CN109022362A - A kind of method for building up of hyperleucocyte acute leukemia PDX model - Google Patents
A kind of method for building up of hyperleucocyte acute leukemia PDX model Download PDFInfo
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Abstract
The invention belongs to animal model technical fields, and in particular to a kind of method for building up of hyperleucocyte acute leukemia PDX model specifically includes: screening suitable hyperleucocyte acute leukemia clinical patients；Using leucocyte in COM.TEC blood cell separator separation peripheral blood；It isolates and purifies the mononuclearcell of leucocyte in peripheral blood collected and carries out cell culture, obtain leukaemia cell's suspension；Prepare hyperleucocyte acute leukemia PDX model: first by mouse χ-ray radiation, then leukaemia cell's suspension is injected to mouse tail vein, it is detected after a week by randomly selecting mousetail blood, whether judgment models are successfully established according to testing result.The characteristics of modeling success rate of hyperleucocyte acute leukemia PDX model of the present invention is high, and the disease performance of constructed mouse PDX model and cellular morphology can directly reflect hyperleucocyte acute leukemia cell.
Description
Technical field
The invention belongs to animal model technical fields, and in particular to a kind of foundation of hyperleucocyte acute leukemia PDX model
Method.
Background technique
Leukaemia is the most common malignant tumour of hematological system, is the clonal expansion disease of a kind of candidate stem cell exception
Disease, poor prognosis.Hyperleucocyte acute leukemia (Hyperleukocytic Leukemia, HLL) refers to peripheral white blood cells exception
Increase (>=100 × 109/ L) high-risk types of leukemia encountered.Leukocyte disorder, which increases, generates Leukostasis and vascular wall infiltration,
It is particularly easy to that Respiratory Distress Syndrome(RDS) occurs and intracranials hemorrhage, and chemotherapy remission rate is low, and is easy to appear tumor lysis syndrome.
However, being still within the starting stage for the correlative study of hyperleucocyte acute leukemia at present, we have found in clinical position,
Certain conditions of patients grade malignancies with hyperleucocyte acute leukemia are high, and easy to recur, chemicotherapy effect is poor, these preciousnesses are faced
Bed case can provide fabulous basis for the scientific research of leukaemia.Mouse is the ideal animals model for studying leukaemia, closely
Nian Lai has been reported that human leukemia cell being implanted into Mice Body building PDX mouse model, but since cell number is few, cell
The disadvantages such as purity is low, it is very low at mould rate.In addition, if long-term cultivation expand leukaemia cell, be easy to cause cytogene and
Immunophenotype variation.Therefore, it there is no the report for establishing hyperleucocyte acute leukemia PDX model both at home and abroad at present.
Summary of the invention
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of hyperleucocyte acute leukemia PDX model is built
Cube method.
For achieving the above object, the technical solution adopted by the present invention are as follows:
A kind of method for building up of hyperleucocyte acute leukemia PDX model, includes the following steps:
(1) suitable hyperleucocyte acute leukemia clinical patients are selected；
(2) acquisition step (1) described hyperleucocyte acute leukemia clinical patients are separated using COM.TEC blood cell separator
Peripheral blood in leucocyte；
(3) mononuclearcell of leucocyte in peripheral blood collected is separated, further magnetic bead sorting purification of individual core is thin
Born of the same parents carry out cell culture, obtain leukaemia cell's suspension；
(4) hyperleucocyte acute leukemia PDX model is prepared: first by mouse χ -- ray radiation, it then will step
Suddenly (3) culture gained leukaemia cell's suspension be injected to mouse tail vein, after a week by randomly select mousetail blood into
Whether row detection, be successfully established according to inspection result judgment models.
In above scheme, the standard of step (1) the hyperleucocyte acute leukemia clinical patients are as follows: a) age 18-60 years old,
Leukaemic；B) ECOG KPS scale is 0-2 points；C) hepatic and renal function is normal；D) leucocyte WBC in peripheral blood >=
100×109cells/ml。
In above scheme, the major parameter setting of step (2) the blood cell separator separation acquisition are as follows:
A) it total white blood cells (WBC): sets to maximum value as 120 × 10E3/UL；
B) every circulating blood volume (V (Cycle)): first circulation is set as 180ml；Second circulation starts: first according to white thin
Born of the same parents' sum sets WBC > 300,000,80-100ml according to following empirical value；Then tunica albuginea is observed in tunica albuginea collection phase, if at this time
There is remaining tunica albuginea in disengagement chamber, then lower " V ", lower 10ml every time, until not observing tunica albuginea；It is on the contrary then raise " V ", every time
10ml is raised, but is no more than upper limit 180ml；
C) tunica albuginea pump output (V (Sopillover)): 25ml；
D) tunica albuginea collecting amount (V (Buffy Coat)): being first set to 10ml, subtracts 1-2ml if collection tail portion is thin out, or
Collection is manually controlled by point by " stop phase "；
E) cycle-index (No.Cycle): processing botal blood volume is at least made to reach 2 times of TBV；
F) whole blood flow velocity (Blood Flow): when Hct > 30%, 50-60ml/min is no more than 70ml/min；20% < Hct
When < 30%, 40ml/min is not exceeded；When Hct < 20%, 30ml/min is not exceeded；
G) anti-coagulants and blood (ACD:Blood) volume ratio are 1:9；
H) centrifuge speed (RPM): default value 1500 increases by 200 on the basis of machine default value；If necessary to reduce
Platelet loss then uses default value.
In above scheme, anti-coagulants ACD.
In above scheme, the method for step (3) the separation mononuclearcell are as follows: will be white in peripheral blood obtained by step (2)
Cell blood sample is added on the liquid level of separating liquid, and after centrifuge separation, blood sample is from top to bottom divided into four layers: first layer is plasma layer,
The second layer is cyclic annular milky mononuclearcell layer, and third layer is transparent separation liquid layer, and the 4th layer is red blood cell layer, draws second
The cyclic annular milky mononuclearcell layer of layer, as isolated mononuclearcell.
In above scheme, the separating liquid is people's whole blood mononuclearcell separating liquid.
In above scheme, the culture medium of mononuclearcell is cultivated described in step (3) are as follows: fetal calf serum containing 10wt%
1640 cultures of (Gibco, lifetechnologiesTM, Australia), 100U/ml penicillin and 100 μ g/ml streptomysins
Base.
In above scheme, the condition of culture of mononuclearcell is cultivated described in step (3) are as follows: be seeded to mononuclearcell
On culture medium, it is placed in 37 DEG C, 5%CO2Incubator in cultivate 24-48h.
In above scheme, mouse described in step (4) is the B-NSG female mice of 4-5 week old；χ-ray the radiation
Dosage be 1.5-3.5Gy；Leukaemia cell's suspension is injected after irradiation in 6-12h to mouse tail vein；It is described be injected to it is small
Contain 1 × 10 in leukaemia cell's suspension of caudal vein7-2×107A human leukaemia cell.
In above scheme, the project that extraction mousetail blood described in step (4) is detected includes: 1. blood routine inspection
Survey peripheral blood blood change；2. blood film and bone marrow smear detect initial cell ratio；3. immune according to Flow cytometry
Parting and tissue infiltration's characteristic of histopathology detection leukaemia cell.
In above scheme, the testing result is shown: 1) finding leukaemia cell in peripheral blood and bone marrow smear；2) streaming
Cell instrument detected in the peripheral blood, spleen and marrow blood of mouse with Patient leukemic's immunophenotype population of stem cells (with
For AML, Testing index CD34+, CD33+, CD117+)；3) Histopathological Studies extensive people into mouse tissue organ
The infiltration of quasi-leukemia cell then judges to model successfully.
Beneficial effects of the present invention are as follows: the present invention passes through blood cell collection machine for the leukaemic with high leukocytic
Again by external purifying and culture after leukaemia cell's acquisition of periphery, it is thin to obtain the leukaemia that purity is higher, number is more
Born of the same parents, and it is implanted into building hyperleucocyte acute leukemia PDX model in Mice Body；Hyperleucocyte acute leukemia of the present invention
For the mouse model that the method for building up of PDX model is transplanted compared with conventional cell culture, a large amount of leukaemia cells are capable of providing, are reduced
Growth cycle, reduces the interference of external condition, and the success rate of modeling is high；Experimental data shows mouse PDX constructed by the present invention
The characteristics of disease performance of model and cellular morphology can directly reflect hyperleucocyte acute leukemia cell, the model are immunized
Phenotype and hereditary feature etc. can reflect the correlated characteristic of hyperleucocyte acute leukemia cell, the white blood of high leukocytic of the present invention
The method for building up of sick PDX model provides important basis for the scientific research of hyperleucocyte acute leukemia.
Detailed description of the invention
Fig. 1 is hyperleucocyte acute leukemia PDX method for establishing model schematic diagram.
Fig. 2 is the result judgment criteria that hyperleucocyte acute leukemia PDX model is successfully established, wherein Fig. 2A is respectively to suffer from
Person's bone marrow smear, mouse blood film (8 days), mouse bone marrow cells smear (20 days/29 days)；Fig. 2 B Flow cytometry mouse periphery
Blood system type (by taking AML as an example, Testing index CD45+, CD34+, CD33+, CD117+)；Fig. 2 C histopathology mouse
Liver,spleen,kidney is dirty leukemiacell infiltration.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
Embodiment 1
A kind of method for building up of hyperleucocyte acute leukemia PDX model includes six steps: (1) selection of clinical patients；
(2) leucocyte in COM.TEC blood cell separator separation peripheral blood；(3) mononuclearcell isolates and purifies in leucocyte；(4)
The culture of mononuclearcell；(5) preparation of hyperleucocyte acute leukemia PDX model；(6) detection and identification of mouse PDX model.
Specific operating process is as follows:
(1) selection of clinical patients
The inclusion criteria of 1.1 clinical patients:
A. age 18-60 years old；
B. the cell smear and cytochemistry inspection of peripheral blood and marrow meet the diagnosis of the leukaemia of FBA and WHO publication
Standard；And there are the testing results such as flow cytomery, chromosome detection, genetic test to prompt for leukaemic；
C.ECOG KPS scale is 0-2 points；
D. hepatic and renal function is normal: hemobilirubin≤34.2 μm ol/L, AST/ALT are at 2 times of Upper Limit of Normal Value hereinafter, blood
Creatinine≤222 μm ol/L；Heart function is normal: EF > 50%；Lung function: indoor oxygen saturation >=95%；
E. high leukaemia mass formed by blood stasis: leucocyte WBC >=100 × 10 in peripheral blood9cells/ml；
F. the informed consent form of patient or family members' signature are obtained.
The exclusion criteria of 1.2 clinical patients:
A. there are previously or simultaneously other malignant tumours；
B. other clinical tests are being participated in；
C. heart function, lung function and hepatic and renal function are obviously abnormal, exceed inclusion criteria；
D. there are the clinical symptoms or serious psychotic disorder of functional disorders of brain.
1.3 couples of subjects that can be included in research establish archives, carry out doctor patient communication, according to Declaration of Helsinki human medical
The codes of ethics of research sign informed consent form.
(2) using leucocyte in COM.TEC blood cell separator separation peripheral blood:
If preoperative Hct < 20% of 2.1 patients, answers transfusions of red cells up to 20% or more；
2.2 Procedure for selection: autoMNC (PIYA)；
2.3, which are initially separated preceding sample sack subsidiary from blood sampling pipe, leaves and takes blood sample inspection blood routine；
2.4 major parameter setting methods
A. it total white blood cells (WBC): sets to maximum value (120 × 10E3/UL)
B. every circulating blood volume (V (Cycle)):
First circulation: it is set as 180ml；
Second circulation starts: first being set according to total white blood cells according to following empirical value；
WBC > 300,000,80-100ml；
Then tunica albuginea is observed in tunica albuginea collection phase, if there is remaining tunica albuginea in disengagement chamber at this time, lowers " V ", every time
10ml is lowered, until not observing tunica albuginea；It is on the contrary then raise " V ", 10ml is raised every time, but is no more than upper limit 180ml；
C. tunica albuginea pump output (V (Sopillover)): 25ml
D. tunica albuginea collecting amount (V (Buffy Coat)): being first set to 10ml, if collect tail portion it is thin out if subtract 1-2ml, or
Collection is manually controlled by point by " stop phase "；
E. cycle-index (No.Cycle): at least make to handle botal blood volume and reach 2 times of TBV；
F. whole blood flow velocity (Blood Flow): when Hct > 30%, 50-60ml/min is no more than 70ml/min；20% < Hct
When < 30%, 40ml/min is not exceeded；When Hct < 20%, 30ml/min is not exceeded；
G. anti-coagulants and blood (ACD:Blood) volume ratio are 1:9；
H. centrifuge speed (RPM): machine default value is 1500, increases by 200 on the basis of machine default value；If necessary
Platelet loss is reduced, then uses default value；
It after 2.5 separation, please checks at once: a. blood routine at once；B. the white blood cell count(WBC) in finished product (notes: needing
Inspection or especially informing laboratory again after 20-40 times of dilution)
(3) human peripheral blood single nucleus cell separates: the people's whole blood list provided according to the Tianjin ocean Hao biological products scientific & technical corporation
The mononuclearcell of gained sample, tool in a nucleus separating liquid specification (TBD, lot:LDS1075) separation above-mentioned steps two
Body method is as follows:
A. a 50ml centrifuge tube is taken, people's whole blood mononuclearcell separating liquid with blood sample equivalent is first added；
B. the careful draw blood sample of suction pipe is added on the liquid level of separating liquid, 650-1100g, centrifugation 20-30min (note:
Centrifugal condition is determined according to blood sample amount, blood sample amount is more, and centrifugal force is bigger, and centrifugation time is longer)；
C. after being centrifuged, from top to bottom it is divided into four layers in centrifuge tube at this time, first layer is plasma layer, and the second layer is cyclic annular milky white
Color mononuclearcell layer, third layer are transparent separation liquid layer, and the 4th layer is red blood cell layer；
D. second layer ring-type milky mononuclearcell layer is carefully drawn with suction pipe into another 15ml centrifuge tube, to gained
10ml cleaning solution is added in centrifuge tube, mixes cell；
E. parameter of noncentricity is arranged, and: 250g is centrifuged 10min, abandons supernatant；Gained cell is resuspended with 5ml cleaning solution with suction pipe, if
Setting parameter of noncentricity: 250g is centrifuged 10min, abandons supernatant；
F. step E is repeated, abandons after supernatant and cell is resuspended with respective liquid needed for 0.5ml subsequent experimental.
(4) above-mentioned cell the cell sorting of mononuclearcell and culture: is obtained into CD34+ table by the method for magnetic bead sorting
The leukaemia cell reached, by the cell culture after isolating and purifying in 37 DEG C when, 5%CO2Incubator in, culture medium be containing 10%
Fetal calf serum (Gibco, lifetechnologiesTM, Australia), 100U/m penicillin and 100 μ g/m streptomysins 1640
Culture medium；Leukaemia cell's suspension is obtained after 24~48h of culture, contains 1 × 10 in leukaemia cell's suspension7A human
Leukaemia cell.
(5) the PDX model of leukaemia B-NSG mouse is prepared:
B-NSG female mice 20 for buying 5 week old, after mouse is raised 5 days, with the χ-ray radiation of 2.5Gy dosage,
The 1 × 10 of the above-mentioned culture of 6h tail vein injection after irradiation7A Human leukaemia cell, cell first uses 0.2mm before injecting cell
Cell filter filtering, 200 μ l of injection are resuspended in the cell suspension of PBS, observation mouse mental condition, appetite, weight etc..
(6) detection and identification after model foundation: after about 1 week, randomly selecting 2 mousetail blood and detected, institute
Stating detection includes: blood routine detection peripheral blood blood change, and blood film and bone marrow smear detect initial cell, flow cytometry and
Histopathology detection, according to testing result parting etc., testing result is shown: 1) finding that leukaemia is thin in peripheral blood and bone marrow smear
Born of the same parents；2) flow cytometer detects dry with Patient leukemic's immunophenotype in the peripheral blood, spleen and marrow blood of mouse
Cell mass (by taking AML as an example, Testing index CD34+, CD33+, CD117+)；3) Histopathological Studies are into mouse tissue organ
The infiltration of extensive human leukaemia cell, then judge to model successfully.Specifically, hyperleucocyte acute leukemia PDX model success
The result judgment criteria of foundation is as shown in Figure 2, wherein Fig. 2A is respectively Bone Marrow of Patients smear, mouse blood film (8 days), mouse
Bone marrow smear (20 days/29 days)；Fig. 2 B Flow cytometry mouse peripheral blood parting (by taking AML as an example, Testing index CD45
+,CD34+,CD33+,CD117+)；Fig. 2 C histopathology Mouse Liver, spleen, kidney have leukemiacell infiltration.
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection scope of the invention.
Claims (10)
1. a kind of method for building up of hyperleucocyte acute leukemia PDX model, which comprises the steps of:
(1) suitable hyperleucocyte acute leukemia clinical patients are screened；
(2) using the outer of COM.TEC blood cell separator separation acquisition step (1) described hyperleucocyte acute leukemia clinical patients
Leucocyte in all blood；
(3) mononuclearcell of leucocyte in peripheral blood collected is separated, and the mononuclearcell after magnetic bead sorting is carried out
Cell culture obtains leukaemia cell's suspension；
(4) hyperleucocyte acute leukemia PDX model is prepared: first by mouse χ-ray radiation, then by step (3)
Culture gained leukaemia cell's suspension is injected to mouse tail vein, is examined after a week by randomly selecting mousetail blood
It surveys, whether judgment models are successfully established according to testing result.
2. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that step (1)
The screening criteria of the hyperleucocyte acute leukemia clinical patients are as follows: a) age 18-60 years old, leukaemic；B) ECOG behavior
Condition grading is 0-2 points；C) hepatic and renal function is normal；D) leucocyte WBC >=100 × 10 in peripheral blood9cells/ml。
3. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that step (2)
The major parameter setting of leucocyte in the blood cell separator separation acquisition peripheral blood are as follows:
A) total white blood cells are set to maximum value as 120 × 10E3/UL；
B) every circulating blood volume: first circulation is set as 180ml；Second circulation starts, first according to total white blood cells according to following
Empirical value setting: then total white blood cells > 300,000,80-100ml observes tunica albuginea in tunica albuginea collection phase, if disengagement chamber at this time
Inside there is remaining tunica albuginea, then lower " V ", lower 10ml every time, until not observing tunica albuginea；It is on the contrary then raise " V ", it raises every time
10ml, but it is no more than upper limit 180ml；
C) tunica albuginea pump output: 25ml；
D) tunica albuginea collecting amount: being first set to 10ml, subtracts 1-2ml if collection tail portion is thin out, or manual by " stop phase "
Control is collected by point；
E) cycle-index: processing botal blood volume is at least made to reach 2 times of TBV；
F) whole blood flow velocity: when Hct > 30%, 50-60ml/min is no more than 70ml/min；When 20% < Hct < 30%, do not exceed
40ml/min；When Hct < 20%, 30ml/min is not exceeded；
G) volume ratio of anti-coagulants and blood is 1:9；
H) centrifuge speed: default value 1500 increases by 200 on the basis of machine default value；If necessary to reduce blood platelet damage
It loses, then uses default value.
4. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that step (3)
The method of the separation mononuclearcell are as follows: leucocyte blood sample obtained by step (2) is added on the liquid level of separating liquid, centrifugation point
From rear, blood sample is from top to bottom divided into four layers: first layer is plasma layer, the second layer is cyclic annular milky mononuclearcell layer, third
Layer be transparent separation liquid layer, the 4th layer be red blood cell layer, draw second layer ring-type milky mononuclearcell layer, cleaning solution progress
After cleaning, carries out magnetic bead sorting and obtain the mononuclearcell of CD34+.
5. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 4, which is characterized in that described point
Chaotropic is people's whole blood mononuclearcell separating liquid.
6. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that step (3)
Described in cultivate mononuclearcell culture medium are as follows: fetal calf serum containing 10wt%, 100U/ml penicillin and 100 μ g/ml streptomysins
1640 culture mediums.
7. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that step (3)
Described in cultivate mononuclearcell condition of culture are as follows: mononuclearcell is seeded on culture medium, 37 DEG C, 5%CO are placed in2's
24~48h is cultivated in incubator.
8. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that step (4)
Described in mouse be 4~5 week old B-NSG female mice；The dosage of the χ-ray radiation is 1.5~3.5Gy, 6 after irradiation
Leukaemia cell's suspension is injected in~12h to mouse tail vein；In the leukaemia cell's suspension for being injected to mouse tail vein
Contain 1 × 107~2 × 107A Human leukaemia cell.
9. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that step (4)
Described in the project that is detected of extraction mousetail blood include: 1. blood routine detection peripheral blood blood change；2. blood film
Initial cell ratio is detected with bone marrow smear；3. thin according to Flow cytometry immunophenotyping and histopathology detection leukaemia
Tissue infiltration's characteristic of born of the same parents.
10. the method for building up of hyperleucocyte acute leukemia PDX model according to claim 1, which is characterized in that the inspection
It surveys as the result is shown: 1) finding leukaemia cell in peripheral blood and bone marrow smear；2) peripheral blood, spleen of the flow cytometer in mouse
With the population of stem cells with Patient leukemic's immunophenotype is detected in marrow blood；3) Histopathological Studies are to mouse tissue organ
In extensive human leukaemia cell infiltration, then model success.
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