CN103969371B - A kind of blood degraded obtains and detects the application of method in cancer detection of monose - Google Patents

A kind of blood degraded obtains and detects the application of method in cancer detection of monose Download PDF

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CN103969371B
CN103969371B CN201410211976.3A CN201410211976A CN103969371B CN 103969371 B CN103969371 B CN 103969371B CN 201410211976 A CN201410211976 A CN 201410211976A CN 103969371 B CN103969371 B CN 103969371B
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cancer
blood
monose
carcinoma
obtains
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CN103969371A (en
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张丽娟
曾璇
兰莹
邱培菊
曾洋洋
徐玲玲
周紫婧
韩章润
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Ocean University of China
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Abstract

The invention belongs to field of medicaments, relating to sugar chain degradations all in blood is the application of method in cancer detection that monose carries out detecting again.Described degraded obtains and the sample detected in the method for monose is blood, and described cancer comprises lung cancer, cancer of the stomach, oophoroma, carcinoma of penis, the cancer of the esophagus, carcinoma of mouth, cholangiocarcinoma, breast cancer, carcinoma of ampulla, the carcinoma of the rectum, carcinoma of urinary bladder.The features such as it is easy to learn that this detection method has operation steps, and method is easily popularized, and detect consuming time short, low to instrument requirements, testing cost is low, and blood consumption is few.In acquired results display blood, the content of 8 kinds of monose not only can distinguish normal person and cancer patient, also can distinguish various cancers.This gaining knowledge sugared group is simplified and the detection being applied to hematologic cancers biomarker belongs to pioneering in world wide.

Description

A kind of blood degraded obtains and detects the application of method in cancer detection of monose
Technical field
The invention belongs to field of medicaments, relate to the degraded of a kind of blood and obtain and detect the application of method in cancer detection of monose.
Background technology
Cancer is one of primary cause of the death in the whole world.2004, number of cancer deaths reached 7,400,000 (accounting for 13% of all death tolls), and lung cancer, cancer of the stomach, colon cancer, liver cancer and breast cancer are the arch-criminals of annual most of cancer mortality.Estimate, if do not intervened, 8,400 ten thousand people during 2005 to 2015, will be had to die from cancer according to World Health Organization.Cancer is the uncontrolled growth of cell and diffusion.It can affect the almost any part of human body.Tumour usually invades the tissue of surrounding and can be transferred to other position.By operation, radiotherapy or chemotherapy, cancer can be cured, if found especially in early days greatly.Just be found before cancer cell diffusion for those and meet subject early stage of lung cancer patient, the average survival time rate is satisfactory; But 70% ~ 80% cancer card patient when occurring that clinical symptoms is gone to a doctor be in, late period, lose healing chance, survival rate is very low.
The diagnostic method of cancer comprises imaging examination, pathological examination etc., be all fully develop in cancer, tumour enough large time, can effectively diagnose.Therefore, the result for the treatment of improving cancer, the key reducing mortality ratio are early detection.Optimal early detection is the blood analysis not needing biopsy.Be that the theoretical analysis of foundation is pointed out with experimental data, rely on tumors secrete to carry out cancer diagnosis to the cancer cell biomarker in blood at present and require that tumour is not less than 20.44mm 3, now tumour has the growth history of 10.1.Therefore, optimal state does not also grow to 20.44mm in tumour 3time just by routine physical examination blood drawing carry out early stage blood testing.Improve the detection sensitivity of cancer biomarker in blood, early detection is become then may need new approaches.
Biological information transmittance process is again to albumen from DNA to RNA.The number of human gene is about 25000, and each gene correspond to corresponding albumen.After albumen is formed, junction saccharogenesis, fat and various compound.Conversely, albumen is subject to again the modification of planting sugar, fat and various compound 200 more.Many functions of albumen determined by these modifications.Group is learned research and have found the possible cancer markers of series, but also has distance from clinical practice.Detect MicroRNA in cancer plasma and kinds cancer is demonstrated to the selectivity of height.But end-stage patients be can not show a candle to the sensitivity of early-stage cancer patient, perhaps this can limit the application of the method at early diagnosis of cancer.On the whole, group Epidemiological Analysis is more comprehensive than existing biomarker for cancer, but in general complicated, expensive and need be a large amount of data processing, also do not reach simple at present, fast, stable, reliable, cheap clinical requirement.
The report of existing kinds cancer blood sugar biomarker at present, but utilize the Isolation and ldentification technology of existing multi-step from all to meet the blood biomarker of the early diagnosis of cancer of clinical requirement very difficult more than finding out sensitivity and selectivity 7000 sugared structures, be difficult to there is clinical value in a short time.
Summary of the invention
Given this, the object of the invention is to: provide the degraded of a kind of blood obtain and detect the application of method in cancer detection of monose, especially for the content detecting cancer patient or the rear gained monose of normal human blood degraded.Monose in Simultaneously test blood, as glucose, and the monose produced after degraded, comprise glucose, mannose, galactose, Glucosamine, amine-galactose, wood sugar, fucose, glucuronic acid, the sugared structure change information of more fully cancer plasma can be obtained, normal person and cancer patient can be distinguished, also can distinguish various cancers.
Object of the present invention is achieved through the following technical solutions:
1. blood degraded obtains and detects the application of method in cancer detection of monose, and described degraded obtains and the method step detecting monose comprises:
(1) sample thief is to ampoule bottle, and often pipe adds 1mL2M C 2hF 3o 2;
(2) tube sealing, 105 DEG C of oil bath 6h, obtain hydrolyzation sample solution;
(3) hydrolyzation sample solution is cooled to room temperature, condensation is concentrated except C 2hF 3o 2;
(4) often pipe adds 100uL methyl alcohol, and centrifugal concentrating is except residual C 2hF 3o 2, in triplicate;
(5) dry sample obtained is every effective water-soluble, then adds NH 3h 2o adjusts pH to 8-14, fully at room temperature leaves standstill after mixing, then adds 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) water-bath;
(6) take out reactant and be cooled to room temperature, add HAc neutralization;
(7) often pipe adds CHCl 3extraction Hou Qi lower floor CHCl 3layer, in triplicate, crosses 0.22um filter membrane by the water layer obtained, is ready to use in efficient liquid phase chromatographic analysis;
Chromatographic condition
Chromatographic column: ZORBAX SB-C18,0.1M phosphate (13.6gKH 2pO 4, 1.8gNaOH) and (pH6.7) damping fluid-acetonitrile (volume ratio 83: 17) isocratic elution, flow velocity: 1.0mL/min, determined wavelength: 245nm, sampling volume: 20uL;
Described degraded obtains and the sample detected in the method for monose is blood, described is applied as the sugar chain degradation in cancer patient or normal human blood to be monose, to detect contents of monosaccharides.
2. blood degraded obtains and detects the application of method in cancer detection for monose, and it is characterized in that, described degraded obtains and the sample detected in the method for monose is serum or blood plasma.
3. a blood degraded obtains and detects the application of method in cancer detection of monose, it is characterized in that, described cancer comprises lung cancer, cancer of the stomach, oophoroma, carcinoma of penis, the cancer of the esophagus, carcinoma of mouth, cholangiocarcinoma, breast cancer, carcinoma of ampulla, the carcinoma of the rectum, carcinoma of urinary bladder.
Blood degraded to be obtained and the method detecting monose is applied to the beneficial effect of cancer:
1. operation steps is easy to learn, and method is easily popularized, and is applicable to the detection of blood clinically.
2. detect consuming time short.
3. pair instrument requirements is low, and testing cost is low, does not need Large expensive instrument and expensive reagent just can complete detection.
4. blood consumption is few, each blood volume that only need be less than 1mL.
5. the content detecting gained 8 kinds of monose after degraded in blood not only can distinguish normal person and cancer patient, also can distinguish various cancers, be expected to find hematologic cancers biomarker by the method.
The content of gained monose after degrading with the methods analyst normal person and cancer plasma that analyze monose, today of cancer threat is subject to increasing people, using value is huge, testing result can supply doctor's reference, more patient is helped to break away from the torment of cancer, find cancer as early as possible, early find early treatment.
Accompanying drawing explanation
Fig. 1 is 0.1mg/mL monose standard solution and carcinoma of mouth patient blood monosaccharide composition analysis high-efficient liquid phase chromatogram.
Fig. 2 is 11 kinds of cancer patients and normal human blood 8 kinds of contents of monosaccharides distribution plans.
Embodiment:
All blood samples that embodiment 2 to 13 relates to provide by Qingdao Municipal Hospital and Qingdao Hai Ci hospital.
Embodiment 1
(1) preparation is containing the solution of the variable concentrations (0.5mg/mL, 0.25mg/mL, 0.1mg/mL, 0.05mg/mL, 0.025mg/mL, 0.01mg/mL) of 8 kinds of monose standard items, then adds NH 3h 2o adjusts pH to 8-14, fully at room temperature leaves standstill after mixing, then adds 0.5M PMP water-bath.
(2) take out reactant and be cooled to room temperature, add HAc neutralization.
(3) often pipe adds chloroform extraction Hou Qi lower floor chloroform layer, in triplicate.The water layer obtained is crossed 0.22um filter membrane, is ready to use in liquid phase analysis.
Chromatographic condition
Chromatographic column: ZORBAX SB-C18 (5um, 4.6mm × 150mm), 0.1M phosphate (13.6gKH 2pO 4, 1.8gNaOH) and (pH6.7) damping fluid-acetonitrile (volume ratio 83: 17) isocratic elution, flow velocity: 1.0mL/min, determined wavelength: 245nm, sampling volume: 20uL.
Obtain the liquid chromatogram of 8 kinds of monose standard items, see Figure 1A.
Can find out that 8 kinds of monose separate completely from Figure 1A, not interfere with each other.The direct reference standards Figure 1A of cancer patient blood sample spectrogram, can learn the monose composition of sample, by variable concentrations standard items spectrogram integration drawing standard curve, can calculate the content of each monose in sample.
Embodiment 2
(1) the centrifugal 5min separation of serum of 42 routine normal human blood 1mL, 3000r/min or blood plasma is got.
(2) get blood plasma 100uL to ampoule bottle, often pipe adds 1mL2M C 2hF 3o 2.
(3) tube sealing, 105 DEG C of oil bath 6h, obtain hydrolyzation sample solution.
(4) hydrolyzation sample solution is cooled to room temperature, is transferred to 1.5mL centrifuge tube ,-4 DEG C of condensations concentrate 5h except C 2hF 3o 2.
(5) often pipe adds 100uL methyl alcohol, and centrifugal concentrating is except residual C 2hF 3o 2, in triplicate.
(6) dry sample obtained is every effective water-soluble, then adds NH 3h 2o adjusts pH to 8-14, fully at room temperature leaves standstill after mixing, then adds 0.5MPMP water-bath.
(7) take out reactant and be cooled to room temperature, add HAc neutralization.
(8) often pipe adds CHCl 3extraction Hou Qi lower floor CHCl 3layer, in triplicate, crosses 0.22um filter membrane by the water layer obtained, is ready to use in efficient liquid phase chromatographic analysis.
Chromatographic condition
Chromatographic column: ZORBAX SB-C18 (5um, 4.6mm × 150mm), 0.1M phosphate (13.6gKH 2pO 4, 1.8gNaOH) and (pH6.7) damping fluid-acetonitrile (volume ratio 83: 17) isocratic elution, flow velocity: 1.0mL/min, determined wavelength: 245nm, sampling volume: 20uL.
Embodiment 2 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 3
Get 52 routine lung cancer patient blood 1mL, all the other steps are with embodiment 2, and embodiment 3 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 4
Get 4 routine Patients with Gastric Cancer blood 1mL, all the other steps are with embodiment 2, and embodiment 4 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 5
Get 4 routine human ovarian cancer patients's blood 1mL, all the other steps are with embodiment 2, and embodiment 5 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 6
Get 1 routine carcinoma of penis patient blood 1mL, all the other steps are with embodiment 2, and embodiment 6 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 7
Get 3 routine cancer of esophagi human blood 1mL, all the other steps are with embodiment 2, and embodiment 7 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 8
Get 1 routine carcinoma of mouth patient blood 1mL, all the other steps are with embodiment 2, and embodiment 8 repeats 3 times, and statistic mixed-state result, is shown in Figure 1B, and Fig. 1 C, Fig. 1 D, is shown in Fig. 2.
Embodiment 9
Get 1 routine cholangiocarcinoma patients blood 1mL, all the other steps are with embodiment 2, and embodiment 9 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 10
Get 5 routine breast cancer disease human blood 1mL, all the other steps are with embodiment 2, and embodiment 10 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 11
Get 1 routine carcinoma of ampulla patient blood 1mL, all the other steps are with embodiment 2, and embodiment 11 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.Embodiment 12
Get 2 routine Patients With Rectal Carcinoma blood 1mL, all the other steps are with embodiment 2, and embodiment 12 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Embodiment 13
Get 2 routine bladder cancer's blood 1mL, all the other steps are with embodiment 2, and embodiment 13 repeats 3 times, and statistic mixed-state result, is shown in Fig. 2.
Sum up Fig. 2 result to table 1,8 kinds of contents of monosaccharides analysis results are in table 2.
Table 1, the comparable trend of gained 8 kinds of monose and normal person after 11 kinds of cancer plasma degrade
Lung cancer Cancer of the stomach Breast cancer The cancer of the esophagus Oophoroma Other cancers
Glucose ↓* - ↓* ↓* ↓ ↓*
Fucose ↑* ↑* ↑* ↑* ↑* ↑*
Wood sugar ↑ ↑ ↑ ↑ ↑ ↑
Galactose ↑* ↑ ↑ ↑ ↑* ↑
Glucuronic acid ↑ ↑ ↑ - ↑ ↑
Galactosamine - ↓* ↓ ↓* ↑ ↓
Mannose ↑* - ↑ - ↑* ↑
Gucosamine - ↓ ↓ ↓ - -
Annotation: "-" representative is unchanged compared with normal person, " ↑ * " and " ↓ * " represent respectively and significantly rise or decline compared with normal person, " ↑ " and " ↓ " represent respectively rises or decline compared with normal person, and " other cancers " represents the result of comprehensive carcinoma of penis, carcinoma of mouth, cholangiocarcinoma, carcinoma of ampulla, the carcinoma of the rectum and carcinoma of urinary bladder.
Table 2,8 kinds of monose composition testing results in 11 kinds of cancers and normal human blood
Normal person Lung cancer Cancer of the stomach Breast cancer The cancer of the esophagus Oophoroma Other cancers
Glucose 5.303±1.157 3.523±1.422 5.413±2.517 3.704±0.662 3.234±0.523 4.873±0.822 3.785±1.123
Fucose 0.242±0.059 0.626±0.177 0.634±0.207 0.542±0.172 0.628±0.098 0.438±0.073 0.617±0.140
Wood sugar 0.218±0.027 0.302±0.356 0.332±0.057 0.304±0.012 0.291±0.020 0.246±0.046 0.322±0.069
Galactose 2.137±0.353 2.898±1.112 2.459±0.411 2.405±0.449 2.286±0.407 2.973±0.310 2.518±0.382
Glucuronic acid 0.001±0.001 0.031±0.049 0.021±0.033 0.030±0.054 0.002±0.003 0.020±0.034 0.046±0.064
Galactosamine 0.436±0.081 0.441±0.222 0.217±0.047 0.317±0.102 0.120±0.018 0.498±0.156 0.319±0.164
Mannose 2.381±0.458 3.112±1.399 2.498±0.401 2.666±0.431 2.314±0.384 3.407±0.452 2.601±0.554
Gucosamine 6.201±0.862 6.348±2.132 5.897±1.519 5.376±1.487 5.262±1.288 6.000±1.094 6.008±1.393
Annotation: " other cancers " represents the result of comprehensive carcinoma of penis, carcinoma of mouth, cholangiocarcinoma, carcinoma of ampulla, the carcinoma of the rectum and carcinoma of urinary bladder.
As can be seen from Figure 1, blood degraded obtains and the method detecting monose can apply to Monosaccharide analysis in normal person and cancer plasma, and 8 kinds of monose high performance liquid chromatography obtain good separation, have feasibility.
From Fig. 2, table 1, table 2 can be found out, after only having lung cancer and human ovarian cancer patients's blood sample to degrade, the content of galactose is apparently higher than normal person; The content of the galactosamine of cancer of the stomach and Patients With Carcinoma of Esophagus is starkly lower than normal person and other cancer patients; After only having cancer of esophagi human blood sample to degrade, glucuronic acid content is suitable with normal person; Fucose content rising is the common trait of all cancer patients.
Above result all shows: the method operation steps is easy to learn, and method is easily popularized, and is applicable to detecting for cancer plasma clinically; Detect consuming time short, low to instrument requirements, testing cost is low, does not need Large expensive instrument and expensive reagent just can complete detection; Blood consumption is few, each blood volume that only need be less than 1mL; After detecting blood degraded, the content of gained monose not only can distinguish normal person and cancer patient, also can distinguish various cancers, be expected to find hematologic cancers biomarker by the method.

Claims (3)

1. blood degraded obtains and detects the application of method in cancer detection of monose, and described degraded obtains and the method step detecting monose comprises:
(1) sample thief is to ampoule bottle, and often pipe adds 1mL 2M C 2hF 3o 2;
(2) tube sealing, 105 DEG C of oil bath 6h, obtain hydrolyzation sample solution;
(3) hydrolyzation sample solution is cooled to room temperature, condensation is concentrated except C 2hF 3o 2;
(4) often pipe adds 100 μ L methyl alcohol, and centrifugal concentrating is except residual C 2hF 3o 2, in triplicate;
(5) dry sample obtained is every effective water-soluble, then adds NH 3h 2o adjusts pH to 8-14, fully at room temperature leaves standstill after mixing, then adds the water-bath of 1-phenyl-3-methyl-5-pyrazolones ketone;
(6) take out reactant and be cooled to room temperature, add HAc neutralization;
(7) often pipe adds CHCl 3extraction Hou Qi lower floor CHCl 3layer, in triplicate, crosses 0.22 μm of filter membrane, is ready to use in efficient liquid phase chromatographic analysis by the water layer obtained;
Chromatographic condition
Chromatographic column: ZORBAX SB-C18, organic phase is acetonitrile, and aqueous phase is the 0.1M phosphate buffer of pH6.7, wherein containing 13.6gKH 2pO 4, 1.8gNaOH, organic phase and aqueous phase volume ratio are 17: 83, isocratic elution, flow velocity: 1.0mL/min, determined wavelength: 245nm, sampling volume: 20 μ L;
It is characterized in that, described degraded obtains and the sample detected in the method for monose is blood, described is applied as the sugar chain degradation in cancer patient or normal human blood to be monose, to detect contents of monosaccharides.
2. a kind of blood degraded as claimed in claim 1 obtains and detects the application of method in cancer detection of monose, and it is characterized in that, described degraded obtains and the sample detected in the method for monose is serum or blood plasma.
3. a kind of blood degraded as claimed in claim 1 obtains and detects the application of method in cancer detection of monose, it is characterized in that, described cancer comprises lung cancer, cancer of the stomach, oophoroma, carcinoma of penis, the cancer of the esophagus, carcinoma of mouth, cholangiocarcinoma, breast cancer, carcinoma of ampulla, the carcinoma of the rectum, carcinoma of urinary bladder.
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CN108956792B (en) 2017-05-29 2020-06-16 青岛大学附属医院 High performance liquid chromatography detection of serum free mannose and glucose
CN110346500B (en) * 2018-04-04 2021-11-30 青岛大学附属医院 Detection method for detecting monosaccharide content in serum based on microwave acid hydrolysis and anion exchange chromatography-pulse amperometry
CN110261494A (en) * 2018-11-01 2019-09-20 青岛大学附属医院 A kind of method and its detection kit for identifying thyroid malignancy biomarker
CN110261495A (en) * 2018-11-01 2019-09-20 青岛大学附属医院 A kind of method and its detection kit for identifying colorectal cancer biomarker

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