CN103399151A - Application of CXCL8 cytokine detection reagent in preparation of papillary thyroid cancer diagnostic reagent - Google Patents
Application of CXCL8 cytokine detection reagent in preparation of papillary thyroid cancer diagnostic reagent Download PDFInfo
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- CN103399151A CN103399151A CN2013102544994A CN201310254499A CN103399151A CN 103399151 A CN103399151 A CN 103399151A CN 2013102544994 A CN2013102544994 A CN 2013102544994A CN 201310254499 A CN201310254499 A CN 201310254499A CN 103399151 A CN103399151 A CN 103399151A
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Abstract
Relating to the technical field of medicines, the invention in particular relates to application of a CXCL8 cytokine detection reagent in preparation of a papillary thyroid cancer diagnostic reagent. The steps include: subjecting a papillary thyroid cancer (PTC) specimen to isolation and conducting culture to obtain a primary tumor-associated macrophage (TAM), according to the hypothesis that a macrophage acts on a tumor cell through a secreted cytokine, culturing the primary TAM isolated from PTC, collecting the conditional medium, and using the conditional medium to stimulate a papillary thyroid cancer cell line. Experiments prove that: the TAM can promote the growth of papillary thyroid cancer cells. Then, the papillary thyroid cancer cell line, a normal people PBMC (peripheral blood mononuclear cell), the TAM and the like are detected. Experiments prove that: CXCL8 is highly expressed in TAM. Therefore, application of the CXCL8 cytokine in preparation of a papillary thyroid cancer diagnostic reagent can enhance the diagnostic rate of papillary thyroid cancer, and papillary thyroid cancer patients can avoid missing the best treatment time.
Description
Technical field
The present invention relates to medical technical field, the particularly application of CXCL8 cytokines measurement reagent in the diagnostic reagent of preparation thyroid papillary carcinoma.
Background technology
Thyroid cancer is modal endocrine tumors, incidence of disease rapid development in recent years, become one of modal malignant tumour, its incidence of disease is up to the 5th in American Women's, in China has also reached women's malignant tumour the 7th, wherein 90% is thyroid papillary carcinoma (papillary thyroid cancer, PTC).Although the mortality ratio of PTC is compared with other malignant tumours, mortality ratio is lower, but the PTC rate of transform is very high, rate of lymph-node metastasis is up to 30%-50%, if PTC shifts and recurrence, patient could not in time diagnose, and result is lost best opportunity of operation, mortality ratio significantly raises, and is the important indicator of prognosis mala.
In recent years, the function and significance of tumor microenvironment in tumorigenesis gradually paid attention to, and the biological behaviour of tumour is not only by tumour cell decision itself, and each cell component in tumor microenvironment all participates in the links of tumor development.TAM (tumor-associatedmacrophage, TAM) be one of cellular component important in tumor microenvironment, the meta-analysis discovery, all there are positive correlation in the density of TAM and the poor prognosis of dissimilar tumour, as lymthoma, breast cancer, prostate cancer etc.; The expression of the cell factor relevant with chemotactic with the macrophage differentiation is also relevant with the poor prognosis of tumour; In body experiment also prove the increase of TAM quantity or reduce can the appreciable impact tumour growth and transfer.
A large amount of clinical datas and experimental result have all very clearly pointed out macrophage can promote the generation of tumour and the malignant progression of tumour.In the different phase and state of tumour, macrophage can play an important role, about being closely connected between macrophage and tumour and interactional molecular mechanism, also have made some progress achievement, most important composition is cell factor in these contacts.Research shows: as an inherent immunity cell, macrophage can be secreted a large amount of cell factors, promote in the Mechanism Study of tumor development at macrophage, the cell factor that macrophage produces is extremely important effector molecule, and macrophage mainly, by the cell factor of its secretion, comprises growth factor, inflammatory factor and chemotactic factor (CF) etc., as EGF, VEGF, MMP etc. promote growth and the progress of tumour.TAM can played the part of important role aspect the growth that promote tumour and transfer by autocrine and the paracrine approach of complexity in general.
Cell factor with the secretion of the macrophage in the thyroid papillary carcinoma tumor tissues, as the judgment criteria that detects thyroid papillary carcinoma, apply it in the diagnostic reagent of thyroid papillary carcinoma, up to the present, also there is no the documents and materials about this respect.
Summary of the invention
The purpose of this invention is to provide the application of CXCL8 cytokines measurement reagent in the diagnostic reagent of preparation thyroid papillary carcinoma.
In the growth of tumour, kinds of tumors comprises breast cancer, and kidney, the result of study of carcinoma of endometrium etc. confirm, and TAM (TAM) infiltrates density and tumor growth is proportionate.A large amount of interior animal experiments and In vitro cell experiment studies confirm that TAM may be by secretion epidermal growth factor (EGF), TGF-β, basic fibroblast growth factor (bFGF), the growth factors such as hepatocyte growth factor (HGF), PDGF (PDGF), promote the propagation of tumour cell and resist apoptosis.The macrophage culture supernatant that tumour cell and macrophage are cultivated altogether or collected on tumour cell in common cultured cells experiment stimulates, and all can promote the growth of tumour cell.
In order to study the growth relationship of TAM and Papillary Thyroid Carcinoma, the present invention cultivated 24 hours after the macrophage in the thyroid papillary carcinoma tumor tissues is separated, collect its culture supernatant, the TAM conditioned medium that obtains is carried out the cell factor chip analysis, obtain the cell factor spectrum of TAM secretion in thyroid papillary carcinoma comprehensively, find high expressed cytokine profiles in the TAM culture supernatant in thyroid papillary carcinoma,, as TNF-α, TNF-β, MIP, CXCL8, TGF-β etc., wherein with CXCL8, express the most remarkable.
In the thyroid papillary carcinoma sample, separate and turn out primary tumour macrophage, pass through the Cytokine of secretion in the hypothesis of tumour cell according to macrophage, the primary TAM that separates from PTC has been carried out cultivating in 24 hours collecting obtained its culture supernatant (conditional medium, CM), CM is stimulated papillary thyroid carcinoma cells strain K1, PC-1 and BCPAP cell, observe the change of culture supernatant to the invasive ability of papillary thyroid carcinoma cells.Experimental results show that: TAM can promote the growth of papillary thyroid carcinoma cells, can significantly improve the invasive ability of PTC tumour cell; The primary tumour macrophage of thyroid papillary carcinoma, papillary thyroid carcinoma cells strain, normal pbmc and TAM have been carried out the expression of realt-time PCR detection CXCL8mRNA, and the culture supernatant of cell line and primary TAM is carried out ELISA measure the CXCL8 expressing quantity, experimental results show that: CXCL8 high expressed in TAM, so, the CXCL8 cell factor can be applied in the diagnostic reagent of thyroid papillary carcinoma, can improve the diagnosis of thyroid papillary carcinoma, avoid patients with papillary thyroid carcinoma to miss best treatment time.
Beneficial effect of the present invention is:
1, TAM can promote the growth of papillary thyroid carcinoma cells, can significantly improve the invasive ability of PTC tumour cell, and in TAM the CXCL8 high expressed, so, CXCL8 cytokines measurement reagent can be applied in the diagnostic reagent of preparation thyroid papillary carcinoma, can improve the diagnosis of thyroid papillary carcinoma, avoid patients with papillary thyroid carcinoma to miss best treatment time.
2, the CXCL8 cell factor is the functional effect cellular elements of TAM secretion, high expressed in TAM, therefore use the CXCL8 cell factor as the effective judge cell factor in the diagnostic reagent of thyroid papillary carcinoma, its rate of correct diagnosis is high.
Description of drawings
Fig. 1 is the graph of a relation that affects of TAM culture supernatant and papillary thyroid carcinoma cells, illustrates that TAM can directly act on tumour cell by the cell factor of its secretion, and can significantly improve the invasive ability of PTC tumour cell.
Fig. 2 is the cell factor chip schematic diagram of the TAM that separates in thyroid papillary carcinoma, and high expressed in CXCL8 separates in thyroid papillary carcinoma TAM is described in figure.
Fig. 3 is the horizontal comparison diagram of mRNA of CXCL8 in the TAM that separates in thyroid papillary carcinoma and PTC clone (K1, TPC-1, BCPAP), illustrates in figure, and in TAM, the CXCL8mRNA expression is significantly higher than PTC clone.
Fig. 4 is the horizontal comparison diagram of mRNA of CXCL8 in the other normal thyroid tissue of cancer of the TAM that separates in thyroid papillary carcinoma and PTC cell and PTC tissue and pairing, illustrates in figure, and in TAM, the CXCL8mRNA expression is significantly higher than primary PTC cell and PTC tissue.
Fig. 5 is the CXCL8 expressing quantity comparison diagram of the culture supernatant of PTC clone (K1, TPC-1, BCPAP) and primary TAM, illustrates in figure, and the TAM high expressed CXCL8 factor in thyroid papillary carcinoma, CXCL8 is the effector molecule of TAM.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
The collection of embodiment 1, thyroid papillary carcinoma sample
the specimens from pri of thyroid papillary carcinoma (PTC) is sent to immediately pathology department and carries out the frozen section pathological diagnosis after the excision parathyroid tissue by the operation room work personnel within half an hour, if preliminary pathological examination is the histological type of thyroid papillary carcinoma, in the situation that being organized in, thyroid papillary carcinoma cancer kitchen range do not affect follow-up complete pathological diagnosis, instruct and help to collect the fresh surgical tissue specimen of thyroid papillary carcinoma by Pathologis, cutting tumor tissues under aseptic condition loses rapidly in the aseptic centrifuge tube of 50mL that hyclone and antibiotic D-Hank ' s liquid are housed, be sent to immediately laboratory and carry out further test experience.In this experiment, collect altogether 60 routine thyroid papillary carcinomas, for separating of with cultivate primary TAM.
The relation that affects of embodiment 2, TAM (TAM) culture supernatant and papillary thyroid carcinoma cells
In the thyroid papillary carcinoma sample in embodiment 1, separate and turn out primary tumour macrophage, pass through the Cytokine of secretion in the hypothesis of tumour cell according to macrophage, we have carried out cultivating in 24 hours collection to the primary TAM that separates from PTC and have obtained its culture supernatant (conditional medium, CM), CM is stimulated papillary thyroid carcinoma cells strain K1, PC-1 and BCPAP cell, observe the change of culture supernatant to the invasive ability of papillary thyroid carcinoma cells.Concrete outcome as shown in Figure 1, compare with the blank group by experimental group, can significantly promote the invasion and attack of PTC cell.Under the stimulation of TAM cells and supernatant, the K1 cell penetrates the cell quantity increase by 50% of matrigel to lower floor from Boyden chamber upper strata, TPC-1 and BCPAP cell increase respectively 53.7% and 73%, illustrate that TAM can directly act on tumour cell by the cell factor of its secretion, and can significantly improve the invasive ability of PTC tumour cell.Wherein, * represents that experimental group compares p＜0.05 with control group, and * * represents that experimental group compares p＜0.01 with contrast, and each tests triplicate at least.
The expression of CXCL8 cell factor in embodiment 3, TAM
The primary tumour macrophage of thyroid papillary carcinoma, papillary thyroid carcinoma cells strain, normal pbmc and TAM have been carried out realt-time PCR detected the expression of CXCL8mRNA, and the culture supernatant of cell line and primary TAM has been carried out ELISA measure the CXCL8 expressing quantity.Concrete outcome is as Figure 2-Figure 5:
Fig. 2 is the cell factor chip schematic diagram of the TAM that separates in thyroid papillary carcinoma,, with the TAM that separates in thyroid papillary carcinoma, uses
Human cell factor antibody chip 5 detects Cytokine expression profile.Cell factor strong positive point marks with numeral, wherein 1 representative: positive control, and 2 representatives: negative control, 3 represent CXCL8,4 representatives: macrophage inflammatory protein 1 β cell factor illustrates high expressed in CXCL8 separates in thyroid papillary carcinoma TAM in figure.
Fig. 3 is the horizontal comparison diagram of mRNA of CXCL8 in the TAM that separates in thyroid papillary carcinoma and PTC clone (K1, TPC-1, BCPAP), uses healthy volunteer PBMC to do correction.As we know from the figure, in TAM, the CXCL8mRNA expression is significantly higher than PTC clone.
Fig. 4 is the horizontal comparison diagram of mRNA of CXCL8 in the other normal thyroid tissue of cancer of the TAM that separates in thyroid papillary carcinoma and PTC cell and PTC tissue and pairing, uses healthy volunteer PBMC to do correction.As can be seen from Figure, in TAM, the CXCL8mRNA expression is significantly higher than primary PTC cell (PTC cell) and PTC tissue (PTC tissue).
Fig. 5 is that the culture supernatant of PTC clone (K1, TPC-1, BCPAP) and primary TAM is carried out ELISA mensuration CXCL8 expressing quantity, experiment confirm, and the certain high expressed CXCL8 of the TAM factor in thyroid papillary carcinoma, CXCL8 is the effector molecule of TAM.
In sum, TAM (TAM) promotes the growth of papillary thyroid carcinoma cells, can significantly improve the invasive ability of PTC tumour cell, and in TAM the CXCL8 high expressed, so, CXCL8 cytokines measurement reagent can be applied in the diagnostic reagent of thyroid papillary carcinoma, can improve the diagnosis of thyroid papillary carcinoma, avoid patients with papillary thyroid carcinoma to miss best treatment time.
Claims (1)
1.CXCL8 the application of cytokines measurement reagent in the diagnostic reagent of preparation thyroid papillary carcinoma.
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Application publication date: 20131120 |