Application of TACSTD2 protein in preparation of product for detecting and identifying human amniotic epithelial cells

Abstract

The invention discloses application of TACSTD2 protein in preparation of a product for detecting and identifying human amniotic epithelial cells, and belongs to the technical field of biological medicine. The invention provides a protein marker for detecting and identifying human amniotic epithelial stem cells. The protein marker comprises a TACSTD2 protein, or a combination of the TACSTD2 protein and one or more selected from the group consisting of CD29, CD166, CD73, CD90, CD105, CD324, CD326, SSEA4, Netin and Vimentin. The invention further provides application of the reagent for detecting TACSTD2 protein expression in preparation of products for detecting and identifying human amniotic epithelial cells, and the reagent has high specific expression in human amniotic epithelial stem cells and can be used for detecting and identifying the human amniotic epithelial cells independently or in combination with other cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of TACSTD2 protein in the preparation of products for detecting and identifying human amniotic epithelial cells. Background Art

[0002] Human amniotic epithelial stem cells (hAESCs) originate from the innermost layer of the placenta closest to the fetus, and are differentiated and developed from the embryonic inner cell mass. They have the characteristics of expressing multiple embryonic stem cell markers and a relatively comprehensive multidirectional differentiation potential.

[0003] The human amnion is a translucent, avascular membrane attached to the fetal surface of the placenta. It consists of five layers: epithelial layer, basement membrane, dense layer, fibroblast layer, and spongy layer (the last three layers are collectively referred to as the matrix layer). Human amnion epithelial stem cells originate from the innermost layer of the placental amnion, and are differentiated from the cell mass in the blastocyst on the eighth day of development from the fertilized egg. These cells are located in the innermost layer of the amnion and are in direct contact with the amniotic fluid. Studies have confirmed that human amnion epithelial cells have the potential to differentiate into the three germ layers, can 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 multiple 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, etc.; in addition, they highly express multiple 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 detection and identification of human amniotic epithelial stem cells can be carried out by combining multiple cell markers, but there is a lack of cell markers dedicated to human amniotic epithelial stem cells. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide the use of TACSTD2 protein in the preparation of products for detecting and identifying human amniotic epithelial cells. TACSTD2 protein is specifically and highly expressed 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 object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a protein marker for detecting and identifying human amniotic epithelial stem cells, which includes TACSTD2 protein.

[0008] Preferably, the protein marker is a combination of TACSTD2 protein and one or more selected from CD29, CD166, CD73, CD90, CD105, CD324, CD326, SSEA4, Nestin, and Vimentin.

[0009] Preferably, the protein marker is a combination of TACSTD2 protein and one or more selected from CD29, CD166, and CD73.

[0010] Preferably, the protein marker is a combination of TACSTD2 protein and CD324 and / or CD326.

[0011] Preferably, the protein marker is a combination of TACSTD2 protein and CD73.

[0012] The second aspect of the present invention provides the use of a reagent for detecting TACSTD2 protein expression in the preparation of a product for detecting and identifying human amniotic epithelial cells.

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

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

[0015] Preferably, the reagent for detecting the expression of TACSTD2 protein includes a reagent for the combined expression of TACSTD2 protein and one or more selected from CD324 and CD326.

[0016] Preferably, the reagent for detecting the expression of TACSTD2 protein includes a reagent for detecting the combined expression of TACSTD2 protein and CD73.

[0017] Compared with the prior art, the beneficial effect of the present invention is that: the present invention provides a protein marker TACSTD2 that is specifically and highly expressed in human amniotic epithelial stem cells, which can specifically identify human amniotic epithelial cells by combining with other cells, and provides a technical route for the quality detection and identification of human amniotic epithelial stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a cell marker of human amniotic epithelial stem cells (hAESCs) in the prior art.

[0019] Figure 2 The figure shows the flow cytometry detection result of TACSTD2 protein in human amniotic epithelial stem cells (AM23034EP1) in the example.

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

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

[0022] Figure 5 The figure shows the flow cytometry detection result of TACSTD2 protein in human peripheral blood mononuclear cells (PBMC00000670) in the example. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments and drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them, and all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[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 expressed at a low level in normal tissues. It plays a role in multiple cell signal transduction pathways and is related to cell proliferation and differentiation.

[0025] Example 1: Proteomic 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. More than 5×10^6 cells in each batch were sent for proteomic testing. The testing unit was Shanghai Luming Biotechnology Co., Ltd., and the detection method was 4D-DIA detection.

[0027] like Figure 1As shown in Table 1, the cell markers related to human amniotic epithelial stem cells in the proteomic test results are shown in Table 1, where AESC is human amniotic epithelial cells; NSC is human neural stem cells; MSC is human umbilical cord mesenchymal stem cells. From the proteomic results, it can be seen that the cell markers TACSTD2, CD324, and CD326 have certain specificity in the expression of human amniotic epithelial stem cells, and TACSTD2 has a higher expression level and better specificity.

[0028] Table 1: Proteomic related cell marker detection results

[0029]

[0030]

[0031] Example 2: Flow cytometric detection of relevant cell markers

[0032] 1. Flow cytometric detection of cell markers related to human amniotic epithelial stem cells (hAESCs) and human umbilical cord mesenchymal stem cells (MSCs)

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

[0034] Human amniotic epithelial stem cells and human mesenchymal stem cells were used for flow cytometric detection of relevant cell markers. The flow cytometric antibodies used were all 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 1st to 3rd samples are human umbilical cord mesenchymal stem cells, the 4th to 6th samples are human amniotic epithelial stem cells, the 7th sample is human amniotic epithelial stem cells that have undergone mesenchymalization, and the 8th sample is human amniotic epithelial stem cells that have completely undergone mesenchymalization.

[0035] The results are shown in Table 2. Figure 2 and Figure 3As shown, the expression percentage of TACSTD2 in human amniotic epithelial stem cells is over 90%, but it is not expressed in human umbilical cord mesenchymal stem cells; the expression difference of TACSTD2 between human amniotic epithelial stem cells and human umbilical cord mesenchymal stem cells is very obvious, and it is completely able to distinguish the two types of cells, and can more sensitively reflect the mesenchymal transformation of human amniotic epithelial stem cells.

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

[0037]

[0038]

[0039] 2. Flow cytometry detection of TACSTD2 expression in other cells

[0040] Human neural stem cells and human peripheral blood mononuclear cells (PBMC) were used for TACSTD2 flow cytometry phenotyping. 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. The TACSTD2 marker is not expressed 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. It can be used as an important marker for the detection and identification of human amniotic epithelial stem cells. It can be used alone or with other related cell markers for the detection and identification of human amniotic epithelial stem cells with good results and high specificity.

[0044] The above is only a preferred embodiment of the present invention, but the present invention should not be limited to the contents disclosed in this embodiment. Any equivalent or modification completed without departing from the spirit disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A protein marker for detecting and identifying human amniotic epithelial stem cells, comprising TACSTD2 protein.

2. The protein marker for detecting and identifying human amniotic epithelial stem cells according to claim 1, characterized in that: The protein marker is a combination of TACSTD2 protein and one or more selected from CD29, CD166, CD73, CD90, CD105, CD324, CD326, SSEA4, Nestin, and Vimentin.

3. The protein marker for detecting and identifying human amniotic epithelial stem cells according to claim 1, characterized in that: The protein marker is a combination of TACSTD2 protein and one or more selected from CD29, CD166, and CD73.

4. The protein marker for detecting and identifying human amniotic epithelial stem cells according to claim 1, characterized in that: The protein marker is a combination of TACSTD2 protein and CD324 and / or CD326.

5. The protein marker for detecting and identifying human amniotic epithelial stem cells according to claim 1, characterized in that: The protein marker is a combination of TACSTD2 protein and CD73.

6. Application of a reagent for detecting TACSTD2 protein expression in the preparation of products for detecting and identifying human amniotic epithelial cells.

7. The use according to claim 6, characterized in that: The reagent is a reagent for detecting the expression of TACSTD2 protein and one or more combinations selected from CD29, CD166, CD73, CD90, CD105, CD324, CD326, SSEA4, Nestin, and Vimentin.

8. The use according to claim 6, characterized in that: The reagent is a reagent for detecting the expression of TACSTD2 protein in combination with one or more selected from CD29, CD166, and CD73.

9. The use according to claim 6, characterized in that: The reagent is a reagent expressed in combination with TACSTD2 protein and CD324 and / or CD326.

10. The use according to claim 6, characterized in that: The reagent is a reagent for detecting the combined expression of TACSTD2 protein and CD73.

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