CN105994125A - Method for evaluating immunodeficiency degree of immunodeficient mice model - Google Patents

Abstract

The invention relates to the field of biology, and discloses a method for evaluating immunodeficiency degree of an immunodeficient mice model. The method comprises: through transplanting neoplastic hematologic disorder cells and solid tumor cells in different orders of magnitudes into an immunodeficient mice model, establishing a tumor mice model, detecting the proportion of tumor cells GPB in internal and external blood of the tumor mice model, the proportion of tumor cells GBM in bone marrow, and the proportion of tumor cells GSP in spleen, according to proportion parameter indexes, evaluating the immunodeficiency degree of the immunodeficient mice model. The method is advantaged in that the method can quantitatively evaluate defect level of the immunodeficient mice, the method relates to simplify and summarize a mathematic model and a calculation method which are obtained through relations between tumor transplanting efficiency and mice immunodeficiency degree, and through scores of immunodeficiency degree, defect difference degree among the immunodeficient mice can be compared. The method is simple and feasible, and is easy to operate, and the method has good application prospect.

Description

A kind of method of the immunodeficiency degree evaluating immunodeficient mouse model

Technical field

The present invention relates to biological field, particularly to the side of a kind of immunodeficiency degree evaluating immunodeficient mouse model Method.

Background technology

With immunodeficient mouse as host, the heteroplastic transplantation model in the humanized mouse model of structure and patient source is people Body immune system and the important tool of human tumor pathomechanism research.1963, scientist developed nude mice (Nude Mice), this mice Foxn1 gene mutation, thymus deteriorates or disappearance, and blocking t cell is grown, caused T cell to reduce sharply in a large number, immunity System defect.But nude mice has B cell and NKT (NK, natural killer) cell, it is unfavorable for maintaining human body cell Growth in Mice Body.Nineteen eighty-three, Severe combined immunodeficient mice (scid, severe combined Immunodeficiency) occur, this mice Prdkc genetic flaw, cause its disappearance B cell and T cell.RAG2 mice (recombination activating 2deficient) Rag2 gene delection, B cell and T cell disappearance.With scid mice Equally, RAG2 mice yet suffers from substantial amounts of NK cell, limits the transplanting of human body cell to a certain extent.Nineteen ninety-five, research Personnel, by Prdkcscid gene mutation is imported NOD (non-obese diabetic) inbred mouse, set up NOD-scid (NOD.Cg-Prkdcscid) mice.This mice not only lacks B cell and T cell, and the most a small amount of NK cell, inherent immunity System injury, is a milestone of immunodeficient mouse.And the important breakthrough that humanization mice builds is Il2Rg-/-mice and the research and development of other immunodeficient mouse based on this mice, such as NOG (NOD/Shi-Prkdc^scid Il2rc^tm1Sug/Jic)、NSG(NOD/LtSz-Prkdc^scidIl2rc^tm1Wjl/ J) .IL2Rc is IL-2, IL-4, IL-7, IL-15 With the total receptor of IL-21 cytokine, and these cytokines have important shadow to the growth of the immunocytes such as T, B, NK Ring.So, IL2Rc-/-immunodeficient mouse disappearance T, B, NK cell, the simultaneously macrophage of this mice and dendritic cell merit Can be weakened.Contrasting with other immunodeficient mouses, NOG, NSG mice shows high-caliber transplantation donor characteristic: this two Planting in IL2R γ c deficient mice strain, human hematopoietic stem cell is transplanted and is shown high transplanting efficiency, and these Hematopoietic Stems are thin Born of the same parents can be divided into multiple hematopoietic lineage cells, sun T cell as mono-in CD4 and CD8；The cancerous cell transplanted can fast-growth in this mice And after repeatedly passing on, keep PD characteristic.Although but immunodeficient mouse and correlational study thereof get more and more, but lacking Immunodeficient mouse is estimated by weary unified standard, then cause the research of further immunodeficient mouse and application to lack ginseng The standard examined and instruct.

Immune system is the disease system of defense that a series of biological structure and process are formed in organisms, it is impossible to Sentence with single or simple combination immune indexes such as T, B, NK cell quantity or the expression such as interferon, chemotactic factor Fixed immunity intensity.In like manner, mouse immune defect level also cannot pass through these single or immunocyte of simple combination or inflammation The defect of the factor is assessed.

In tumor microenvironment, the immune system of tumor cell and body interacts, and influences each other.Substantial amounts of clinical number It is easier to spontaneous tumor according to the individuality showing immunodeficiency.Such as, with not having the patient of immunodeficiency to contrast, there is immunodeficiency Patient is more likely to obtain leukemia.With the patient of immunodeficiency is similar, immunodeficient mouse suffers from the likelihood ratio immunity of spontaneous tumor The normal mice of system is much higher.Therefore, in immunodeficient mouse body, immune extreme defect can cause largely Tumor proliferation.Tumour immunity be extensively mobilize body immune system to neoplasm growth and the process of growth, so based on swollen The mouse immune defect level assessment that tumor is transplanted is comprehensive.Tumour immunity includes removing, balance and escape three Main Stage. And the stage of removing can be subdivided into four-stage, one: initial anti-tumor immune response, the immunocyte identification of innate immune system Just carry out matrix remodeling, cause the tumor cell of tissue injury.Then, call up by release inflammatory signals and gather innate immune The immunocyte of system, such as natural killer cell (NK), natural killer T cells (NKT) macrophage and dendritic cell.Collection The NK cell tied and come and NKT cell release interferon-alpha (IFN-α).Two: interferon-alpha effect, newly synthesized interferon-alpha can lure Lead tumor death, and promote the generation of Chemokines CC XCL10, CXCL9 and CXCL11.These chemotactic factors can be by blocking The generation of neovascularity and suppress tumor growth.More immunocyte will by chemotactic factor chemotactic, concentrate at tumor locus.These rank Section, after dendritic cell phagocytosis tumor cell, will migrate to lymph node.Three: expand effect, NK cell and macrophage by releasing Put interferon-alpha and IL-12 mutually activates.These cells will further facilitate tumor death and active oxygen, the generation of nitrogen intermediate. In lymph node, the tumour-specific dendritic cell after phagocytosis tumor can start the differentiation of helper T lymphocyte, then promotes swollen Tumor killer T cell i.e. CD8⁺The growth of T cell.Four: specificity remove, fully-developed tumour-specific CD4+T cell and CD8+T cell migration is to tumor locus, and kills the tumor cell of the antigen tolerance still retained.Equilibrium stage and escaping phase, Tumor cell mutations body survived in the removing stage, and entered equilibrium stage.In equilibrium stage, lymphocyte and α interference Element can give selective clearing pressure, and tumor cell can carry out gene mutation adaptation, and enters escaping phase.Tumour immunity is wide The general scope relating to inherent immunity and adaptive immunity, immunodeficiency scale evaluation based on Replanting model mice can reflect comprehensively Mouse immune defect level.Heteroplastic tumor cell is more difficult to adapt to the body environment of immune pressure relatively, and combination of the same race is different The Replanting model mice assessment planted can reflect mouse immune defect level more really.

Now, along with various novel extensive gene Knockouts and the appearance of genomic modification instrument, such as TALEN (Transcription Activator-Like Effector Nuclease) and Cas (Type II Clustered, Regularly Interspaced, Short Palindromic Repeat (CRISPR) associated system) technology Deng, the efficiency building various immunodeficient mouse strain constantly improves.The present inventor also utilizes TALEN technology, success to exist Under NOD-scid mouse species background, knock out IL2Rc gene, it is thus achieved that NOD-scid IL2Rg-/-(NSI) mouse species (patent Application number: CN201310229629).But, along with the continuous appearance of immunodeficient mouse, little still without an assessment at present The standard method of Mus immunodeficiency degree, is difficult to assess objectively the immunodeficiency degree of genetic modification mouse species, will cause The blindness of immunodeficient mouse builds and uses, and is also unfavorable for that the standard that immunodeficient mouse is applied is unified and promotes.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of immunodeficiency degree evaluating immunodeficient mouse model Method, its purpose realized is the immunodeficiency degree that can quantify immunodeficient mouse according to the inventive method, thus by being somebody's turn to do Method can simply, accurately obtain the tumor cell effect to immunodeficient mouse model, and then can preferably apply Build in immunodeficient mouse model.

For achieving the above object, the technical solution used in the present invention is, a kind of evaluation immunodeficiency disclosed by the invention is little The method of the immunodeficiency degree of mouse model, comprises the steps of

By transplanting the blood tumor cell of varying number level and solid tumor cell in immunodeficient mouse model, structure After building tumor mouse model, tumor cell in the ratio of tumor cells in peripheral blood GPB, bone marrow in detection tumor mouse model The ratio of tumor cell GSP in the ratio of GBM and spleen, according to described scale parameter metrics evaluation immunodeficient mouse mould The immunodeficiency degree of type.

Further, described tumor cell is of the same race or xenogenesis kind, uses the inventive method to be possible not only to evaluate animal sources The tumor cell effect to immunodeficient mouse, it is also possible to for evaluating the humanized tumor cell work to immunodeficient mouse With.

Further, the varying number level of tumor cell transplantation is L:1 × 10⁴Individual, M:1 × 10⁵Individual, H:1 × 10⁶Individual, should The selection of the order of magnitude based on less than this order of magnitude, then cannot obtain the tumor cell effect index to immunodeficient mouse model, Higher than this order of magnitude, immunodeficient mouse model mice effect is difficult to differentiate between out by tumor cell.

Patent of the present invention provides a kind of method quantifying immunodeficient mouse strain immunodeficiency degree, for immunodeficiency The assessment of mice provides standard, and helps scientific research personnel to select suitable mice offer foundation, and is immunodeficient mouse Research and application provide reference and the standard of guidance.

Concrete application such as, assists scientific research personnel's definition and the immunodeficient mouse of the new research and development of assessment；Select for scientific research personnel Immunodeficient mouse carries out zoopery provides foundation；The results of animal analysis of immunodeficient mouse is utilized for scientific research personnel Reasonable reference is provided；Related immune ability for the different immunodeficient mouse of assessment provides a kind of mathematical model referred to and reality Test setting.

The present inventor to the immune degree that relates generally to, proposes of the same race by transplanting in mice according to tumour immunity Or the tumor cell of xenogenesis, observation mice body, to the of the same race or repulsion degree of xenograft tumor cell, reflects exempting from of mice with this Epidemic disease defect level.Described tumor cell is preferably the tumor cell in people source, and wherein human blood tumor cell is preferably behaved chronic marrow Being K562 leukemic cells system, people's solid tumor cell is preferably typeⅡ pneumocyte system；It is highly preferred that described tumor cell For Mus source tumor cell, blood tumor cell preferred mouse lymphoma cell system RMA, the preferred mouse melanin of solid tumor cell Oncocyte system B16-F10.

Further, according in the ratio of tumor cells in peripheral blood GPB, bone marrow in the tumor mouse model that obtains of detection The ratio of tumor cell GSP in the ratio of tumor cell GBM and spleen, evaluates the immunodeficiency of immunodeficient mouse model The method of degree comprises the steps, it is little to immunodeficiency that (1) uses formula to obtain transplanting each order of magnitude blood tumor cell The action index HTEI of Mus,Gi is GPB, GBM, GSP sum, and Di is for be implanted into immunity by blood tumor cell Start, to immunodeficient mouse model effect interlude, to respectively obtain each quantity to tumor cell in deficient mice model Level blood tumor cell the action index HTEI (H) to immunodeficient mouse, HTEI (M), HTEI (L), try to achieve average the most again HTEI；

(2) formula is used to obtain the action index transplanting each order of magnitude solid tumor cell to immunodeficient mouse model STEI,Wn is the weight that entity tumor is implanted in immunodeficient mouse model the entity tumor formed, Dn For starting the interval time to immunodeficient mouse model effect to tumor cell in entity tumor is implanted into immunodeficiency, point Do not obtain each order of magnitude solid tumor cell action index STEI (H) to immunodeficient mouse, STEI (M), STEI (L), Finally try to achieve average STEI again；

(3) average HTEI, the average STEI obtained according to step (1) and step (2), uses equation below to obtain total tumor Action indexThis total function of tumor index and immunodeficiency degree are negative correlation.

Present invention firstly provides mathematical model and the computational methods of an immunodeficiency degree quantifying mice, by this Mathematical model, can the defect level degree of adaptive immune deficient mice quantitatively.Further, obtained by contrast immunodeficiency degree Divide and can assess the defect difference degree between immunodeficient mouse.Research worker can utilize this mathematical model, picks out Suitably immunodeficient mouse is specifically tested.

Accompanying drawing explanation

Fig. 1 is 7 kinds of mouse immune defect level block diagrams；

Fig. 2 shows 7 kinds of mice same tumor immunodeficiency degree block diagrams.

Detailed description of the invention

The invention discloses a kind of method quantifying mouse immune defect level, those skilled in the art can use for reference herein Content, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change are to art technology Being apparent from for personnel, they are considered as being included in the present invention.The method of the present invention has passed through preferred embodiment Be described, related personnel substantially can in without departing from present invention, spirit and scope to method described herein and should With being modified or suitably changing and combine, realize and apply the technology of the present invention.

Embodiment one: be embodied as step:

The structure of the human blood oncocyte system of tape label gene

For the ease of the promotion and application of the invention, lack using people chronic myelogenous leukemia cell system K562 as immunity Fall into standard (xenogenesis) the human blood tumor cell of mathematical model, using human lung adenocarcinoma cell line A549 as immunodeficiency mathematical model Standard (xenogenesis) people's solid tumor cell, and in K562 cell line import marker gene (as GFP green fluorescence gene, Tdtomato red fluorescent gene, Luciferase luciferase etc.).GFP green fluorescence gene transfection method introduced below, packaging Lenti-GFP virus infected tumor cell line, after the infected cell of amplification culture, it is thin that successful GFP positive tumor is infected in sorting Born of the same parents, it is thus achieved that GFP positive cell purity is more than 98%.

Building and the calculating of HTEI of blood tumor mouse model

By varying number level (10⁶(H),10⁵(M),10⁴(L)) the blood tumor cell line (K562-GFP) of tape label gene is moved Implanting specific immunodeficiency strain experiment mice (each order of magnitude is transplanted with 5 experiment mices) to be assessed, transplantation tumor is thin After born of the same parents, observe mice state every day.When mice disease is sent out, the time-to-live (D of record mice_i) and mouse blood, spleen, bone marrow In tumor cell (GFP positive cell) ratio (G_PB、G_SP、G_BM), use equation below to calculate each order of magnitude respectively HTEI (H), HTEI (M), HTEI (L),

Gi is G_PB、G_BM、G_SPSum, it is noted that, Gi, D_iBe all parallel repeat to test repeatedly obtain Meansigma methods, this embodiment have employed that 5 mices are parallel repeats to test 5 times, finally obtains, further according to HTEI (H), HTEI (M), HTEI (L) tries to achieve average HTEI；

Building and the calculating of STEI of solid tumor mouse model

Transplant varying number level (10⁶(H),10⁵(M),10⁴(L)) solid tumor cell system (A549 is implanted into spy to be assessed Determining immunodeficiency strain experiment mice (each order of magnitude is transplanted with 5 experiment mices), after implanted tumor cells, every day observes little Mus state.After Transplanted Human solid tumor cell system's certain time, (observing time of different tumor cell lines is different, A549 cell line Then after transplanting 30 days, carry out observed and recorded), weigh and record quality (Wn, unit: g), and the general of the entity tumor of formation D_n=30 and Wn substitute into formula,Obtain STEI (H), STEI (M), STEI (L) respectively, finally try to achieve average STEI。

Assess the immunodeficiency degree of this Strains of Mouse

The average HTEI that abovementioned steps is calculated and average STEI formula to be entered Finally calculating this strain immunodeficient mouse model total function of tumor index TEI, this index and immunodeficiency degree are negative Close.

TEI numerical value is the highest, then represent that the immunodeficiency degree of this Strains of Mouse is the most serious；TEI numerical value is the lowest, represents this product The immune system being mice is the most complete.Methods set forth above is comprehensive assessment mouse immune defect level, for concrete Immune system situation, such as T cell or B cell or the quantity of NK cell and function, the immune system defect infected for inhibition of HIV Degree etc., TEI is only provided that reference role；

For concrete immune system assessment of scenario, the mathematical model introduced in above-mentioned formula is equally used to combine animal The method of model is estimated, it is only necessary to sets experiment such as tumour transplatation experiment, tumor cell, the transplanting order of magnitude, obtain assessment Parameters etc. make certain amendment.

Embodiment two:

The method utilizing mouse immune scale evaluation of the present invention, comprehensive assessment NSI (NOD-scid IL2rg-/-) mice Immunodeficiency degree (TEI_NSI), specifically comprise the following steps that

1.1.HTEI_NSINumerical computations: transplant H respectively, M, the K562-GFP cell of L number level enter in NSI Mice Body, often It observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 1.NSI mice-human blood Replanting model mice parameter list

Above parameter substitution formula is obtained:

${\mathrm{HETI}}_{(NSI,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.023$

${\mathrm{HETI}}_{(NSI,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.012$

${\mathrm{HETI}}_{(NSI,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.007$

HTEI_NSI=average (HTEI_{(NSI, H)}, HTEI_{(NSI, M)}, TEI_{(NSI, L)})=0.014

1.2.STEI_NSINumerical computations: transplant H respectively, M, the A549-GFP cell of L number level enter in NSI Mice Body, often It observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 2.NSI mice-people's entity tumor transplantation model parameter list

Above parameter substitution formula is obtained:

${\mathrm{STEI}}_{(NSI,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.059$

${\mathrm{STEI}}_{(NSI,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.051$

${\mathrm{STEI}}_{(NSI,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.012$

STEI_NSI=average (STEI_{(NSI, H)}, STEI_{(NSI, M)}, STEI_{(NSI, L)})=0.040

1.3.NSI the assessment of mouse immune defect level, TEI_NSINumerical computations:

By HTEI achieved above_NSIAnd STEI_NSINumerical value substitute into formula obtain

${\mathrm{TEI}}_{strain}=\frac{{\mathrm{HTEI}}_{NSI}+{\mathrm{STEI}}_{NSI}}{2}=0.027$

Can be obtained from above, the immunodeficiency degree of NSI mice must be divided into 0.027.

Embodiment three

The method utilizing mouse immune scale evaluation of the present invention, the immunity of comprehensive assessment NOD-scid (writing a Chinese character in simplified form: NS) mice Defect level (TEI_NS), specifically comprise the following steps that

1.4.HTEI_NSNumerical computations: transplant H respectively, M, the K562-GFP cell of L number level enter NOD-scid Mice Body In, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 3.NOD-scid mice-human blood Replanting model mice parameter list

Above parameter substitution formula is obtained:

${\mathrm{HETI}}_{(NS,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.012$

${\mathrm{HETI}}_{(NS,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.003$

${\mathrm{HETI}}_{(NS,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

HTEI_NS=average (HTEI_{(NS, H)}), HTEI_{(NS, M)}, TEI_{(NS, L)})=0.009

1.5.STEI_NSNumerical computations: transplant H respectively, M, the A549-GFP cell of L number level enter NOD-scid Mice Body In, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 4.NOD-scid mice-people's entity tumor transplantation model parameter list

Above parameter substitution formula is obtained:

${\mathrm{STEI}}_{(NS,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.050$

${\mathrm{STEI}}_{(NS,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.020$

${\mathrm{STEI}}_{(NS,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

STEI_NS=average (STEI_{(NS, H)}, STEI_{(NS, M)}, STEI_{(NS, L)})=0.023

1.6.NOD-scid the assessment of mouse immune defect level, TEI_NSNumerical computations:

By HTEI achieved above_NSAnd STEI_NSNumerical value substitute into formula,

${\mathrm{TEI}}_{NS}=\frac{{\mathrm{HTEI}}_{NS}+{\mathrm{STEI}}_{NS}}{2}=0.016$

Can be obtained from above, the immunodeficiency degree of NOD-scid mice must be divided into 0.016.

2. embodiment four:

The method utilizing mouse immune scale evaluation of the present invention, comprehensive assessment CB-17scid (writing a Chinese character in simplified form: scid) mice Immunodeficiency degree (TEI_scid), specifically comprise the following steps that

2.1.HTEI_scidNumerical computations: transplant H respectively, M, the K562-GFP cell of L number level enter CB-17scid mice Internal, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 5.CB-17scid mice-human blood Replanting model mice parameter list

Above parameter substitution formula is obtained:

${\mathrm{HETI}}_{(scid,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.003$

${\mathrm{HETI}}_{(scid,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(scid,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

HTEI_scid=average (HTEI_{(scid, H)}, HTEI_{(scid, M)}, TEI_{(scid, L)})=0.001

2.2.STEI_scidNumerical computations: transplant H respectively, M, the A549-GFP cell of L number level enter CB-17scid mice Internal, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 6.CB-17scid mice-people's entity tumor transplantation model parameter list

Above parameter substitution formula is obtained:

${\mathrm{STEI}}_{(scid,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.021$

${\mathrm{STEI}}_{(scid,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.012$

${\mathrm{STEI}}_{(scid,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

STEI_scid=average (STEI_{(scid, H)}, STEI_{(scid, M),}STEI_{(scid, L)})=0.011

2.3.CB-17scid the assessment of mouse immune defect level, TEI_scidNumerical computations:

By HTEI achieved above_scidAnd STEI_scidNumerical value substitute into formula,

${\mathrm{TEI}}_{scid}=\frac{{\mathrm{HTEI}}_{scid}+{\mathrm{STEI}}_{scid}}{2}=0.006$

Can be obtained from above, the immunodeficiency degree of CB-17scid mice must be divided into 0.006.

3. embodiment five:

The method utilizing mouse immune scale evaluation of the present invention, the immunity of comprehensive assessment IL2rg-/-(writing a Chinese character in simplified form: IL2) mice Defect level (TEI_IL2), specifically comprise the following steps that

3.1.HTEI_IL2Numerical computations: transplant H respectively, M, the K562-GFP cell of L number level enter IL2rg-/-Mice Body In, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 7.IL2rg-/-mice-human blood Replanting model mice parameter list

Above parameter substitution formula is obtained:

${\mathrm{HETI}}_{(IL2,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.001$

${\mathrm{HETI}}_{(IL2,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(IL2,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

HTEI_IL2=average (HTEI_{(IL2, H)}, HTEI_{(IL2, M)}, TEI_{(IL2, L)})=0.0003

3.2.STEI_IL2Numerical computations: transplant H respectively, M, the A549-GFP cell of L number level enter IL2Rg-/-Mice Body In, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 8.IL2Rg-/-mice-people's entity tumor transplantation model parameter list

Above parameter substitution formula is obtained:

${\mathrm{STEI}}_{(IL2,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.006$

${\mathrm{STEI}}_{(IL2,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

${\mathrm{STEI}}_{(IL2,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

STEI_IL2=average (STEI_{(IL2, H)}, STEI_{(IL2, M)}, STEI_(IL2L))=0.002

The assessment of 3.3.IL2Rg-/-mouse immune defect level, TEI_IL2Numerical computations:

By HTEI achieved above_IL2And STEI_IL2Numerical value substitute into formula obtain

${\mathrm{TEI}}_{IL2}=\frac{{\mathrm{HTEI}}_{IL2}+{\mathrm{STEI}}_{IL2}}{2}=0.001$

Can be obtained from above, the immunodeficiency degree of IL2Rg-/-mice must be divided into 0.001.

4. embodiment six:

The method utilizing mouse immune scale evaluation of the present invention, the immunity of comprehensive assessment Rag2-/-(writing a Chinese character in simplified form: Rag2) mice Defect level (TEI_Rag2), specifically comprise the following steps that

4.1.HTEI_Rag2Numerical computations: transplant H respectively, M, the K562-GFP cell of L number level enter Rag2-/-Mice Body In, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 9.Rag2-/-mice-human blood Replanting model mice parameter list

Above parameter substitution formula is obtained:

${\mathrm{HETI}}_{(Rag2,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(Rag2,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(Rag2,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

HTEI_Rag2=average (HTEI_{(Rag2, H)}, HTEI_{(Rag2, M)}, TEI_{(Rag2, L)})=0.000

4.2.STEI_Rag2Numerical computations: transplant H respectively, M, the A549-GFP cell of L number level enter Rag2-/-Mice Body In, every day observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 10.Rag2-/-mice-people's entity tumor transplantation model parameter list

Above parameter substitution formula is obtained:

$\mathrm{STEI}{(Rag2,H)}_{}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.015$

${\mathrm{STEI}}_{(Rag2,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

$S{\mathrm{TEI}}_{(Rag2,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

STEI_Rag2=average (STEI_{(Rag2, H)}, STEI_{(Rag2, M)}, STEI_{(Rag2, L)})=0.005

The assessment of 4.3.Rag2-/-mouse immune defect level, TEI_Rag2Numerical computations:

By HTEI achieved above_Rag2And STEI_Rag2Numerical value substitute into formula obtain

${\mathrm{TEI}}_{\mathrm{Rag}2}=\frac{{\mathrm{HTEI}}_{\mathrm{Rag}2}+{\mathrm{STEI}}_{\mathrm{Rag}2}}{2}=0.0025$

Can be obtained from above, the immunodeficiency degree of Rag2-/-mice must be divided into 0.0025.

5. embodiment seven

The method utilizing mouse immune scale evaluation of the present invention, the immunodeficiency of comprehensive assessment nude (writing a Chinese character in simplified form: Nu) mice Degree (TEI_Nu), specifically comprise the following steps that

5.1.HTEI_NuNumerical computations: transplant H respectively, M, the K562-GFP cell of L number level enter in nude Mice Body, often It observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 11.nude mice-human blood Replanting model mice parameter list

Above parameter substitution formula is obtained:

${\mathrm{HETI}}_{(NU,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(NU,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(NU,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

HTEI_Nu=average (HTEI_{(Nu, H)}, HTEI_{(Nu, M),}TEI_{(Nu, L)})=0.000

5.2.STEI_NuNumerical computations: transplant H respectively, M, the A549-GFP cell of L number level enter in nude Mice Body, often It observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 12.nude mice-people's entity tumor transplantation model parameter list

Above parameter substitution formula is obtained:

${\mathrm{STEI}}_{(Nu,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.018$

${\mathrm{STEI}}_{(Nu,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.004$

${\mathrm{STEI}}_{(Nu,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

STEI_Nu=average (STEI_{(Nu, H)}, STEI_{(Nu, M)}, STEI_{(Nu, L)})=0.008

5.3.nude the assessment of mouse immune defect level, TEI_NuNumerical computations:

By HTEI achieved above_NuAnd STEI_NuNumerical value substitute into formula 1,

${\mathrm{TEI}}_{Nu}=\frac{{\mathrm{HTEI}}_{Nu}+{\mathrm{STEI}}_{Nu}}{2}=0.004$

Can be obtained from above, the immunodeficiency degree of nude mice must be divided into 0.004.

6. embodiment eight:

The method utilizing mouse immune scale evaluation of the present invention, the immunity of comprehensive assessment C57BL/6 (writing a Chinese character in simplified form: B6) mice lacks Fall into degree (TEI_B6), specifically comprise the following steps that

6.1.HTEI_B6Numerical computations: transplant H respectively, M, the K562-GFP cell of L number level enter in nude Mice Body, often It observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 13.C57BL/6 mice-human blood Replanting model mice parameter list

Above parameter substitution formula is obtained:

${\mathrm{HETI}}_{(B6,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(B6,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

${\mathrm{HETI}}_{(B6,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{G_n}{D_i}=0.000$

HTEI_B6=average (HTEI_{(Nu, H)}, HTEI_{(Nu, M)}, TEI_(Nu_{, L)})=0.000

6.2.STEI_B6Numerical computations: transplant H respectively, M, the A549-GFP cell of L number level enter in nude Mice Body, often It observes mice, obtains and record experiment parameter such as following table when mice disease is sent out:

Table 14.B6 mice-people's entity tumor transplantation model parameter list

Above parameter substitution formula is obtained:

${\mathrm{STEI}}_{(B6,H)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

${\mathrm{STEI}}_{(B6,M)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

${\mathrm{STEI}}_{(B6,L)}=\frac{1}{5}\underset{n=1}{\overset{5}{\Σ}}\frac{W_n}{D_n}=0.000$

STEI_B6=average (STEI_{(B6, H)}, STEI_{(B6, M)}, STEI_{(B6, L)})=0.000

6.3.B6 the assessment of mouse immune defect level, TEI_B6Numerical computations:

By HTEI achieved above_B6And STEI_B6Numerical value substitute into formula obtain

${\mathrm{TEI}}_{B6}=\frac{{\mathrm{HTEI}}_{B6}+{\mathrm{STEI}}_{B6}}{2}=0.000$

Can be obtained from above, the immunodeficiency degree of B6 mice must be divided into 0.000.

From embodiment two to eight, immunodeficiency degree contrast (below figure) of seven kinds of Strains of Mouses is: NSI ＞ NOD- Scid ＞ scid ＞ nude ＞ Rag2-/-＞ IL2rg-/-＞ C57BL/6 (WT), this builds experiment for research worker animal model Suitable immunodeficient mouse is selected to provide important reference frame；Meanwhile, as shown in Figure 1, NSI mice has the strongest exempting from Epidemic disease defect level, therefore when research worker research and development Novel immune deficient mice, should be new using NSI mice as reference standard assessment The immunodeficiency degree of research and development mouse species.

7. embodiment nine

Immune system will be employed greatly, so tumor can be passed through because resisting, killing heteroplastic tumor cell The method (as set forth above) of xenotransplantation combined mathematical module, the immunodeficiency degree of comprehensive assessment mice.And need certain The assessment of the immunodeficiency degree such as one specific function, a certain certain immune cells, then need to test Replanting model mice with relative The mathematical model answered carries out certain amendment, to assess certain specific immune system defect degree, as the present embodiment utilizes swollen The method of tumor homotransplantation models coupling mathematical model, assesses tumor homoioplastic immunodeficiency degree.As follows:

8.1 mathematical model parameter implication is revised:

HTEI_{(strain, allograft)}, blood tumor transfer ability score of the same race, certain strain (Strain) representing to be assessed is little Mus is transplanted mouse blood oncocyte and rebuilds the ability score of neoplastic hematologic disorder (Hematologic Tumor), at this with mouse lymph Oncocyte system RMA transplants as standard.

Solid tumor transfer ability score of the same race, it is thin that certain strain (Strain) mice representing to be assessed transplants mouse entity tumor Born of the same parents rebuild the ability score of entity tumor (Solid Tumor), at this with mouse melanin tumor cell system B16-F10 as standard.

Represent certain strain (Strain) mice allogeneic tumor transplantation ability score, assess this Strains of Mouse to of the same race swollen with this The immunodeficiency degree of tumor.

8.2 are embodied as step:

8.2.1 the structure of the mouse blood oncocyte system of tape label gene

In RMA cell line import marker gene (as GFP green fluorescence gene, Tdtomato red fluorescent gene, Luciferase luciferase etc.).As a example by GFP green fluorescence gene, introduce marker gene introduction method below, pack Lenti- GFP virus infected tumor cell line, after the infected cell of amplification culture, successful GFP positive tumor cell is infected in sorting, it is thus achieved that GFP positive cell purity is more than 98%.

8.2.2 the structure of blood tumor mouse model and HTEI_strainCalculating

By varying number level (10⁶(H), 10⁵(M), 10⁴(L)) the blood tumor cell line (RMA-GFP) of tape label gene is moved Implanting specific immunodeficiency strain experiment mice (each order of magnitude is transplanted with 5 experiment mices) to be assessed, transplantation tumor is thin After born of the same parents, observe mice state every day.When mice disease is sent out, the time-to-live (D of record mice_n) and mouse blood, spleen, bone marrow In tumor cell (GFP positive cell) ratio (G_PB、G_SP、G_BM), and for people's formulaCalculate

8.2.3 the structure of solid tumor mouse model and STEI_strainCalculating

Transplant varying number level (10⁶(H), 10⁵(M), 10⁴(L)) solid tumor cell system (B16F10-GFP) is implanted into be evaluated The specific immunodeficiency strain experiment mice (each order of magnitude is transplanted with 5 experiment mices) estimated, after implanted tumor cells, every day Observe mice state.After Transplanted Human solid tumor cell system's certain time (observing time of different tumor cell lines is different, B16F10 cell line then carries out observed and recorded after transplanting 20 days), weigh and record the quality (W of the entity tumor of formation_n, Unit: g), and by D_n=20 and W_nSubstitute into formulaCalculate

8.2.4 immunodeficiency degree TEI of this Strains of Mouse is assessed_strain

By above-mentioned that calculate and to be entered formulaFinally calculate this strain little Mus allogeneic tumor transplantation ability score, and this numerical value is defined as this Strains of Mouse immunodeficiency degree to same tumor.

8.2.5 the allogeneic tumor transplantation ability score of 7 kinds of Strains of Mouses is assessed

By the inventive method, obtain respectively NSI, IL2rg-/-, NOD-scid, scid, Rag2-/-, nude, C57BL/ H, M, L of 6 seven kinds of mices transplants HTEI and the STEI numerical value of the order of magnitude, and finally obtains the TEI of different lines_allograftScore (such as table 15 and Fig. 2).

Table 15. same tumor immunodeficiency degree TEI

Can be obtained by embodiment nine result, the immunodeficiency degree contrast to same tumor is: NSI > IL2rg-/-> NOD- Scid > scid > Rag2-/-> nude > C57BL/6 (WT), NSI mice is the highest to the immunodeficiency degree of allogeneic tumor transplantation.

Of the same race, the height sequence of tumour immunity defect level is roughly the same with combined immune deficiency degree, it is seen then that comprehensively exempt from The immune system defect scale evaluation specific to mice of assessing of epidemic disease defect level has certain reference value.

Identify and kill that same tumor is more needs to employ inherent immunity system particularly NK natural killer cell, and IL2rg-/-higher with the NK cell defect degree of Rag2-/-mice, so IL2rg-/-and the same tumor of Rag2-/-mice The sequence of immunodeficiency degree is moved relatively forward.It follows that the method for zoopery combined mathematical module cannot be only used for combining Close the assessment of immunodeficiency degree, and can the specific immune system defect degree of accurate evaluation mice.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (6)

1. the method evaluating the immunodeficiency degree of immunodeficient mouse model, comprises the steps of

By transplanting the blood tumor cell of varying number level and solid tumor cell in immunodeficient mouse model, build and swell After tumor mouse model, tumor cells in peripheral blood G in detection tumor mouse model_PBRatio, tumor cell G in bone marrow_BMRatio Tumor cell G in example and spleen_SPRatio, according to the immunity of described scale parameter metrics evaluation immunodeficient mouse model Defect level.

Method the most according to claim 1, it is characterised in that described tumor cell is of the same race or xenogenesis.

Method the most according to claim 1, it is characterised in that the varying number level of tumor cell transplantation is L:1 × 10⁴It is individual, M:1 × 10⁵Individual, H:1 × 10⁶Individual.

Method the most according to claim 1, it is characterised in that the tumor cell in described xenograft tumor cell behaviour source, its Middle human blood tumor cell is preferably people's chronic myelogenous leukemia K562 cell line, and people's solid tumor cell is preferably people's pulmonary carcinoma A549 cell line；.

Method the most according to claim 1, it is characterised in that described same tumor cell is Mus source tumor cell, blood Tumor cell preferred mouse lymphoma cell system RMA, solid tumor cell preferred mouse melanin tumor cell system B16-F10.

Method the most according to claim 3, it is characterised in that according in peripheral blood in the tumor mouse model that detection obtains Tumor cell G_PBRatio, tumor cell G in bone marrow_BMRatio and spleen in tumor cell G_SPRatio, evaluate immunity The method of the immunodeficiency degree of deficient mice model comprises the steps, (1) uses formula to obtain transplanting each order of magnitude blood The liquid tumor cell action index HTEI to immunodeficient mouse,Gi is G_PB、G_BM、G_SPSum, Di is by blood Liquid tumor cell transplantation starts immunodeficient mouse model effect interval to tumor cell in entering immunodeficient mouse model Time, respectively obtain each order of magnitude blood tumor cell action index HTEI (H) to immunodeficient mouse, HTEI (M), HTEI (L), tries to achieve average HTEI the most again；

(2) formula is used to obtain the action index transplanting each order of magnitude solid tumor cell to immunodeficient mouse model STEI,Wn is the weight that entity tumor is implanted in immunodeficient mouse model the entity tumor formed, Dn For starting the interval time to immunodeficient mouse model effect to tumor cell in entity tumor is implanted into immunodeficiency, point Do not obtain each order of magnitude solid tumor cell action index STEI (H) to immunodeficient mouse, STEI (M), STEI (L), Finally try to achieve average STEI again；

(3) average HTEI, the average STEI obtained according to step (1) and step (2), uses equation below to obtain total function of tumor Index TEI,

This total function of tumor index and immunodeficiency degree are negative correlation.