CN104515840A - Method for evaluating therapeutic effect of anti-leukemia drug - Google Patents
Method for evaluating therapeutic effect of anti-leukemia drug Download PDFInfo
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- CN104515840A CN104515840A CN201410772022.XA CN201410772022A CN104515840A CN 104515840 A CN104515840 A CN 104515840A CN 201410772022 A CN201410772022 A CN 201410772022A CN 104515840 A CN104515840 A CN 104515840A
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Abstract
The invention provides a method for evaluating therapeutic effect of anti-leukemia drugs. The method comprises the steps of: a, collecting and separating a blood sample to obtain blood corpuscle corresponding to the types of leukemia; b, detecting surface roughness, height and hardness of the cells obtained in the step a to obtain evaluation index values; and c, comparing the evaluation index values before and after drug therapy to obtain the results of drug therapeutic effect evaluation. The method is simple, more intuitive and accurate.
Description
Technical field
The present invention relates to a kind of method evaluating anti-leukemia medicine curative effect, particularly a kind of diseased cells microscopic feature by observing, detecting the forward and backward leukaemic for the treatment of evaluates the method for result for the treatment of.
Background technology
Leukaemia is the malignant tumour of hemopoietic system, also known as " leukemia ", is one of modal malignant tumour.Along with improving constantly of medical level, by rational comprehensive therapy, leukemic prognosis is taken on a new look greatly, and the state of an illness of a lot of patient is controlled, and even cures.Leukaemia takes leave of the epoch of " incurable disease " gradually.
The clinical treatment to leukaemia (as acute leukemia, chronic lymphocytic leukemia etc.) at present adopts anti-leukemia medicine to carry out chemotherapy more, therapeutic evaluation after its chemotherapy is for clinical rational drug use, and the generation reducing bad reaction and drug resistance is significant.But existing Testing index and the many complexity of evaluation method and poor accuracy, may may there is larger difference with truth to the reflection of result for the treatment of, thus affect the setting of therapeutic scheme, intensity, cause treatment not reach optimum efficiency.
Clinically to the therapeutic evaluation of leukaemia medication, need to 1. clinical manifestation: as with or without anaemia, hemorrhage, infect and leukemiacell infiltration, 2. blood picture: haemoglobin, total white blood cells, classification is without juvenile cell, platelet count, 3. bone marrow smear etc. are comprehensively analyzed, and can understand change of illness state trend.This assessment method not only complex operation, labor intensive, material resources, financial resources, the more important thing is that the intuitive of its reflection result for the treatment of and accuracy neither be very desirable.In addition, this assessment method is more that the analysis of relative presentation is inferred, cannot be directly acquainted with the situation of change of source tumour cell.Easily because evaluation deviation causes that the specific aim for the treatment of is poor, even delay treatment best period, thus cause serious consequence.
Therefore, be badly in need of a kind of simple to operate, result anti-leukemia medicine method for estimating curative effect accurately, to determine the medication therapeutic scheme of leukaemic in time, accurately, improve leukemic result for the treatment of further.
Summary of the invention
For in prior art to anti-leukemia medicine therapeutic evaluation aspect exist deficiency, inventor is through a large amount of experimental studies, find to there is close association between the microscopic feature of leukaemia and patient's condition, thus establish the method evaluating anti-leukemia medicine effect with the variation tendency of cell surface microscopic feature and mechanical property, the method is easy, intuitively, accurately and reliably, doctor can be helped to understand patient treatment situation in time, exactly, to determine therapeutic regimen, reach patient's restorative object early.
The invention provides a kind of method evaluating anti-leukemia medicine curative effect, steps of the method are:
A. blood sample is separated through gathering, and obtains the haemocyte corresponding with corresponding type of leukemia;
B. the roughness of step a gained cell, height and hardness are detected, obtain evaluation index value;
C. the evaluation index value before and after drug therapy is compared, draw the evaluation result of curative effect of medication.
When the patient of anti-leukemia medicine treatment is myelocytic leukemia, above-mentioned steps a obtains bone marrow cell for gathering marrow and being separated.
The height of above-mentioned cell is the long axis length of phalangeal cell, and specifically, when cell is circular (spherical), being highly its diameter, when cell is oval (olive shape), is highly the length of its major axis.
Find in inventor's experimentation, there is notable difference in the microscopic pattern of the diseased cells of leukaemic cell corresponding to normal person, the physical features such as structure and mechanical property in various degree, and the development of its microcosmic character and the state of an illness has correlativity, in regular change.As the shape of cell and the degree of roughness on surface thereof, increasing the weight of with the state of an illness, cell can gradually become olive shape from original spheroidal and surface roughness significantly increases; Otherwise through effectively treating, cell can recover spheroidal gradually, surface is smooth-out; For another example, the physical strength of cell, that is, the hardness of cell or elasticity, can increasing the weight of and reduce with the state of an illness.According to the corresponding relation of These parameters cell microscopic feature and leukaemia patient's condition, we have established evaluation method of the present invention.Adopt the method can recognize variation tendency and the degree of diseased cells treatment front and back intuitively, exactly, thus judge the curative effect of the patient's condition that patient is current and medicine used, treat rationally and effectively for next step and provide important guiding.Avoid the deviation because of therapeutic evaluation that therapeutic process is taken an unnecessary way, and extend the course of disease, even delay treatment causes the adverse consequences being difficult to retrieve.Leukaemia, be often divided into the types such as acute lymphoblastic leukemia, acute myelocytic cells leukaemia, chronic myelocytic leukemia, chronic lymphocytic leukemia clinically, dissimilar leukemic diseased cells is not quite similar.The present invention only needs to carry out extraction to diseased cells and detects, and just can reach and separate the object of medication effect, method is easy, visual result, accurately.In addition, the method testing cost is low, for sufferer reduces medical burden.
The detection of described step b, can select the analytical instrument carrying out accurately image to the ultrastructure of cell and mechanical property, preferably adopt atomic force microscope (AFM).
The evaluation result of described step c judges to draw by following standard:
Cure: after drug therapy, the roughness of cell, height and hardness number all return in normal range;
Effective (Be very effective); Before comparatively treating after drug therapy, the roughness of cell and the elevation amplitude of fall highly and cell hardness are not less than 30%;
Effectively (have certain effect): before comparatively treating after drug therapy, the roughness of cell and the elevation amplitude of fall highly and cell hardness are 15 ~ 30%;
Invalid: before comparatively treating after drug therapy, the roughness of cell and the elevation amplitude of fall highly and cell hardness are lower than 15%;
The roughness of the cell in normal range, height and hardness number are:
When cell is leukaemia diseased cells, the evaluation index value of described step b is:
These parameters value adopts a large amount of primary leukemia cell (namely, the diseased cells of leukaemic) obtain through experiment, at present, be all adopt non-primary cell to the research of leukaemia mostly, namely, through manually modified leukaemia, as K562 cell, Raji cell etc., experiment shows, there is notable difference in two kinds of iuntercellulars, Comparatively speaking, primary cell more can reflect the feature of diseased cells truly, exactly, more can describe the problem.
The extraction separation method of the neutrophil leucocyte in described step a, lymphocyte and bone marrow cell, the detection method of described step b, all can adopt and well known to a person skilled in the art that conventional method realizes.
Inventor illustrates evaluation method of the present invention further by specific experiment.
Reiterate: following experiment is the illustrative experiment in development process of the present invention in numerous test, do not contained with limit all experiments that invention people does for the present invention, object is only to set forth the intuitive of evaluation method of the present invention and accuracy etc. by those data.
One, chronic myelocytic leukemia primary cell, acute myelocytic leukemia primary cell, B-lineage Acute Lymphocyte Leukemia primary cell, the surface microscopic feature difference of the neutrophil leucocyte of the normal human corresponding with it, bone marrow cell, bone-marrow-derived lymphocyte:
1, experimental technique:
(1) extraction of ill primary cell and cultivation
Get the blood of chronic myelocytic leukemia patient, through Ficoll (1.077g/cm
3) density gradient centrifugation, collect MNC layer, wash 2 times with RPMI1640 medium centrifugal, obtain chronic myelocytic leukemia primary cell.
Get the marrow of acute myelocytic leukemia patient, be separated through lymphocyte separation medium process and obtain acute myelocytic leukemia primary cell.
Get the blood of B-lineage Acute Lymphocyte Leukemia patient, be separated through lymphocyte separation medium process and obtain bone-marrow-derived lymphocyte Leukemic cells.
Above-mentioned three kinds of cell chulture, in containing in the RPMI1640 nutrient solution of 10% serum, are blown and beaten after cell is suspended for detecting.
(2) Normocellular extraction and isolation
The extraction of neutrophil leucocyte
The extraction of human peripheral liquid neutrophil leucocyte: get 50ml polyethylene pipe, aseptic collection human peripheric venous blood, the citric acid saline solution adding 4.4ml3.8% or 5% is settled to 40ml, above-mentioned whole blood 300g × 20min, centrifugal, room temperature; Hematoblastic supernatant is rich in sucking-off, 2500g × 15min, prepares PFP (platelet-poor Plasma, PPP); Remaining sedimentation whole blood adds 5ml 6%Dextran (dextran) (molecular weight 500,000, prepare by stroke-physiological saline solution), and regulate final volume to 50ml with physiological saline (0.9%), mix gently, thoroughly, room temperature sedimentation 30min; Liquid after sedimentation, leukocytic upper liquid is rich in sucking-off, 275g × 6min; The cell 8mlPPP-physiological saline (1:4) of precipitation is resuspended, and moves on in 15ml centrifuge tube; Add above suspension cell 3mlFicoll-Hypaque (ficoll-thypaque sodium) (density 1.077), 750g × 5min, room temperature; Neutrophil leucocyte and red blood cell layer are rich in absorption, with 0.155M NH4Cl re-suspended cell, make erythrocyte splitting, then neutrophil leucocyte is with containing 0.25%BSA Hanks equilibrium liquid (HBSA, without calcium) wash 2 times, finally use HBSA (calcic) resuspended.Get and carry out cell count and vitality test a little.
The extraction and isolation of bone-marrow-derived lymphocyte
Aseptic aspiration venous blood 2ml, removes the sterile test tube of syringe needle injection containing heparin and shakes up, add 2ml Hanks solution and carry out two-fold dilution, mixing; In being slowly added to containing 2ml lymphocyte separation medium test tube by suction pipe draw blood along test tube wall (blood: Hanks liquid: the ratio of hedge cell separation liquid is 1:1:1), the dilute blood flowed down along tube wall is superimposed upon on parting liquid, and forms obvious interface with parting liquid; Through the centrifugal 2000r/min of horizontal, carefully take out test tube; The middle level buffy coat vaporific in white is carefully drawn with flat mouth suction pipe, with Hanks solution washing twice, each centrifugal 1500r/min × 10min; Abandon and reset and add RPMI-1640 nutrient solution 1ml and mix, get and carry out cell count and vitality test a little.
The extraction and isolation of bone marrow cell
Illustrate by kit and carry out (Lang Dun bio tech ltd, Shanghai).
(3) atomic force microscope detects cell-surface engineering feature
Centrifugal for cell suspension rear PBS is diluted to concentration about 1 × 10
4the cell liquid of/ml, drops in above-mentioned cell liquid and is coated with on the microslide of poly-D-lysine in advance, and be coated with uniform residence 1-2min, suck liquid unnecessary on slide, drips 1-2 and drip paraformaldehyde and be fixed on slide.After this sample standing and drying 60min, with super-ionic water cleaning 2-3 time, nitrogen dries up, and is placed in by sample on AFM objective table and carries out cell-surface engineering scanning.
2, experimental result:
Pass through atomic force microscope, clearly can observe the generation significant change compared with normal cell of diseased cells surface microscopic feature, its surfaceness is apparently higher than its corresponding normal cell, simultaneously, the height of cell and diameter (length of cell minor axis) all have increase, and especially the height of cell obviously increases, because the ratio of cell height and diameter obviously increases, thus the shape of cell is changed to olive shape by spheroidal, and cell microscopic feature quantitative analysis results can see table 1.This laboratory test results comes from 200 cell samples of 20 people (often kind of patient and each 20 people of normal person), has statistical significance.
Table 1 three kinds of diseased cells and corresponding Normocellular microscopic feature quantitative statistics table thereof
(Ra: average roughness; Rq: peak value roughness; Hight: cell height; Size: cell dia)
Two, chronic myelocytic leukemia primary cell, acute myelocytic leukemia primary cell, B-lineage Acute Lymphocyte Leukemia primary cell, the mechanical property difference of the neutrophil leucocyte of the normal human corresponding with it, bone marrow cell, bone-marrow-derived lymphocyte:
1, experimental technique
Several cells that above-mentioned experiment one is adopted are after centrifugal treating, with Hank ' s liquid, cell is resuspended, and getting 1-2ml concentration is 1 × 10
6the cell liquid of/ml, be added dropwise in the Tissue Culture Dish in advance with the 70mm of poly-D-lysine process, after placing 1min, then add 8-10mlHank ' s solution wherein, this sample being placed on AFM objective table and obtaining cell force curve, showing hardness by calculating Young modulus reflection cell.
2, experimental result
Between normal cell, Young modulus is very close, and diseased cells its Young modulus compared with normal cell obviously increases, and is Normocellular more than 4 times, shows that ill its skin hardness rear of cell obviously declines.Refer to table 2.
Table 2 different cell surface hardness (Young modulus) quantized result
Three, differentiating inducer all-trans retinoic acid is to the inhibited proliferation of chronic myelocytic leukemia primary cell:
1, experimental technique
(1) medicine ordinance and give with
Take all-trans retinoic acid, it fully dissolved with DMSO, final concentration is 100mM, and room temperature keeps in Dark Place.Before administration, be diluted to desired concn with nutrient solution.
(2) mtt assay investigates medicine to the inhibited proliferation of cell
Chronic myelocytic leukemia primary cell is with 3 × 10
4the density of/ml is inoculated in 96 orifice plates, and every hole adds 100 μ l cell suspensions.Cell disposes living cell counting after 96h and 120h through variable concentrations all-trans retinoic acid (ATRA), the inhibiting rate of cell growth after calculating variable concentrations all-trans retinoic acid effect 96h and 120h, and adopt SPSS data software calculation of half inhibitory concentration (IC
50).Each experiment repetition 3 times.
2, experimental result
Result shows, ATRA can be the propagation (see accompanying drawing 1) of the suppression chronic myelocytic leukemia primary cell of concentration and time dependence.96h and 120h, ATRA are to the IC of chronic myelocytic leukemia primary cell in effect
50be respectively and 13.5 and 5.7 μMs.
Accompanying drawing illustrates:
Fig. 1 is the Proliferation Ability curve map of all-trans retinoic acid to chronic myelocytic leukemia primary cell;
Horizontal ordinate is all-trans retinoic acid concentration, and ordinate is inhibiting rate.
Embodiment
Embodiment 1
Evaluate the method for all-trans retinoic acid to treatment for chronic myelocytic leukemia effect, steps of the method are:
Chronic myelocytic leukemia patient 10 example, by 80mg/m
2d oral dose ATRA, every day twice, takes 30 days continuously.Within before administration and after administration the 15th day, the 30th day, get blood respectively, through Ficoll (1.077g/cm
3) density gradient centrifugation, collect MNC layer, wash 2 times with RPMI1640 medium centrifugal, the ill neutrophil leucocyte before and after obtaining medical treatment; Each group of random selecting 10 cells, carry out observation by atomic force microscope to cell and detect, obtain cell surface microscopic appearance and skin hardness parameters.ATRA being treated forward and backward evaluation index compares known, and within oral ATRA15 days, effectively can treat chronic myelocytic leukemia, the result for the treatment of of oral ATRA30 days is more remarkable, refers to table 3.
Table 3:ATRA is to the therapeutic evaluation table of chronic myelocytic leukemia
More than be intended to further illustrate the present invention, scope of the present invention do not limited.Those skilled in the art can not depart from improvement and the change of scope and spirit to embodiment disclosed herein.
Claims (6)
1. evaluate a method for anti-leukemia medicine curative effect, it is characterized in that, steps of the method are:
Blood sample is separated through gathering, and obtains the haemocyte corresponding with corresponding type of leukemia;
The surfaceness of step a gained cell, height and hardness are detected, obtains evaluation index value;
Evaluation index value forward and backward for drug therapy is compared, draws the evaluation result of curative effect of medication.
2. the method for claim 1, is characterized in that, described step a obtains haemocyte and bone marrow cell for collection blood is separated with marrow.
3. the method for claim 1, is characterized in that, the detection of described step b, adopts atomic force microscope.
4. the method as described in any one of claim 1-3, is characterized in that, the evaluation result of described step c judges to draw by following standard:
Cure: after drug therapy, the roughness of cell, height and hardness number all return in normal range;
Effective; Before comparatively treating after drug therapy, the roughness of cell and the elevation amplitude of fall highly and cell hardness are not less than 30%;
Effective: before comparatively treating after drug therapy, the roughness of cell and the elevation amplitude of fall highly and cell hardness are 15 ~ 30%;
Invalid: before comparatively treating after drug therapy, the roughness of cell and the elevation amplitude of fall highly and cell hardness are lower than 15%.
5. method as claimed in claim 4, is characterized in that, the roughness of the cell in normal range, height and hardness number are:
Average roughness μm peak value roughness μm cell height μm skin hardness kPa
Neutrophil leucocyte 1.38-2.26 1.32-2.78 7.57-9.61 8.66-10.42
Bone-marrow-derived lymphocyte 0.91-2.31 0.85-2.57 8.16-12.54 8.38-9.92
Bone marrow cell 0.97-1.21 1.41-1.89 4.77-7.15 12.36-13.8.
6. the method for claim 1, is characterized in that, when cell is leukaemia diseased cells, the evaluation index value of described step b is:
Average roughness μm peak value roughness μm cell height μm skin hardness kPa
Neutrophil leucocyte >=3.04 >=4.62 >=15.14 < 3.32
Bone-marrow-derived lymphocyte >=3.35 >=4.18 >=19.82 < 3.17
Bone marrow cell >=2.07 >=2.67 >=11.0 < 3.27.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045715A (en) * | 2011-10-11 | 2013-04-17 | 上海交通大学医学院 | Medicament target for treating acute promyelocytic leukemia and inhibitor thereof |
| CN103237901A (en) * | 2010-03-01 | 2013-08-07 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarkers for theranostics |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103237901A (en) * | 2010-03-01 | 2013-08-07 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarkers for theranostics |
| CN103045715A (en) * | 2011-10-11 | 2013-04-17 | 上海交通大学医学院 | Medicament target for treating acute promyelocytic leukemia and inhibitor thereof |
Non-Patent Citations (2)
| Title |
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| 田国栋等: "全反式维甲酸治疗白血病74例临床研究", 《陕西医学杂志》 * |
| 黄训等: "原子力显微镜对中性粒细胞与K562细胞超微结构及机械性能的探测", 《分析测试学报》 * |
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