CN105920041A - Method for constructing model of treating triple negative breast cancer with ulinastatin - Google Patents
Method for constructing model of treating triple negative breast cancer with ulinastatin Download PDFInfo
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Abstract
The invention discloses a method for constructing a model of treating triple negative breast cancer with ulinastatin. The method for constructing the model comprises the following steps: constructing a TNBC (triple negative breast cancer) cell model of CCL22 over-expression and knockdown; constructing a mice tumor-bearing model by virtue of 4T1 cells of CCL22 over-expression and knockdown; and constructing a mice transplantation tumor model by virtue of the 4T1 cells. Meanwhile, the invention discloses a method for detecting influence and mechanism of the ulinastatin on treating the triple negative breast cancer, wherein the method comprises the following steps: detecting inhibition of the ulinastatin on the amount of chemotactic factors CCL22 secreted by TNBC cells; detecting recruitment of the CCL22 on treg cells around the tumor cells; and detecting the expression of GITR on the surface of the treg cells. The research of the invention on the therapeutic value of the ulinastatin on treating the breast cancer is the first case at home and abroad; and the research on the mechanism of the drug affecting tumor microenvironment can offer an application basic research for the clinical application of the drug in the treatment of the breast cancer, in particular the treatment of the triple negative breast cancer.
Description
Technical field
The invention belongs to breast cancer treatment field, particularly relate to a kind of ulinastatin and treat three negative breast cancer
Model building method.
Background technology
Inventor is found by previous research work:
1) ulinastatin suppresses Cells Proliferation of Human Breast Cancer, promotes apoptosis, and its mechanism may be with ulinastatin
Reduce immunity/inflammatory factor TNF-a, IL-6 with IL-10 expression with regulation immunity microenvironment relevant.
2) ulinastatin and taxotere all can significantly inhibit breast cancer cell MDA-MB-231 invasion and attack, transfer, crow
Si Tading can strengthen the inhibitory action of taxotere, and mechanism may suppress blood vessel shape with the expression of downward TGF-β
Becoming, epithelium mesothelium converts relevant, meanwhile, also lowers the expression of breast cancer cell CXCR4 with ulinastatin
Relevant.
3) ulinastatin associating taxotere suppression breast carcinoma propagation and transfer, may be with regulation breast carcinoma microenvironment
Middle CD4+CD25+Foxp3+T/CD4+T is correlated with.
Research shows, ulinastatin has suppression breast carcinoma and includes three negative breast cancer (triple negative
Breast cancer, TNBC) breed the effect shifted, wherein to the shadow of Treg in tumour immunity microenvironment
Sound may play pivotal role.
Treg promotes that tumor generation immunologic escape is that a large amount of Chemokines CC CL22 produced by tumor cell are recruited
The raise Treg of high expressed chemokine receptors CCR4, to around tumor, on the one hand makes tumor tissues be in immunity and presses down
In environment processed, thus promote the propagation of tumor cell；On the other hand, Treg is gathered in tumor tissues week in a large number
Enclose, formed rich in the cellular layer of Treg, can effectively hinder NKT sexual cell (natural killer cell,
And the lymphocyte attack to tumor cell such as Tc NK).And exist during tumors secrete Chemokines CC CL22
TGF-β-microRNA-34a-CCL22 axle, the TGF-β of activation can suppress the table of microRNA-34a gene
Reach, increase so that Chemokines CC CL22 produces, transforming growth factor TGF-β and Chemokines CC CL22 are described
It is proportionate, it can be assumed that ulinastatin affects tumor by TGF-β microRNA-34a-CCL22 axle
The CCL22 quantity that cell produces.And the research of early stage and document all point out ulinastatin can reduce tumor cell
And the TGF-β of lymphocytic emiocytosis.Therefore previous work finds ulinastatin reduce around tumor tissues and
Treg in tumor, it may be possible to by acting on TGF-β microRNA-34a-CCL22 axle, reduces tumor cell
The quantity of secretion CCL22, thus reduce the gathering of Treg around tumor tissues, reduce tumor generation immunologic escape,
Suppression tumor proliferation, invasion and attack and metastasis.Separately there are research display, Treg surface inhibition immunity receptor sugar
Tumor Necrosis Factor Receptors (the glucocorticoid-inducedtumor necrosis of 17-hydroxy-11-dehydrocorticosterone induction
Factorreceptor, GITR), can be combined with the GITR part on DC surface, promote the propagation of Treg, and
The quantity of suppression DC surface GITR part so that it is decline with the adhesion of other T effector cells, depression effect
The function of property T cell.As can be seen here, the GITR on CCL22 and the Treg surface of tumor cell secretion, is regulation
The potential target spot of breast carcinoma immunity microenvironment.
May be by suppressing tumor cell secretion CCL22 currently, with respect to ulinastatin, and reduce Treg surface
The expression of GITR, suppresses quantity and the function of regulatory T cells in three negative breast cancer microenvironments, controls swollen
The data of tumor growth and transfer is not yet reported.
Summary of the invention
It is an object of the invention to provide ulinastatin and treat the model building method of three negative breast cancer, it is intended to
Study and may be by suppressing tumor cell secretion CCL22 about ulinastatin, and reduce Treg surface GITR
Expression, suppress quantity and the function of regulatory T cells in three negative breast cancer microenvironments, control tumor growth
And transfer, for the problem that effectively treatment provides new direction of three negative breast cancer.
The present invention is achieved in that
A kind of ulinastatin treats the model building method of three negative breast cancer, described ulinastatin treatment three
The model building method of negative breast cancer includes:
Build CCL22 process LAN and strike low TNBC cell model；
With CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model；
Use 4T1 cell construction mice-transplanted tumor model.
Another object of the present invention is to provide a kind of ulinastatin that detects to treat impact and the machine of three negative breast cancer
The method of system, the method for impact and mechanism that described detection ulinastatin treats three negative breast cancer includes following
Step:
Step one, by building CCL22 process LAN and strike low TNBC cell model, use quantitative PCR with
The suppression that the Chemokines CC CL22 of TNBC emiocytosis is measured by Western blot detection ulinastatin；
Step 2, by with CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model, use stream
Formula cell art and Western blot method detect the CCL22 recruitment near tumor cells treg cell, checking
The CCL22 impact on tumour immunity microenvironment；
Step 3, by use 4T1 cell construction mice-transplanted tumor model, application Western blot method detection
The expression of treg cell surface GITR, checking ulinastatin is raw to tumor cell by the change of Treg cell function
The impact that thing scholarship and moral conduct is.
Further, the suppression that the Chemokines CC CL22 of TNBC emiocytosis is measured by described detection ulinastatin,
The method used is:
By TNBC cell model detection checking ulinastatin to the Chemokines CC CL22 of secretion in TNBC cell
The change of amount；
Application ulinastatin processes TNBC cell, extracts cell RNA, confirms RNA mass；
Changed in the expression of mRNA level in-site by real-time fluorescence quantitative PCR detection CCL22；
Application ulinastatin processes TNBC cell, extracts total protein of cell, confirms albumen quality；
The protein expression situation in corresponding cell is detected, by relatively clear and definite ulinastatin by Western blot
CCL22 is affected in the expression of protein level；
Application ulinastatin processes TNBC cell, collects protein in culture medium, confirms protein quality；
By the level of CCL22 in ELISA detection culture medium, checking ulinastatin is secreted in TNBC cell
The change of amount of CCL22.
Further, the described detection CCL22 recruitment near tumor cells treg cell, checking CCL22 is to swollen
The method of the impact of tumor immunity microenvironment is:
Build CCL22 process LAN and strike low TNBC cell model,
With CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model,
Take the amount of Treg in tumor tissues, Flow cytometry tumor tissues and tumor peripheral lymph node, checking
The detection CCL22 recruitment effect to Treg；Detection gross tumor volume size, the immunity that in tumor tissues, Treg expresses
Factor IL-10, the differential expression of TGF-β, and tumor metastasis situation, by analyzing the number of detection Treg
Amount is on tumor growth and the impact of transfer.
Further, the expression of described detection treg cell surface GITR, checking ulinastatin is by Treg cell merit
The method on the impact of cellular biology of tumor behavior that changes of energy is:
By using 4T1 cell construction mice-transplanted tumor model, processing with ulinastatin, comparison of tumor growth turns
Shift one's love condition, study the ulinastatin impact on transplanted tumor；
Take the amount of Treg in tumor tissues, Flow cytometry tumor tissues and tumor peripheral lymph node, simultaneously
With the expression of GITR in western blot method detection Treg cell；
By analyzing detection ulinastatin to the Expression modulation effect of GITR and the impact on the recruitment of Treg cell
Impact with tumor biological behavior.
Further, described analysis uses SPSS.19.0 statistics software data processing, data withRepresent,
Use one factor analysis of variance to compare between carrying out organizing more, between group, compare employing LSD-t or Tamhane method two-by-two,
Inspection level α=0.05.
The research that the present invention is directed to ulinastatin therapeutic value in breast carcinoma the most all belongs to the first, pin
Research (e.g., up-to-date valuable discovery: ulinastatin is combined to this drug influence tumor microenvironment mechanism
Chemotherapeutics can reduce the ratio of CD4+CD25+FoxP3+/CD4+T cell in breast cancer tumour microenvironment)
Application base will be provided in the Clinical practice that breast cancer treatment particularly three negative breast cancer is treated for this medicinal application
Plinth is studied.
Accompanying drawing explanation
Fig. 1 is that the detection ulinastatin that the present invention provides treats the impact of three negative breast cancer and the method for mechanism
Flow chart.
Detailed description of the invention
For the summary of the invention of the present invention, feature and effect can be further appreciated that, hereby enumerate following example, and
Accompanying drawing is coordinated to describe in detail as follows.
Pass through the present invention:
1), ulinastatin and cyclophosphamide or taxotere compatibility, suppression breast cancer cell MCF-7/DA-MB-231
Propagation, its mechanism may reduce immunity, the expression of inflammatory factor TNF-a, IL-6 and IL-10 with ulinastatin
Relevant with regulation immunity microenvironment.
2), ulinastatin and taxotere all can significantly inhibit breast cancer cell MDA-MB-231 invasion and attack, transfer,
Ulinastatin can strengthen the inhibitory action of taxotere, and mechanism may be with downward VEGF-C, EGFR, AKT, TGF-
The expression of β, anti-angiogenesis, epithelium mesothelium converts relevant, also lowers breast cancer cell with ulinastatin
The expression of CXCR4 is correlated with.
3), ulinastatin and taxotere all can significantly inhibit breast cancer cell MDA-MB-231 invasion and attack, mechanism can
Can be suppressed with ERK, uPA, uPAR, MMP-9 down-regulated expression is relevant.
4), ulinastatin suppression Cells Proliferation of Human Breast Cancer, promote apoptosis, may with by suppression JNk-2
Reduce IGF-1R, PDGFA with NF-κ B path and generate relevant.
5), ulinastatin associating docetaxel suppresses growth of breast cancers and transfer, with reduction tumor microenvironment
The ratio of middle CD4+CD25+FoxP3+/CD4+T cell is relevant.
Below in conjunction with the accompanying drawings the present invention is described in detail.
A kind of ulinastatin treats the model building method of three negative breast cancer, described ulinastatin treatment three
The model building method of negative breast cancer includes:
Build CCL22 process LAN and strike low TNBC cell model；
With CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model；
Use 4T1 cell construction mice-transplanted tumor model.
As shown in Figure 1: a kind of ulinastatin that detects treats the impact of three negative breast cancer and the method for mechanism, institute
The method stating impact and mechanism that detection ulinastatin treats three negative breast cancer comprises the following steps:
S101: by building CCL22 process LAN and striking low TNBC cell model, use quantitative PCR with
The suppression that the Chemokines CC CL22 of TNBC emiocytosis is measured by Western blot detection ulinastatin；
S102: by with CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model, use streaming thin
Born of the same parents' art detects the CCL22 recruitment near tumor cells treg cell with Western blot method, verifies CCL22
Impact on tumour immunity microenvironment；
S103: by using 4T1 cell construction mice-transplanted tumor model, application Western blot method detection treg
The expression of cell surface GITR, checking ulinastatin is biological to tumor cell by the change of Treg cell function
The impact that scholarship and moral conduct is.
The suppression that the Chemokines CC CL22 of TNBC emiocytosis is measured by described detection ulinastatin, the side of employing
Method is: by building CCL22 process LAN and striking low TNBC cell model,
Application ulinastatin processes TNBC cell, extracts cell RNA, confirms RNA mass；
Changed in the expression of mRNA level in-site by real-time fluorescence quantitative PCR detection CCL22；
Application ulinastatin processes TNBC cell, extracts total protein of cell, confirms albumen quality；
The protein expression situation in corresponding cell is detected, by relatively clear and definite ulinastatin by Western blot
CCL22 is affected in the expression of protein level；
Application ulinastatin processes TNBC cell, collects protein in culture medium, confirms protein quality；
By the level of CCL22 in ELISA detection culture medium, checking ulinastatin is secreted in TNBC cell
The change of amount of CCL22.
The described detection CCL22 recruitment near tumor cells treg cell, CCL22 is micro-to tumour immunity in checking
The method of the impact of environment is:
Build CCL22 process LAN and strike low TNBC cell model,
By with CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model, take tumor tissues, stream
The amount of Treg in formula cell art detection tumor tissues and tumor peripheral lymph node, checking detection CCL22 is to Treg
Recruitment effect；Detection gross tumor volume size, the immune factor IL-10 of Treg expression, TGF-β in tumor tissues
Differential expression, and tumor metastasis situation, and turned tumor growth by the quantity analyzing detection Treg
The impact moved.
The expression of described detection treg cell surface GITR, the change by Treg cell function of the checking ulinastatin
On the method for the impact of cellular biology of tumor behavior it is:
By using 4T1 cell construction mice-transplanted tumor model, processing with ulinastatin, comparison of tumor growth turns
Shift one's love condition, study the ulinastatin impact on transplanted tumor；
Take the amount of Treg in tumor tissues, Flow cytometry tumor tissues and tumor peripheral lymph node, simultaneously
With the expression of GITR in western blot method detection Treg cell；
By analyzing detection ulinastatin to the Expression modulation effect of GITR and the impact on the recruitment of Treg cell
Impact with tumor biological behavior.
Described analysis uses SPSS.19.0 statistics software data processing, data withRepresent, use single
Analysis of variance compares between carrying out organizing more, compares employing LSD-t or Tamhane method two-by-two, check water between group
Quasi-α=0.05.
The research that the present invention is directed to ulinastatin therapeutic value in breast carcinoma the most all belongs to the first, pin
Research to this drug influence tumor microenvironment mechanism will be cloudy in breast cancer treatment particularly three for this medicinal application
Property breast cancer treatment Clinical practice provide applied basic research.
The present invention is further described in conjunction with the embodiments.
The impact that CCL22 is expressed in TNBC cell by checking ulinastatin includes with the concrete grammar of mechanism:
Step one, application blank and ulinastatin (point low concentration 400UI/ml, middle concentration 800UI/ml,
High concentration 1600UI/ml) process TNBC cell (4T1 and MDA-MB-231), in the 12nd, 24,36,48
Hour extracting cell RNA, detection RNA concentration also confirms that RNA mass is good；
Step 2, take above-mentioned RNA by reverse transcription construction cDNA library, detected by real-time fluorescence quantitative PCR
CCL22, in the expression of mRNA level in-site, compares ulinastatin and processes and blank group differential expression and variable concentrations
The difference that between ulinastatin packet, CCL22 expresses, specifies the impact that CCL22mRNA is expressed by ulinastatin；
Step 3, application blank and variable concentrations ulinastatin process TNBC cell (4T1 and
MDA-MB-231), different time extracts total protein of cell, and application BCA test kit detection protein concentration also confirms
Albumen quality is good；
Step 4, take above-mentioned albumen and carry out Western blot analysis, by CCL22 antibody recognition, detect CCL22
In the expression of protein level, compare ulinastatin and process and blank group differential expression and variable concentrations crow department
The difference that between his fourth packet, CCL22 expresses, specifies the ulinastatin impact on CCL22 protein expression.
Step 5, application blank and ulinastatin (point low concentration 400UI/ml, middle concentration 800UI/ml,
High concentration 1600UI/ml) process TNBC cell (4T1 and MDA-MB-231), in the 24th, 48,72 hours
Collecting cell culture medium, albumen in enrichment medium, application BCA test kit detection protein concentration also confirms albumen
Quality is good；
Step 6, take above-mentioned albumen and carry out elisa assay, by CCL22 antibody recognition, detection CCL22's
Amount, compares CCL22 between ulinastatin process and blank group differential expression and the packet of variable concentrations ulinastatin
The difference of amount, specifies the ulinastatin impact on TNBC cell exocrine CCL22 protein content.
Inquire into CCL22 and pass through the immune induction impact on tumor biological behavior, method particularly includes:
Step one, structure CCL22 process LAN plasmid and siRNA interference plasmid, packed by viral vector, sense
Dye TNBC cell (4T1 cell), obtains CCL22 process LAN by screening and strikes low cell strain (4T1-CCL22
With 4T1-CCL22-down cell), by real-time fluorescence quantitative PCR and Western blot at mRNA and albumen
Level verification process LAN and interference effect；
Step 2, cultivation 4T1 cell, 4T1-CCL22 and 4T1-CCL22-down cell, collect cell, OK
Subcutaneous injection builds mouse-borne tumor model, and every treated animal is 8-10；
Step 3, observation in every 3 days, measure tumor volume, draw growth of xenografted curve.To transplanted tumor diameter
About putting to death mice during 1cm, take tumor tissues, half paraffin embedding is for HE dyeing and SABC detection, partly
The amount of Treg cell around quantitative analysis tumor tissues；It is quantitative by flow cytometry that half makes cell suspension
Analyze the amount of Treg in tissue, by the above-mentioned experimental verification CCL22 recruitment effect to Treg.Take mice simultaneously
Viscera tissue, analyzes tumor metastasis situation；
Step 4, with above-mentioned paraffin-embedded tissue cut into slices, use Immunohistochemical Method detection tumor tissues in Treg
The immune factor IL-10 that expresses, the differential expression of TGF-β etc., by with tumor size and metastasis situation
Correlation analysis confirms that the quantity of Treg is on tumor growth and the impact of transfer.
Further, ulinastatin is inquired on the impact that GITR in Treg cell expresses method particularly includes:
Step one, cultivating above-mentioned 4T1 cell, collect cell, row subcutaneous injection builds mouse-borne tumor model, meter
Draw and use animal 24；
The ulinastatin of step 2, point blank and variable concentrations processes laboratory animal, and often group 8-10 is the least
Mus, shares 2 weeks.Within every 3 days, observing, measure tumor volume, draw growth of xenografted curve, comparison of tumor is raw
Long transfer case, by the impact on transplanted tumor of the comparative analysis clear and definite ulinastatin；
Step 3, to putting to death mice during transplanted tumor diameter about 1cm, take tumor tissues, half paraffin embedding is used
In HE dyeing and SABC detection, the amount of Treg cell around semi-quantitative analysis tumor tissues；Half makes
Cell suspension is by the amount of Treg in flow cytometry quantitative analysis tissue, by above-mentioned experimental verification CCL22 pair
The recruitment effect of Treg.Take mice viscera tissue simultaneously, analyze tumor metastasis situation；
Step 4, with above-mentioned paraffin-embedded tissue cut into slices, use Immunohistochemical Method semi-quantitative analysis tumor tissues
The expression of GITR in middle Treg cell, regulation GITR expressed by correlation analysis clear and definite ulinastatin, with
Time carry out correlation analysis with tumor growth situation and transfer case, inquire into ulinastatin by regulation GITR to shifting
Plant the impact of tumor biological behaviour.
The immunoregulation effect of internal checking ulinastatin method particularly includes:
Step one, in clinic select TNBC patient with breast cancer 80 example, random packet, with placebo (n=40)
Comparison and ulinastatin (1600U/ person/day) (n=40) process 2 weeks；
Step 2, medication adopt Venous Blood 10ml after 3-4 week, and by the dense poly-hemocyte of centrifuge, streaming is thin
Born of the same parents instrument patient detects the amount of Treg cell in blood；
Step 4, compare the different disposal impact on patient's Treg cell, the clearest and the most definite ulinastatin pair
The regulation effect of Treg cell.
The research that the present invention is directed to ulinastatin therapeutic value in breast carcinoma the most all belongs to the first, pin
Research to this drug influence tumor microenvironment mechanism will be cloudy in breast cancer treatment particularly three for this medicinal application
Property breast cancer treatment Clinical practice provide applied basic research.
The above is only to presently preferred embodiments of the present invention, not makees the present invention any pro forma
Limiting, any simple modification made for any of the above embodiments, equivalent are become by every technical spirit according to the present invention
Change and modify, belonging in the range of technical solution of the present invention.
Claims (6)
1. a ulinastatin treats the model building method of three negative breast cancer, it is characterised in that described
Ulinastatin is treated the model building method of three negative breast cancer and is included:
Build CCL22 process LAN and strike low TNBC cell model；
With CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model；
Use 4T1 cell construction mice-transplanted tumor model.
2. the impact utilizing the detection ulinastatin of method described in claim 1 to treat three negative breast cancer with
The method of mechanism, it is characterised in that described detection ulinastatin treats impact and the mechanism of three negative breast cancer
Method comprise the following steps:
Step one, by building CCL22 process LAN and strike low TNBC cell model, use quantitative PCR with
The suppression that the Chemokines CC CL22 of TNBC emiocytosis is measured by Western blot detection ulinastatin；
Step 2, by with CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model, use stream
Formula cell art and Western blot method detect the CCL22 recruitment near tumor cells treg cell, checking
The CCL22 impact on tumour immunity microenvironment；
Step 3, by use 4T1 cell construction mice-transplanted tumor model, application Western blot method detection
The expression of treg cell surface GITR, checking ulinastatin is raw to tumor cell by the change of Treg cell function
The impact that thing scholarship and moral conduct is.
3. detection ulinastatin as claimed in claim 2 treats the impact of three negative breast cancer and the side of mechanism
Method, it is characterised in that described detection ulinastatin is to pressing down that the Chemokines CC CL22 of TNBC emiocytosis measures
System, the method for employing is: by building CCL22 process LAN and striking low TNBC cell model,
Application ulinastatin processes TNBC cell, extracts cell RNA, confirms RNA mass；
Changed in the expression of mRNA level in-site by real-time fluorescence quantitative PCR detection CCL22；
Application ulinastatin processes TNBC cell, extracts total protein of cell, confirms albumen quality；
The protein expression situation in corresponding cell is detected, by relatively clear and definite ulinastatin by Western blot
CCL22 is affected in the expression of protein level；
Application ulinastatin processes TNBC cell, collects protein in culture medium, confirms protein quality；
By the level of CCL22 in ELISA detection culture medium, checking ulinastatin is secreted in TNBC cell
The change of amount of CCL22.
4. detection ulinastatin affecting and the method for mechanism in three negative breast cancer as claimed in claim 2,
It is characterized in that, the described detection CCL22 recruitment near tumor cells treg cell, checking CCL22 is to swollen
The method of the impact of tumor immunity microenvironment is:
Build CCL22 process LAN and strike low TNBC cell model,
With CCL22 process LAN and strike low 4T1 cell construction mouse-borne tumor model,
Take the amount of Treg in tumor tissues, Flow cytometry tumor tissues and tumor peripheral lymph node, checking
The detection CCL22 recruitment effect to Treg；Detection gross tumor volume size, the immunity that in tumor tissues, Treg expresses
Factor IL-10, the differential expression of TGF-β, and tumor metastasis situation, by analyzing the number of detection Treg
Amount is on tumor growth and the impact of transfer.
5. detection ulinastatin affecting and the method for mechanism in three negative breast cancer as claimed in claim 2,
It is characterized in that, the expression of described detection treg cell surface GITR, checking ulinastatin is by Treg cell merit
The method on the impact of cellular biology of tumor behavior that changes of energy is:
By using 4T1 cell construction mice-transplanted tumor model, processing with ulinastatin, comparison of tumor growth turns
Shift one's love condition, study the ulinastatin impact on transplanted tumor；
Take the amount of Treg in tumor tissues, Flow cytometry tumor tissues and tumor peripheral lymph node, simultaneously
With the expression of GITR in western blot method detection Treg cell；
By analyzing detection ulinastatin to the Expression modulation effect of GITR and the impact on the recruitment of Treg cell
Impact with tumor biological behavior.
6. the detection ulinastatin as described in claim 4-5 any one is at the impact of three negative breast cancer and machine
The method of system, it is characterised in that described analysis uses SPSS.19.0 statistics software data processing, data
WithRepresent, use one factor analysis of variance to compare between carrying out organizing more, between group, compare employing LSD-t two-by-two
Or Tamhane method, inspection level α=0.05.
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