CN103740639A - Method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model - Google Patents
Method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model Download PDFInfo
- Publication number
- CN103740639A CN103740639A CN201310391969.1A CN201310391969A CN103740639A CN 103740639 A CN103740639 A CN 103740639A CN 201310391969 A CN201310391969 A CN 201310391969A CN 103740639 A CN103740639 A CN 103740639A
- Authority
- CN
- China
- Prior art keywords
- mouse
- cell
- nod
- mouse model
- scid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model, and the method comprises the following steps: acquisition of leukemia cells, pretreatment of NOD/SCID mouse and implantation of leukemia cells, and implantation of leukemia cells. Specifically, the method comprises: 60Co irradiation of sub-lethal dose combined with a pretreatment with rat anti-mouse CD122 monoclonal antibody, and injection of Ph+ALL bone marrow mononuclear cells from knee joint marrow cavity of NOD/SCID mouse, thereby a humanized Ph+ALL mouse model is established with high molding efficiency and good repeatability; cell sorting is not needed, and the molding process is easy to be operated; the method provides an experiment platform for carrying out mankind Ph+ALL research on the level of living animals, and will promote deep research of domestic Ph+ALL.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method that builds humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model.Foundation has the disease model of the clinical Ph chromatin-positive acute lymphoblastic leukemia feature of simulation, and the pathogenesis, diagnosis and treatment method and the new drug development that can be research Ph chromatin-positive acute lymphoblastic leukemia provide research platform in Mice Body.
Background technology
Ph karyomit(e), as the modal chromosome abnormalty of adult acute lymphoblastic leukemia (English name Acute lymphoblastic leukemia, abbreviation ALL), has become the sign of ALL prognosis mala.Along with the widespread use of tyrosine kinase inhibitor, (English name Philadelphia chromosome-positive acute lymphoblastic leukemia, Ph chromatin-positive acute lymphoblastic leukemia is abridged Ph in recent years
+aLL) curative effect has obtained obvious improvement, but recurrence remains Ph
+aLL treats failed major cause, has a strong impact on patient's long-term survival.Therefore, try to explore Ph
+the Mechanisms of recurrence of ALL and research and development newtype drug are to treatment Ph
+aLL is significant, also will make more Ph
+aLL patient is benefited.Accept the immunodeficient mouse of mankind's Transplanted cells as a humanization mouse important kind of (being called again mouse-people mosaic), owing to can overcoming social ethics that human trial causes and the restriction of technical factor, become the important tool of human leukemia pathogenesis and Mechanisms of recurrence research and new drug development and therapeutic evaluation.Therefore, setting up good xenotransplantation system efficiently responsive, that can support the long-term implantation of Humanized cell, is the basis and key of leukemia being carried out to functional study in body.
Since nineteen nineties, adult (8-12 age in week) non-obese diabetic-severe combined immunodeficiency (English name non-obese diabetic/severe combined immunodeficiency, abbreviation NOD/SCID) mouse model has been widely used in mankind's normal hematopoiesis stem cell (English name Hematopoietic stem cells, abbreviation HSCs) and the body of acute human myelocytic leukemia (English name Acute myeloid leukemia, abbreviation AML) in research.But because NOD/SCID mouse life is shorter, remaining active natural killer (English name Natural killer to a certain degree, abbreviation NK) cell, and there is experiment to confirm that NK cell remaining in Mice Body is high to xenogenesis leukemia cell's rate of rejection, cause xenogenesis leukemia cell lower at the implantation rate of NOD/SCID mouse model, the report that successfully utilizes this model to carry out functional study to acute human Lymphocytic leukemia (English name Acute lymphoblastic leukemia, abbreviation ALL) is very limited.
2004, doctor Shultz in U.S. Jackson laboratory waited and has taken the lead in setting up in the world advanced person's NOD/SCID/IL2R γ the most
nullimmunodeficient mouse model, this model is owing to there being IL-2R γ defect, suppressed with the signal transduction pathway that common cytokine receptor high-affinity is combined, and causes the multiple innate immune function defects such as NK cell.Successfully confirm, compared with adult NOD/SCID mouse, NOD/SCID/IL2r γ
nullmouse model can be supported the implantation of mankind's normal hematopoiesis system and AML and B-ALL cell more efficiently.
But, domestic NOD/SCID/IL2r γ
nullthe source of mouse only can rely at present from the U.S. or Japan and buy, and not only expense is high, and mouse purchase, declaration and Isolation Quarantine supervisor are loaded down with trivial details, and only can buy monosexual NOD/SCID/IL2r γ
nullmouse, does not allow to breed at home, has seriously restricted NOD/SCID/IL2r γ
nullstructure, the application of mouse leukemia model and carry out Ph by this model
+functional study in the body of ALL.And preparing humanization Ph
+during ALL mouse model, adopt ordinary method to carry out after pre-treatment NOD/SCID mouse, in Mice Body, the remaining NK of having cytoactive can cause xenogenesis leukemia cell implantation rate lower, after having on the other hand experiment to confirm to adopt conventional tail vein injection method to carry out leukemia cell's infusion, xenogenesis leukemia cell is being subject to the rate of going back to the nest of mouse marrow lower, thereby existing humanization Ph
+the modeling success ratio of ALL mouse model is low, and poor reproducibility has hindered domestic to Ph
+the research of pathogenesis, diagnosis and treatment method and the new drug development of ALL.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method that builds humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model, it adopts the method adaptive immune defect NOD/SCID mouse more completely of combined pretreatment, and by implantation means with leukemia cell go back to the nest the higher method of rate replaced tradition tail vein injection, constructed Ph
+aLL mouse model success ratio is high, favorable reproducibility, modeling process are simple, for carrying out mankind Ph in living animal
+aLL research provides experiment porch.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
Build a method for humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model, comprise the taking of leukemia cell, the pre-treatment of NOD/SCID mouse and leukemia cell's implantation, specifically comprise the following steps:
Steps A, Ph
+the preparation of ALL Bone Marrow of Patients mononuclearcell: get first visit Ph
+aLL Bone Marrow of Patients sample, anticoagulant heparin, application human lymphocyte parting liquid separates prepares BMNC, with the RPMI1640 nutrient solution containing 10% foetal calf serum (English name Fetal bovine Serum, abbreviation FBS)
resuspendedcell to the whole density of cell is 1 ~ 8 × 10
8/ mL, makes Ph
+the cell suspension of ALL BMNC;
The pre-treatment of step B, NOD/SCID mouse: get no-special pathogen (English name Specific Pathogen Free, abbreviation SPF) level NOD/SCID female mice, carry out sublethal dose
60co irradiation is only injected anti-mouse CD122 monoclonal antibody 150 ~ 250 μ g/ subsequently in mouse peritoneal;
The cell suspension of preparing in step C, implantation step A: after pre-treatment in 24h, the cell suspension that NOD/SCID female mice is prepared in knee joint medullary space injecting step A, every injected in mice amount is 0.1mL, modeling in 4 ~ 8 weeks success after implanting;
Preferably, also comprise that the leukemia mouse model to setting up in step C identifies in aforesaid method, described evaluation comprises:
(1) adopt people CD45 in polychrome Flow cytometry mouse bone marrow cells or spleen cell
+cD19
+leukemia cell's content and immunophenotype thereof;
(2) adopt people bcr/abl gene content in real-time quantitative polymerase chain reaction detection by quantitative mouse bone marrow cells and spleen;
(3) adopt people t (9 in fluorescence in situ hybridization detection mouse bone marrow cells and spleen; 22) (q34; Q11);
(4) adopt the dyeing of Hematorylin-Yihong and immunohistochemical staining to detect people Ph in mice organs
+the situation that ALL cell migration infiltrates.
In technique scheme, by application sublethal dose
60co irradiation is combined anti-mouse CD122 monoclonal antibody pre-treatment NOD/SCID mouse, not only can guarantee that NOD/SCID mouse has T cell and B cellular immunity deficiency, and can effectively remove NK cytoactive in Mice Body, thereby obtained immune deficiency immunodeficient mouse more completely, for improving heterocellular transplanting succeed rate, laid a good foundation.The present invention also adopts Ph
+aLL BMNC replaces traditional NOD/SCID mouse tail vein injection method through the injection of NOD/SCID mouse knee joint medullary space, can make xenogenesis leukemia cell avoid the loss of pulmonary circulation to infusion cell and directly go back to the nest in being subject to mouse marrow, thereby further improve xenogenesis leukemia cell's implantation rate.
The beneficial effect that adopts technique scheme to produce is: (1) mouse model of the present invention has the clinical Ph of simulation
+the disease feature of ALL; (2) build Ph
+aLL model efficiency is high, reproducible; (3) do not need cell sorting, modeling process is simple; (4) made up traditional through NOD/SCID mouse tail vein injection Method Modeling people Ph
+the defect that ALL implantation rate is low, and solved immune deficiency degree NOD/SCID/IL2r more completely
γnull mouse only can rely on the formality of foreign procurement loaded down with trivial details and expensive, thereby for to carry out mankind Ph in living animal level
+aLL research provides experiment porch, will promote domestic Ph
+the further investigation of ALL.
Accompanying drawing explanation
Figure 1A and 1B are respectively the internal anatomys of experimental mice (left side) and control group mice (right side) spleen;
Fig. 2 A and 2B are respectively people source leukemia cell's content and Immunophenotype analysis figure in experimental mice and control group mice marrow;
Fig. 3 is experimental mice and control group mice medullary cell BCR-ABL%;
Fig. 4 is that in marrow, brain, liver, spleen and the kidney of experimental mice and control group mice, people source leukemia cell moves the pathological biopsy HE colored graph of infiltration;
Fig. 5 is the anti human CD 19 immunohistochemical methods figure of people source leukemia cell in marrow, brain, liver, spleen and the kidney of experimental mice and control group mice;
Fig. 6 is the antihuman CD 34 immunohistochemical methods figure of people source leukemia cell in marrow, brain, liver, spleen and the kidney of experimental mice and control group mice;
Fig. 7 A and Fig. 7 B carry out people t (9 by fluorescence in-situ hybridization method to experimental mice and control group mice medullary cell; 22) (q34; Q11) detection figure.
Embodiment
The present invention tests NOD/SCID mouse used all purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., anti-mouse CD122 monoclonal antibody is purchased from purchased from U.S. Bioxcell company (BE0030), and in Flow cytometry, monoclonal antibody information as shown in Table 1.This experiment has repeated 10 patients, each Ph
+aLL patient B MMNCs transplants to 3 NOD/SCID mouse simultaneously, and totally 30 mouse, have all obtained similar results.
table 1 streaming monoclonal antibody information
The present embodiment, with preferred dose construction humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model, comprises the following steps:
Steps A, Ph
+the preparation of ALL Bone Marrow of Patients mononuclearcell: get first visit Ph
+aLL Bone Marrow of Patients sample, anticoagulant heparin, application human lymphocyte parting liquid separate prepare BMNC, and with the RPMI1640 nutrient solution containing 10% foetal calf serum
resuspendedcell to the whole density of cell is 5 × 10
8/ mL, makes Ph
+the cell suspension of ALL BMNC.Concrete steps are as follows:
1, in 15mL centrifuge tube, add 5mL lymphocyte separation medium;
2, taking heparin anti-freezing people marrow 10 mL and equivalent Hank's liquid fully mix, and are slowly added on laminated fluid level with dropper along tube wall, note keeping clearly interface, horizontal centrifugal 600g × 20 minute;
3, after centrifugal, see in pipe and be divided into three layers, upper strata is blood plasma and PBS damping fluid, and lower floor is mainly red corpuscle and granulocyte, and middle level is lymphocyte separation medium, has one take mononuclearcell as the main narrow band of tunica albuginea layer in upper, interface, middle level;
4, with dropper, be inserted into tunica albuginea layer, draw mononuclearcell, insert in another 15mL centrifuge tube, add the PBS damping fluid of 5 times of volumes, 300g × 5 are minute centrifugal, washed cell 2 times; After last is centrifugal, abandon supernatant, with containing 10%(weight ratio) the RPMI1640 nutrient solution re-suspended cell of FBS, adjust cell density to 5 × 10
8/ mL is standby.
The pre-treatment of step B, NOD/SCID mouse: get SPF level NOD/SCID female mice, carry out sublethal dose
60co irradiation, then only injects anti-mouse CD122 monoclonal antibody 200 μ g/ to mouse peritoneal.Concrete steps are as follows:
Test and within first 1 week, get SPF level NOD/SCID female mice in 4 week age, raise in SPF level laminar flow laboratory, feed and tap water are all through aseptically process.Mouse is implanted Ph
+aLL patient B MMNCs same day, the 0th day time above all represented with "-" as the 0th day.In-1 day, NOD/SCID mouse is carried out to pre-treatment, pretreating scheme is: 2.1cGy sublethal dose
60co irradiation, the anti-mouse CD122 of abdominal injection monoclonal antibody (U.S. Bioxcell company) 200 μ g/ are only subsequently.
The cell suspension of preparing in step C, implantation step A: after pre-treatment in 24h, the cell suspension that NOD/SCID female mice is prepared in knee joint medullary space injecting step A, every injected in mice dosage is 0.10mL, modeling in 4 ~ 8 weeks success after implanting.
Concrete, in this step for experimental result is compared, pretreated NOD/SCID mouse has been divided into two groups, one group is experimental group, the cell suspension of preparing in implantation step A, another group for control group, is raised according to identical method, and different is, and what to implant is 1 × PBS damping fluid.The Ph that the 0th day every experimental mice prepares in knee joint medullary space injecting step A
+aLL patient's BMMNCs 0.1mL, containing 5 × 10
7individual cell, every control group mice is through knee joint injection 1 × PBS damping fluid 0.1mL; At least two weeks continuously.Routine observation mouse spirit, hair, body weight, active situation etc. after transplanting.
Step D, the mouse model of setting up in step C is identified.Specifically comprise: (1) adopts people CD45 in polychrome Flow cytometry mouse bone marrow cells or spleen cell
+cD19
+leukemia cell's content and immunophenotype thereof;
(2) adopt people bcr/abl gene content in real-time quantitative polymerase chain reaction detection by quantitative mouse bone marrow cells and spleen;
(3) adopt fluorescence in situ hybridization detection to be subject to people t (9 in mouse marrow and spleen; 22) (q34; Q11);
(4) adopt the dyeing of Hematorylin-Yihong and immunohistochemical staining to detect people Ph in mice organs
+the situation that ALL cell migration infiltrates.
Concrete authentication method and step are referring to embodiment 4.
As different from Example 1, in steps A, the whole density of cell is 8 × 10
8/ mL;
Step B, to get pre-treatment the last week be the NOD/SCID female mice in 6 week age,
60co irradiation dose is 2.4 cGy, only injects anti-mouse CD122 monoclonal antibody 250 μ g/.
As different from Example 1, in steps A, the whole density of cell is 1 × 10
8/ mL;
Step B, to get pre-treatment the last week be the NOD/SCID female mice in 6 week age,
60co irradiation dose is 1.8 cGy, only injects anti-mouse CD122 monoclonal antibody 150 μ g/.
take the mouse model of embodiment 1 as example, authentication step and the method for mouse model is described below.
One, adopt people CD45 in polychrome Flow cytometry mouse bone marrow cells or spleen cell
+cD19
+leukemia cell's content and immunophenotype thereof
1.1, preparation pH 10 × PBS damping fluid that is 7.2 ~ 7.4, and dilute 10 times and become 1 × PBS damping fluid, compound method: take Na
2hPO
412H
2o 26.3g, NaH
2pO
42H
2o 3.0g and NaCl 85.0g are in volumetric flask, and adding distil water is to 1000mL: get that to pack test kit into after the buffer reagent encapsulation of supporting capacity stand-by.
1.2, get blood cytolysate (U.S. company BD), and with 10 times of 1 × PBS damping fluid dilutions,
1.3, get calf serum and be added in 1 × PBS damping fluid, preparation is containing the PBS solution of 0.5wt.% ~ 2wt.% serum.
The tight survival state that is subject to mouse of observing, in transplanting latter 8 weeks, is subject to mouse after ether inhalation anesthesia moribund condition, put to death by the de-cervical vertebra of mouse, aseptic separation bilateral femur and shin bone, go out medullary cell with RPMI1640 nutrient solution, then make single cell suspension by No. 4 syringe needles; 70 μ m nylon wires (U.S. Falcon company product) filter, and are prepared into bone marrow cell suspension.Under aseptic condition, get the spleen that is subject to mouse, (internal anatomy is referring to Figure 1A and 1B, compared with control group mice, the spleen of experimental mice obviously increases) be placed in the glass dish that contains in right amount the RPMI1640 nutrient solution of 2%FBS is housed, carefully grind with aseptic flour milling slide glass, collect splenocyte and cross 70 μ m sterile filters, be prepared into spleen cell suspension.By marrow and 0.4% trypan blue solution-dyed for spleen cell suspension, carry out mononuclearcell counting, wherein viable cell >=95%.
3.1 in same Special test tube, adds respectively successively fluorescently-labeled monoclonal antibody as follows: 3 μ L CD58FITC, 10 μ L CD10PE, 1 μ L CD34 PerCP, 2 μ LCD38APC, 5 μ L CD19APC-Cy7 and 1.3 μ L CD45Pacific Blue; Then (every pipe is containing being no less than 1 × 10 to add 500 μ L bone marrow cell suspensions or spleen cell suspension
6individual marrow or spleen cell), mix, room temperature lucifuge is placed and is hatched for 15 minutes,
3.2, add the blood cytolysate 2mL after dilution in step 1.2, mix rear lucifuge, room temperature is placed 8 minutes, lysed erythrocyte,
3.3,1500 revs/min centrifugal 5 minutes, abandon supernatant liquor, add the PBS solution containing 0.5wt.% ~ 2wt.% serum of 2mL in step 1.3, mix,
3.4,1500 revs/min centrifugal 5 minutes, abandon supernatant liquor, add 0.3mL1 × PBS damping fluid in step 1.1, mix, make test sample book;
two,adopt the dyeing of Hematorylin-Yihong and immunohistochemical staining to detect people Ph in mice organs
+the situation that ALL cell migration infiltrates
1, fixing: to get each organs and tissues and be cut into small pieces, in 10% neutral formalin solution, fix 24 hours; 1 × PBS damping fluid is washed 5 times, each 5 minutes;
2, dehydration: the tissue sample fixing is dewatered step by step, be followed successively by 70% ethanol 1 hour, 80% ethanol 1 hour, 95% ethanol I 1 hour, 95% ethanol II 2 hours, 95% ethanol III 2 hours, 100% ethanol I 1 hour and 100% ethanol 2 hours;
3, transparent: sample after dehydration is placed respectively 10 minutes and 20 minutes in dimethylbenzene I and dimethylbenzene II;
4, waxdip: the paraffin that the tissue after transparent is put into fusing soaks into, 3 times repeatedly, each 0.5-1 hour;
5, embedding: pour the new paraffin of fusing in embedding device, put into rapidly tissue block before it solidifies, will distinguish each face of tissue before putting into, by required section down; While being embedded with cavity tissue, tissue is kept flat or stood up;
6, section: cut the section that thickness is 4 μ m on slicing machine, launch 37 ℃ of water-baths, and drag on the special slide of immunohistochemical methods; 60 ℃ of oven for baking 4 hours;
7, dewaxing: section is positioned over to dimethylbenzene I, II each 5 minutes;
8, water inlet: be positioned over successively in 100%, 95%, 85%, 75%, 50% ethanol each 5 minutes cutting into slices, be positioned in tap water 5 minutes;
9, hematoxylin-eosin (English name Hematoxylin-Eosin staining, abbreviation HE) dyeing: section is put in Hematorylin dye liquor to 5 minutes, 1% aqueous hydrochloric acid differentiation several seconds (3～4 seconds), flowing water is positioned in 0.5% Yihong ethanolic soln 1 minute after rinsing;
10, dehydration: section is positioned over to 85%, 95% each several seconds of ethanol, each 1 minute of 100% ethanol I, II;
11, in dimethylbenzene, place the several seconds transparent, adopt neutral gum mounting;
12, optical microphotograph Microscopic observation HE dyeing tissue slice, found that in leukemia mouse liver, spleen and kidney all visible a large amount of heteromorphism cellular infiltrations, especially obvious with liver and spleen tissue.Heteromorphism cell extensively moves and infiltrates to being subject in the histoorgans such as the renal glomerulus surrounding tissue of mouse liver portal area, nephrolithotomy and spleen, and abnormal cells diffusivity in above-mentioned each histoorgan is assembled, and causes the normal histological structure of each internal organs to change; And the each internal organs of control group mice still keep normal configuration, referring to Fig. 4;
13, application anti human CD 19 or CD34 monoclonal antibody are carried out immunohistochemical methods detection to tissue slice, with HE dyeing, equally first paraffin section are dewaxed and are intake;
14, antigen retrieval: the 0.01mol citrate buffer solution (pH6.0) that preheating is put in section boils 20 minutes, naturally cooling 20 minutes;
15, with 3% hydrogen peroxide at room temperature, hatch 20 minutes, to eliminate the activity of endogenous peroxydase;
16, the section of application distilled water flushing, soaks in PBS damping fluid 5 minutes;
17, remove PBS damping fluid, every section adds 1 or the normal nonimmune animal serum of 50 μ L, sealing, under room temperature, hatch 10 minutes, the serum deprivation that inclines, does not wash, the anti human CD 19 or the CD34 monoclonal antibody (U.S. Abcam company) that drip dilution in 1: 100,4 ℃ of wet boxes spend the night;
18, PBS damping fluid rinses section, each 5 minutes, totally 3 times;
19, drip Envision working fluid, in room temperature, place 30 minutes
20, PBS damping fluid is washed section, and each 5 minutes, totally 3 times;
21, remove PBS damping fluid, every section adds 2 or the freshly prepared DAB solution of 100 μ L, micro-Microscopic observation 3-10 minute;
22, tap water rinses, and Hematorylin is redyed 1 minute, and tap water rinses and returns indigo plant;
23, section is positioned over to 85%, 95% each several seconds of ethanol, each 1 minute of 100% ethanol I, II, place the several seconds transparent in dimethylbenzene, adopt neutral gum mounting;
24, the CD19 positive or CD34 positive human leukemia cell's Infiltrating in the each section of optical microphotograph Microscopic observation; Observations is found in spleen, liver and kidney all visible a large amount of CD19 positives or CD34 positive human leukemiacell infiltration, especially maximum with spleen and liver.Result is referring to Fig. 4-6.
three,adopt quantitatively (English name Real-time Quantitative Polymerase Chain Reaction, the abbreviation of real-time quantitative polymerase chain reaction
rQ-PCR) detect people bcr/abl gene content in mouse bone marrow cells;
1, RNA extracts
1.1 by one times of 1 × PBS damping fluid dilution for bone marrow cell suspension, add to gently on Ficoll-Hypaque lymphocyte separation medium, the centrifugal 20min of 1800rpm, the careful mononuclearcell layer of drawing, wash twice with 1 × PBS damping fluid, for mononuclearcell layer, containing the more sample of red corpuscle, with deionized water, dissolve red corpuscle, by approximately 1 × 10
7cell is transferred in EP pipe;
1.2 flick the cell precipitation in EP pipe, add 1mL TRIzol(Invitrogen, the U.S.); with rifle point, thoroughly pressure-vaccum is extremely even repeatedly, then adds 200 μ L chloroform/primary isoamyl alcohol solution (V/V=24/1), and thermal agitation mixes; room temperature is placed 5min, 4 ℃ of centrifugal 15min of 12000g;
The 1.3 careful upper strata waters of drawing, add 600 μ L aqueous isopropanols, mix, and room temperature is placed 10min or-20 ℃ of refrigerator overnight, and 4 ℃ of centrifugal 15min of 12000g are to precipitate total RNA;
1.4 outwell aqueous isopropanol, add 1 mL75% ethanolic soln, and RNA precipitation is upspring fully to wash RNA precipitation, and the centrifugal 5min of 7500g, outwells ethanolic soln, drying at room temperature;
The 1.5 deionized water dissolving RNA that add appropriate DEPC to process, survey concentration and A260/A280 ratio, and this ratio is answered >1.6.
reverse transcription (RT) synthesizes cDNA
2.1 preparation RT public systems: 20 μ L public systems comprise 5 × RT damping fluid, 4 μ L, dNTP (each 2.5mmol, Pharmacia, the U.S.) 4 μ L, MMLV reversed transcriptive enzyme (200U/ μ L, Promega, the U.S.) 1 μ L, random hexamers (0.5 μ g/ μ L, Promega, the U.S.) 0.2 μ L, RNAsin (50U/ μ L, Promega, the U.S.) 0.4 μ L.
2.2 packing RT public systems, every pipe adds first 65 ℃ of 5min of RNA1 ~ 2 μ g(RNA, ice bath 5min).37 ℃ of reaction 1h, 95 ℃ of 5min deactivation reversed transcriptive enzymes.
、RQ-PCR
The making of 3.1 typical curves: first prepare abl and bcr-abl plasmid standard.Adopt the primer sequence of European anticancer project and we the design positive sample cDNA that increases respectively, PCR product is inserted respectively in PGEM-T plasmid vector, through bacterium, transform, screen, identify, increase, extract the plasmid standard that DNA obtains respectively reference gene abl and various goal gene, survey concentration, calculate copy number, utilize 10 times of serial dilutions of plasmid diluent to become 10
6～10
2individual copy, for making the typical curve of abl and various goal gene.The making of typical curve at least needs six points (10
6, 10
5, 10
4, 10
3each pipe, 10
2make 2 parallel pipes);
3.2 carry out RQ-PCR on ABI Prism7500 (U.S. A BI company) fluorescence real-time quantitative PCR instrument: reference gene is selected abl.Utilize Primer express 2.0 softwares (American AB I company product) design bcr-abl gene primer and probe sequence as follows:
Upstream primer is: 5 '-CCGCTGACCATCAATAAG-GAA-3 ',
Downstream primer is: 5 '-CTCAGACCCTGAGGCTCAAAGT-3 ',
Probe is: 5 '-FAM-AGCCCTTCAGCGGC-CAGTAGCATCT-TAMRA-3 '.
1) reaction system is 20 μ l, comprises the each 0.3 μ mol of upstream and downstream primer, TaqMan probe 0.2 μ mol, 2 × TaqMan universal PCR Mastermix, 10 μ l (ABI company, the U.S.), cDNA 2 μ L;
2) PCR reaction conditions: 50 ℃ of 2min, 1 circulation; 95 ℃ of 10min, 1 circulation; 95 ℃ of 15s, 62 ℃ of 1min, 40 circulations;
3) every crowd of RQ-PCR experiment all carry out two positive controls (high copy and low copy) simultaneously, without template blank (H
2o) and without correlate template negative control (not expressing the cDNA of goal gene) and for the amplification of the plasmid standard of production standard curve.The goal gene of every part of sample and ABL all make 2 parallel pipes, and copy number is got the mean value of two times result.If the large (C of parallel pipe result difference
tthe different >1.0 of value difference), also need again to increase;
4) while analyzing every batch of RQ-PCR result, threshold value is all fixed as 0.08%.
The calculating of 3.3 sample bcr-abl gene expression doses: SDS software utilizes the C of sample
tbe worth (English name Cycle at threshold, refers in pcr amplification process, and fluorescent signal starts to be entered by background the corresponding cycle number of flex point of people's exponential growth phase), according to typical curve, calculate respectively the copy number of each part of sample abl and bcr-abl.If abl copy number>=3 × 10
4, think that this sample can use.For guaranteeing accuracy, for the sample of bcr-abl copy number <500, all carry out the 2nd amplification, bcr-abl copy number is got the mean value of two times result.According to typical curve, calculate respectively the copy number of each part of sample ABL and goal gene.
gene level(%)=bcr-abl gene copy number/abl gene copy number × 100%.
In final quantitative Analysis marrow, people bcr/abl gene content result as shown in Figure 3.
four, by FISH, detect and be subject to people t (9 in mouse medullary cell; 22) (q34; Q11)
step 1: film-making
1.1 slide processing: methyl alcohol soaked overnight;
1.2 is centrifugal: bone marrow cell suspension 1000rpm/min, 10min;
1.3 sheets: abandon supernatant, add suitable fresh stationary liquid, make the cell suspension that concentration is suitable, suction pipe blows gently evenly draws a small amount of upper strata suspension along tube wall afterwards, highly drips down to one end and tilts on the cleaning of the 30 ° of left and right slide without fat, every 2 ~ 3 in one meter, make to be paved with full wafer, be placed in by (gauze of paving one deck immersion) on 40 ℃ of damp and hot roasting sheet machines of preheating, after the water smoke of surface of glass slide disperses, can take off seasoning;
1.4 roasting sheets: 80 ℃ of roasting sheet machines 4 hours.
step 2: FISH operation steps
2.1 fresh bone marrow cell sample preprocessor
2.1.1 freshly prepd karyomit(e) slide need be at 56 ℃ spend the night under aging 30-60 minute or room temperature aging;
2.1.2 slide is hatched 30 minutes in 37 ℃ of 2XSSC solution;
2.1.3 slide is placed in successively to the dehydration in each 3 minutes of 70% ethanol, 85% ethanol and 100% ethanol.Seasoning slide;
2.1.4 slide is heated to 56 ℃;
2.2 carry out FISH experiment according to FISH operation steps
2.2.1 probe preparation: under room temperature, following liquid is joined in Eppendorf tube;
| Title | (μ l) for volume |
| Hybridization buffer | 7.0 |
| Deionized water | 1.0 |
| Probe | 2.0 |
| ∑ | 10.0 |
2.2.2 centrifugal 1-3 second, again of short duration centrifugal after vortex mixes;
2.2.3 10 μ L probe mixture are dripped in slide hybridization region, add a cover immediately cover glass, use rubber adhesive edge;
2.2.4 prepare hybridization instrument, slide is put into hybridization instrument sex change hybridization.Denaturing altogether: 78 ℃, 5 minutes, hybridization conditions: 42 ℃, 16 hours.
slide washing
Attention: before using, 0.3%NP-40/2 × SSC is placed in to 67 ℃ of water baths at least 30 minutes, makes solution reach temperature required;
2.3.1 remove cover glass, slide is placed in to 0.3% NP-40/0.4 × SSC solution, vibrate 1-3 second, rinsing 2 minutes.Special instruction: the temperature and time of 0.3% NP-40/0.4 in washing scheme fast × SSC washings need to regulate according to differences between samples, and 67 ℃ is recommendation temperature;
2.3.2 under room temperature, slide is placed in to 0.1% NP-40/2 × SSC, vibrates 1-3 second, rinsing 30 seconds;
2.3.3 under room temperature, slide is placed in to 70% ethanol, rinsing 3 minutes.
redye
2.4.1 dark place seasoning slide;
2.4.2 15 μ L DAPI counterstains are dripped in hybridization regional location, immediately covered.Placed after 10-20 minute dark place, selects suitable filter set to observe slide under fluorescent microscope.Result is referring to Fig. 7, and experimental mice can detect people t (9; 22) (q34; Q11) positive signal, the negative signal of control group mice.
In sum, the present invention is by application sublethal dose
60co irradiation is combined anti-mouse CD122 monoclonal antibody pre-treatment and Ph
+aLL patient B MMNCs replaces traditional NOD/SCID mouse tail vein injection method to set up a kind of humanization Ph through the injection of NOD/SCID mouse knee joint medullary space
+aLL mouse model, its modeling success ratio is high, favorable reproducibility.
Claims (7)
1. build a method for humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model, comprise the taking of leukemia cell, the pre-treatment of NOD/SCID mouse and leukemia cell's implantation, it is characterized in that specifically comprising the following steps:
Steps A, Ph
+the preparation of ALL Bone Marrow of Patients mononuclearcell: get first visit Ph
+aLL Bone Marrow of Patients sample, anticoagulant heparin, application human lymphocyte parting liquid separates prepares BMNC, with the RPMI1640 nutrient solution containing 10% foetal calf serum
resuspendedcell to the whole density of cell is 1 ~ 8 × 10
8/ mL, makes Ph
+the cell suspension of ALL BMNC;
The pre-treatment of step B, NOD/SCID mouse: get SPF level NOD/SCID female mice, carry out sublethal dose
60co irradiation, only injects anti-mouse CD122 monoclonal antibody 150 ~ 250 μ g/ with backward mouse peritoneal;
The cell suspension of preparing in step C, implantation step A: after pre-treatment in 24h, the cell suspension that NOD/SCID female mice is prepared in knee joint medullary space injecting step A, every injected in mice amount is 0.1mL, modeling in 4 ~ 8 weeks success after implanting.
2. the method for structure humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model according to claim 1, it is characterized in that NOD/SCID mouse in step B adopts pre-treatment before 1 week, the 4-6 NOD/SCID female mice in age in week, raise in SPF level laminar flow laboratory, feed and tap water are all through aseptically process.
3. the method for structure humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model according to claim 2, is characterized in that in step B
60the irradiation dose of Co is 1.8 ~ 2.4cGy.
4. the method for structure humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model according to claim 1, is characterized in that implanting after cell suspension in step C spirit, hair, body weight and the active situation of routine observation mouse.
5. the method for structure humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model according to claim 1, is characterized in that also comprising in the step of present method the evaluation of the leukemia mouse model to completing in step C, specifically comprises:
(1) adopt people CD45 in polychrome Flow cytometry mouse bone marrow cells or spleen cell
+cD19
+leukemia cell's content and immunophenotype thereof;
(2) adopt people bcr/abl gene content in real-time quantitative polymerase chain reaction detection by quantitative mouse bone marrow cells and spleen;
(3) adopt people t (9 in fluorescence in situ hybridization detection mouse bone marrow cells and spleen; 22) (q34; Q11);
(4) adopt the dyeing of Hematorylin-Yihong and immunohistochemical staining to detect people Ph in mice organs
+the situation that ALL cell migration infiltrates.
6. the method for structure humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model according to claim 5, while it is characterized in that adopting polychrome flow cytometry, fluorescently-labeled monoclonal antibody is respectively: CD58FITC, CD10PE, CD34 PerCP, CD38APC, CD19APC-Cy7 and CD45Pacific Blue.
7. the method for structure humanization Ph chromatin-positive acute lymphoblastic leukemia mouse model according to claim 5, is characterized in that upstream primer used in real-time quantitative polymerase chain reaction is:
5’-CCGCTGACCATCAATAAG-GAA-3’，
Downstream primer is: 5 '-CTCAGACCCTGAGGCTCAAAGT-3 ',
Probe is: 5 '-FAM-AGCCCTTCAGCGGC-CAGTAGCATCT-TAMRA-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310391969.1A CN103740639A (en) | 2013-09-02 | 2013-09-02 | Method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310391969.1A CN103740639A (en) | 2013-09-02 | 2013-09-02 | Method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103740639A true CN103740639A (en) | 2014-04-23 |
Family
ID=50497706
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310391969.1A Pending CN103740639A (en) | 2013-09-02 | 2013-09-02 | Method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103740639A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105210981A (en) * | 2015-09-15 | 2016-01-06 | 中国科学院生物物理研究所 | Set up method and the application thereof of the ferret model that can be applicable to human diseases research |
| CN107773573A (en) * | 2016-08-30 | 2018-03-09 | 张超 | Leukemia disease model and its construction method |
| CN109022362A (en) * | 2018-08-02 | 2018-12-18 | 武汉大学 | A kind of method for building up of hyperleucocyte acute leukemia PDX model |
| CN111149767A (en) * | 2020-02-24 | 2020-05-15 | 中南大学湘雅二医院 | Humanized skin type lupus erythematosus mouse model and construction method and application thereof |
| CN112899233A (en) * | 2021-03-25 | 2021-06-04 | 杭州医学院 | Method for constructing acute lymphocytic leukemia mouse model |
| CN113728972A (en) * | 2021-08-17 | 2021-12-03 | 武汉大学中南医院 | PDX model of low-risk acute myelogenous leukemia CIT and construction method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101896200A (en) * | 2007-12-06 | 2010-11-24 | Csl有限公司 | The method that suppresses leukemic stem cells |
| WO2011066660A1 (en) * | 2009-12-04 | 2011-06-09 | University Health Network | Lsc and hsc signatures for predicting survival of patients having hematological cancer |
| US20120302517A1 (en) * | 2008-06-12 | 2012-11-29 | Assistance Publique - Hopitaux De Paris | Tet2 as a new diagnostic and pronostic marker in hematopoietic neoplasms |
-
2013
- 2013-09-02 CN CN201310391969.1A patent/CN103740639A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101896200A (en) * | 2007-12-06 | 2010-11-24 | Csl有限公司 | The method that suppresses leukemic stem cells |
| US20120302517A1 (en) * | 2008-06-12 | 2012-11-29 | Assistance Publique - Hopitaux De Paris | Tet2 as a new diagnostic and pronostic marker in hematopoietic neoplasms |
| WO2011066660A1 (en) * | 2009-12-04 | 2011-06-09 | University Health Network | Lsc and hsc signatures for predicting survival of patients having hematological cancer |
Non-Patent Citations (3)
| Title |
|---|
| H. JOSEF VOMOOR: "Malignant stem cells in childhood acute lymphoblastic leukkemia: The stem cell concept revisited", 《CELL CYCLE》 * |
| Y.Z. QIN 等: "Different kinetic patterns of BCR-ABL transcript levels in imatinib-treated chronic myeloid leukemia patients after achieving complete cytogenetic response", 《INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY》 * |
| 孔圆 等: "人急性B淋巴细胞白血病-NOD/SCID/IL2rynull新生小鼠异种移植模型的建立", 《中国实验血液学杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105210981A (en) * | 2015-09-15 | 2016-01-06 | 中国科学院生物物理研究所 | Set up method and the application thereof of the ferret model that can be applicable to human diseases research |
| CN107773573A (en) * | 2016-08-30 | 2018-03-09 | 张超 | Leukemia disease model and its construction method |
| CN109022362A (en) * | 2018-08-02 | 2018-12-18 | 武汉大学 | A kind of method for building up of hyperleucocyte acute leukemia PDX model |
| CN109022362B (en) * | 2018-08-02 | 2022-01-25 | 武汉大学 | Method for establishing PDX (PDX) model of high-leucocytic leukemia |
| CN111149767A (en) * | 2020-02-24 | 2020-05-15 | 中南大学湘雅二医院 | Humanized skin type lupus erythematosus mouse model and construction method and application thereof |
| CN112899233A (en) * | 2021-03-25 | 2021-06-04 | 杭州医学院 | Method for constructing acute lymphocytic leukemia mouse model |
| CN112899233B (en) * | 2021-03-25 | 2023-03-14 | 杭州医学院 | Method for constructing acute lymphocytic leukemia mouse model |
| CN113728972A (en) * | 2021-08-17 | 2021-12-03 | 武汉大学中南医院 | PDX model of low-risk acute myelogenous leukemia CIT and construction method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103740639A (en) | Method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model | |
| CN1354657A (en) | EGF-R antagonists for treating hypersecretion of mucus in lungs | |
| CN106265740B (en) | Umbilical cord mesenchymal stem cells combine application of the astragalus polyose in treatment hyperglycaemia and medicine for treating diabetic nephropathy is prepared | |
| CN110604743B (en) | Application of neutrophils in preparation of medicine for treating and/or preventing autoimmune hepatitis | |
| CN111713450B (en) | Method for establishing PDX model of chronic granulocytic leukemia | |
| CN109674819A (en) | Placenta mesenchyma stem cell preparation and its purposes for treating hardening illness | |
| CN103301154B (en) | Application of umbilical cord mesenchymal stem cells in preparation of formulation for treating lupus erythematosus | |
| CN106074604A (en) | For repairing the therapeutic agent that body function is aging and delays organ function to fail | |
| CN109646458A (en) | Use the method for placenta mesenchyma stem cell preparation for treating hardening illness | |
| CN103881975A (en) | Human prostate cancer cell as well as passage separation culture and subculturing culture method and application thereof | |
| CN109868274A (en) | siRNA, siRNA plasmid and lentivirus for targeted inhibition of EGFL9 gene expression, and construction method and application thereof | |
| CN109136273A (en) | Prepare the method and its application of the rat of immune deficiency | |
| Kenney et al. | ASPS-1, a novel cell line manifesting key features of alveolar soft part sarcoma | |
| CN106963780A (en) | Purposes of the PMBC activation inhibitor in the medicine for preparing treatment acute hepatic failure | |
| CN103911343B (en) | A kind of multiple organ shifts human bladder cancer cell | |
| CN103349670A (en) | Application of Japan schistosome egg extractive to preparing anti-hepatic fibrosis medicine | |
| Lee et al. | Effects of stromal vascular fraction on breast cancer growth and fat engraftment in NOD/SCID mice | |
| CN102433307A (en) | Cell strain from human lung adenocarcinoma and preparation method thereof | |
| CN107789361A (en) | A kind of preparation method of medicine of magnetic target therapy tumour and application | |
| CN107828789A (en) | Application of the inhibitor and its application and KAP1 of targeted therapy of lung cancer as drug targets in the medicine of screening anti-lung cancer | |
| CN114304066A (en) | Method for constructing colorectal cancer PDX model | |
| CN103881974B (en) | A kind of low aggressive transitional cell bladder carcinoma cell line of people | |
| CN106701886A (en) | Method for detecting influence of triple negative breast cancer cell on endothelial cell secretion function in epithelial-mesenchymal transition process | |
| CN109929804B (en) | Human ovarian cancer cell line and preparation method and application thereof | |
| Tang et al. | Isolation and characterization of peritoneal microvascular pericytes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140423 |
|
| RJ01 | Rejection of invention patent application after publication |