sorted based on Liquid phase antibodies is used for diagnosing cancer of liver and early screening.
Description of the drawings
Fig. 1 is detection level of the sCD40L marks in normal population, chronic hepatitis, cirrhosis and the class cross section crowd of liver cancer four；
Fig. 2 is detection levels of the AFP in normal population, cirrhosis and the class crowd of liver cancer three；
Fig. 3 is patient's sCD40L diagnostic analysis of radio-frequency ablation of liver cancer minimally-invasive treatment recurrence；
Fig. 4 is the transplanting Plasma Before And After sCD40L horizontal analysis of the patient of liver Transplantation for Hepatocellular Carcinoma；
Fig. 5 is cirrhosis dynamic screening results；
Fig. 6 is that early liver cancer, liver cancer make a definite diagnosis a blood plasma sCD40L detection level；
Fig. 7 is that early liver cancer, liver cancer make a definite diagnosis plasma A FP detection level a little.
Specific embodiment
In order to be more readily understood the technical scheme of present invention offer, hereafter by the preferred embodiment of the present invention, and schema is coordinated to be described below in detail：
Embodiment 1：
The sCD40L in testing sample is detected using nano immune microballoon.
1st, in serum or blood plasma sCD40L detection：
（1）50 μ l sample diluting liquids are separately added in each test tube（0.02M PBS, containing 0.1% Tween 20, pH=7.4）,
（2）50 μ l measuring samples are added,
（3）50 μ l genetic engineering sCD40L solution are added in Positive control wells（Buffer solution is 0.1M PBS）,
（4）50 μ l normal human serums are added in negative control hole,
（5）Sample diluting liquid is not added with blank well.
（6）By in test tube or hole different material mix, 37 DEG C reaction 15 minutes after take out,
（7）Each test tube is separately added into 50 μ l magnetic immuno-microsphere solution（Blank well is not added with）, mix, 37 DEG C reaction 15 minutes after take out,
（8）Each test tube is separately added into 50 μ l enzyme mark sCD40L and resists more（Blank well is not added with）, mix, 37 DEG C are reacted 15 minutes,
（9）Magnetic Isolation, abandons supernatant, and often pipe adds 400 μ l cleaning solutions（0.02M Tris-HCl, containing 0.02% Tween 20, PH=7.4）, so wash 3 times.
（10）50 μ l nitrite ions A and 50 μ l nitrite ion B are separately added in per test tube, rearmounted 37 DEG C of lucifuges are mixed and is reacted 5 minutes,
（11）100 μ l 2M H are added in per test tube2SO4Carry out terminating reaction,
（12）Magnetic Isolation 2 minutes, takes the μ l of each test tube supernatant 100 and moves in ELISA Plate hole,
（13）The absorbance in each hole is measured at wavelength 450nm with ELIASA（OD）Value（Reference wavelength is 630nm）.
2nd, the detection of sCD40L can also adopt chemoluminescence method in serum or blood plasma：
（1）50 μ l sample diluting liquids are separately added in each test tube（0.02M PBS, containing 0.1% polysorbas20, PH=7.4）,
（2）50 μ l measuring samples are added in detection hole,
（3）50 μ l genetic engineering sCD40L antigenic solutions are added in Positive control wells（Buffer solution is 0.1M PBS）,
（4）50 μ l normal human serums are added in negative control hole,
（5）Sample diluting liquid is not added with blank well, only adds conventional Chemoluminescent substrate, main component is luminol, NaOH and hydrogen peroxide.
（6）Mix, 37 DEG C of reactions are taken out after 15 minutes, and each test tube is separately added into 50 μ l magnetic immuno-microspheres（Blank well is not added with）, mix, 37 DEG C reaction 15 minutes after take out,
（7）Each test tube is separately added into 50 μ l enzyme mark sCD40L and resists more（Blank well is not added with）, mix, 37 DEG C are reacted 15 minutes,
（8）Magnetic Isolation, abandons supernatant, and often pipe adds 400 μ l cleaning solutions（0.02M Tris-HCl, containing 0.02% Tween 20, PH=7.4）, Magnetic Isolation abandons supernatant, so washes 3 times.
（9）Each pipe adds 200 μ l chemiluminescent substance AMPPD solution,
（10）The optical signalling for producing is detected with chemiluminescence detector.
The above, only presently preferred embodiments of the present invention is not intended to limit protection scope of the present invention.Every impartial change done according to present invention and modification, are encompassed by the scope of the claims of the present invention.
Claims (7)
1. a kind of new detection mark sCD40L is found, for liver cancer（HCC）Diagnosis, it is characterised in that the mark is a kind of blood plasma molecular marker by intra platelet free calcium.
2. according to claim 1 for liver cancer（HCC）Diagnosis, it is characterised in that can be used for liver cancer（HCC）Early diagnosis, recurrence early diagnosis and liver cancer population at risk（Liver cirrhosis patient）Liver cancer early screening.
3. the blood plasma molecular marker of intra platelet free calcium according to claim 1, it is characterized in that, sCD40L is an enzymatic reaction process from intra platelet free calcium during liver cancer, there is lasting Cascaded amplification effect, therefore these liver cancer secreting type blood plasma molecular markers are compared with AFP, GP73 etc., and Sensitivity and Specificity greatly strengthens.
4. blood plasma molecular marker according to claim 1, it is characterized in that, sCD40L is by intra platelet free calcium to blood plasma, cirrhosis is compared with normal population, platelet levels are remarkably decreased because of hypersplenia, therefore the background level of sCD40L is significantly reduced in liver cirrhosis patient compared with normal population, therefore higher sensitiveness is had when being occurred by cirrhosis to liver cancer.
5. two kind novel liver cancer examinations and early diagnosis detection kit of the invention based on sCD40L, it is characterised in that kit sorts the detection architecture of magnetic bead based on magnetic bead chemoluminescence method and based on Liquid phase antibodies.
6. diagnostic test kits according to claim 5, characterized in that, can be used for the clinic of liver cancer high risk population and the recurrence early diagnosis of scientific research dynamic examination, liver cancer patient clinical diagnosis and liver cancer patient liver transfer operation, liver cancer minimally-invasive treatment and Post hepatectomy of liver cancer.
7. a kind of antibody for detecting sCD40L, it is characterised in that the antibody is monoclonal antibody, with high immunological activity.
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| CN109374897A (en) * | 2018-11-22 | 2019-02-22 | 大连大学 | A kind of kit for liver cancer detection |
| CN109557312A (en) * | 2018-12-14 | 2019-04-02 | 北京艾克伦医疗科技有限公司 | Hepatocarcinoma early diagnosis method and diagnostic kit |
| CN112147336A (en) * | 2019-09-18 | 2020-12-29 | 首都医科大学附属北京佑安医院 | Marker for detecting and evaluating liver regeneration and application thereof |
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Cited By (4)
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| CN107966565A (en) * | 2017-11-15 | 2018-04-27 | 北京市肝病研究所 | A kind of liver cancer early screening and early diagnosis marker sCD40L |
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| CN112147336A (en) * | 2019-09-18 | 2020-12-29 | 首都医科大学附属北京佑安医院 | Marker for detecting and evaluating liver regeneration and application thereof |
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