CN102053150B - Human osteosarcoma stem-cell related antigenic marker and application thereof - Google Patents
Human osteosarcoma stem-cell related antigenic marker and application thereof Download PDFInfo
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- CN102053150B CN102053150B CN 200910197959 CN200910197959A CN102053150B CN 102053150 B CN102053150 B CN 102053150B CN 200910197959 CN200910197959 CN 200910197959 CN 200910197959 A CN200910197959 A CN 200910197959A CN 102053150 B CN102053150 B CN 102053150B
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Abstract
The invention discloses a human osteosarcoma stem-cell related antigenic marker stage-specific embryonic antigen-4 (SSEA-4) and an application thereof in a targeted therapy of osteosarcoma. The SSEA-4 is a specific osteosarcoma stem-cell surface marker which is almost not expressed in each normal adult tissue. The surface marker is easy for antibody labeling without affecting the cell viability, and applicable to the function tests after separation, therefore, a reliable molecular tracer is provided for further researching the function of stem cells in the origin, occurrence and development processes of osteosarcoma, and a foundation for explaining the clinical osteosarcoma drug resistance as well as transferring and looking for cellular and molecular targets for the targeted therapy is laid.
Description
Technical field
The present invention relates to the oncobiology field, more particularly, relate to a kind of people's osteogenic sarcoma stem cell related antigen label and application thereof.
Background technology
In recent years, along with to the deepening continuously of tumor stem cell theoretic knowledge, require us that origin and generation, the development of tumour are had more clearly understanding.Especially tumor stem cells such as leukaemia, breast cancer, human glioma and colorectal cancer are separated and identify the report of work, not only confirm and consolidated the tumor stem cell theory, also making becomes focus to the evaluation of other tumor stem cells and the research of inherent molecular mechanism feature.At present, few about the report of osteogenic sarcoma stem cell both at home and abroad, people such as external Gibbs adopted the in vitro culture mode in 2005, from clone, isolate the sarcoma cell ball (sarcosphere) that contains the stem-like cell osteosarcoma cell first, express surface marker (as Stro-1, CD44 and CD105) and the embryonic stem cell characterizing gene (Nanog and Oct-4) of multiple mescenchymal stem cell; There is the SP cell in the human osteosarcoma cell colony of discovery in vitro culture such as Colleen Wu in 2007, but also fails to separate concrete, the specific osteogenic sarcoma stem cell surface label of evaluation.Of paramount importance is that these work all are not based on the research work of people's tumor tissues of former generation, do not carry out inoculation test in strict cell separation and the body.
People such as Gibbs in 2005 isolate sarcoma cell ball and people such as Colleen Wu in 2007 of containing the stem-like cell osteosarcoma cell first and find the SP cell from the sarcoma patient specimens.On the one hand, above-mentioned scholar's work mainly is confined to cell in vitro cultivation level, fails to be created as in vivo phenotype and the function (and this be the goldstandard of tumor stem cell research) of osteosarcoma cell heteroplastic transplantation model to identify the osteogenic sarcoma stem cell; On the other hand, the separation method of SP cell exists hoechst33342 dyestuff toxicity, low, the more high limitation of instrument configuration of cell viability.Thereby separate and identify that specific osteogenic sarcoma stem cell surface label is the technical foundation of carrying out the research of tumor stem cell specific aim.
Existing research and the application of SSEA-4: SSEA-4 is stage specific embryonic antigen4, be belong to the phase specificity embryonal antigen a kind of, glycolipid class antigen as a kind of body early embryo, surface marker as embryonic stem cell and mescenchymal stem cell commonly used, but the evaluation of tumor stem cell and screening never, the present invention for the first time with SSEA-4 as surface marker, as the evaluation of osteogenic sarcoma stem cell.
Summary of the invention
The purpose of this invention is to provide a kind of people's osteogenic sarcoma stem cell related antigen label.
Second purpose of the present invention provides the application of described people's osteogenic sarcoma stem cell related antigen label SSEA-4 in the osteogenic sarcoma targeted therapy.
19 people's osteogenic sarcomas that the present invention utilizes set up early stage are striden species transplantation model and free serum culture technology, in the body and external two aspects, separate and identify the osteogenic sarcoma surface marker SSEA-4 with mescenchymal stem cell and embryonic stem cell double characteristic coexisting with tumor stem cell colony.Surface marker SSEA-4 is easy to antibody labeling, and mark does not influence cell viability, the functional experiment after being fit to separate; Simultaneously SSEA-4 not only points out osteogenic sarcoma may originate from the mescenchymal stem cell stage, for follow-up mechanism research provides concrete minute subobject and model platform.Clinically, the expression degree in a part of patient can be pointed out its grade malignancy, transfer and prognosis situation, is the potential molecular target that can be used as targeted therapy.
Because it is lower that osteogenic sarcoma patient sample of former generation is striden the species transplanting succeed rate, so we have attempted a large amount of clinical samples, obtains 3 animal models at last.In addition, we have detected a plurality of have mescenchymal stem cell and embryonic stem cell surface markers, mark of correlation thing according to expression and article report, the SSEA-4 that selection has mescenchymal stem cell and an embryonic stem cell double characteristic as the osteosarcoma surface marker after, then confirmed, we verify the specificity of label in conjunction with clone and patients clinical sample at last.
SSEA-4 is a specific osteogenic sarcoma stem cell surface label, expresses hardly in each normal adult tissue.This surface marker is easy to antibody labeling, do not influence cell viability, functional experiment after being fit to separate, provide a reliable molecule tracer for further studying stem cell in the effect of origin, generation and the evolution of osteogenic sarcoma, for cell and the molecular target of explaining clinical osteogenic sarcoma resistance, transfer and searching targeted therapy are laid a good foundation.
Description of drawings
Below in conjunction with Figure of description, the present invention is described in more detail.
Fig. 1: the screening of human osteosarcoma cell immunophenotype.Select mescenchymal stem cell and embryonic stem cell surface marker CD105, CD166, CD117, CD90, CD44, STRO-1, SSEA-4, SSEA-3 etc. commonly used.
Fig. 2 A: former generation human osteosarcoma cell expression SSEA-4 and Oct-4; Fig. 2 B: the human osteosarcoma cell of foundation is that 5well expresses SSEA-4 and Oct-4.
Fig. 3: osteogenic sarcoma Pathologic specimen SABC shows that SSEA-4, nanog, oct-4 express.
Fig. 4: the osteogenic sarcoma primary cell forms sphere under external free serum culture condition.
Embodiment
I, we in early-stage Study, adopt former generation sample of just sending out the osteogenic sarcoma patient, be digested to single cell suspension after, utilize free serum culture technology and NOD/SCID mouse successfully to set up the human osteosarcoma cell ball and stride the species transplantation model.By the flow cytometry technology, the human osteosarcoma cell of taking from into the knurl mouse is carried out phenotype analytical, find that these cells often express that SSEA-3, SSEA-4, CD184, CD105, CD166 and CD49d etc. are a plurality of to have mescenchymal stem cell and an embryonic stem cell surface marker.
II, SSEA-4 is carried out more detailed checking: utilize clone, adopt immunofluorescence and SABC to get method, carry out phenotypic evaluation and checking, test expression etc. in former generation sample.
III, by to differentiation more careful between the different surfaces label and comparison, choose the strongest label of sub-fraction specificity, carry out the immunofluorescence antibody labeling, utilize the airflow classification technology that the human osteosarcoma cell of different expressions is sorted out, mode with gradient dilution is implanted into the NOD/SCID mouse respectively, by to becoming the comparative analysis of knurl situation, select the special surface marker the most relevant with osteogenic sarcoma tumorigenesis ability, i.e. SSEA-4.
IV, with the osteogenic sarcoma primary cell that obtains under external free serum culture condition, add cell factors such as FGF, N2B27, can be at external formation human osteosarcoma cell ball (sphere).
Claims (1)
1.SSEA-4 the application in preparation people osteogenic sarcoma stem cell related antigen label.
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| CN110476891B (en) * | 2019-09-09 | 2022-09-23 | 中南大学湘雅二医院 | Construction method and application of synovial sarcoma xenograft mouse model |
| CN110495424B (en) * | 2019-09-09 | 2022-09-27 | 中南大学湘雅二医院 | Construction method of synovial sarcoma xenograft mouse model |
| CN110476892A (en) * | 2019-09-09 | 2019-11-22 | 中南大学湘雅二医院 | A kind of construction method of osteosarcoma xenograft mouse model and application |
| CN110402895A (en) * | 2019-09-09 | 2019-11-05 | 中南大学湘雅二医院 | A kind of construction method of osteosarcoma xenograft mouse model |
| CN110495422B (en) * | 2019-09-23 | 2022-12-20 | 中南大学湘雅二医院 | Construction method and application of malignant schwannoma xenograft mouse model |
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| CN1331140A (en) * | 2000-06-26 | 2002-01-16 | 上海博德基因开发有限公司 | Polypeptide-human retinoblastome bindin (RBBP9) 25.52 and polynucleotide for coding it |
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| EP1963849A2 (en) * | 2005-11-14 | 2008-09-03 | Bayer Healthcare, LLC | Methods for prediction and prognosis of cancer, and monitoring cancer therapy |
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| CA2013544A1 (en) * | 1989-03-31 | 1990-09-30 | Yuen Kai Fung | Retinoblastoma gene product antibodies and uses therefor |
| CN1331140A (en) * | 2000-06-26 | 2002-01-16 | 上海博德基因开发有限公司 | Polypeptide-human retinoblastome bindin (RBBP9) 25.52 and polynucleotide for coding it |
Non-Patent Citations (4)
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Inventor after: Zhang Wu Inventor after: Zhu Jiang Inventor after: Zhang Weibin Inventor before: Zhang Wu Inventor before: Zhu Jiang Inventor before: Zhang Weibin |