CN109752559A - The detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease - Google Patents
The detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease Download PDFInfo
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Abstract
The invention belongs to enteropathy detection technique fields, disclose a kind of detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease, experimental animal and grouping；Model preparation and interfering effects of drug；Sample is collected and processing；Statistical analysis.The anti-MIF monoclonal antibody of first passage of the present invention realizes the blocking to the signal path excessive activation of TLR4/NF- κ B signal access excessive activation; the influence of antiblock and anti-MIF monoclonal antibody to downstreams proinflammatory factors such as MIF, NF- κ B and TNF-α, IL-6 is analysed in depth, determines the blocking effect of anti-MIF monoclonal antibody and to ulcerative colitis protective effect；The blocking effect of monoclonal antibody and to ulcerative colitis protective effect；The microcosmic point of cell factor grid system from UC, determine in the microcosmic point of cell factor grid system set out, determine pivotal role of the NF- κ Factor B in UC inflammatory factor and its be associated with MIF, TNF-α, IL-6.
Description
Technical field
The invention belongs to enteropathy detection technique field more particularly to anti-macrophage migration inhibition factor Antybody therapy inflammation
The detection method of property enteropathy.
Background technique
Currently, the prior art commonly used in the trade is such that
IBD is a kind of enteron aisle chronic nonspecific inflammation disease, including ulcerative colitis (UC) and Crohn disease
(CD), the illness rate of IBD is on the rise at home in recent years, but its pathogenesis not yet illustrates completely so far, there is no sensitivity
Diagnosis index and effective remedy measures.In recent years research thinks that the imbalance between proinflammatory factor and anti-inflammatory factors is both at home and abroad
An important link in IBD pathogenesis, MIF is the important proinflammatory cytokine of one kind, the morbidity for taking part in IBD, with disease
The activity of disease is related, and anti-MIF antibody may open up a new way for the treatment of IBD.And proinflammatory cytokine and anti-inflammatory thin in IBD
The induction and activation of process and nuclear-κB (nuclear factorKappaB, NF- κ B) that intracellular cytokine network is unbalance have
It closes.NF- κ BDNA combines activity significantly raised in existing UC patient's core, NF- κ B be activated and have after entering in core it is very strong and
The premise and key that the ability that gene kB site combines is cell factor transcription and is discharged, it is close with the Disease activity of UC
Correlation may play pivotal role in the disease activity of UC.And most cytokine gene promoters or enhancing sub-portion
Position have NF- κ B binding site, activate into core NF- κ B can transcription in combination and promoting these cell factors, NF- κ B
Being activated can promote a variety of proinflammatory factor expression to increase, such as TNF-α, IL-1 β, IL-6, IL-8, IL-12, existing, NF- κ B's
Activation causes the transcription of TNF-α, IL-1 β to increase, and the release of TNF-α, IL-1 β further activate NF- κ B.The latter is by just
Feedback further increases the secretion of TNF-α, IL-1, while the expression for making other cell factors such as IL-6, IL-8 etc. also increases.By
This generates cascade reaction, makes inflammation constantly amplification-formation " water fall effect ".
NF- κ B and inhibition kappa albumen (inhibitorKappaB, I- κ B) are the weights of inflammatory cytokine gene expression
Want regulatory factor.I- κ B is the obstruction albumen that a molecular weight is 36KD, the nuclear localization signal of NF- κ B can be sheltered, to make it
It is anchored in cytoplasm in the form of inactive complex.Under quiescent condition, NF- κ B with inactive form there are in cytoplasm,
Trimer compositions are formed by p50/p65 and I- κ B, activation relies on the I- kappa b kinase of ubiquitin protein after cell is stimulated, makes I-
2 serine phosphorylations of the end κ B α N- 32 and 36, the I- κ B α after phosphorylation is by ubiquitin protein in multiple site ubiquitin
Change, the I- κ B α of last ubiquitination exposes the nuclear localization signal on Rel albumen by 26S proteasomal degradation, and then activates
NF- κ B enters NF- κ B rapid translocation in core, in conjunction with target gene kB site, induces the transcription of target gene rapidly.
TNF-α and IL-6 are also the important proinflammatory cytokine for participating in IBD morbidity, are clinically successfully applied at present
Anti-TNF-α antibody and anti-IL-6 antibodies treat CD, and there is not been reported for the curative effect country of the anti-MIF antibody to IBD at present, external
It reports rare.MIF is found in delayed immune response earliest, is a kind of important proinflammatory cytokine, is being adjusted
It is played a crucial role in immune and inflammatory reaction, domestic at present is mainly MIF in autoimmune disease, inflammatory disease
The basic research in the directions such as disease, cancer, graft rejection, still shortage clinical data, while MIF is in IBD disease development
In research report it is still few, anti-MIF antibody mainly pass through block MIF expression, reduce the infiltration of macrophage and work, resist
MIF antibody it is expensive, domestic at present is also mainly research report of the antibody in the diseases such as liver cancer, colorectal cancer, breast cancer
Road, and prompt anti-MIF antibody that can inhibit tumor vessel by lowering vascular endothelial growth factor, reducing the approach such as microvessel density
Generation；But in addition to the sent the documents chapter of this seminar, domestic basis or clinical application for anti-MIF antibody in IBD is showed no
Report；Although there are the research of MIF Yu IBD onset relation in foreign countries, it was also proposed that latent effect of the MIF in IBD is treated, about
Research of the anti-MIF antibody in the application and treatment curative effect in the disease lacks report, so we exist about anti-MIF antibody
The basic research of therapeutic effect in IBD not only demonstrates MIF important function in IBD pathogenesis, and it is anti-more to demonstrate anti-MIF
Body therapeutic effect in the disease, and anti-MIF antibody is likely to become applied to the theoretical basis of clinical disease treatment and research foundation stone.
In conclusion problem of the existing technology is: clinically successfully having applied anti-TNF-α antibody at present and resisted
IL-6 Antybody therapy CD, and there is not been reported for the curative effect country of the anti-MIF antibody to IBD at present, external report is rare.
Solve the difficulty and meaning of above-mentioned technical problem:
Difficulty: although the present invention has affirmed anti-MIF antibody to the treatment curative effect of IBD, because being only to a certain extent
Basic research rests on zooscopy level, lacks clinical foundation, and there are the risks such as immunological rejection, and it is still necessary to determine antibody
The safety and stability used.
Influence of the clearly anti-MIF antibody of the present invention to the inflammatory factor level of IBD finds the novel targets of IBD treatment, is
The clinical treatment of IBD provides reliable basic data.
MIF is the index of IBD disease activity situation and therapeutic effect, and MIF is expected to target as the drug therapy of IBD.This
The method for building up of animal model used in inventing is reproducible, and the experimental technique ELISA and immunohistochemistry technique of use are
Mature method, is easy expanded application, there is preferable popularization and application foreground.
Summary of the invention
In view of the problems of the existing technology, the present invention provides anti-macrophage migration inhibition factor Antybody therapy inflammation
The detection method of property enteropathy.
The invention is realized in this way a kind of detection of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease
Method, have the following steps are included:
Step 1: experimental animal and grouping: SPF grades of male BALB/c mouses 40, after adaptable fed 1 week, random point
For Normal group, UC model group, anti-MIF monoclonal antibody intervention group, physiological saline group, every group 10；
Step 2: model preparation and progress interfering effects of drug during modeling and administration, observe and record the change of each group mouse weight
The ordinary circumstances such as change, the state of mind, hair color, fecal character, and according to the classical methods of marking of Cooper, it carries out disease activity and refers to
Number (DiseaseActivity Index, DAI) scoring；
Step 3: being collected sample and handle, and eyeball of mouse takes blood to detect MIF, TNF-a, IL-6 using ELISA；
Exposure colon carries out anatomical lesion scoring to colon；Under the microscope, 3 not overlapped views are chosen under high power lens, are occurred with cytoplasm
Brown color or brown granular are positive cell, count each visual field positive cell rate；
Step 4: statistical analysis, the data obtained data indicates in the form of (mean ± SD), using SPSS18.0 software into
Row comparative analysis, the comparison of mean uses one-way analysis of variance (One-wayANOVA) between multiple groups.
Further, in step 1, the age of mouse of mouse 8~9 weeks, weight week, weight (24 ± 2) g.
Further, in step 2, model preparation and interfering effects of drug, specifically:
Normal group free water, excess-three group drink the continuous 7d of 5%DSS solution daily；Anti- MIF monoclonal antibody (4mg/mL)
After the dilution of physiological saline 1:3 volume ratio, anti-MIF monoclonal antibody intervention group and physiological saline group are injected intraperitoneally respectively in 1,2,4,6d
Anti- MIF monoclonal antibody and 0.2mL/ pcs/day of physiological saline.
Further, in step 3, sample is collected, and is specifically included:
(1) the 8th day disconnected neck puts to death mouse, and eyeball of mouse takes blood, use to be measured in minus 80 DEG C of preservations after centrifuging and taking supernatant
ELISA detects MIF, TNF-a, IL-6；
(2) exposure colon, the entire intestinal segment of interception cap end to anus are cut along the mesenterium longitudinal axis, and physiology salt is pre-chilled
After water rinses, filter paper is blotted, and carries out anatomical lesion scoring to colon.
Further, in step 3, sample disposal is specifically included:
(1) a part of, it is saved after being placed in cryopreservation tube liquid nitrogen flash freezer in minus 80 DEG C, detects NF- κ for real time PCR
B；
(2) another part, row HE dyes microscopic observation each group mouse Colon histo pathological change parallel organization damage respectively
Wound scoring；
(3) expression of the proinflammatory factors such as immunohistochemical staining microscopic observation MIF, TNF-α, IL-6, NF- κ B, I κ B.
Further, in step 4, compare two-by-two between group and examined using t；Think there is significant difference in p < 0.05.
In conclusion advantages of the present invention and good effect are as follows:
After DSS modeling, UC model group mouse hair, fecal character blood degree spirit states model group mouse hair, excrement
Just character blood degree spirit states model group mouse hair, fecal character blood degree spirit states model group mouse hair, fecal character
Blood degree spirit state and colonic tissue HE dyeing, immunohistochemistry microscopic observation etc., have notable difference to mention compared with normal control
Show that UC mouse model is successfully prepared.
Anti- MIF monoclonal antibody has significant protective effect to the morbidity of mouse UC, may be subtracted by inhibiting NF-k signal beta access
The expression of few proinflammatory factor plays a role.
The anti-MIF monoclonal antibody of first passage of the present invention is realized excessive to the signal path of TLR4/NF- κ B signal access excessive activation
The blocking of activation analyses in depth antiblock, analyzes anti-MIF monoclonal antibody to downstreams proinflammatory factors such as MIF, NF- κ B and TNF-α, IL-6
Influence, determine the blocking effect of anti-MIF monoclonal antibody and to ulcerative colitis protective effect；The blocking effect of monoclonal antibody and to ulcer
Property colitis protective effect.
The microcosmic point of present invention cell factor grid system from UC, determine in cell factor grid system it is micro-
Sight level is set out, and is determined pivotal role of the NF- κ Factor B in UC inflammatory factor and its is associated with MIF, TNF-α, IL-6.
Detailed description of the invention
Fig. 1 is the detection of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease provided in an embodiment of the present invention
Method flow diagram.
Fig. 2 is the detection of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease provided in an embodiment of the present invention
Method and technology route schematic diagram.
Fig. 3 is mouse Colon tissue HE pathological section (HEx100) schematic diagram provided in an embodiment of the present invention.
Fig. 4 is expression (SP x200) signal of mouse Colon histogenic immunity histochemical staining MIF provided in an embodiment of the present invention
Figure.
Fig. 5 is that the expression (SP x200) of mouse Colon histogenic immunity histochemical staining TNF-α provided in an embodiment of the present invention is shown
It is intended to.
Fig. 6 is that the expression (SP x200) of mouse Colon histogenic immunity histochemical staining IL-6 provided in an embodiment of the present invention is shown
It is intended to.
Fig. 7 is the schematic diagram of expression SP × 200 of mouse Colon tissue NF- κ B provided in an embodiment of the present invention.
Fig. 8 is the schematic diagram of expression SP × 200 of mouse Colon tissue I κ B provided in an embodiment of the present invention.
Fig. 9 is the relative expression quantity schematic diagram of each group mouse NF-k β p65mRNA provided in an embodiment of the present invention；
In figure: A: Normal group；B:UC model group；C: anti-MIF monoclonal antibody intervention group；D: physiological saline group.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention carries out modeling using 5%DSS, using BALB/c mouse, has been successfully established UC mouse model, and to mouse
Carry out mouse activity index (ADI) scoring, colon substantially with histological score.
The present invention establishes rat colitis model using DSS induction, by observing the Clinical symptoms of animal and the damage of colon
Situation is done harm to judge the severity of colitis, is detected control group respectively using ELISA and Immunohistochemical Method, colitis group, is resisted
MIF, TNF-α, the level of IL-6 in MIF Antybody therapy group animal blood serum and colonic tissue, analysis proinflammatory cytokine MIF,
The inhibition of proinflammatory factor MIF, TNF-α, IL-6 are made in the effect and anti-MIF antibody of TNF-α, IL-652 in IBD morbidity
With the Clinical symptoms of IBD and the improvement situation of colonic damage, with clearly anti-MIF antibody to the therapeutic effect of IBD, for facing for IBD
Bed treatment provides reliable basic data.
Application principle of the invention is further elaborated with reference to the accompanying drawing；
As shown in Figure 1, anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease provided in an embodiment of the present invention
Detection method, have the following steps are included:
S101: experimental animal and grouping: SPF grades of male BALB/c mouses 40 are randomly divided into after adaptable fed 1 week
Normal group, UC model group, anti-MIF monoclonal antibody intervention group, physiological saline group, every group 10；
S102: model preparation and progress interfering effects of drug during modeling and administration, observe and record the change of each group mouse weight
The ordinary circumstances such as change, the state of mind, hair color, fecal character, and according to the classical methods of marking of Cooper, it carries out disease activity and refers to
Number (DiseaseActivity Index, DAI) scoring；
S103: being collected sample and handle, and eyeball of mouse takes blood to detect MIF, TNF-a, IL-6 using ELISA；Cruelly
Reveal colon, anatomical lesion scoring is carried out to colon；Under the microscope, 3 not overlapped views are chosen under high power lens, palm fibre occur with cytoplasm
Yellow or brown granular are positive cell, count each visual field positive cell rate；
S104: statistical analysis, the data obtained data are indicated in the form of (mean ± SD), are carried out using SPSS18.0 software
Comparative analysis, the comparison of mean uses one-way analysis of variance (One-wayANOVA) between multiple groups.
In step S101, the age of mouse of mouse provided in an embodiment of the present invention 8~9 weeks, weight week, weight (24 ± 2)
g。
In step S102, model preparation provided in an embodiment of the present invention and interfering effects of drug, specifically:
Normal group free water, excess-three group drink the continuous 7d of 5%DSS solution daily；Anti- MIF monoclonal antibody (4mg/mL)
After the dilution of physiological saline 1:3 volume ratio, anti-MIF monoclonal antibody intervention group and physiological saline group are injected intraperitoneally respectively in 1,2,4,6d
Anti- MIF monoclonal antibody and 0.2mL/ pcs/day of physiological saline.
In step S103, sample provided in an embodiment of the present invention is collected, and is specifically included:
(1) the 8th day disconnected neck puts to death mouse, and eyeball of mouse takes blood, use to be measured in minus 80 DEG C of preservations after centrifuging and taking supernatant
ELISA detects MIF, TNF-a, IL-6；
(2) exposure colon, the entire intestinal segment of interception cap end to anus are cut along the mesenterium longitudinal axis, and physiology salt is pre-chilled
After water rinses, filter paper is blotted, and carries out anatomical lesion scoring to colon.
In step S103, sample disposal provided in an embodiment of the present invention is specifically included:
(1) a part of, it is saved after being placed in cryopreservation tube liquid nitrogen flash freezer in minus 80 DEG C, detects NF- κ for real time PCR
B；
(2) another part, row HE dyes microscopic observation each group mouse Colon histo pathological change parallel organization damage respectively
Wound scoring；
(3) expression of the proinflammatory factors such as immunohistochemical staining microscopic observation MIF, TNF-α, IL-6, NF- κ B, I κ B.
In step S104, compares two-by-two between provided in an embodiment of the present invention group and examined using t；Think there is conspicuousness in p < 0.05
Difference.
As shown in Fig. 2, anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease provided in an embodiment of the present invention
Detection method technology path schematic diagram.
Application principle of the invention is further elaborated below with reference to specific experiment；
Experiment 1；
1, experimental method:
1. experimental animal and grouping
SPF grades of male BALB/c mouses 40, age of mouse 8~9 weeks, weight (24 ± 2) g was reached purchased from Hunan Si Laike scape
Experimental animal company.After adaptable fed 1 week, it is randomly divided into Normal group, UC model group, anti-MIF monoclonal antibody intervention group, physiology
Salt water group, every group 10.
2. model preparation and pharmaceutical intervention
Normal group free water, excess-three group drink the continuous 7d of 5%DSS solution daily.Anti- MIF monoclonal antibody (4mg/mL)
After the dilution of physiological saline 1:3 volume ratio, anti-MIF monoclonal antibody intervention group and physiological saline group are injected intraperitoneally respectively in 1,2,4,6d
Anti- MIF monoclonal antibody and 0.2mL/ pcs/day of physiological saline.During modeling and administration, the variation of each group mouse weight, spirit are observed and recorded
The ordinary circumstances such as state, hair color, fecal character, and according to the classical methods of marking of Cooper, carry out disease activity index
(Disease Activity Index, DAI) scoring.
3. sample is collected and processing
8th day disconnected neck puts to death mouse, and eyeball of mouse takes blood, to be measured in minus 80 DEG C of preservations after centrifuging and taking supernatant, using ELISA
Detect MIF, TNF-a, IL-6.Exposure colon, the entire intestinal segment of interception cap end to anus are cut, in advance along the mesenterium longitudinal axis
After cold saline rinses, filter paper is blotted, and carries out anatomical lesion scoring to colon.A part be placed in after cryopreservation tube liquid nitrogen flash freezer in
Minus 80 DEG C of preservations, detect NF- κ B for real time PCR；Row HE dyes microscopic observation each group mouse knot to another part respectively
Intestinal tissue pathological change parallel organization Injury score；Immunohistochemical staining microscopic observation MIF, TNF-α, IL-6, NF- κ B, I κ B
The expression of equal proinflammatory factors.3 not overlapped views are chosen under high power lens, brown color occur with cytoplasm or brown granular is
Positive cell counts each visual field positive cell rate (positive cell/visual field total number of cells x100%), as a result the table in the form of ± s
Show.
4. statisticalling analyze
The data obtained data is indicated in the form of (mean ± SD), is compared analysis, multiple groups using SPSS18.0 software
Between mean comparison use one-way analysis of variance (One-way ANOVA)；Compare two-by-two between group and is examined using t；P < 0.05 is recognized
To there is significant difference.
As a result:
(1) mouse ordinary circumstance:
Normal group mouse hair color is glossy, smooth, reacts fast, and weight increases in various degree, and excrement is in strip, nothing
Hematochezia and diarrhea phenomenon；It is compared with Normal group, UC model group and physiological saline group mouse hair color mix, are matt, is serious
Person's hair is withered, and lags in response, mobility obviously lowers, and hogback occurs, flock together phenomenon, and weight loss is obvious, loose and watery stool sample, secondary
Number increases, hematochezia is serious；Anti- MIF monoclonal antibody intervention group mouse hair color is slightly dull compared with Normal group, part is slightly aobvious mixed and disorderly, but is not so good as
Model group and physiological saline group are obvious, and the state of mind is good, and weight loss amplitude is small, and slight hematochezia phenomenon occur in some animals.
(2) mouse DAI scores:
Compared with Normal group, excess-three group difference is statistically significant (P < 0.05), shows that model is successfully prepared；With
Anti- MIF monoclonal antibody intervention group is compared, and UC model group and physiological saline group and its difference all have statistical significance (P < 0.05)；And UC
No significant difference (P > 0.05) between model group and physiological saline group.Each group scoring is shown in Table 1.
(3) colon anatomical lesion scores:
Visually observe Normal group mouse Colon without congestion and edema, without blutpunkte, intestinal wall is uniform and smooth, and blood vessel is clear；
Model group and physiological saline group colon intestinal wall congestion and edema, color are dark and gloomy, and obvious ulcer occurs in part, with erosion, with surrounding
Adhesion is organized, tube wall has stiff sense；Anti- MIF monoclonal antibody intervention group mouse Colon intestinal wall color is dim, congestion and edema, and appearance is small out
Blood point, but its range, degree, ulcer number are significant not as good as model group and physiological saline group.Each group scoring is shown in Table 1；
1 mouse DAI of table scores and colon anatomical lesion scores (N=10)
Note: * P < 0.05vs Normal group；#P < 0.05vs UC model group；▲The anti-MIF monoclonal antibody intervention group of P < 0.05vs
(4) HE stained tissue scores:
Normal group mouse Colon mucous membrane and submucosa structural integrity N/D, epithelial cell marshalling, body of gland
Form rule, placenta percreta blood vessel is without congested phenomenon；UC model group colonic mucosa epithelial erosion is serious, mucosa lamina propria major part gland
Body structure is destroyed, and granulation tissue is formed, and it is thin by a large amount of inflammation that prompt may once have fester, mucosa lamina propria and submucosa
Born of the same parents' infiltration；Anti- MIF monoclonal antibody intervention group colonic mucosa epithelium is without erosion, and gland structure is normal, intravascular erythrocyte aggregation, in hyperemia
Phenomenon, mucosa lamina propria inflammatory cell infiltration, but inflammatory cell quantity and invasive depth are not as good as UC model group and physiological saline group；
Physiological saline group colonic mucosa epithelial defect, fracture, slight rotten to the corn, inflammatory cell is concentrated mainly on mucosa lamina propria.As a result see
Fig. 3 and table 3.
As shown in figure 3, mouse Colon tissue HE pathological section schematic diagram provided in an embodiment of the present invention.
(5) ELISA detects mice serum MIF, TNF-α, the concentration of IL-6:
UC model group mice serum MIF, TNF-α, the concentration of IL-6 are apparently higher than anti-MIF monoclonal antibody group and Normal group,
With significant difference (p is < 0.05)；The above-mentioned scoring of anti-MIF monoclonal antibody group mouse is also higher than Normal group, and difference has significant
Property (p < 0.05).It is shown in Table 2.
2 mice serum MIF of table, TNF-α, the concentration of IL-6 (mean ± SD, pg/mL)
Note:#For UC model group and anti-MIF monoclonal antibody comparison among groups；*For UC model group and normal control comparison among groups；$ is anti-
MIF monoclonal antibody group and normal control comparison among groups；P is < 0.05.
(6) Use immunohistochemistrySP SP intestinal mucosa galandular epithelium MIF, TNF-α, the level of IL-6:
Each group mouse Colon mucous membrane galandular epithelium does not express MIF, TNF-α, IL-6.Normal group colonic mucosa inflammation is thin
Born of the same parents do not express or denier expression；And in UC model group and physiological saline group, the equal strongly expressed of colonic mucosa inflammation cell；It is anti-
Colonic mucosa inflammation cell MIF in MIF monoclonal antibody intervention group, TNF-α, IL-6 are in weak expression, between Normal group and remaining two
Between group.As a result see Fig. 4,5,6 and table 3.
As shown in figure 4, expression (the SP x of mouse Colon histogenic immunity histochemical staining MIF provided in an embodiment of the present invention
200) schematic diagram.
As shown in figure 5, expression (the SP x of mouse Colon histogenic immunity histochemical staining TNF-α provided in an embodiment of the present invention
200) schematic diagram.
As shown in fig. 6, expression (the SP x of mouse Colon histogenic immunity histochemical staining IL-6 provided in an embodiment of the present invention
200) schematic diagram.
3 mouse Colon histological score of table and immunohistochemistry positive cell rate (N=10)
Note: * P < 0.05vs Normal group；#P < 0.05vs UC model group；▲The anti-MIF monoclonal antibody intervention group of P < 0.05vs
(7) Use immunohistochemistrySP SP detects NF- κ B P65, expression of the I κ B α in colitis mice colonic mucosal tissue:
1. mouse Colon NF- κ B P65 is mainly expressed in mucosal epithelium, monocyte and macrophage, normal control group colon
Mucosal inflammation cell is expressed in cytoplasm on a small quantity, and in UC model group and physiological saline group, colonic mucosa inflammation cell is strong
It expresses, based on karyon；The expression of colonic mucosa inflammation cell is reduced in anti-MIF monoclonal antibody intervention group, is expressed in cytoplasm.Such as Fig. 7 institute
Show.
As shown in fig. 7, the schematic diagram of expression SP × 200 of mouse Colon tissue NF- κ B provided in an embodiment of the present invention.
2. I κ B α is not expressed in each group mouse Colon epithelial tissue, but is expressed in inflammatory cell.Control group colon I
κ B α is expressed in the cytoplasm of mucosal inflammation cell, and in mucous membrane, has table in the inflammatory cell of submucosa and lamina propria
It reaches；And in UC model group and physiological saline group, the inflammatory cell expression of mouse Colon mucous membrane significantly reduces, and I κ B α is in colon group
Expression in knitting also significantly reduces and (has the significance difference opposite sex, p < 0.05 compared with the control group)；Colonic mucosa in anti-MIF monoclonal antibody group
Inflammatory cell expression increases, and expression expression in colonic tissue of the I κ B α in colonic tissue also increased significantly (with model group
Compare with sex differernce, p < 0.05).As shown in Figure 8.
As shown in figure 8, the schematic diagram of expression SP × 200 of mouse Colon tissue I κ B provided in an embodiment of the present invention.
(8) Real-time PCR result:
1. RNA purity and concentration:
Extracted colonic tissue total serum IgE measures OD260/OD280 between 1.8~2.0 through ultraviolet specrophotometer,
Illustrate that extracted total serum IgE purity is higher；Gel imaging system shows more bright 18S after 1% agarose gel electrophoresis
With two light belts of 28S, no hangover and miscellaneous band occur, and illustrate no non-specific amplification and primer dimer, and 28S strip width and
Brightness is approximately 2 times of 18S band, illustrates that total serum IgE is not yet degraded.The success of preceding description colonic tissue Total RNAs extraction, Ke Yijin
Row subsequent experimental.
2. the expression of NF- κ B:
UC model group mouse Colon tissue NF-k β p65mRNA relative expression quantity (2.011 ± 0.137) is significantly higher than normally
Control group (1.01 ± 0.018) and anti-MIF monoclonal antibody intervention group (0.84 ± 0.211), difference all have statistical significance (P <
0.05)；Compared with UC model group, the relative expression quantity of anti-MIF monoclonal antibody intervention group is lower (P < 0.05), and physiological saline group
(2.00 ± 0.12) and its no significant difference (P > 0.05).As shown in Figure 9.
As shown in figure 9, the relative expression quantity schematic diagram of each group mouse NF-k β p65mRNA provided in an embodiment of the present invention.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (7)
1. a kind of animal model of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease, which is characterized in that described
The animal model of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease are as follows:
SPF grades of male BALB/c mouses 40, after adaptable fed 1 week, are randomly divided into Normal group, UC model group, anti-MIF
Monoclonal antibody intervention group, physiological saline group, every group 10；
Normal group free water, excess-three group drink the continuous 7d of 5%DSS solution daily；Anti- MIF monoclonal antibody 4mg/mL and physiology
After the dilution of salt water 1:3 volume ratio, it is mono- that anti-MIF is injected intraperitoneally in 1,2,4,6d in anti-MIF monoclonal antibody intervention group and physiological saline group respectively
Resist and 0.2mL/ pcs/day of physiological saline；During modeling and administration, the variation of each group mouse weight, the state of mind, hair are observed and recorded
Color, fecal character.
2. a kind of animal model using anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease described in claim 1
Anti- macrophage migration inhibition factor Antybody therapy inflammatory bowel disease detection method, which is characterized in that the anti-macrophage
The detection method of cell migration inhibition factor Antybody therapy inflammatory bowel disease, have the following steps are included:
Step 1: experimental animal and grouping: SPF grades of male BALB/c mouses 40 are randomly divided into just after adaptable fed 1 week
Normal control group, UC model group, anti-MIF monoclonal antibody intervention group, physiological saline group, every group 10；
Step 2: model preparation and carry out interfering effects of drug, modeling and administration during, observe and record each group mouse weight variation,
The ordinary circumstances such as the state of mind, hair color, fecal character, and according to the classical methods of marking of Cooper, carry out disease activity index
(DiseaseActivity Index, DAI) scoring；
Step 3: being collected sample and handle, and eyeball of mouse takes blood to detect MIF, TNF-a, IL-6 using ELISA；Exposure
Colon carries out anatomical lesion scoring to colon；Under the microscope, 3 not overlapped views are chosen under high power lens, are occurred with cytoplasm pale brown
Color or brown granular are positive cell, count each visual field positive cell rate；
Step 4: statistical analysis, the data obtained data is indicated in the form of (mean ± SD), is compared using SPSS18.0 software
Compared with analysis, the comparison of mean uses one-way analysis of variance (One-wayANOVA) between multiple groups.
3. the detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease as claimed in claim 2,
It is characterized in that, in the step 1, the age of mouse of mouse 8~9 weeks, weight week, weight (24 ± 2) g.
4. the detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease as claimed in claim 2,
It is characterized in that, in the step 2, model preparation and interfering effects of drug, specifically:
Normal group free water, excess-three group drink the continuous 7d of 5%DSS solution daily；Anti- MIF monoclonal antibody (4mg/mL) and life
After managing the dilution of salt water 1:3 volume ratio, anti-MIF is injected intraperitoneally in 1,2,4,6d in anti-MIF monoclonal antibody intervention group and physiological saline group respectively
Monoclonal antibody and 0.2mL/ pcs/day of physiological saline.
5. the detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease as claimed in claim 2,
It being characterized in that, in the step 3, sample is collected, it specifically includes:
(1) the 8th day disconnected neck puts to death mouse, and eyeball of mouse takes blood, to be measured in minus 80 DEG C of preservations after centrifuging and taking supernatant, using ELISA
Detect MIF, TNF-a, IL-6；
(2) exposure colon, the entire intestinal segment of interception cap end to anus are cut along the mesenterium longitudinal axis, pre- cold saline punching
After washing, filter paper is blotted, and carries out anatomical lesion scoring to colon.
6. the detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease as claimed in claim 2,
It is characterized in that, in the step 3, sample disposal is specifically included:
(1) a part of, it is saved after being placed in cryopreservation tube liquid nitrogen flash freezer in minus 80 DEG C, detects NF- κ B for real time PCR；
(2) another part, row HE dyes the damage of microscopic observation each group mouse Colon histo pathological change parallel organization and comments respectively
Point；
(3) expression of the proinflammatory factors such as immunohistochemical staining microscopic observation MIF, TNF-α, IL-6, NF- κ B, I κ B.
7. the detection method of anti-macrophage migration inhibition factor Antybody therapy inflammatory bowel disease as claimed in claim 2,
It is characterized in that, in the step 4, compares two-by-two between group and examined using t；Think there is significant difference in p < 0.05.
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