Use of tacstd2 protein in the preparation of a product for detecting and identifying human amnion epithelial cells

Abstract

The application discloses application of TACSTD2 protein in preparation of products for detecting and identifying human amniotic epithelial cells, and belongs to the technical field of biological medicines. The application proposes a protein marker for detecting and identifying human amniotic epithelial stem cells, which comprises TACSTD2 protein, or TACSTD2 protein combined with one or more selected from CD29, CD166, CD73, CD90, CD105, CD324, CD326, SSEA4, Nestin and Vimentin. The application further proposes application of a reagent for detecting TACSTD2 protein expression in preparation of products for detecting and identifying human amniotic epithelial cells, which is specifically highly expressed in human amniotic epithelial stem cells and can be used alone or in combination with other cells for detecting and identifying human amniotic epithelial cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of TACSTD2 protein in preparation of products for detecting and identifying human amniotic epithelial cells. BACKGROUND

[0002] Human amniotic epithelial stem cells (hAESCs) are derived from the innermost layer of the placenta closest to the fetus and are developed from the embryonic inner cell mass, and have the characteristics of expressing various embryonic stem cell markers and comprehensive multilineage differentiation potential.

[0003] Human amniotic membrane is a translucent avascular thin film attached to the fetal surface of the placenta, and is composed of five layers of structure: epithelial layer, basement membrane, dense layer, fibroblast layer and sponge layer (the last three layers are collectively referred to as the stroma layer). Human amniotic epithelial stem cells are derived from the innermost layer of the placental amniotic membrane and are differentiated from the cell mass in the blastocyst developed from the fertilized egg to the 8th day. These cells are located in the innermost layer of the amniotic membrane and directly contact with the amniotic fluid. Studies have confirmed that human amniotic epithelial cells have the potential to differentiate into cells of the three germ layers, and have the characteristics of immune exemption or low immunogenicity, which makes them have broad application prospects in the field of regenerative medicine.

[0004] Human amniotic epithelial stem cells express various embryonic stem cell markers such as SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and also express pluripotent stem cell transcription factors such as Oct-4, Sox-2, Nanog and REX-1; In addition, they also highly express various cell markers such as CD29, CD166, CD44, CD73, CD90, CD105, CD324, CD326, etc., but do not express cell markers such as CD14, CD34, CD45, CD79 and HLA-DR. In the prior art, the quality of human amniotic epithelial stem cells can be detected and identified by various combinations of cell markers, but there is a lack of cell markers specific to human amniotic epithelial stem cells. SUMMARY

[0005] In view of this, the purpose of the present application is to provide application of TACSTD2 protein in preparation of products for detecting and identifying human amniotic epithelial cells, which is highly expressed specifically in human amniotic epithelial stem cells and can be used alone or in combination with other cells to detect and identify human amniotic epithelial cells.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] In a first aspect of the present application, a protein marker for detecting and identifying human amniotic epithelial stem cells is provided, which comprises TACSTD2 protein.

[0008] As preferred, the protein marker is TACSTD2 protein in combination with one or more selected from CD29, CD166, CD73.

[0009] As preferred, the protein marker is TACSTD2 protein in combination with one or more selected from CD29, CD166, CD73.

[0010] As preferred, the protein marker is TACSTD2 protein in combination with one or more selected from CD324 and / or CD326.

[0011] As preferred, the protein marker is TACSTD2 protein in combination with CD73.

[0012] In the second aspect of the present application, there is provided use of a reagent for detecting expression of TACSTD2 protein in the preparation of a product for detecting and identifying human amniotic epithelial cells.

[0013] As preferred, the reagent for detecting expression of TACSTD2 protein comprises a reagent for detecting expression of TACSTD2 protein in combination with one or more selected from CD29, CD166, CD73, CD90, CD105, CD324, CD326, SSEA4, Nestin, Vimentin.

[0014] As preferred, the reagent for detecting expression of TACSTD2 protein comprises a reagent for detecting expression of TACSTD2 protein in combination with one or more selected from CD29, CD166, CD73.

[0015] As preferred, the reagent for detecting expression of TACSTD2 protein comprises a reagent for detecting expression of TACSTD2 protein in combination with one or more selected from CD324, CD326.

[0016] As preferred, the reagent for detecting expression of TACSTD2 protein comprises a reagent for detecting expression of TACSTD2 protein in combination with CD73.

[0017] Compared with the prior art, the present application has the beneficial effect that the present application provides a protein marker TACSTD2 which is specifically highly expressed in human amniotic epithelial stem cells, and can be used to specifically identify human amniotic epithelial cells in combination with other cells, thereby providing a technical route for quality detection and identification of human amniotic epithelial stem cells. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The present application provides a cell marker for human amniotic epithelial stem cells (hAESCs) in the prior art. The present application provides a cell marker for human amniotic epithelial stem cells (hAESCs) in the prior art.

[0019] Figure 2 Flow cytometry results of TACSTD2 protein in human amniotic epithelial stem cells (AM23034EP1) in the examples.

[0020] Figure 3 Flow cytometry results of TACSTD2 protein in human mesenchymal stem cells (UC20015MP5) in the examples.

[0021] Figure 4 Flow cytometry results of TACSTD2 protein in human neural stem cells (NSC230102MP9) in the examples.

[0022] Figure 5 Flow cytometry results of TACSTD2 protein in human peripheral blood mononuclear cells (PBMC00000670) in the examples. DETAILED DESCRIPTION

[0023] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely below in conjunction with the examples and drawings. Obviously, the described examples are part of the embodiments of the present application, rather than all the embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative effort fall within the protection scope of the present application.

[0024] Tumor-associated calcium signal transducer 2 (TACSTD2), also known as human trophoblast cell surface glycoprotein antigen 2 (TROP-2), is a cell surface glycoprotein encoded by the TACSTD2 gene. TACSTD2 is usually not expressed or lowly expressed in normal tissues and plays a role in multiple cell signal transduction pathways, and is related to cell proliferation and differentiation.

[0025] Example 1: Proteome detection

[0026] Three batches of human amniotic epithelial stem cells P1 from different donors, three batches of human umbilical cord mesenchymal stem cells P3 from different donors, and three batches of human neural stem cells P9 from different donors were taken, and more than 5×10^6 cells per batch were sent for proteome detection. The detection unit was Shanghai Luming Biological Technology Co., Ltd., and the detection method was 4D-DIA detection.

[0027] As Figure 1The cell markers related to human amniotic epithelial stem cells in the proteomic detection results are shown in Table 1, wherein: AESC is human amniotic epithelial cells; NSC is human neural stem cells; and MSC is human umbilical cord mesenchymal stem cells. As can be seen from the proteomic results, the cell markers TACSTD2, CD324 and CD326 have certain specificity in the expression of human amniotic epithelial stem cells, and the expression amount of TACSTD2 is higher and the specificity is better.

[0028] Table 1: Proteomic detection results of cell markers

[0029]

[0030]

[0031] Example 2: Flow detection of related cell markers

[0032] I. Flow detection of human amniotic epithelial stem cells (hAESCs) and human umbilical cord mesenchymal stem cells (MSCs) related cell markers

[0033] Human amniotic epithelial stem cells and human umbilical cord mesenchymal stem cells are derived from discarded placental tissue, and the outer skin of the amnion and the umbilical cord is connected. In addition to cell morphology, it is necessary to distinguish the two cells in terms of cell markers.

[0034] Human amniotic epithelial stem cells and human mesenchymal stem cells were taken for flow detection of related cell markers. The flow antibodies used were Biolegend products: (1) CD146, 361016; (2) CD29, 303004; (3) CD166, 343904; (4) CD324, 324108; (5) CD326, 324204; (6) SSEA4, 330406; (7) CD73, 344006; (8) CD90, 328108; (9) CD105, 800504; (10) CD34, 343504; (11) CD45, 304008; (12) HLA-DR, 307604; (13) TACSTD2, 363804; The first to third samples are human umbilical cord mesenchymal stem cells, the fourth to sixth samples are human amniotic epithelial stem cells, the seventh sample is human amniotic epithelial stem cells with mesenchymal differentiation, and the eighth sample is completely mesenchymal human amniotic epithelial stem cells.

[0035] The results are shown in Table 2, Figure 2 and Figure 3As shown, the percentage of TACSTD2 expression in human amniotic epithelial stem cells is more than 90%, while no expression in human umbilical cord mesenchymal stem cells; TACSTD2 shows a very obvious difference between human amniotic epithelial stem cells and human umbilical cord mesenchymal stem cells, which can completely distinguish the two kinds of cells, and can be more sensitive to reflect the case of human amniotic epithelial stem cell interstitialization.

[0036] Table 2: Flow cytometry results of human amniotic epithelial cell and human umbilical cord mesenchymal stem cell related cell markers (%)

[0037]

[0038]

[0039] II. Flow cytometry detection of TACSTD2 expression in other cells

[0040] Human neural stem cells and human peripheral blood mononuclear cells (PBMC) were taken for TACSTD2 flow cytometry detection, and the results are shown in Table 3, Figure 4 and Figure 5 As shown: the first sample is human amniotic epithelial stem cells, the second sample is human umbilical cord mesenchymal stem cells, the third to fifth samples are human neural stem cells, and the sixth to eighth samples are human peripheral blood mononuclear cells. TACSTD2 marker does not express in human neural stem cells and human peripheral blood mononuclear cells.

[0041] Table 3: Expression of TACSTD2 in human neural stem cells and human peripheral blood mononuclear cells (%)

[0042] Sample No. TACSTD2 AM23034EP1 99.53 UC20015MP5 0.27 NSC230809CP9 0.43 NSC230814HP9 0.23 NSC230102MP9 0.42 PBMC00000670 0.04 PBMC00001086 0.1 PBMC00001223 0.08

[0043] In summary, TACSTD2 protein is highly expressed in human amniotic epithelial stem cells, and has specificity, and can be used as an important marker for detection and identification of human amniotic epithelial stem cells. It can be used alone or with other related cell markers for detection and identification of human amniotic epithelial stem cells, with good effect and high specificity.

[0044] The above is only a preferred embodiment of the present application, but the present application should not be limited to the disclosed content of the embodiment. Any equivalent or modification made without departing from the disclosed spirit of the present application falls within the scope of protection of the present application.

Claims

1. Use of a reagent for detecting expression of TACSTD2 protein in the manufacture of a product for detecting and identifying human amnion epithelial stem cells, said reagent being a reagent for detecting expression of TACSTD2 protein in combination with one or more selected from the group consisting of CD29, CD166, CD73, CD90, CD105, CD324, CD326, SSEA4, Nestin, Vimentin.

2. Use according to claim 1, characterized in that, said reagent being a reagent for detecting expression of TACSTD2 protein in combination with one or more selected from the group consisting of CD29, CD166, CD73.

3. Use according to claim 1, characterized in that, said reagent being a reagent for detecting expression of TACSTD2 protein in combination with one or more selected from the group consisting of CD324 and CD326.

4. Use according to claim 1, characterized in that, said reagent being a reagent for detecting expression of TACSTD2 protein in combination with CD73.

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