CN104698060A - Acute myelogenous leukemia tumor marker and application thereof - Google Patents
Acute myelogenous leukemia tumor marker and application thereof Download PDFInfo
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- CN104698060A CN104698060A CN201310647095.1A CN201310647095A CN104698060A CN 104698060 A CN104698060 A CN 104698060A CN 201310647095 A CN201310647095 A CN 201310647095A CN 104698060 A CN104698060 A CN 104698060A
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Abstract
The invention relates to an acute myelogenous leukemia tumor marker and application thereof. The acute myelogenous leukemia tumor marker is Lacto series glycosphingolipid. Specifically, the structure formula of the Lacto series glycosphingolipid is shown as the following one item or multi terms: Gal-alpha4-Gal-beta4-Galbeta-Cer; Gal-beta3-GlcNAc-beta3-Gal-beta4-Glcbeta-Cer; Gal-beta4-GlcNAc-beta3-Gal-beta4-Glcbeta-Cer; wherein Gal represents galactose, GlcNAc represents N-acetyl glucose, Glc represents glucose, and Cer represents N-fatty acyl-sphingosine. The acute myelogenous leukemia tumor marker can be used for the clear characterization of acute myelogenous leukemia.
Description
Technical field
The present invention relates to tumor markers technical field, particularly relate to a kind of acute myeloid leukemia tumor markers and application thereof.
Background technology
Acute myeloid leukemia (Acute Myeloid Leukemia, AML) be a kind of heterogeneous myelocyte tumour, also claim acute nonlymphocytic leukemia, normal progress rapidly, is characterized in being cancerated by candidate stem cell and the initial cell clone that formed instead of normal bone marrow hematopoiesis.Because leukaemia instead of normal plasma cell, they do not possess the function of normal plasma cell, cause anaemia thus, and blood platelet and granulocyte reduce and cause a series of complication.If without treatment, survival period is generally no more than half a year.Some cases are from diagnosis to death, and even only one week, main causes of death were hemorrhage and infect.AML is modal acute leukemia, and patient is generally adult, and its incidence of disease increases with advancing age simultaneously.Although leukaemia is a kind of relatively rare disease (accounting for greatly number of cancer deaths's 1.2%), its incidence of disease may increase along with the increase of aging population.Leukemic symptom be due to normal marrow cell substitute by leukaemia, the hazards that some leukaemia are relevant and chromosome abnormality are determined, but concrete pathogenic factor it be unclear that.Nowadays, the first step of primary treatment scheme of nearly all treatment AML is inductive treatment, and it is that the level of instigating leukaemia's quantity to reduce to can't detect is to reach the object of complete incidence graph AML.Current standard chemotherapy can obtain complete incidence graph (CompleteRemission, the CR) rate of about 70 ~ 80%, but the patient after most of CR is recurred at last, and even a part of patient is difficult to reach CR and becomes Refractory Leukemia, fails to respond to any medical treatment and dead.
Leukaemia is the malignant disease of a class hematopoietic cell exception, and leukaemia loses the ability continuing differentiation and maturation and is stuck in cytocerastic different phase, and continuous hyperplasia affects body normal function.So, whether the glycosyl sphingolipid of leukaemic has difference compared with Healthy People is the focus that everybody pays close attention to, Cooling etc. be once reported in acute non lymphocytic leukemia peripheral blood in patients be separated the unconventionality expression having glycosyl sphingolipid in the leukaemia obtained, and to relate in people's myelocyte maturation the change of relevant glycosyl sphingolipid.In vitro, drug-induced Leukemia Cell Lines HL-60, the NB4 of can causing such as ATRA, PMA produces differentiating phenomenon, glycosyl sphingolipid change before and after the external drug-induced generation leukaemia of people focus attentions equally on breaks up, these experimental results show, glycosyl sphingolipid all plays an important role in leukemic whole pathogenic process, and even these glycosyl sphingolipids that significant change occurs can as leukemic biomarker.Therefore, find and determine that the tumor markers of acute myeloid leukemia has important value for the Diagnosis and Treat of this disease.
Summary of the invention
The present inventor finds that Lacto series glycosyl sphingolipid exists the phenomenon of high expressed in the myeloid tissue and serum of Patients with Acute Myeloid Leukemia after deliberation, therefore, it is possible to as the tumor marker of acute myeloid leukemia, the present invention is based on above-mentioned discovery and complete.
The object of the present invention is to provide a kind of acute myeloid leukemia tumor markers, it is as the significant material of Diagnosing Acute Myeloid Leukemia, can the clear and definite generation clearly characterizing acute myeloid leukemia disease.
The present invention includes following content:
The invention provides a kind of acute myeloid leukemia tumor markers, it is Lacto series glycosyl sphingolipid.
As preferably of the present invention, the following any one of structural formula of described Lacto series glycosyl sphingolipid or multinomial shown in:
Gal-α4-Gal-β4-Galβ-Cer；
Gal-β3-GlcNAc-β3-Gal-β4-Glcβ-Cer；
Gal-β4-GlcNAc-β3-Gal-β4-Glcβ-Cer；
Wherein, Gal represents galactose, and GlcNAc represents N-acetyl glucosamine, and Glc represents glucose, and Cer represents N fatty acyl sphingosine.
The Lacto series glycosyl sphingolipid that structure above represents is called Lc3, Lc4 and nLc4 successively.
As preferably of the present invention, the structure and/or composition of described Lacto series glycosyl sphingolipid is determined by mass spectrometry method.
The present invention also provides above-mentioned tumor markers preparing and the application in the specificity junction mixture of its combination.
Described specificity junction mixture can be such as the monoclonal antibody or polyclonal antibody etc. of anti-described tumor markers.Described monoclonal antibody or polyclonal antibody can be complete antibody, also can be the partial antibody fragments with binding site.
Preferably, described specificity junction mixture is the antibody of anti-described tumor markers.That is, acute myeloid leukemia tumor markers of the present invention can use as the antigen of the antibody of the anti-described acute myeloid leukemia tumor markers of preparation.
Beneficial effect of the present invention is: find that Lacto series glycosyl sphingolipid exists the phenomenon of specificity overexpression in the myeloid tissue and serum of Patients with Acute Myeloid Leukemia, and there is no this specificity overexpression phenomenon in the normal tissue, therefore, it is possible to as the tumor markers of acute myeloid leukemia, the generation of acute myeloid leukemia fast, accurately and clearly can be determined by the expression of Mass Spectrometer Method Lacto series glycosyl sphingolipid.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates of Lacto series glycosyl sphingolipid under first mass spectrometric, wherein A is Patients with Acute Myeloid Leukemia sample of bone marrow glycosyl sphingolipid first mass spectrometric collection of illustrative plates, B is normal control sample of bone marrow glycosyl sphingolipid first mass spectrometric collection of illustrative plates, wherein m/z 1100 and 1120 is LacCer glycosyl sphingolipid, m/z 1214.3 and 1326.3 is Gb3 glycosyl sphingolipid, m/z 1255.3 and 1368.3 is Lc3 glycosyl sphingolipid, and m/z 1460.0 is Lc4 or nLc4 glycosyl sphingolipid.
Fig. 2 is the result figure (wherein * * * represents p<0.001) of Lacto series glycosyl sphingolipid high expressed in patient AML.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand the present invention better, thus should not be considered as limiting scope of the present invention.For a person skilled in the art, the present invention can have various modifications and variations, within the spirit and principles in the present invention all, and any amendment done, equivalent replacement or improvement etc., all should be included within protection scope of the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method; Experiment material used, if no special instructions, is and is purchased available from routine biochemistry chemical reagent work.
Collect clinical Bone Marrow sample or blood sample, get identical amount and be placed in glass test tube, 1mL extraction agent A(methylene chloride is added: methyl alcohol=1:1(v/v in test tube)), vortex oscillation makes it mix, and ultrasonic extraction 1h, then by centrifugal for test tube 2000rpm 10min, careful absorption supernatant is placed in new clean tube, remaining precipitation continues to add 1mL extraction agent A, ultrasonic extraction 1h, repeats 4 times.Above-mentioned remaining precipitation is continued add 1mL extraction agent B(isopropyl alcohol: normal hexane: water=55:25:20(v/v/v)), vortex oscillation makes it mix, be placed in ultrasonic equipment ultrasonic extraction 1h, the centrifugal 10min of same 2000rpm, careful absorption supernatant is placed in new clean tube, remaining precipitation continues to add 1mL extraction agent B ultrasonic sound and extracts 1h, repeats 4 times.The above-mentioned supernatant collected is carried out drying by Vacuum Concentration dryer, finally obtains the solid of trace.
Utilize Sephadex chromatography method Separation of Neutral, acidic glycosphingolipids.Sephadex filler being installed to (column volume is about 3mL) in glass column, with 15mL chloroform: methyl alcohol: water=30:60:10(v/v/v) mixed solvent washes post, with above-mentioned dissolution with solvents sample, on a small quantity, 500 μ about L.The sample of dissolving is joined on pillar, adds the above-mentioned solvent elution of 10mL equally, collect and be neutral glycosphingolipid.Neutral glycosphingolipid just can after methylation reaction Mass Spectrometer Method, acidic glycosphingolipids need carry out methylation reaction Mass Spectrometer Method again after dialysis desalting.
The glycosyl sphingolipid solid of gained drying is added 150 μ L DMSO to dissolve, avoid moisture to enter, moisture can affect methylation reaction.The NaOH solid of grind into powder is joined in test tube, requires rapidly, to avoid the solid NaOH powder moisture absorption bottom covering test tube at once.Add 150 μ L iodomethane fast, use aluminium foil immediately, sealed membrane seals, and avoids the moisture in air to enter impact reaction.Be placed on horizontal shaker and run 1h with maximum (top) speed, then add 1mL ultrapure water cessation reaction, precipitation is dissolved completely.In test tube, add 2mL methylene chloride, can see that liquid is divided into two-layer, lower floor is methylene chloride, and last time is water, piping and druming liquid, makes two-phase liquid mix, aqueous phase discarded, add clean ultrapure water again, the operation before repetition more than at least ten times, cleaning methylene chloride mutually in impurity.Clean methylene chloride and aqueous phase are separated as far as possible, then that methylene chloride is dry, add methyl alcohol and dissolve and can carry out Mass Spectrometer Method.
Electron spray linear ion trap mass spectrometer is used to carry out MS and MS to methylated glycosyl sphingolipid in the positive-ion mode
nanalyze, the methanol solution of direct injection glycosyl sphingolipid sample detects, parameters is set to: flow velocity 0.30 μ L/min, capillary temperature 230 DEG C, sheath gas velocity 2arb, spray voltage 3.50kV, capillary voltage 28.00kV, inject time 100.00ms, soak time 30ms, activate Q value 0.250, m/z and isolate width 1.5.The normalized energy using forerunner's ion to reach minimum abundance is collided, and detected ion is all Na ion complex.The first mass spectrometric collection of illustrative plates of Lacto series glycosyl sphingolipid as shown in Figure 1, A is Patients with Acute Myeloid Leukemia sample of bone marrow glycosyl sphingolipid first mass spectrometric collection of illustrative plates, B is normal control sample of bone marrow glycosyl sphingolipid first mass spectrometric collection of illustrative plates, wherein m/z 1100 and 1120 is LacCer glycosyl sphingolipid, m/z 1214.3 and 1326.3 is Gb3 glycosyl sphingolipid, m/z 1255.3 and 1368.3 is Lc3 glycosyl sphingolipid, and m/z 1460.0 is Lc4 or nLc4 glycosyl sphingolipid.Through carrying out employing Student-Newman-Keulsa to the mass-spectrogram of 16 routine normal bone marrow samples and 106 routine patients AML, b check analysis data, to take statistics analysis with GraphPad Prism 5 software (GraphPadSoftware Inc.), find it is have statistical significance (p<0.001, Fig. 2) between them.Fig. 2 shows the phenomenon that Lacto series glycosyl sphingolipid exists specificity overexpression in the myeloid tissue and serum of Patients with Acute Myeloid Leukemia, and there is no this specificity overexpression phenomenon in the normal tissue, point out the generation of this analogues of glycosphingolipids at acute myeloid leukemia and developing vital role.Further, using a kind of diagnosis marker of the content of this analogues of glycosphingolipids as acute myeloid leukemia, early prevention and the treatment of acute myeloid leukemia can be contributed to, prognosis be also very helpful.Therefore Lacto series glycosyl sphingolipid of the present invention as the tumor markers of acute myeloid leukemia, can fast, accurately and clearly can determine the generation of acute myeloid leukemia by the expression of Mass Spectrometer Method Lacto series glycosyl sphingolipid.
Applicant states, the present invention illustrates detailed features of the present invention and method detailed by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method detailed, namely do not mean that the present invention must rely on above-mentioned detailed features and method detailed could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, concrete way choice etc. that the present invention selects component, all drops within protection scope of the present invention and open scope.
Claims (5)
1. an acute myeloid leukemia tumor markers, it is Lacto series glycosyl sphingolipid.
2. tumor markers according to claim 1, is characterized in that, the following any one of structural formula of described Lacto series glycosyl sphingolipid or multinomial shown in:
Gal-α4-Gal-β4-Galβ-Cer；
Gal-β3-GlcNAc-β3-Gal-β4-Glcβ-Cer；
Gal-β4-GlcNAc-β3-Gal-β4-Glcβ-Cer；
Wherein, Gal represents galactose, and GlcNAc represents N-acetyl glucosamine, and Glc represents glucose, and Cer represents N fatty acyl sphingosine.
3. tumor markers according to claim 1 and 2, is characterized in that, the structure and/or composition of described Lacto series glycosyl sphingolipid is determined by mass spectrometry method.
4. the tumor markers as described in any one of claim 1-3 is in preparation and the application in the specificity junction mixture of its combination.
5. application according to claim 4, is characterized in that, described specificity junction mixture is the antibody of anti-described tumor markers.
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| 王征: "急性髓细胞白血病中鞘糖脂的表达及其功能研究", 《CNKI中国优秀硕士学位论文全文数据库》 * |
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