CN103114074B - Method for building cGVHD (chronic graft-versus-host disease) model of mouse allogene after bone marrow transplantation - Google Patents

Abstract

The invention provides a method for building a cGVHD (chronic graft-versus-host disease) model of a mouse allogene after bone marrow transplantation. Marrow and splenocyte of an allogene CB6F1(H-2bxd) male donor mouse are prepared and are transferred into a BALB/c(H-2d) female receptor mouse in an intravenous infusion manner, and modelling is carried out to obtain the cGVHD model. The model building method provided by the invention has the characteristic of simulating occurrence and development of cGVHD after clinically allogeneic hematopoietic stem cell transplantation; a process is simple and practicable, model building efficiency is high, repeatability is good, and no similar report exists at home and abroad; and especially typical pathologic change of lung and skin can be simulated, and an experimental platform is provided for research of prevention and treatment of human cGVHD in living animals.

Description

The establishment method of cGVHD model after a kind of Mouse Allogeneic Bone Marrow Transplantation

Technical field

The invention belongs to biological technical field, relate to the establishment method of cGVHD model after a kind of Mouse Allogeneic Bone Marrow Transplantation.Set up and have the disease model of simulating clinical development of chronic graft-versus-host disease after allogeneic haematopoietic stem cell transplantation feature, the mechanism and the diagnoses and treatment that can be chronic graft versus host disease provide research platform.

Background technology

Chronic graft versus host disease is major complications after Allogeneic Hematopoietic Stem Cell Transplantation and the cause of death, becomes the key factor affecting patients ' life quality and long-term survival.Further clear and definite cGVHD mechanism, studies active and effective methods for the treatment of, reduces or alleviates it and occur and the course of disease, have important practical significance.Therefore, the foundation of cGVHD animal model is the important foundation of translational medicine research.Current most of cGVHD Animal Model is transplanted between H-2 antigen half-matched or the small mouse do not conformed to, and main manifestations is lupoid acne (SLE-cGVHD), and target organ of getting involved is mainly skin, kidney, liver and small intestine; And sclerderm sample (Scl-cGVHD), target organ of getting involved is mainly skin.The model of lung cGVHD once had indivedual report, and because needs use endoxan in preprocessing process, consider the detrimental effect of medicine to lung itself, this model need further optimization.This research uses H-2 antigen half-matched mouse, and for mouse, all derive from domestic conventional mouse species by mouse, method is easy; And in this research for by mouse pairing mode from domestic and international similar study report different, infusion splenocyte comparatively small amt, mould time and symptom is become to meet the generation development characteristic of clinical development of chronic graft-versus-host disease after allogeneic haematopoietic stem cell transplantation, the performance of lung and skin cGVHD is very typical, and liver also has the performance of part cGVHD.Process provides the new model of research cGVHD, the correlative studys such as cGVHD pathogenesis, drug test, cellular immunotherapy can be carried out for most domestic research centre and lay the foundation.

Summary of the invention

The object of this invention is to provide the establishment method of cGVHD model after a kind of Mouse Allogeneic Bone Marrow Transplantation, realized by following steps:

(1) with the female mouse (H-2 of C57BL/6 ^b) mouse (H-2 male with BALB/c ^d) hybridize the CB6F1(H-2 ^bxd) for male mice as mouse;

(2) with BALB/c(H-2 ^d) female mice is as by mouse;

(3), after accepting full-body exposure pre-treatment by mouse, be injected to available from the medullary cell BMC suspension and splenocyte SpC suspension that supply mouse by mouse.

Preferably, describedly mouse in 8 week age is all adopted for mouse and by mouse.

Specifically, in described step (3), for being prepared as follows of murine myeloid cells BMC suspension: put to death for after mouse with cervical dislocation, putting into 75% ethanol and soak 3 ~ 5min, aseptic stripping femur and shin bone, medullary space is rinsed with RPMI1640 nutrient solution, the washing fluid collected containing marrow makes single cell suspension, and abolish red corpuscle with erythrocyte cracked liquid after filtration, RPMI1640 washs several times, count and use RPMI1640 re-suspended cell, being adjusted to 5 × 10^7/mL cell concn for subsequent use.

Specifically, in described step (3), for being prepared as follows of mice spleen cell SpC suspension: put to death for after mouse with cervical dislocation, put into 75% ethanol and soak 3 ~ 5min, aseptic taking-up spleen, remove mesenteric tissue around spleen, move in plate with RPMI1640 nutrient solution washing several times, mill, collecting cell washing fluid makes single cell suspension, abolishes red corpuscle, wash several times with RPMI1640 after filtration with erythrocyte cracked liquid, count and use RPMI1640 re-suspended cell, being adjusted to 2.5 × 10^7/mL cell concn for subsequent use.

Specifically, in described step (3), transplant and accepted linear accelerator 700cGy single full-body exposure pre-treatment by mouse the same day, supplement immediately after irradiation and drink water, have a rest after 4 ~ 6 hours through medullary cell BMC suspension and splenocyte SpC suspension that tail vein injection final volume is 0.4mL.

Preferably, in described step (3), the consumption be administered to by the medullary cell BMC suspension of mouse is 1 × 10^7BMC, and the consumption of splenocyte SpC suspension is 5 × 10^6.

The present invention, by the marrow of application H-2 antigen half-matched generation mice and splenocyte, transplants the mouse of the parent after to lethal irradiation but different sexes, sets up cGVHD model after a kind of allogeneic bone marrow transplantation.It has following features: (1) this model has the advantages that to simulate development of chronic graft-versus-host disease after allogeneic haematopoietic stem cell transplantation clinically; (2) established model efficiency is high, reproducible; (3) needed for modeling, mouse easily obtains, and process is simple; (4) there is typical pathologic simultaneously and change in the multiple organ such as lung, skin, liver; (5) the mouse survival time after transplanting is long, and the symptom closer to mankind cGVHD shows, for the research carrying out preventing and treating mankind cGVHD in living animal provides new experiment porch.

Accompanying drawing explanation

Fig. 1 is the clinical score schematic diagram (in legend, the scoring of each time point represents with mean ± standard deviation, n=5) of cGVHD experimental mice

Fig. 2 is the clinical manifestation of cGVHD experimental mice;

Fig. 3 is+135d chromosome map after blank group and cGVHD experimental mice are transplanted;

Fig. 4 is the pathological biopsy HE colored graph transplanting the lung, skin and the liver that are separated after control group is put to death with cGVHD experimental mice 135d.

Embodiment

The present invention is further described in conjunction with the accompanying drawings and embodiments.This tests mouse used all purchased from Guangzhou Zhongshan University Experimental Animal Center.

Embodiment one

This example is the foundation example of chronic graft versus host disease model after Mouse Allogeneic Bone Marrow Transplantation of the present invention, operates according to the following steps:

One, prepare for mouse

1, no-special pathogen (Specefic pathogen Free, SPF) level C57BL/6 female mice (H-2 ^b) 8-10 age and SPF level BALB/c male mice (H-2 in week ^d) 8-10 week age, the mating of 2:1 ratio.

2, heterozygote F1 generation is selected, male, raise to 8 week age, as subsequent use for mouse.

Two, prepare by mouse

SPF level BALB/c mouse (H-2 ^d), female, 8 week age, as being subject to mouse.4 groups will be divided at random by mouse, often organize 5; Wherein,

A, Normal group: synchronously raise, without intervening;

B, radiocontrast group: TBI+RPMI1640, namely carry out full-body exposure (TBI) process, through tail vein injection PRMI1640 nutrient solution to mouse in group;

C, transplanting control group: TBI+1 × 10^7BMC, namely carry out full-body exposure (TBI) process, through the medullary cell BMC suspension of tail vein injection through the process of PRMI1640 nutrient solution to mouse in group;

D, cGVHD group: TBI+1 × 10^7BMC+5 × 10^6SpC, namely carries out full-body exposure (TBI) process, through tail vein injection through the medullary cell BMC suspension of PRMI1640 nutrient solution process and splenocyte SpC suspension to mouse in group.Three, according to dividing into groups above, following step is adopted to carry out transplantation experiments operation:

1, allogeneic bone marrow transplantation carries out INTESTINAL CLEANSING to the sterilizing drinking-water that each group is subject to mouse to accept to add microbiotic (erythromycin 250mg/L, gentamicin 320,000 U/L) in laminar flow animal room the last week, and is maintained until transplanting latter 3 weeks.

2, transplant the same day (0d), by mouse, full-body exposure (TBI) pre-treatment of linear accelerator 700cGy single is accepted to B, C, D group.Supplement drinking-water immediately after irradiation, have a rest after 4 ~ 6 hours through transplanted cells suspension that tail vein injection final volume is 0.4mL.

Every only from tail venoclysis RPMI1640 nutrient solution 0.4mL in control group B group mouse;

Every only from tail venoclysis RPMI1640 cell suspension 0.4mL in control group C group mouse, comprise 1 × 10^7BMC;

Every only from tail venoclysis RPMI1640 cell suspension 0.4mL in D group mouse, comprise 1 × 10^7BMC and 5 × 10^6SpC.

Wherein, medullary cell (BMC) suspension preparation manipulation is as follows: adopt cervical dislocation to put to death for mouse, puts into 75% ethanol immediately and soaks 3 ~ 5min; Aseptic stripping femur and shin bone in Bechtop, medullary space is rinsed with RPMI1640 nutrient solution, the washing fluid collected containing marrow makes single cell suspension by No. 4 syringe needles, filter, red corpuscle is abolished with erythrocyte cracked liquid, RPMI1640 washs 3 times, counts and uses RPMI1640 re-suspended cell, being adjusted to 5 × 10^7/mL cell concn for subsequent use.

Splenocyte (SpC) suspension preparation manipulation is as follows: aseptic taking-up spleen, mesenteric tissue around careful removal spleen, move in plate and wash 3 times with RPMI1640 nutrient solution, then be placed in 200 order stainless steel mesh 2mL plungers to mill gently, collecting cell washing fluid makes single cell suspension by No. 4 syringe needles, filters, red corpuscle is abolished with erythrocyte cracked liquid, wash 3 times with RPMI1640, count and use RPMI1640 re-suspended cell, being adjusted to 2.5 × 10^7/mL cell concn for subsequent use.

After transplanting, every day observes mouse general state, and comprise body weight, fash, depilation, incrustation, the back of a bow, diarrhoea etc., transplant and within after 14 days every 3 days, carry out a clinical score, standard is see table 1; Dying sacrifice is drawn materials, and its survival time, note was to putting to death next day; Experimental endpoints is for transplanting rear+135d.Leukocyte counts: often organize mouse and count WBC monitoring hematopoietic reconstitution situation in+7d ,+10d and+14d.Method of counting: blood 10 μ L is got in docking, adds in 190 μ L white corpuscle diluents and mixes, be then added drop-wise on cell counting count board, counts cell count (N) in 4 block plaid after horizontal rest 1min, WBC=(N ÷ 20) × 10^9/L.

Transplanting observed and recorded shows: A group mouse survive is to experimental endpoints; B group mouse meta is survived 12.5 days, all dead in 14 days; C, D group mouse survival is to experimental endpoints.More than record shows that C, D group is all transplanted successfully, and the survival time reaches more than 100 days, and the survival time of setting up than existing similar disease animal model is long.

Embodiment 2

This example is mouse allogenic bone marrow implantation test example.

Get and transplant detecting by mouse of rear+135d, carry out according to the following steps:

1, colchicine process: doing first 3 ~ 4 hours of experiment, to the colchicine (2-4 μ g/g) of mouse peritoneal injection 0.1%.2, get femur: during experiment, with cervical dislocation rapidly by sacrifice, cut off skin and the muscle of hind leg immediately with scissors, take off both sides femur together with joint, pick clean muscle on it.

3, extracting marrow cell: draw 6ml physiological saline with syringe, syringe needle is inserted one end of medullary space, go out medullary cell to centrifuge tube with physiological saline, rinses repeatedly until bone chamber bleaches.Put into whizzer by after the medullary cell solution equilibria collected, with the centrifugal 10min of 1000r/min, suck supernatant liquor.

4, hypotonic: how many according to cell concentration, add 0.075mol/L KCl hypotonic medium 5-6ml, blow and beat mixing gently with suction pipe, hypotonic 20-30 minute in 37 DEG C of constant water bath box.

5, pre-fix: hypotonic complete, add the Carnoy stationary liquid that 1-2ml newly prepares immediately, gently after piping and druming, with the centrifugal 10min of 2000r/min, careful suction pipe sucks supernatant liquor.

6, fixing: slowly to add stationary liquid 7ml along centrifugal tube wall, blow and beat into cell suspension with suction pipe.The centrifugation of 2000rpm 10 minutes, incline supernatant.

7, fix again: repeating step 6 to be operated to supernatant substantially colourless clear.

8, cell suspension is prepared: add appropriate stationary liquid in throw out, blow and beat into cell suspension gently with suction pipe.

9, drip borneol: the slide glass taking out precooling from refrigerator, sheet is downwards dripped in hand-held suction pipe about 10cm place above slide glass, and 2-3 drips, make suspension evenly spread with slide on, unnecessary liquid can carefully incline.

10, dry, flame is fixed: slide is under spirit lamp flame envelope is several back and forth rapidly.

11, slide is aging: be placed in 37 ° of C incubators after 1-2 days, 24h in 65 ° of C baking boxs.

12, dye: lower concentration trysinization, clear water is cleaned, and dyes about 10 minutes under Giemsa dye liquor room temperature.Clear water rinses, at room temperature seasoning.

13, microscopy: first find a good split coil method region with low power lens, high power lens of then converting is observed.

As shown in Figure 3, for coming from the chromosome map of A group (blank group) and D group (cGVHD group) mouse, arrow is depicted as Y chromosome.In figure, chromosomal change also shows that D group mouse is transplanted successfully further.

Embodiment 3

This example is the clinical pathology test example of cGVHD group mouse.

One, D group mouse is carried out to the clinical score of chronic GVHD, its standards of grading are as shown in table 1:

The clinical score standard of table 1 chronic GVHD

Score	0 point	1 point	2 points	3 points
					Depilation	Nothing	<1cm 2	1～2cm 2	>2cm 2
Lose weight	Without or alleviate≤2%	2％～8％	≥8％
					Back of a bow figure	Nothing	Do not affect motion	Impact motion

Note: (a) ear, tail, sole often locate decortication or incrustation note 0.3 point, skin clinical manifestation minimum 0 point, the highest 3.9 points.B () skin score is considered as more than 0.6 point GVHD occurs; Also be considered as GVHD occurs even if resolution of symptoms, mouse natural death or human factor cause death; The asymptomatic death of mouse is considered as without GVHD.

Survival observes at least+100d.Dying sacrifice is drawn materials, and its survival time, note was to putting to death next day.

Fig. 1 is the clinical score schematic diagram of D group mouse.(in legend, the scoring of each time point represents with mean ± standard deviation, n=5)

Fig. 2 is the clinical manifestation of D group mouse, and A shows the back of a bow; B shows depilation; C shows tail incrustation; Form a scab in D display depilation and ear.

Two, the C group of right+135d and D group mouse carry out the pathology detection of chronic GVHD, and pathological score standard is as shown in table 2 below:

The pathological score standard of table 2 chronic GVHD

Note: (l) score is considered as more than 2 GVHD occurs.

C group and the D group of getting+135d after transplanting detect by after mouse execution, carry out according to the following steps:

1, fixing: to get each organs and tissues and be cut into small pieces, 10% neutral formalin solution internal fixtion 24 hours;

2, following program completes in the full-automatic enclosed water extracter of Shandon:

2.1 tissue samples fixed respectively at 80% ethanol 50min, 90% ethanol 50min, each 40min of 95% ethanol I II, each 40min of 100% ethanol I II;

Sample after dehydration is placed 30min by 2.2 respectively in dimethylbenzene I, II, III;

Place each 25min(temperature in 2.3 low melt point paraffins I, II, III and be set to 62 degrees Celsius);

2.4 inspissated wax I cylinder 25min(temperature are set to 64 degrees Celsius);

3, after tissue handling procedure terminates, take out sample, be placed in the wax pan of embedding machine, carry out embedding (temperature is set to 62 degrees Celsius);

4, cut into slices: on slicing machine, cut 4 μm of sections, float on clean water face, launch in 50-57 degree Celsius of warm water, then tissue slice is picked up; At 70 degrees Celsius of oven for baking 15-20min;

5, from baking box, take out section, room temperature slightly cools, and section be put on dyeing machinery and carry out full-automatic HE dyeing, dyeing procedure is as follows: place each 7min in dimethylbenzene I, II, III, 100% ethanol I II, 95% ethanol I II, 80% ethanol, each 1min of distilled water; .In phenodin dye liquor 15 minutes, washing, 0.5% hydrochloride alcohol 10s; 2% lithium carbonate aqueous solution 1min; Running water 10min, 80% ethanol, 95% ethanol I II, 80% ethanol, 100% ethanol I II III, each 1min of dimethylbenzene I, II;

6, take out section, section is hung up Shandon mounting machine and carry out mounting.

7, optical microphotograph Microscopic observation HE staining tissue slides, found that mouse lungs show as bronchiolitis, massive inflammatory cells infiltrated in bronchiole, blocks bronchiole chamber, and tube wall smooth muscle layer is destroyed, and pulmonary consolidation; Tail skin ulcer, a large amount of collagen deposition of skin corium; Liver cell granular degeneration, oedema, a small amount of inflammatory cell infiltration in portal area; Result is see Fig. 4 and table 3.Fig. 4 is the pathological biopsy HE colored graph transplanting the lung, skin and the liver that are separated after control group is put to death with cGVHD experimental mice 135d.Table 3 is the detected result of each group of multiple experiment mice chronic GVHD.

Result shows, and transplants the lung of control group, tail skin and liver healthy tissues (× 100).CGVHD experimental group: B1/B4 bronchiolitis, massive inflammatory cells infiltrated in bronchiole, block bronchiole chamber, tube wall smooth muscle layer is destroyed (× 100), B7 pulmonary consolidation (× 100); B2 tail skin ulcer (× 100), a large amount of collagen deposition of B5 tail dermal layer of the skin (× 200), B8 tail skin inflammation cellular infiltration (× 200); The a small amount of inflammatory cell infiltration in B3 liver portal area (× 100), B6 liver cell granular degeneration, oedema (× 200), a small amount of inflammatory cell infiltration in B9 liver portal area (× 200).

The pathology detection result of table 3 chronic GVHD

Detected result shows, cGVHD experimental mice all shows cGVHD symptom in skin, liver and lung, closely similar with the cGVHD symptom showed after mankind's allotransplantation.

Claims (2)

1. the establishment method of cGVHD model after Mouse Allogeneic Bone Marrow Transplantation, is realized by following steps:

(1) with C57BL/6 mouse (H-2 ^b) and BALB/c mouse (H-2 ^d) hybridize the F1 generation male mice that as mouse;

(2) with BALB/c (H-2 ^d) female mice is as by mouse;

(3), after accepting full-body exposure (TBI) pre-treatment by mouse, will be injected to by mouse available from the medullary cell BMC suspension and splenocyte SpC suspension that supply mouse,

Wherein, for being prepared as follows of murine myeloid cells BMC suspension: put to death for after mouse with cervical dislocation, put into 75% ethanol and soak 3 ~ 5min, aseptic stripping femur and shin bone, rinse medullary space with RPMI RPMI-1640, the washing fluid collected containing marrow makes single cell suspension, abolish red corpuscle with erythrocyte cracked liquid after filtration, RPMI 1640 washs several times, counts and uses RPMI 1640 re-suspended cell, be adjusted to 5 × 10^7/mL cell concn for subsequent use

For being prepared as follows of mice spleen cell SpC suspension: put to death for after mouse with cervical dislocation, put into 75% ethanol and soak 3 ~ 5min, aseptic taking-up spleen, remove mesenteric tissue around spleen, move in plate with RPMI1640 nutrient solution washing several times, mill, collecting cell washing fluid makes single cell suspension, abolishes red corpuscle after filtration with erythrocyte cracked liquid, several times are washed with RPMI 1640, count and use RPMI 1640 re-suspended cell, being adjusted to 2.5 × 10^7/mL cell concn for subsequent use

Transplant and accepted linear accelerator 700cGy single full-body exposure pre-treatment by mouse the same day, drinking-water is supplemented immediately after irradiation, have a rest after 4 ~ 6 hours through medullary cell BMC suspension and splenocyte SpC suspension that tail vein injection final volume is 0.4mL, the consumption of medullary cell BMC suspension is 1 × 10^7 cell count, and the consumption of splenocyte SpC suspension is 5 × 10^6 cell count.

2. method according to claim 1, is characterized in that: describedly all adopt mouse in 8 week age for mouse and by mouse.