CN109507161A - Application of the TIMP-1 in the kit and suppression cancer drug of preparation Prognosis in Breast Cancer diagnosis - Google Patents
Application of the TIMP-1 in the kit and suppression cancer drug of preparation Prognosis in Breast Cancer diagnosis Download PDFInfo
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- CN109507161A CN109507161A CN201811441062.0A CN201811441062A CN109507161A CN 109507161 A CN109507161 A CN 109507161A CN 201811441062 A CN201811441062 A CN 201811441062A CN 109507161 A CN109507161 A CN 109507161A
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Abstract
The invention discloses application of the TIMP-1 in the kit and suppression cancer drug of preparation Prognosis in Breast Cancer diagnosis, and kit includes the reagent for detecting the TIMP-1 expression quantity of neutrophil leucocyte in tumor epithelial cell；In addition, TIMP-1 neutralizing antibody can inhibit the transfer of breast cancer cell.Kit of the present invention assists existing Prognosis, to improve the survival rate of patient；The present invention also provides a kind of new breast cancer treatment regimens.
Description
Technical field
The invention belongs to the kits that biomedicine technical field more particularly to TIMP-1 are diagnosed in preparation Prognosis in Breast Cancer
With the application in suppression cancer drug.
Background technique
Breast cancer is the highest malignant tumour of women worldwide disease incidence.In the coastal developed regions such as the Guangdong Province in China,
Due to the increase of living-pattern preservation and social pressures, the disease incidence of women with breast cancer is much higher than world's balanced growth speed
Degree.About 50% patient can cure after operative treatment, but transfer or palindromia may occur for remaining patient.Therefore, it predicts
High-risk patient, and the appropriate adjuvant treatment of progress helps to improve the survival rate of patient as early as possible.Currently, prognostic indicator includes tumour
Whether size, lymph node occur transfer, Pathologic Grading, Expression of Estrogen and Progesterone Receptors state etc..Studies have shown that Expression of Estrogen and Progesterone Receptors is positive
Property Patients on Recurrence rate it is low, survival rate is high, and prognosis is preferable；Ki-67 is a kind of nucleoprotein relevant to proliferation, and Ki-67 overexpression is
The important risk factor of tumor by local recurrence.With the understanding to breast cancer disease development process and mechanism, prognosis is therefrom found
Index and the new approaches for seeking breast cancer treatment.
Neutrophil leucocyte is one of most common cell of peripheral blood.It is primarily involved in cell as inflammatory immune response,
It also plays an important role in the morbidity development of breast cancer.In breast cancer it is reported that neutrophil leucocyte and lymph in patient with breast cancer
The ratio of cell increases related to poor prognosis.But in tumor epithelial cell, neutrophil leucocyte is related to prognosis unclear.
It is one of important mechanisms of Metastasis in Breast Cancer that tumour cell Epithelial and stromal, which converts (EMT), and the tumour cell of EMT occurs
Migration and invasive ability significantly improve.Interaction may influence the diffusion of tumour between tumor microenvironment and tumour cell.It is more
Studies have shown that occur EMT tumour cell can refigure tumor microenvironment, so that it is further deepened tumour cell
The degree of EMT promotes transfer etc..Therefore, it is necessary to a kind of drug be designed for EMT, to inhibit the transfer of breast cancer cell.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, and TIMP-1 is provided in preparation Prognosis in Breast Cancer diagnosis
Kit and suppression cancer drug in application, the kit be included in detection tumor epithelial cell infiltration neutrophil leucocyte in TIMP-1
The reagent of expression quantity, kit can assist existing Prognosis, to improve the survival rate of patient；In addition, TIMP-1 is neutralized
Antibody can inhibit the transfer of breast cancer cell.
To achieve the above object, the technical scheme adopted by the invention is as follows: it is a kind of for Prognosis in Breast Cancer diagnosis kit,
It includes the reagent for detecting TIMP-1 expression quantity in the neutrophil leucocyte of tumor epithelial cell infiltration.
As an improvement of the above technical solution, kit includes for detecting neutrophil leucocyte CD66b expression quantity and TIMP-
The reagent of 1 expression quantity.
As a further improvement of the above technical scheme, described for detecting the reagent of neutrophil leucocyte CD66b expression quantity
CD66b secondary antibody including CD66b primary antibody and fluorescent marker, it is described for detecting the reagent packet of neutrophil leucocyte TIMP-1 expression quantity
Include the TIMP-1 secondary antibody of TIMP-1 primary antibody and fluorescent marker, the fluorescence of CD66b secondary antibody label and the fluorescence of TIMP-1 secondary antibody label
Not identical, CD66b primary antibody, CD66b secondary antibody, TIMP-1 primary antibody and TIMP-1 secondary antibody are binding antibody.
Further improvement as above-mentioned technical proposal, the reagent further include antigen retrieval buffers and hydrogen peroxide.
In addition, the present invention also provides a kind of for inhibiting the drug of breast cancer comprising TIMP-1 neutralizing antibody.
As an improvement of the above technical solution, the drug further includes pharmaceutically acceptable auxiliary material.
The present invention also provides a kind of detection methods of kit comprising following steps:
1) collect patient breast cancer tissue sample, by dehydration, embedding, slice and etc. histotomy is made；
2) 1) histotomy in is dewaxed, aquation, antigen retrieval, closing nonspecific sites, to CD66b mono-
Anti-, TIMP-1 primary antibody and histotomy are incubated for；
3) 2) the CD66b secondary antibody of histotomy and fluorescent marker, the TIMP-1 secondary antibody of fluorescent marker in are incubated for；
4) 3) histotomy in and dapi are incubated for, fluorescence microscopy under the microscope, to neutrophil leucocyte in tumor epithelial cell
TIMP-1 expression quantity recorded；Related data is analyzed by SPSS software.
The beneficial effects of the present invention are: the present invention provides kit and suppression of the TIMP-1 in preparation Prognosis in Breast Cancer diagnosis
Application in cancer drug, kit of the present invention mainly for detection of neutrophil leucocyte in tumor epithelial cell TIMP-1 expression quantity, in
Property granulocyte TIMP-1 expression quantity and patient with breast cancer's prognosis have strong correlation, the TIMP-1 expression quantity of neutrophil leucocyte
It can be used as the index of patient with breast cancer's prognosis；1) kit of the present invention, which has the advantage that, is suitable for all patient with breast cancers；
2) easy to detect, Testing index has obvious related to prognosis, and Testing index is single, easy to operate；3) all reagents mostly compared with
To be common, cost control is in feasible region.In addition, the present invention also provides a kind of new breast cancer treatment regimens: TIMP-1
Neutralizing antibody can inhibit the transfer of breast cancer cell.
Detailed description of the invention
Fig. 1 shows the Infiltrating of neutrophil leucocyte in tumor epithelial cell；Wherein, it is thin only to iris out part neutrality grain for black ellipse
Born of the same parents；
Fig. 2 shows the Infiltrating of neutrophil leucocyte and the relationship of survival, wherein ordinate Disease-Free
Survival indicates disease-free survival rate, and ordinate Overall Survival indicates overall survival；Top in each small figure
The sample standard deviation of curve has infiltrated the patient of less neutrophil leucocyte from tumor epithelial cell, the sample of the curve of lower section in each small figure
Originally the patient that tumor epithelial cell has infiltrated more neutrophil leucocyte is derived from；The quantity of digital representation sample behind curve bracket；
The case where migration of Fig. 3 tumor cells showed and invasion；Wherein, blank control refers to thinless with other in tumour cell
Born of the same parents co-culture；
After Fig. 4 statistics co-cultures, the tumour cell quantity of migration and invasion occurs；Wherein, MCF-7, MB-231 and BT-
474 be 3 kinds of breast cancer cells ,-indicating that tumour cell is not co-cultured with it, neu indicates that the neutral grain of addition is thin in tumour cell
Born of the same parents, TINs indicate the neutrophil leucocyte (following similar) that tumor epithelial cell infiltration is added in tumour cell；* * indicates that difference is very aobvious
It writes；
Fig. 5 counts the expression quantity of the TIMP-1 of neutrophil leucocyte；Wherein, * * indicates significant difference；* * indicates difference very
Significantly；
Fig. 6 display recombination TIMP-1 (rTIMP-1) to e-cadherin in MCF-7 cell (calcium adhesion protein E) and
The influence of vimentin (vimentin) expression quantity；Indicate to be that rTIMP-1 is not added in MCF-7 cell；
Fig. 7 shows influence of the TIMP-1 neutralizing antibody to tumor cell migration and invasion；Wherein, Ctrl indicates tumour cell
Individually culture, TINs co-culture the neutrophil leucocyte for indicating that tumor epithelial cell infiltration is added in tumour cell ,-indicate that other are not added
Component, IgG indicate to be additionally added IgG when tumor cell culture, and TIMP-1 neutralizing antibody indicates to be additionally added when tumor cell culture
TIMP-1 neutralizing antibody；
Fig. 8 shows that TIMP-1 neutralizing antibody significantly inhibits the thin transfer of mouse breast cancer；Wherein, picture blur does not know it
Place indicates the transfer of breast cancer cell；
The luminous flux of breast cancer cell in Fig. 9 statistics lung, liver；Wherein, * * indicates significant difference；
Figure 10 shows the result of sufferer breast cancer tissue center dye, TIMP-1 dyeing and CD66B dyeing；Wherein, high table
Show that the expression quantity of TIMP-1 is more, low indicates that the expression quantity of TIMP-1 is less；
Figure 11 shows the expression quantity of TIMP-1 and the relationship of survival；Wherein, the curve of top in each small figure
Sample standard deviation expresses the patient of less TIMP-1 from the neutrophil leucocyte of tumor epithelial cell infiltration, the curve of lower section in each small figure
Sample standard deviation express the patient of more TIMP-1 from the neutrophil leucocyte of tumor epithelial cell infiltration；Digital table behind curve bracket
The quantity of sample sheet；
Figure 12 shows the result of e-cadherin dyeing, CD90 dyeing and CK dyeing in sufferer breast cancer tissue；
The CD90 expression quantity of Figure 13 tumor cells showed and the relationship of survival, wherein ordinate Disease-Free
Survival indicates disease-free survival rate, and ordinate Overall Survival indicates overall survival；Top in each small figure
The sample standard deviation of curve expresses the patient of less CD90 from tumour reality cell, and the sample standard deviation of the curve of lower section comes in each small figure
The patient of more CD90 is expressed derived from tumour reality cell；The quantity of digital representation sample behind curve bracket；
Figure 14 shows that CD90 neutralizing antibody significantly inhibits the thin transfer of mouse breast cancer；Wherein, the transfer of breast cancer cell
It has irised out；
The transfer stove quantity of breast cancer cell in Figure 15 display statistics lung, liver；Wherein, * * * indicates difference highly significant.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiment, subordinate list and attached
The invention will be further described for figure.
Specification is needed, it is routine experiment method that the present invention, which is not mentioned to experimental method,；In addition, the present invention is used for
Antibody is divided into binding antibody and neutralizing antibody, and the antibody for detecting antigenic profile is binding antibody, is used as drug
Antibody is neutralizing antibody.
The Infiltrating of neutrophil leucocyte is related to prognosis in tumor epithelial cell
Hospital's breast cancer patients tissue specimen is collected, sample is diagnosed as breast cancer through pathological diagnosis.By being dehydrated, wrapping
Bury, be sliced and etc. histotomy is made；The steps such as pathological section dewaxed, aquation, antigen retrieval, closing nonspecific sites
Suddenly, histotomy successively carries out incubation processing with CD66b primary antibody and CD66b secondary antibody；Then, it develops the color to slice, record is swollen
The quantity of the neutrophil leucocyte of the CD66b positive in tumor essence.
As shown in Figure 1, being impregnated with neutrophil leucocyte in tumor epithelial cell.As shown in Fig. 2, coming from the souvenir of Zhongshan University's sun yat-sen
The neutrophil leucocyte of the CD66b positive in 341 patients of hospital, respectively in test group (170 patients), internal verification group (171
Patient) and external certificate group (105 patients) in analyze the quantity of tumor epithelial cell neutrophil leucocyte and the relationship of Clinical Outcome,
Test group are as follows: 95%CI HR=2.88-8.68, p < 0.001；Internal verification group be 95%CI HR=2.07-6.14, p <
0.001；External certificate group are as follows: 95%CI HR=2.27-11.33, p < 0.001；It can be seen that the neutrality of tumor epithelial cell infiltration
Granulocyte quantity is obviously related to prognosis: the neutrophil leucocyte infiltrated in tumor epithelial cell is fewer, and survival is higher.
Neutrophil leucocyte promotes breast cancer cell migration and invasion
As shown in Figure 3 and Figure 4, it is tested by the migration of transwell gradient serum-concentration, discovery is at the same time
Interior, the tumour cell after co-culturing with the neutrophil leucocyte of tumor epithelial cell infiltration passes through transwell faster and more, obtains
The neutrophil leucocyte of stronger transfer ability, this prompt tumor epithelial cell infiltration promotes the migration of breast cancer cell；In addition it invades
Experiment has obtained same result: the neutrophil leucocyte of tumor epithelial cell infiltration promotes the invasion of breast cancer cell.
The expression of the neutrophil leucocyte TIMP-1 (neutrophil leucocyte matrix metalloproteinase inhibitors) of tumor epithelial cell infiltration
It is significant to increase
Hospital's breast cancer patients tissue specimen is collected, the neutrophil leucocyte that tumor epithelial cell infiltrates is separately cultured, training is taken
Feeding supernatant detects TIMP-1.As shown in figure 5, with untreated normal human peripheral blood's neutrophil leucocyte and do not occur EMT (on
Skin mesenchymal transformation) the neutrophil leucocyte that co-cultures of breast cancer cell MCF-7 compare, the neutrophil leucocyte of tumor epithelial cell infiltration
The TIMP-1 content of supernatant is significantly high.
The TIMP-1 of the neutrophil leucocyte release of tumor epithelial cell infiltration can promote tumour cell that EMT occurs
As shown in fig. 6, handling breast cancer cell MCF-7 with TIMP-1 recombinant protein, significant epithelium occurs for MCF-7 cell
Change and change: e-cadherin expression reduces, vimentin expression rises；As shown in fig. 7, the generation with unprocessed/IgG processing
The tumour cell of EMT is compared, and after the processing of TIMP-1 neutralizing antibody, tumor cell migration, invasive ability are substantially reduced.
TIMP-1 neutralizing antibody inhibits mouse mastopathy cell to be transferred to lung, in liver
As shown in Figure 8 and Figure 9, it in breast cancer BALB/c tumor-bearing mice, compared with untreated and IgG processing mouse, adopts
After being handled with TIMP-1 neutralizing antibody, breast cancer cell is substantially reduced in mouse lung, hepatic metastases, and TIMP-1 neutralizing antibody obviously presses down
Metastasis in Breast Cancer has been made into lung, liver.
The expression of the neutrophil leucocyte TIMP-1 of tumor epithelial cell infiltration is related to Prognosis in Breast Cancer
Hospital's breast cancer patients tissue specimen is collected, sample is diagnosed as breast cancer through pathological diagnosis.By being dehydrated, wrapping
Bury, be sliced and etc. histotomy is made；The steps such as pathological section dewaxed, aquation, antigen retrieval, closing nonspecific sites
Suddenly, it is incubated for by CD66b primary antibody, TIMP-1 primary antibody and histotomy；Slice in succession with the immunohistochemistry secondary antibody of corresponding kind,
Dapi is incubated for, and records the expression situation of change of tumour cell CD66b and TIMP-1.As shown in Figure 10 and Figure 11, pass through analysis
TIMP-1 high expresses patient and Survival, and discovery TIMP-1 is obviously related to prognosis: the neutrophil leucocyte of tumor epithelial cell infiltration
Fewer TIMP-1 is expressed, survival is higher.
The expression and tumour cell of tumour cell CD90 (member of cell adhesion molecule immunoglobulin superfamily)
EMT degree is related, while related to prognosis
Hospital's breast cancer patients tissue specimen is collected, sample is diagnosed as breast cancer through pathological diagnosis.By being dehydrated, wrapping
Bury, be sliced and etc. histotomy is made；The steps such as pathological section dewaxed, aquation, antigen retrieval, closing nonspecific sites
Suddenly, by CD90 primary antibody (binding antibody), e-cadherin primary antibody (binding antibody), CK (cytokeratin) primary antibody (in conjunction with anti-
Body) it is incubated for histotomy；Slice is incubated for the immunohistochemistry secondary antibody of corresponding kind, dapi in succession, records tumour cell e-
The expression situation of change of cadherin, CD90.As shown in Figure 12 and Figure 13, patient and existence feelings are expressed by analysis CD90 high
Condition, discovery CD90 are obviously related to prognosis: the survival rate of low expression CD90 patient is higher.
CD90 neutralizing antibody is used in breast cancer tumor-bearing mice, and breast cancer cell can obviously be inhibited to be transferred to lung, in liver
As shown in Figure 14 and Figure 15, in breast cancer BALB/c tumor-bearing mice, compared with untreated and IgG processing mouse,
After being handled using CD90 neutralizing antibody, mouse lung, liver turn middle breast cancer cell transfer amount and substantially reduce, and CD90 neutralizing antibody is obvious
Inhibit Metastasis in Breast Cancer into lung, liver turn.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and
Range.
Claims (6)
1. a kind of kit for Prognosis in Breast Cancer diagnosis, which is characterized in that including for detecting in tumor epithelial cell infiltration
The reagent of TIMP-1 expression quantity in property granulocyte.
2. kit as described in claim 1, which is characterized in that including for detect neutrophil leucocyte CD66b expression quantity and
The reagent of TIMP-1 expression quantity.
3. kit as claimed in claim 2, which is characterized in that described for detecting neutrophil leucocyte CD66b expression quantity
Reagent includes the CD66b secondary antibody of CD66b primary antibody and fluorescent marker, described for detecting the examination of neutrophil leucocyte TIMP-1 expression quantity
Agent includes the TIMP-1 secondary antibody of TIMP-1 primary antibody and fluorescent marker, what the fluorescence and TIMP-1 secondary antibody of CD66b secondary antibody label marked
Fluorescence is not identical, and CD66b primary antibody, CD66b secondary antibody, TIMP-1 primary antibody and TIMP-1 secondary antibody are binding antibody.
4. kit as claimed in claim 3, which is characterized in that the reagent further includes antigen retrieval buffers and hydrogen peroxide.
5. a kind of for inhibiting the drug of breast cancer, which is characterized in that including TIMP-1 neutralizing antibody.
6. drug as claimed in claim 5, which is characterized in that further include pharmaceutically acceptable auxiliary material.
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Cited By (1)
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| CN111735944A (en) * | 2019-03-25 | 2020-10-02 | 首都医科大学附属北京世纪坛医院 | Application of CICs in breast tumor tissue in preparation of products for predicting breast cancer prognosis survival |
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