CN100406058C

CN100406058C - Placenta factor, preparation method and application

Info

Publication number: CN100406058C
Application number: CNB2006100786151A
Authority: CN
Inventors: 闫晨华; 陆道培
Original assignee: Peking University Peoples Hospital
Current assignee: Peking University Peoples Hospital
Priority date: 2006-04-28
Filing date: 2006-04-28
Publication date: 2008-07-30
Anticipated expiration: 2026-04-28
Also published as: CN1872333A

Abstract

The present invention discloses a placenta factor, a preparation method and an application for the placenta factor. The placenta factor is prepared by the following methods: (1) the placenta is sheared into pieces and processed through tissue homogenization with centrifugal force of 1770 to 2991.3g to obtain homogenate which is incubated in water bath of 25 to 40 DEG C for 0.5 to 2.5 hours and then processed through centrifugation with centrifugal force of 113.28 to 398.25g at a temperature of 4 to 10 DEG C, and supernatant fluid is taken; (2) the supernatant fluid obtained in procedure (1) is ultrafiltered with interception molecular weight of 10KD to obtain an ultrafiltration product, namely a placenta factor. The present invention has the advantages of simple PF preparation method, abundant raw material, simple preparation method and high stability. The placenta factor of the present invention is a micromolecule polypeptide substance extracted from human placenta tissue, has the advantages of small side effect, safe use and broad application prospective, and can be used for preventing and/or treating graft rejection and a graft versus host disease (GVHD) in organ graft and hematopoietic stem cell graft.

Description

A kind of placenta factor and preparation method thereof and application

Technical field

The present invention relates to a kind of placenta factor and preparation method thereof and application.

Background technology

(graft-versus-host disease is a complication common after the hematopoietic stem cell transplantation GVHD) to graft versus host disease, is that successful major obstacle is transplanted in restriction.Current applied immunosuppressant (as: glucocorticoid, FK506, ciclosporin A and MMF etc.) though and the whole bag of tricks of removing T cell in the graft can alleviate GVHD, but above-mentioned these methods have but improved the incidence rate of opportunistic infection, leukemia relapse and graft failure, and are attended by poisonous side effect of medicine.Therefore, the new method of seeking control GVHD is the major issue that blood educational circles pays close attention to always, also is one of the focus of transplantation immunology research and difficult point.

Fetus and parent be different individualities but can coexist and immunologic rejection do not take place, and this phenomenon is pointed out and may be contained certain composition in our Placenta Hominis and can make fetus and parent produce immunologic tolerance.Domestic scholars is a raw material with the human placenta, from Placenta Hominis, extracted micromolecular polypeptides matter and be placenta factor (placenta factor, PF).At present, the extracting method of PF mainly contains two kinds.The one, dialysis: Placenta Hominis shreds, high-speed homogenization, through multigelation (30 ℃ to 40 ℃ 3 times repeatedly), high speed centrifugation (3000r/min, 25min) after, get supernatant dialysis 48 hours, filtration sterilization obtains PF; The 2nd, ultrafiltration: Placenta Hominis shreds, and adds 2 times of normal saline, tissue homogenate, and (8000r/min 20min), gets supernatant and carries out ultrafiltration and obtain PF through high speed centrifugation.Physicochemical property discovers that the PF outward appearance is little yellow transparent liquid, and the protein qualitative reaction is negative, and is a kind of mixture of micromolecule polypeptide class material, and the SDS-polyacrylamide gel electrophoresis confirms that its molecular weight is 3000-5000D.Further biological activity research is found, PF is in external recovery, the inductive T lymphopoiesis of promotion PHA, promotion lymphocytic emiocytosis IL-2 and the lymphocytic cell surface IL-2 receptor expression that can promote T lymphocytic cell surface sheep erythrocyte receptor; Experiment also finds that PF can strengthen phagocytosis of macrophages, strengthen the NK cell tumor cell K562 killed and wounded, strengthens the inductive T lymphopoiesis of PHA ability in the animal body.Some scholars use it for diseases such as treatment malignant tumor, viral hepatitis, bronchial asthma and allergic rhinitis and have also obtained good curative effect simultaneously.

Summary of the invention

The purpose of this invention is to provide a kind of placenta factor and preparation method thereof.

Placenta factor provided by the present invention can prepare in accordance with the following methods:

1) Placenta Hominis is shredded, carrying out centrifugal force and be 1770～2991.3g (10000～13000 changes/min) tissue homogenate, after placing 25-40 ℃ of water-bath to hatch 0.5～2.5 hour the homogenate that obtains, (supernatant is got in 800～1500 commentaries on classics/min) centrifugal 20～40 minutes at 4-10 ℃ of 113.28～398.25g;

2) supernatant that step 1) is obtained carries out the ultrafiltration that molecular cut off is 10KD, and the ultrafiltration product that obtains is placenta factor.

Described placenta tissue can homogenate in normal saline.

The centrifugal force of described tissue homogenate be preferably 2548.8g (12000 change/min).

Described homogenate preferably places 37 ℃ of water-baths to hatch 1 hour.

Described homogenate is hatched the back in 4-10 ℃ of 113.28g (800 commentaries on classics/min) centrifugal 30 minutes.

Described Placenta Hominis behaviour Placenta Hominis.

In the said method, also comprising step 2) placenta factor that obtains carries out the step of sterilization treatment, as the ultrafiltration product being carried out the filtration sterilization in 220 μ m apertures.

The PF outward appearance of the present invention's preparation is little yellow transparent liquid, pH 6.5～7.5, protein is qualitative to be negative, it is 5.7～6.9mg/g fresh weight Placenta Hominis that the Bradford method is measured the polypeptide quality, SDS-PAGE method measurement result shows that the PF that the present invention prepares mainly contains two kinds of compositions, and molecular weight is respectively 9.187KD and 4.794KD.

Another object of the present invention provides a kind of medicine that prevents and/or treats transplant rejection and graft versus host disease.

The medicine that prevents and/or treats transplant rejection and graft versus host disease provided by the present invention, its active component is the placenta factor of the present invention's preparation.

When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.The dosage form of medicine of the present invention is preferably injection or injectable powder.

The described medicine that prevents and/or treats transplant rejection and graft versus host disease can import body by injecting method.

In preparation method of the present invention, increased by 37 ℃ of steps of hatching, and reduced centrifugal rotational speed (reducing to 800 rev/mins) by high speed centrifugation (as 8000 rev/mins) in the past, these are different with in the past method.PF outward appearance of the present invention is little yellow transparent liquid, and pH 6.5～7.5, and protein is qualitative to be negative, and it is 5.7～6.9mg/g fresh weight Placenta Hominis that the Bradford method is measured the polypeptide quality, and this was to bibliographical information was similar in the past.But SDS-PAGE shows the PF of the present invention's preparation and mainly contains two kinds of compositions that molecular weight is respectively 9.187KD and 4.794KD, this and the past bibliographical information different (Bao Dao PF was the mixtures of polypeptides of molecular weight less than 5KD in the past).This may be because 37 ℃ hatch and low-speed centrifugal more helps keeping the higher active component of molecular weight among the PF.By research, confirm that also the PF that the present invention prepares has and different in the past biologic activity in addition to its biologic activity.It is external in vivo all to have powerful immunosuppressive action to the T lymphocyte, after using in the PF body, does not have tangible T lymphocyte removal effect, but propagation and activation capacity by reducing the T cell, induces CD4 ⁺CD25 ⁺Regulatory T cells and CD8 ⁺CD28 ^-The number of ways such as secretion of the generation of suppressor T lymphocyte, adjusting Th1/Th2 cytokine are induced the immunity of organism tolerance, and its effect is better than traditional immune suppressive cyclosporin A.Simultaneously, PF can improve the quantity of interior NK cell of body and NKT cell, and promotes the NK cell external killing and wounding tumor cell.Subsequently, in mice allogeneic bone marrow transplantation model, confirm further that also PF can prevent the generation of GVHD, the order of severity that alleviates GVHD, reduction to transplant mortality of mice, application helps transplant mice donor hematopoietic cell and lymphocytic implantation in addition in the PF body in addition, and above-mentioned effect all obviously is better than ciclosporin A.These results suggest, PF is in inducing immune tolerance, can not influence the immunologic reconstitution after the transplanting, and may be by improving the killing activity of NK cell, and the quantity of NK cell and NKT cell keeps the resistance of body to pathogen and tumor, simultaneously can also promote donor hemopoietic and lymphocytic implantation, this will provide new approach for the effect that improves clinical organ transplantation and hematopoietic stem cell transplantation.

The present invention prepares the method for PF, abundant raw material, and preparation method is simple, good stability.Placenta factor of the present invention is the micromolecule polypeptide class material that extracts from human placenta, side effect is little, safe in utilization, be with a wide range of applications, can in organ transplantation and hematopoietic stem cell transplantation, be used to prevent and/or treat transplant rejection and graft versus host disease (GVHD).

Description of drawings

Fig. 1 is the SDS-PAGE electrophoresis result of PF

Fig. 2 is that PF is to the inductive lymphopoietic result that influences of PHA

Fig. 3 is the influence result of PF to mixed lymphocyte reaction

Fig. 4 is the influence result of PF to T cell CD69 expression

Fig. 5 is the influence result of PF to NK cell killing tumor cell

Fig. 6 is the influence result of PF to the mice body weight

Fig. 7 is the influence result of PF to the mice peripheral blood leucocyte

Fig. 8 is PF to the result that influences of the mouse spleen lymphocyte propagation of ConA and allogene antigen induction

Fig. 9 is that PF is to T cell, CD4 ⁺And CD8 ⁺T cell quantity and CD4 ⁺/ CD8 ⁺T cell ratio influence the result

Figure 10 is the influence result of PF to mouse spleen T cell and each subgroup CD28 expression thereof

Figure 11 is that PF is to mouse spleen CD4 ⁺CD25 ⁺Regulatory T cells and CD8 ⁺CD28 ^-Suppressor T lymphocyte quantity influence the result

Figure 12 is the influence result of PF to mouse spleen NK cell and NKT cell quantity

Figure 13 is the influence result of PF to IFN-γ and IL-4 in the MLR culture supernatant

Figure 14 is for respectively organizing the incidence rate of mice GVHD after the bone marrow transplantation

Figure 15 is for respectively organizing the survival curve of mice after the bone marrow transplantation

Figure 16 A is three groups of mice GVHD time of origins and the comparison of sending out case load

Figure 16 B is the comparison of three groups of mouse diing times and dead routine number

Figure 17 A is the variation of respectively organizing the mice body weight after the bone marrow transplantation

Figure 17 B is the variation of respectively organizing mice peripheral blood leucocyte counting after the bone marrow transplantation

Figure 18 is three groups of mice donor hematopoietic cells and lymphocytic implantation situation

Figure 19 is 100 times of HE dyeing photos of three groups of mouse livers and small intestinal pathological change

The specific embodiment

Experimental technique among the following embodiment if no special instructions, is conventional method.

Embodiment 1, placenta factor (placenta factor, preparation PF)

Material is healthy puerpera's Placenta Hominis.The antenatal capable hepatitis B virus of puerpera, hepatitis C virus, HIV, toxoplasma and syphilis detect and is negative patient is healthy puerpera.

Fresh human placenta is removed the fascia blood vessel, wash repeatedly,, be cut into 1 * 1cm to remove the blood that residues in the matter fully with normal saline ²The fritter of size, adding be the normal saline of 2 times of volumes of placenta tissue carry out high-speed homogenization (12000 rev/mins, i.e. 2548.8g, 3 minutes/time, 10 times), homogenate places 37 ℃ of water-baths to hatch 1 hour, and 4 ℃ of low-speed centrifugals are (800 rev/mins then, be 113.28g) 30 minutes, supernatant is placed AmiconUltra ultrafiltration pipe (molecular cut off 10KD), carry out centrifugal ultrafiltration, Millipore filter (220 μ m) the filtration sterilization packing of ultrafiltration afterproduct, obtain PF, 4 ℃ of preservations.And prepared PF carried out the research of physicochemical property, the result is as follows:

(1), protein qualitative test

Adopt heating method of acetic acid and 20% sulfosalicylic acid method to carry out the protein qualitative test.The result shows that the PF outward appearance is little yellow transparent liquid, and pH 6.5～7.5.Heating method of acetic acid and 20% sulfosalicylic acid method protein are qualitative negative.

(2), the mensuration of polypeptide quality (Bradford method)

Adopt the Bradford method to carry out the mensuration of polypeptide quality: the protein standard substance of preparation variable concentrations, add the Bradford dye liquor, abundant mixing, room temperature was placed 2 minutes, measured the optical density value OD of 590nm wavelength place solution then _590nmConcentration with protein example is vertical coordinate, and optical density value is an abscissa, draws standard curve.Get testing sample 500 microlitres, become 1ml, add the Bradford dye liquor, measure OD as stated above with distilled water diluting _590nmAnd find the concentration (being the polypeptide mass concentration) of testing sample from standard curve.Polypeptide quality (mg/g)=when mensuration (polypeptide mass concentration * extracting solution cumulative volume/sample volume)/placenta tissue quality).

With standard protein quality sample (bovine serum albumin, OD BSA) _590nmMass concentration mapping to the standard protein quality sample obtains regression equation, polypeptide mass concentration (y)=-0.459+1.608OD _590nm(x), R ²=0.847, p=0.001 is according to the OD of testing protein quality sample _590nm, the polypeptide quality of obtaining PF by regression equation is 5.70～6.86mg/g fresh weight Placenta Hominis (6.28 ± 0.58mg/g).

(3), the mensuration of various component molecular weight

Use the mensuration that SDS-polyacrylamide gel electrophoresis (SDS-PAGE) carries out polypeptide molecular weight.Separation gel, gap glue and concentrated glue are respectively: 15.5%T (T represents acrylamide concentration) 6%C (C represents the acrylamide degree of cross linking) (including 6mol/mL carbamide), 10%T 3%C and 4%T 3%C.Ultra-low molecular amount protein and peptide standard molecule weight range: 14.4KD-3.313KD.

The SDS-PAGE electrophoresis result shows that PF is the mixture of one group of micromolecule polypeptide, and it mainly contains two kinds of compositions (Fig. 1).Migration distance with ultra-low molecular amount protein and peptide standard is mapped to the logarithm of ultra-low molecular amount protein and peptide standard molecular weight, obtains regression equation, molecular weight logarithm (y)=4.286-0.269 (x) (migration distance), R ²=0.950, p=0.025 according to the migration distance of testing protein quality sample, obtains each component relative molecular weight of PF and is respectively 9.187KD and 4.794KD in equation; While is 3.158KD (theoretical value is 3.108KD) through the molecular weight that regression equation calculation goes out alpha-thymulin.Among Fig. 1,1. ultra-low molecular amount protein and peptide standard (molecular weight ranges is 14.4KD to 3.313KD), 2. alpha-thymulin, 3.PF.

The biological activity of the PF of embodiment 2, the present invention's preparation

(1), extracorporeal biology activity research

1, to the inductive lymphopoietic influence of PHA

Human peripheral blood single nucleus cell (PBMC) inoculation 96 orifice plates, the phytohaemagglutinin (PHA) and different preparations of adding final concentration 10 μ g/ml.Test divides three groups: different dilution PF (1/1,1/2,1/4,1/6,1/8 and 1/10 dilution), ciclosporin A (the cyclosporin A of variable concentrations, CsA) (0.8,0.6,0.4,0.2,0.1 and 0.05mg/ml) and normal saline (normal saline NS), sets up culture medium simultaneously and does not add the control wells of PHA.In 37 ℃ of 5%CO ₂Cultivated 68 hours in the incubator, use mtt assay and detect, and calculate inhibitory rate of cell growth (growth inhibition ratio (%)=1-(test hole OD value/non-stimulated hole OD value) * 100).

The result as shown in Figure 2, show that PF has the obvious suppression effect to the inductive lymphopoiesis of PHA, be dose-dependence, increase with the PF dilution factor, inhibitory action weakens (each dosage group and NS group be P＜0.0001 relatively) gradually, wherein 1/1,1/2 and 1/4 3 dosage group and CsA group relatively (is respectively 70.03%vs 67.91%, P=0.401 in P＞0.05; 67.80%vs 67.23%, P=0.744 and 59.33%vs 67.34%, P=0.104).Among Fig. 2, n=10, * represent to compare P＜0.05 with the NS group.The above results shows that PF is suitable at the effect and the CsA of the inductive T cell proliferation of vitro inhibition PHA.

2, to the influence of mixed lymphocyte reaction:

Different volunteers' PBMC is respectively as reacting cells and irritation cell, and irritation cell is handled with ametycin (final concentration 60 μ g/ml), and reacting cells and irritation cell are inoculated 96 orifice plates in 1: 1 ratio, adds different preparations.The grouping situation is with step 1, and sets up the control wells of simple reacting cells, simple irritation cell and simple culture medium.In 37 ℃ of 5%CO ₂Cultivated 6 days in the incubator, use mtt assay and detect, and calculate inhibitory rate of cell growth (same step 1).

The result shows that PF also has significant inhibitory effect to mixed lymphocyte reaction as shown in Figure 3, also is dose-dependence, increases with the PF dilution factor, and inhibitory action weakens (each dosage group and NS group be P＜0.0001 relatively) gradually.Wherein 1/1,1/2,1/4 and 1/6 4 dosage group and CsA group relatively (is respectively 78.90%vs 72.96%, P=0.708 in P＞0.05; 78.70%vs 73.43%, P=0.599; 73.04%vs 73.54%, P=1.000 and 66.02%vs 74.25%, P=0.469).Among Fig. 3, n=10, * represent to compare P＜0.05 with the NS group.The above results shows that PF is suitable at the effect and the CsA of vitro inhibition mixed lymphocyte reaction.

3, Buddhist ripple ester (PMA)+ionomycin stimulates the influence that back T cell activation antigens c D69 expresses:

PBMC inoculates 24 orifice plates, and every hole adds PMA (final concentration 200ng/ml), ionomycin (final concentration 2 μ g/ml) and different preparation.The grouping situation is with step 1, and sets up negative control hole.In 37 ℃ of 5%CO ₂Cultivated in the incubator 16-18 hour, (negative control adds anti-people CD3-FITC and homotype contrast-PE to the by specification traget antibody; All the other each specimen add anti-people CD3-FITC and anti-people CD69-PE), use flow cytometer and detect CD3 ⁺The expression of cell CD69.The expression of CD69 is with CD69 ⁺CD3 ⁺Cell is at CD3 ⁺Percentage ratio in the cell is represented.

The result shows that PF stimulates the expression of back T cell activation antigens c D69 to have tangible downward modulation effect to the PMA+ ionomycin as shown in Figure 4, is dose-dependence, increases with the PF dilution factor, and effect weakens (each dosage group of PF and NS group be P＜0.05 relatively all) gradually.Wherein 1/1,1/2 and 1/4 3 dosage group CD69 expression rate is less than or equal to the CsA group and (is respectively 19.58%vs 39.53%, P=0.002; 35.53%vs 39.75%, P=0.394 and 46.64%vs 39.70%, P=0.230).N=8 among Fig. 4, * represent relatively P＜0.05 of PF and NS group; # represents relatively P＜0.05 of PF and CsA group.The above results shows, but PF and CsA are in external all suppressor T cell activation (PF vs CsA, P＞0.05).

4, to the influence of the killing activity of NK cell killing tumor cell:

PBMC inoculates 24 orifice plates and is the effector lymphocyte, adds different preparations.Test divides three groups: and CsA of different dilution PF (1/1,1/2,1/4,1/6,1/8 and 1/10), variable concentrations (0.2,0.1 and 0.05mg/ml) and normal saline (normal saline, NS).37 ℃ of 5%CO ₂Incubator was cultivated 12 hours, took out the effector lymphocyte, again counting inoculation 96 orifice plates.The K562 cell strain is inoculated 96 orifice plates as target cell, imitates target than 20: 1.And set up the maximum release aperture of the spontaneous release aperture of effector lymphocyte, the spontaneous release aperture of target cell, target cell, volume correction release aperture and culture medium blank hole.All the other step by specifications carry out, and microplate reader detects OD ₄₉₀And calculating cell killing rate.

Kill rate (%)=(A-B-C)/(D-C) * 100.

A=test hole OD value-blank hole OD value

The spontaneous release aperture OD value of B=effector lymphocyte-blank hole OD value

The spontaneous release aperture OD value of C=target cell-blank hole OD value

The maximum release aperture OD of D=target cell value-volume correction hole OD value.

The result shows that PF group NK cell killing rate all is greater than or equal to the NS group as shown in Figure 5, and wherein 1/4,1/6 and 1/8 3 dosage group NK cell killing rate is higher than the NS group and (is respectively 32.32%vs 15.23%, P=0.000; 22.73%vs15.23%, P=0.011 and 21.30%vs 15.23%, P=0.038); And 1/1,1/2 and 1,/10 three dosage group NK cell killing rate and NS group comparing difference not statistically significant (are respectively 14.80%vs 15.23%, P=0.880; 16.48%vs 15.23%, P=0.663 and 18.96%vs 15.23%, P=0.199).And CsA group is starkly lower than the NS group and (is respectively 5.02%vs 15.23%, P=0.000; 6.69%vs 15.23%, P=0.004; 7.88%vs 15.23%, P=0.013).The above results shows that different with CsA, the external application of PF does not influence even can promote NK cell killing and wounding tumor cell.

(2), biological activity research in the body

The Balb/c mice is divided into three groups at random, 12 every group.Difference lumbar injection PF (20ml/kg.d), CsA (30mg/kg.d) and NS (0.4ml//day), continuous 14 days, the 15th day execution mice.Carry out following biological activity assay:

1, PF is to the influence of mice body weight and peripheral blood leucocyte counting

Body weight and the peripheral blood leucocyte counting of mice respectively organized in next day monitoring.The result of variations of body weight shows that application does not have obvious influence to the mice body weight in the PF body as shown in Figure 6, and this group mice body weight fluctuates in 20g always; And cause after using in the CsA body that mice weight loss, mice feed reduce, the movable minimizing and spiritual variation.The result of variations of peripheral blood leucocyte shows and uses in PF and the CsA body all to the obviously influence of mice peripheral blood leucocyte counting nothing as shown in Figure 7.Among Fig. 6 and Fig. 7, n=12.

2, PF is to the influence of the inductive mouse spleen lymphocyte of ConA propagation:

Mice takes off cervical vertebra puts to death, and separating spleen grinds spleen in glass homogenizer, make splenocyte suspension, uses the hemolysin lysed erythrocyte, and adjusting concentration is 1 * 10 ⁶Individual/ml is standby.

Splenocyte is inoculated 96 orifice plates, and adding final concentration is the ConA of 4 μ g/ml, and sets up simple culture medium to reach the control wells that does not add ConA.37 ℃ of 5%CO ₂Incubator was cultivated 68 hours, and mtt assay detects, and calculated stimulation index (SI) (SI=test hole OD value/NS organizes non-stimulated hole OD value).

PF influences the result as shown in Figure 8 to the inductive mouse spleen lymphocyte propagation of ConA, shows under the resting state PF group mouse spleen lymphocyte OD _570nmBe lower than NS group (0.384vs 0.583, p=0.001) and the CsA group (0.384vs 0.424, p=0.408).And after ConA stimulates, PF group stimulation index be lower than the NS group (0.890vs 2.139, p=0.008) and the CsA group (0.890vs 1.312, p=0.423).The The above results explanation, effect and CsA that PF suppresses resting stage and the inductive spleen lymphocyte proliferation activity of ConA in vivo are suitable.Among Fig. 8, n=12; Resting stage, cell-proliferation activity was with OD ₅₇₀Expression; The cell-proliferation activity of ConA and allogene antigen induction is represented with stimulation index (SI); * expression is compared P＜0.05 with the NS group.

3, PF to mixed lymphocyte reaction (mixed lymphocyte reactions, influence MLR):

Balb/c mice (H-2 ^d) and C57BL/6 mice (H-2 ^b) splenocyte inoculates 96 orifice plates, mixing with 1: 1 ratio.Set up simple reacting cells, simple irritation cell and simple culture medium control wells simultaneously.37 ℃ of 5%CO ₂Incubator was cultivated 6 days, and mtt assay detects, and calculated stimulation index (with step 2).The result as shown in Figure 8, show and PF group stimulation index be lower than the NS group (0.586vs 1.651, p=0.000) and CsA group (0.586vs 0.608, and p=0.801), these results show that PF suppresses suitable by the effect and the CsA of the mouse spleen lymphocyte propagation of allogene antigen induction.Among Fig. 8, n=12; Resting stage, cell-proliferation activity was with OD ₅₇₀Expression; The cell-proliferation activity of ConA and allogene antigen induction is represented with stimulation index (SI); * expression is compared P＜0.05 with the NS group.

4, PF is to T cell and each subgroup thereof, suppressor T lymphocyte (CD4 ⁺CD25 ⁺And CD8 ⁺CD28 ^-The T cell), NKT (NK) cell and natural killer T (NKT) cell quantity, the influence that T cell surface costimulatory molecules CD28 expresses:

Adopt fluorescently-labeled specific antibody, splenocyte is analyzed, extracting spleen cell, every pipe contains cell number 2 * 10 ⁶Individual/ml, by specification adds CD3 ε, CD4, CD8 α, CD28, CD25, Pan-NK 1.1 antibody respectively, 4 ℃ of lucifuge 30min, and re-suspended cell can be gone up machine testing, or fixes with 1% paraformaldehyde, and 4 ℃ of preservations detect in the 24h.

PF is to T cell, CD4 ⁺And CD8 ⁺T cell quantity and CD4 ⁺/ CD8 ⁺T cell ratio influence the result as shown in Figure 9, show PF group T cell, CD4 after the medication ⁺And CD8 ⁺T cell number average (is respectively 43.88vs31.78, P=0.004 apparently higher than the CsA group; 29.06vs 18.97, P=0.004; 8.71vs 5.96, P=0.026), and organize zero difference (P＞0.05) with NS.PF organizes CD4 ⁺/ CD8 ⁺T cell ratio also is higher than CsA and NS group (but P＞0.05).The result shows that different with CsA, PF does not have T cell removal effect in vivo, but induces the immunity of organism tolerance by the function of suppressor T cell.Among Fig. 9, n=12; # represents to compare P＜0.05 with the CsA group.

PF influences the result as shown in figure 10 to T cell and each subgroup CD28 expression thereof, PF group T cell, CD4 after the medication ⁺T and CD8 ⁺The expression of T cell surface CD28 all is starkly lower than CsA and the NS group (relatively is respectively with the CsA group: 20.29vs27.16, P=0.006; 13.52vs 18.58, P=0.034; 4.67vs 8.52, P=0.024.And relatively be respectively with the NS group: 20.29vs 30.46, P=0.001; 13.52vs 20.05, P=0.011; 4.67vs 8.71, P=0.017).This result shows the expression that application can be reduced T cell and each cell subset costimulatory molecules thereof in the PF body, thus the inducing T cell incapability, and effect is better than CsA.Among Figure 10, n=12; The expression of CD28 is with CD28 ⁺The absolute quantity (* 10 of cell ⁶Individual/l) expression; * represent relatively P＜0.05 of PF and NS group; # represents relatively P＜0.05 of PF and CsA group.

PF is to CD8 ⁺CD28 ^-Suppressor T lymphocyte and CD4 ⁺CD25 ⁺Regulatory T cells quantity influence the result as shown in figure 11, show PF group CD4 after the medication ⁺CD25 ⁺Regulatory T cells and CD8 ⁺CD28 ^-The quantity of suppressor T lymphocyte is all apparently higher than CsA group and NS group (PF group and NS group relatively, CD4 ⁺CD25 ⁺T and CD8 ⁺CD28 ^-The T cell quantity is respectively: 8.81vs4.95, P=0.002; 3.62vs 1.79, P=0.026), the CsA group then is lower than the NS group and (is respectively: 2.83vs4.95, P=0.052; 1.43vs 1.79, P=0.630).This result shows that different with CsA, PF in vivo can be by increasing CD8 ⁺CD28 ^-Suppressor T lymphocyte and CD4 ⁺CD25 ⁺Regulatory T cells is induced the immunity of organism tolerance.Among Figure 11, n=12; * represent relatively P＜0.05 of PF and NS group; # represents relatively P＜0.05 of PF and CsA group.

PF influences the result as shown in figure 12 to NK cell and NKT cell quantity, all (PF group and NS group are relatively apparently higher than NS and CsA group for the quantity that shows PF group NK cell and NKT cell after the medication, NK cell and NKT cell quantity are respectively: 4.8vs 2.08, P=0.002; 1.96vs 0.81, P=0.000).CsA group NK cell quantity and NS group compare no significant difference, and (2.32vs 2.08, and P=0.682), (0.41vs 0.81, P=0.000) but CsA group NKT cell then is starkly lower than the NS group.Among Figure 12, n=12; * represent relatively P＜0.05 of PF and NS group; # represents relatively P＜0.05 of PF and CsA group.

5, the mensuration of MLR culture supernatant IL-4, IFN-γ level:

MLR the 6th day, the centrifuging and taking supernatant is used the BioSource mouse cell factor ELISA of company test kit and is detected IL-4 and IFN-γ level.The result as shown in figure 13, show PF group IFN-γ level after the medication be the NS group 1/4 (42.33pg/ml vs 179.86pg/ml, P=0.000), and be lower than the CsA group (42.33pg/ml vs51.61pg/ml, P=0.603); And the IL-4 level be 2.5 times of NS group (110.8pg/ml vs 45.47pg/ml, P=0.006), and be higher than the CsA group (110.8pg/ml vs 62.52pg/ml, P=0.031).The result shows that application can obviously reduce the secretion (as IFN-γ) of Th1 cytokines in the PF body, and promotes the secretion (as IL-4) of Th2 cytokines, and the interior Th1 type cell of body is offset to Th2 type cell.Among Figure 13, n=12; * expression is compared P＜0.05 with the NS group; # represents to compare P＜0.05 with the CsA group.

(3), PF is to the influence of mice allogeneic bone marrow transplantation (allo-BMT) back graft versus host disease (GVHD)

Experimental technique is as follows:

1, prepared by Mus before the transplanting

Mice is raised in the Experimental Animal Center SPF of The People's Hospital of Peking University level Animal House.7d begins to drink the antibiotics solution that contains gentamycin (320U/L) and erythromycin (250mg/L) and carries out the intestinal preparation before transplanting, and lasts till and transplants 4 weeks of back.4-6h warp before transplanting ⁶⁰Co-gamma-rays total irradiation (Department Of Medicine, Peking University's radiation chamber), irradiation source are shone accumulated dose 8.0Gy apart from irradiated plane 150cm, and close rate is 2.967Gy/min, irradiation time 2 minutes and 36 seconds.

2, transplant grouping

Be subjected to Mus to be divided into 4 groups of A, B, C and D at random postradiation BaLB/C and organize, except that 7 of A groups, other respectively organize equal 22.B, C and D group mice began the different preparations (PF, CsA and NS) of lumbar injection respectively the same day from transplanting, and continuous 14 days, injected dose was: PF (20ml/kg.d), CsA (30mg/kg.d) and NS (0.4ml//day).

A group (simple irradiation group): the irradiation not all right transplanting in back.

B organizes (PF transplantation group): feed back for Os Mus marrow and splenocyte simultaneously continuous lumbar injection PF 14 days.

C organizes (CsA transplantation group): feed back for Os Mus marrow and splenocyte simultaneously continuous lumbar injection CsA 14 days.

D organizes (NS matched group): feed back for Os Mus marrow and splenocyte simultaneously continuous lumbar injection NS 14 days.

3, supply the separation of Os Mus myelocyte and spleen cell

Take off cervical vertebra for Mus and put to death aseptic clip mice tibia, femur and spleen.Cut off metaphysis, connect No. seven syringe needles with the syringe that RPMI1640 liquid is housed and wash medullary cavity repeatedly, till medullary cavity bleaches, collect medullary cell and cross syringe needle No. four, make bone marrow cell suspension.Get for the Mus spleen and make splenocyte suspension (the same step 2 of method).Above-mentioned medullary cell and spleen cell detect cell viability 〉=95% with 4% trypan blue staining method, and be standby.

4, transplant

Except that simple irradiation group, other is respectively organized every and is subjected to Mus, contains for Mus 2 * 10 through the input of tail vein in 4-6 hour in irradiation back ⁸The medullary cell of individual cell/kg and 2 * 10 ⁸The mixing suspension 0.4～0.5ml of the splenocyte of individual cell/kg.The ratio of medullary cell and splenocyte is 1: 1.

5, existence and dead criterion

Transplant interior death of 2 weeks of back and WBC＜0.5 * 10 ⁹/ L is hemopoietic depletion.

WBC＞1.0 * 10 ⁹/ l, and have asthenia, lose hair or feathers, stick up hair, suffer from diarrhoea, lose weight, the back of a bow even dead, dead mouse liver and small intestinal meet GVHD pathological change person and are the GVHD generation.

Was long term survival with existence greater than 60 days.

6, observation index behind the mice allo-BMT

(1) ordinary circumstance:

Observe and transplant back mice body weight, spirit, the bodily form, position, hair, have or not diarrhoea etc.And each group mice carried out GVHD scoring, but be GVHD greater than person's clinical diagnosis in 4 fens, be severe GVHD greater than 6 fens persons.

(2) life cycle and death:

Except that 7 mices of A group, all the other every group each 16 are used for life cycle and mortality rate observation (observe always and transplanted back 60 days), and the residue mice does medullary cell and the splenocyte chimera detects.

(3) hemopoietic function:

Respectively organize the mice docking next day of transplanting the back and get blood 10 μ l, join in the numeration of leukocyte liquid of 10 times of dilutions, observe and respectively organize the situation that leukocyte recovers in the peripheral blood.

(4) the chimeric detection of bone marrow and splenocyte:

Transplant back each group of execution in the 14th, 21 day and be subjected to Mus (each 3), get bone marrow and spleen, make medullary cell and spleen cell suspension respectively, applying flow cytometry detects H-2D in medullary cell, the spleen cell ^bThe percentage ratio of positive cell can react Mus hematopoietic cell and the lymphocytic implantation situation of being subjected to.

(5) pathomorphism of GVHD:

Each group was observed 60 days altogether, and the mice of GVHD performance occurs and all get liver, intestinal tissue before dying, be that 10% formalin is fixed with volume fraction, routine paraffin wax embedding, section, HE dyeing, light microscopic is observation down.The mice of long-term surviving is got liver before putting to death, intestinal tissue is done pathological observation.

Experimental result is as follows:

1, transplant back survival time of mice, mortality rate and the GVHD observed result that a situation arises and show that the beginning in the 4th day after irradiation of 7 mices of A group is dead, all dead in 12 days, all die from hemopoietic depletion, life span (8.143 ± 1.056) sky.

In the D group mice 11 in transplanting the back began to occur on the 4th day that asthenia, activity obviously reduce, few food, alarm symptoms such as hair, diarrhoea, weight loss and the serious back of a bow (the GVHD scoring is 7.18 ± 1.33), above-mentioned symptom appearred on the 6th day in 5 mices of surplusing after transplanting.The meta disease time of GVHD is for transplanting the back the 4th day, and the incidence rate of transplanting back 14 days GVHD is 100% (Figure 14 and table 1).D group mice is in transplanting back beginning death in the 5th day, and dead middle bit time is 8.5 days, and is all dead in 19 days, life span (10.00 ± 1.268) day (Figure 15 and table 2).

C group mice began to occur above symptom (the GVHD scoring is 6.07 ± 0.83 minutes) on the 6th day in transplanting the back.The meta disease time of GVHD is 6 days, and the incidence rate of transplanting back 14 days GVHD is 100% (seeing Figure 14 and table 1).C group mice is dead in transplanting back beginning in the 8th day, dead middle bit time is 14.5 days, all dead in 25 days, life span (14.938 ± 1.453) sky, (14.94vs 10.00 to be longer than D group mice, but be significantly shorter than B group mice (14.94vs 48.15, P=0.000) (see Figure 15 and table 2) P=0.015).

16 mices of B group have 5 light activity minimizing, weight loss to occur on the 8th day after transplanting, alarm hair (the GVHD scoring is 4 minutes), wherein there are 2 in 29 days, to die from GVHD (the GVHD scoring is 6 minutes when dead, the back of a bow and towering hair when showing as weight loss, light activity minimizing, tranquillization), survive in the observation period for other 3 always, and as time passes, above-mentioned symptom alleviates disappearance gradually.Other has 1 mice to occur above-mentioned symptom (the GVHD scoring is 4 minutes) on the 26th day after transplanting, and with laxativeness, in transplanting back death in the 31st day when dead (GVHD scoring be 5 minutes).The back of a bow reached and alarms a hair phenomenon when all the other had 4 mices slightly depilation of abdominal part, weight loss, light activity minimizing, tranquillization to occur after 30 days, and respectively at the death in the 47th, 58,43 and 40 day of transplanting back (the GVHD scoring is 4.75 ± 1.26 minutes during death).The meta disease time of B group mice GVHD is 46 days, obviously is later than C group and D group (P＜0.0001).Simultaneously B group mice GVHD sickness rate also is starkly lower than C and D group (P＜0.0001), and the incidence rate of transplanting back 14 days GVHD is 0,30 day to be 18.7%, 45 day to be 39.1%, 60 day to be 49.2% (see Table 1 and Figure 14).Have 14/16 mice time-to-live greater than 30 days.6/9 survival time of mice was greater than 45 days, and 2/5 survival time of mice was greater than 60 days.Assessing the visible B group of the survival curves of four groups of mices mice with Kaplan-Meier respectively organizes life span than other and obviously prolongs, mean survival time is (48.145 ± 3.382) sky, and comparing (A, C, D group) difference in twos with other each group has significance (P＜0.0001).B group mice 30 days, 45 days and 60 days survival rates are all apparently higher than C group and D group (P＜0.0001).Wherein 30 days survival rates are respectively 87.5%, 0%, 0%, 45 day survival rate and are respectively 60.9%, 0%, 0%, 60 day survival rate and are respectively 30.5%, 0%, 0% (see Table 2 and Figure 15).

B group GVHD scoring is starkly lower than D group (P＜0.0001).Wherein, transplanting back 7 days is 2.375vs 7.00, P=0.000; Transplant and be 3.125vs 8.111, P=0.000 in back 14 days; Transplant and be 1.5vs 8.5, P=0.000 in back 21 days.And B group GVHD scoring also is starkly lower than C group (P＜0.01), is respectively: transplanting back 7 days is 2.375vs 6.31, P=0.000; Transplant and be 3.125vs 7.38, P=0.000 in back 14 days; Transplant and be 1.5vs 7.86, P=0.000 in back 21 days; Transplant and be 1.875vs 8.5, P=0.001 in back 30 days.And C group GVHD scoring is lower than the D group, but difference not statistically significant (seeing Table 3).

Figure 16 A has shown that time and the routine number of GVHD clinical manifestation appear in three groups of mices, the time that the GVHD symptom appears in visible B group mice among the figure obviously is later than C group and D group, and (the meta disease time of three groups of mice GVHD is respectively: 6 days vs of 46 days vs 4 days) and the time that GVHD appears in C group mice and D organize no significant difference; And the routine number of B group mice generation GVHD obviously is less than the C group and (is respectively: 7vs 16vs 16) with the D group.Figure 16 B has shown that dead time and dead routine number appear in three groups of mices, visible B group mouse diing time obviously is later than C group and D group among the figure, and not obvious (three groups of mice meta death times are respectively: 14.5 days vs of 40 days vs 8.5 days) and C group mouse diing time and D organize difference; And B organizes, and dead mice example number obviously is less than the C group and the D group (is respectively: 7vs 16vs 16).Among Figure 16 A and Figure 16 B, n=16.

Application can obviously reduce generation and the relevant death of GVHD of allogeneic bone marrow transplantation chmice acute GVHD in the The above results prompting, PF body, and alleviates the order of severity that is subjected to Mus GVHD, prolongs to be subjected to Mus life cycle, improves overall survival rate.And above-mentioned effect all is better than CsA (P＜0.01).

Among Figure 14, n=16; * expression is organized (NS transplantation group) relatively P value with B group (PF transplantation group) with D; # represents that B group and C group (CsA transplantation group) compare the P value; ﹠amp; Expression C group compares the P value with the D group.The A group is 7 mices among Figure 15; All the other each groups are 16 mices.* expression is organized (NS transplantation group) relatively P value with B group (PF transplantation group) with D; # represents that B group and C group (CsA transplantation group) compare the P value; ﹠amp; Expression C group compares the P value with the D group.

Respectively organize the mice GVHD comparison that a situation arises (n=16) behind the table 1.Allo-BMT

Annotate: ^*Expression B group (PF transplantation group) is organized (NS transplantation group) relatively P＜0.01 with D, and # represents that B group and C group (CsA transplantation group) compare P＜0.01

Respectively organize mice existence situation relatively behind the table 2.Allo-BMT

Annotate: ^*Expression B group (PF transplantation group) is organized (NS transplantation group) relatively P＜0.01 with D, and # represents that B group and C group (CsA transplantation group) compare P＜0.01.

Respectively organize the comparison (n=16) of mice GVHD scoring behind the table 3.Allo-BMT

2, respectively organize mice body weight change and numeration of leukocyte result after the transplanting shown in Figure 17 A, Figure 17 B and table 4, show that A group mice continues decline, peripheral blood leucocyte＜0.5 * 10 in transplanting the back body weight ⁹/ L, dead successively about 1 week.

B group mice is reopened beginning decline in transplanting back 2 days bodies, reduces to 15.74g gradually by average 20.11g, gos up to 20.7g gradually again since the 10th day.Peripheral blood leucocyte descended since the 2nd day, reached minimum point 0.3 * 10 on the 4th day ⁹/ L gos up afterwards gradually to normal.From transplanting the back the 6th day, B group mice peripheral blood leucocyte counting all is higher than C group and D group (P＜0.05).Application can promote the hemopoietic of transplanting the back mice to recover in this results suggest, PF body.

C group mice is reopened the beginning and descends in transplanting the 2nd day body in back, reduces to minimum (reducing to 14.89g by average 20.14g) about 14 days, gos up gradually afterwards to the 15g.Leukocyte count descended since the 2nd day, reached minimum point 0.35 * 10 on the 4th day ⁹/ L gos up gradually to normal later on.From transplanting the back the 6th day, C group mice peripheral blood leucocyte counting all is higher than the D group, and (the 6th day: 2.06vs 1.39, P=0.01; The 8th day: 5.42vs 3.34, P=0.054; The 14th day: 10.13vs 3.63, P=0.042).

D group mice body weight began to descend in transplanting the back on the 2nd day, reached minimum point on the 6th day to reduce to 14.47g by average 19.95g, increased to about 17g gradually later on.Peripheral blood leucocyte began to descend on the 2nd day, reached minimum point 0.3 * 10 on the 4th day ⁹/ L gos up to normal level afterwards gradually.N=16 in Figure 17 A, Figure 17 B and the table 4, * represent that B group (PF transplantation group) and D organize (NS transplantation group) relatively P＜0.05, and # represents that B group and C group (CsA transplantation group) compare P＜0.05.

After transplanting, table 4. respectively organizes the comparison (n=16) of mice peripheral blood leucocyte counting.

Annotate: * represents that B group (PF transplantation group) and D organize (NS transplantation group) relatively P＜0.05, and # represents that B group and C group (CsA transplantation group) compare P＜0.05

3, the donor hematopoietic cell is implanted situation

After transplanting, used PE-H-2D respectively in 14 and 21 days ^bMonoclonal antibody detects H-2D in bone marrow and the spleen cell ^B+The ratio of cell.Experimental result is transplanted H-2D in the 14th day B group bone marrow cells in mice of back shown in A among Figure 18 and B ^B+(be respectively: 91.55%vs 68.9%vs 65.13%), C group and D group be P=0.004 relatively apparently higher than C and D group (P=0.000) for the ratio of cell; Organize H-2D in the bone marrow cells in mice and transplant the 21st day B in back ^B+The ratio of cell and C group compare zero difference, and (99.08%vs 98.6%, P=0.379).Simultaneously, experiment is also found, transplants H-2D in the 14th day B group mouse spleen lymphocyte of back ^B+The ratio of cell is higher than D group (P=0.001), but compares no significant difference (P=0.147) with the C group, is respectively 97.7%vs 96.94%vs 94.86%; And the 21st day B organizes H-2D in the mouse spleen lymphocyte after transplanting ^B+The ratio of cell and C group compare no significant difference, and (98.02%vs 98.49%, P=0.507).The above results prompting be subjected to successfully to plant work in the Mus body for Mus hematopoietic cell and lymphocyte in the 21st day after the transplanting, and application can obviously promote donor hematopoietic cell and lymphocytic implantation in the PF body.Among Figure 18, n=3, donor implant situation with H-2D ^B+The percentage ratio of cell is represented.* represent that B group (PF transplantation group) and D group (NS transplantation group) compare P＜0.01; # represents that B group (PF transplantation group) and C group (CsA transplantation group) compare P＜0.01.(note: all dead because of transplanting back the 21st day D group mice, so can't detect the implantation situation of D group mice).

4, pathology change

The GVHD clinical manifestation all appears in D group mice, and pathologic finding is seen a plurality of internal organs atrophys, black, and the atrophy of liver spleen is obvious, liver surface visible point lamellar hemorrhagic focus.Pathological section shows swelling of liver cell, degeneration, the hepatic cords disorder, and portal vein and central vein are obviously expanded, congestion, in hepatic sinusoid and the central portal area a large amount of lymphocytic infiltrations are arranged, and stones in intrahepatic bile duct epithelial cell apoptosis, a large amount of lymphocytic infiltrations are (among Figure 19 a); The intestinal villi necrosis comes off, and the crypts structural deterioration disappears, visible many apoptotic bodies in crypts place and non-viable non-apoptotic cell fragment, and the obvious edema of mucosa and tela submucosa, hyperemia, tela submucosa has a large amount of lymphocytic infiltrations (b among Figure 19).The pathological change degree of this group mouse liver and small intestine is consistent, all meets the diagnosis of GVHD.

C group mice pathological change is organized with D.

B group mice pathological change only is slight inflammatory reaction: c among slight hepatic cell degeneration Figure 19).Mucous membrane of small intestine is complete, Mild edema under the mucosa, and small intestinal has a small amount of lymphocytic infiltration (d among Figure 19).

Pathologic finding prompting PF can alleviate liver, the intestinal injury due to the GVHD.

In a word, biological activity research confirms that PF is external in vivo to have powerful immunosuppressive action to the T lymphocyte.After using in the PF body, do not have tangible T lymphocyte removal effect, but propagation and activation capacity by reducing the T cell, induce CD4 ⁺CD25 ⁺Regulatory T cells and CD8 ⁺CD28 ^-The number of ways such as secretion of the generation of suppressor T lymphocyte, adjusting Th1/Th2 cytokine are induced the immunity of organism tolerance, and its effect is better than traditional immune suppressive cyclosporin A.Simultaneously, PF can improve the quantity of interior NK cell of body and NKT cell, and promotes the NK cell external killing and wounding tumor cell.Subsequently, in mice allogeneic bone marrow transplantation model, confirm further that also PF can prevent the generation of GVHD, the order of severity that alleviates GVHD, reduction to transplant mortality of mice, application helps transplant mice donor hematopoietic cell and lymphocytic implantation in addition in the PF body in addition, and above-mentioned effect all obviously is better than ciclosporin A.These results suggest, PF is in inducing immune tolerance, may not can influence the immunologic reconstitution after the transplanting, and may be by improving the killing activity of NK cell, and the quantity of NK cell and NKT cell keeps the resistance of body to pathogen and tumor, simultaneously can also promote donor hemopoietic and lymphocytic implantation, this will provide new approach for the effect that improves clinical organ transplantation and hematopoietic stem cell transplantation.

Claims

1. method for preparing people's placenta factor may further comprise the steps:

1) Placenta Hominis is shredded, carries out the tissue homogenate that centrifugal force is 1770～2991.3g, place 25-40 ℃ of water-bath to hatch 0.5～2.5 hour the homogenate that obtains after, centrifugal 20～40 minutes of 4-10 ℃ of 113.28～398.25g, get supernatant;

2. method according to claim 1 is characterized in that: the centrifugal force of described tissue homogenate is 2548.8g.

3. method according to claim 1 is characterized in that: described homogenate places 37 ℃ of water-baths to hatch 1 hour.

4. method according to claim 2 is characterized in that: described homogenate places 37 ℃ of water-baths to hatch 1 hour.

5. method according to claim 1 is characterized in that: described homogenate is hatched the back centrifugal 30 minutes of 4-10 ℃ of 113.28g.

6. method according to claim 2 is characterized in that: described homogenate is hatched the back centrifugal 30 minutes of 4-10 ℃ of 113.28g.

7. according to any described method of claim 1-6, it is characterized in that: also comprise in the described method step 2) placenta factor that obtains carries out the step of sterilization treatment.

8. by the placenta factor of the described method of arbitrary claim in the claim 1 to 7 preparation; Described placenta factor contains two peptide species, and molecular weight is respectively 9.187KD and 4.794KD.

9. medicine that prevents and/or treats transplant rejection and graft versus host disease, its active component are the placenta factor of the described method preparation of claim 7.

10. medicine according to claim 9 is characterized in that: the dosage form of described medicine is injection or injectable powder.