Type I alveolar cell surface molecular marker CD73 and application thereof

Abstract

The invention discloses an I-type alveolar cell surface molecular marker CD73 and application thereof, and belongs to the technical field of medical bioengineering. The technical problem to be solved by the invention is to provide the surface marker of the type I alveolar cells. The surface marker of the I-type alveolar cell is CD73, the CD73 is highly expressed in the I-type alveolar cell, is almost not expressed in a lung epithelial cell of a non-I-type alveolar cell and is positioned on the surface of the I-type alveolar cell, and the I-type alveolar cell can be separated and purified from a mixed lung epithelial cell by utilizing the CD73 marker. CD73 is adopted as the surface marker, so that whether the cells are I-type alveolar cells or not can be effectively judged, and the I-type alveolar cells can be separated out. The CD73 serving as the I-type alveolar cell surface marker is high in specificity and applicability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical biological engineering, and particularly relates to a type I alveolar cell surface molecule marker CD73 and application thereof. BACKGROUND

[0002] The main function of lung tissue, gas exchange between the body and the environment, depends on its special anatomical structure, involving a variety of different cell types. Gas exchange occurs in the alveolar unit, which is characterized by a layer of capillaries wrapped around the ultra-thin alveolar type I cells (AT1 cells) supported by interstitial cells. Type I alveolar cells cover about 95% of the alveolar surface (Crapo, 1982. Cell number and cell characteristics of the normal human lung. American Review of Respiratory Disease 126, 332-337.), and are very thin, which is conducive to passive gas diffusion, have almost no organelles, and have tight junctions between adjacent cells (Williams, 2003. Alveolar type I cells: molecular phenotype and development. Annual review of physiology 65, 669-695.), and like a collar around the lung cells, a single AT1 cell spans multiple alveoli. In addition, AT1 has a continuous basement membrane that fuses with the basement membrane of alveolar capillary endothelial cells, which are directly located below the alveolar lining cells. The epithelial cells of the capillary, the basement membrane and the endothelial cells constitute the gas exchange barrier of the alveolar wall. Therefore, AT1 is essential to the physiological function of the lung. However, since the discovery of AT1 cells, their study has been far less in-depth than that of type II alveolar cells AT2 due to the difficulty in isolating and sorting methods.

[0003] Currently, obtaining accurate specific surface markers to purify high purity cell types has become the main cell sorting strategy. The previously discovered AT1 cell surface markers are not specific enough, and are also expressed in other cells in the lung. For example: PDPN, as an AT1 cell surface marker, is expressed in basal cells, lymphatic epithelial cells and mesothelial cells in the lung (Williams, 2003. Alveolar type I cells: molecular phenotype and development. Annual review of physiology 65, 669-695.). Similarly, AQP5 is also expressed in endothelial cells and AT2 cells (Nielsen S, 1997. Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat). AGER is also expressed in AT2 cells (Dahlin K, 2004. Identification of genes differentially expressed in rat alveolar type I cells. Am J Respir Cell Mol Biol. 31:309-316). Therefore, it is very difficult to sort AT1 cells with high purity by surface protein. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a surface marker of type I alveolar cells.

[0005] To solve the above technical problems, the present application first provides the application of CD73 protein as a surface marker of type I alveolar cells (AT1 cells).

[0006] The present application also provides at least one of the following applications of a substance for detecting CD73 protein: X1) detecting or assisting in detecting type I alveolar cells; X2) preparing a product for detecting or assisting in detecting type I alveolar cells; X3) identifying or assisting in identifying type I alveolar cells; X4) preparing a product for identifying or assisting in identifying type I alveolar cells; X5) isolating or purifying type I alveolar cells; X6) preparing a product for isolating or purifying type I alveolar cells; X7) labeling type I alveolar cells; X8) preparing a product for labeling type I alveolar cells; X9) locating type I alveolar cells; X10) preparing a product for locating type I alveolar cells.

[0007] The CD73 protein, also known as Nt5e (ecto-5'-nucleotidase), is a cell surface glycosylphosphatidylinositol-anchored glycoprotein belonging to the nucleotide metabolism enzyme family, and mainly catalyzes the hydrolysis of adenosine acid into adenosine and inorganic phosphate. The mouse CD73 protein is encoded by the mouse Nt5e gene (located on mouse chromosome 9, Gene ID: 23959 (updated on 12-Jun-2025)), and consists of 576 amino acids, which is a multifunctional transmembrane glycoprotein anchored on the cell membrane surface.

[0008] The present application also provides at least one of the following applications of the substance for detecting the CD73 protein and the substance for detecting the EpCAM protein: X1) detecting or assisting in detecting type I alveolar cells; X2) preparing a product for detecting or assisting in detecting type I alveolar cells; X3) identifying or assisting in identifying type I alveolar cells; X4) preparing a product for identifying or assisting in identifying type I alveolar cells; X5) isolating or purifying type I alveolar cells; X6) preparing a product for isolating or purifying type I alveolar cells; X7) labeling type I alveolar cells; X8) preparing a product for labeling type I alveolar cells; X9) locating type I alveolar cells; X10) preparing a product for locating type I alveolar cells.

[0009] The EpCAM protein, Gene ID: 17075, updated on 11-Jun-2025.

[0010] In the above, the substance for detecting the CD73 protein can contain a CD73 antibody, or can be a CD73 antibody.

[0011] The substance for detecting the EpCAM protein can contain an EpCAM antibody, or can be an EpCAM antibody.

[0012] In the above, the detection, identification or labeling of type I alveolar cells can be detected, identified or labeled in lung epithelial cells or lung whole cells. The isolation or purification of type I alveolar cells can be isolated or purified from lung epithelial cells or lung whole cells.

[0013] The present application also provides a method for isolating (or purifying) type I alveolar cells, comprising: labeling lung cells with a CD73 antibody, and isolating cells binding to the CD73 antibody to obtain type I alveolar cells.

[0014] The present application also provides a method for isolating (or purifying) type I alveolar cells, comprising: labeling lung cells with a CD73 antibody and an EpCAM antibody, and isolating cells binding to the CD73 antibody and the EpCAM antibody to obtain type I alveolar cells.

[0015] The present application also provides a method for labeling type I alveolar cells, comprising: labeling lung cells with a CD73 antibody, and cells binding to the CD73 antibody are type I alveolar cells.

[0016] The present application also provides a method for labeling type I alveolar cells, comprising: labeling lung cells with a CD73 antibody and an EpCAM antibody, and cells binding to the CD73 antibody and the EpCAM antibody are type I alveolar cells.

[0017] The above method for isolating (or purifying) type I alveolar cells can be used to isolate (or purify) type I alveolar cells by fluorescence-labeled cell sorting, immunomagnetic bead separation or immunoadsorption column separation. These methods all use antibodies of surface markers of AT1 cells (CD73 antibody and / or EpCAM antibody) and secondary antibodies connected to fluorescence labels, magnetic substances or adsorption columns, and through the binding of antibodies and markers on the surface of target cells, the target cells are separated and purified.

[0018] In the present application, the CD73 antibody can be a fluorescence-labeled CD73 antibody. The EpCAM antibody can be a fluorescence-labeled EpCAM antibody.

[0019] In the present application, the lung cells can be lung epithelial cells (or lung epithelial cell mixed cell system, including AT2 cells, airway epithelial cells, ciliated cells, etc.) or lung whole cells.

[0020] The lung cells can be mammalian lung cells. Further, the lung cells can be human lung cells, mouse lung cells, etc.

[0021] The present application finds that CD73 is a surface marker of AT1 cells, is highly expressed in AT1 cells, is almost not expressed in non-AT1 epithelial cells, and is located on the surface of AT1 cells, so that AT1 cells can be separated and purified from mixed lung epithelial cells by using CD73 marker. When CD73 is used as a surface marker, it can effectively determine whether the cells are AT1 cells and separate AT1 cells. CD73 as a surface marker of AT1 cells has strong specificity and strong applicability.

[0022] The present application will be further described in conjunction with the specific embodiments, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 . Flow cytometry sorting of AT1 cells using CD73. Left panel shows the gating of EpCAM and CD73 in lung mixed cells, right panel shows the gating of EpCAM and CD73 in Hopx positive cells. mid CD73 hi Expression of Hopx in cell population.

[0024] Figure 2 . Purity detection of CD73 sorting using AT1 cell specific antibody Hopx. Left panel shows the Hopx positive cells (i.e. AT1 cells) in lung mixed cells, right panel shows the expression of EpCAM and CD73 in Hopx positive cells. DETAILED DESCRIPTION

[0025] In the following examples, the experimental methods are routine methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources, unless otherwise specified.

[0026] The inventors of the present application found that CD73 is a surface marker of mouse AT1 alveolar cells through proteomic data analysis. It was found that CD73 is highly expressed in AT1 cells and specifically locates on the surface of AT1 cells, while there is almost no expression in non-AT1 epithelial cells. Further, the inventors applied the marker CD73 to sort the lung mixed epithelial cell system, and isolated and purified AT1 cells.

[0027] The present application will be further described in conjunction with the specific embodiments, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0028] Example 1, searching for AT1 cell surface markers.

[0029] The application applies self-produced mouse lung cell type proteome data resources to analyze the expression profile of AT1 cells. In the first stage, according to the difference in expression amount, the proteins expressed higher (more than 2 times) in AT1 cells than in non-AT1 cells are obtained, which are defined as AT1 cell-related proteins. In the second stage, functional and localization analysis is performed on these related proteins, and the proteins located on the surface of AT1 cell membrane and related to the function of AT1 cell are obtained, which are defined as AT1 cell candidate surface molecules. In the third stage, combined with literature review and analysis, the candidate surface molecules are subjected to cell level differential expression identification and detection of whether they can be used as markers to sort AT1 cells to obtain new AT1 cell surface marker CD73.

[0030] CD73, also known as Nt5e (ecto-5'-nucleotidase), is a cell surface glycosylphosphatidylinositol-anchored glycoprotein belonging to the nucleotide metabolism enzyme family, which mainly catalyzes the hydrolysis of adenosine acid to adenosine and inorganic phosphate. The mouse CD73 protein is encoded by the mouse Nt5e gene (located on mouse chromosome 9, Gene ID: 23959 (updated on 12-Jun-2025)), which consists of 576 amino acids and is a multifunctional transmembrane glycoprotein anchored on the cell membrane surface.

[0031] Example 2, identification of the specificity of CD73 in AT1 cells This example identifies the specificity of CD73 in the membrane surface localization of AT1 cells by FACS (Fluorescence-Activated Cell Sorting).

[0032] 1. Prepare a single cell suspension containing all cell types of mouse lung.

[0033] Anatomize the mouse (C57 / Bl6 mouse), open the abdominal cavity and chest cavity, and cut the inferior vena cava. Right ventricular perfusion with 10 ml of pre-cooled PBS, try to wash away the blood in the lungs; peel off the muscle around the trachea, expose the trachea, insert the intravenous retention needle into the trachea, link a 2 ml syringe, and slowly push in 2 ml of 37°C preheated enzyme solution (type I collagenase, dissolved in DMEM / F12 culture medium, the content of type I collagenase is 450 U / ml), so that the lung is obviously swollen; quickly change to a 1 ml syringe, slowly push in 0.5 ml of 1% (mass percent) low-solubility agarose (low-solubility agarose: LONZA, 50101) dissolved in 1×PBS preheated at about 65°C; keep the syringe to prevent liquid from flowing out. Slowly drip pre-cooled PBS on the lung on ice to accelerate the solidification of agarose in the lung; after the agarose in the lung solidifies, remove the syringe, intravenous retention needle, and take out the lung and put it into a small dish containing PBS, remove other tissues that may be attached, such as thymus, heart, lymph, etc.; put the lung into 2 ml of 37°C preheated enzyme solution, put it into a shaking bed, shake at 200 rpm at room temperature for 30 min, then filter, filter with a 45 μm cell screen, and get a mouse whole lung single cell suspension.

[0034] 2. Detect the purification effect of CD73 on AT1 cells.

[0035] 1) Centrifuge the mouse whole lung single cell suspension obtained in step 1 at 500g for 5 min at 4°C, remove the supernatant, resuspend the cells with 1×PBS to obtain a cell suspension.

[0036] 2) After step 1) is completed, take out 100 μL of the cell suspension, add immunofluorescence antibody CD73 (Biolegend, 127209) and fluorescent antibody EpCAM (EpCAM is epithelial cell adhesion molecule, BD Biosciences, 563134), the final concentration of CD73 and EpCAM in the single cell suspension is 0.25 µg / million cells, and incubate on ice for 30 minutes.

[0037] 3) After step 2) is completed, then add 4-5 volumes of 1×PBS to the cell suspension, centrifuge at 500g for 5 min at 4°C, remove the supernatant, resuspend the cells with 1×PBS and wash twice, and centrifuge to remove the supernatant.

[0038] 4) After step 3) is completed, the cells are fixed with 4% paraformaldehyde solution (Biosharp, 143174) at room temperature for 5 min, and then the cells are resuspended and washed twice with 1xPBS, centrifuged at 4°C, 500g for 5 min; the cells are permeabilized in 0.1% (v / v) Triton x-100 (Sigma, T8787) aqueous solution on ice for 10 min, and then the cells are resuspended and washed twice with 1xPBS, centrifuged at 4°C, 500g for 5 min; the washed cells are resuspended in 1xPBS, labeled with intracellular antibody Hopx (Senta Cruz Biotechnology, sc-398703) on ice for 30 min, and then the cells are resuspended and washed twice with 1xPBS, centrifuged at 4°C, 500g for 5 min, to obtain the treated cell sample.

[0039] Hopx is a known AT1 cell marker, expressed intracellularly, used to label AT1 cells.

[0040] 5) After step 4) is completed, the treated cell sample is detected by flow cytometry to determine whether the EpCAM mid CD73 hi cell population highly expresses the AT1 cell-specific marker Hopx.

[0041] The results show that the expression of Hopx in the EpCAM mid CD73 hi cell population accounts for 90.10%, i.e. the above method successfully obtained AT1 cells.

[0042] To further prove the specificity of the expression amount of CD73 in AT1 cells, the inventors sorted the AT1 cells that highly express Hopx, and then detected EpCAM and CD73 antibodies, and the antibody labeling operation process is as follows 1) -4): First, add immunofluorescence antibodies CD73 and EpCAM, and then label intracellular antibody Hopx after fixing and permeabilizing. Then the treated cell sample is detected and analyzed by flow cytometry, first the cell population that highly expresses Hopx is gated, i.e. the AT1 cell population, and then the AT1 cell population obtained by gating is detected and analyzed for the expression of CD73 and EpCAM, and the results show that 61.60% of the AT1 cells are EpCAM mid CD73 hi cells.

[0043] The above results show that CD73 is a marker expressed on the surface of AT1 cells, which can be used to isolate or label AT1 cells, and can also be used to identify AT1 cells.

[0044] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Application of CD73 protein as a surface marker of type I alveolar cells.

2. At least one of the following uses of a substance for detecting CD73 protein: X1) Detection or auxiliary detection of type I alveolar cells; X2) Preparation of products for detecting or assisting in the detection of type I alveolar cells; X3) Identify or assist in identifying type I alveolar cells; X4) Preparation of products that identify or assist in the identification of type I alveolar cells; X5) Isolation or purification of type I alveolar cells; X6) Prepare products for isolating or purifying type I alveolar cells; X7) marks type I alveolar cells; X8) Preparation of products for labeling type I alveolar cells; X9) localization of type I alveolar cells; X10) Prepare products targeting type I alveolar cells.

3. The use according to claim 2, characterized in that: The substance for detecting CD73 protein contains CD73 antibody.

4. At least one of the following uses of a substance for detecting CD73 protein and a substance for detecting EpCAM protein: X1) Detection or auxiliary detection of type I alveolar cells; X2) Preparation of products for detecting or assisting in the detection of type I alveolar cells; X3) Identify or assist in identifying type I alveolar cells; X4) Preparation of products that identify or assist in the identification of type I alveolar cells; X5) Isolation or purification of type I alveolar cells; X6) Prepare products for isolating or purifying type I alveolar cells; X7) marks type I alveolar cells; X8) Preparation of products for labeling type I alveolar cells; X9) localization of type I alveolar cells; X10) Prepare products targeting type I alveolar cells.

5. The use according to claim 4, characterized in that: The substance for detecting CD73 protein contains CD73 antibody; the substance for detecting EpCAM protein contains EpCAM antibody.

6. A method for isolating type I alveolar cells, comprising: Lung cells are labeled with CD73 antibodies, and cells that bind to the CD73 antibodies are isolated to obtain type I alveolar cells.

7. A method for isolating type I alveolar cells, comprising: Lung cells are labeled with CD73 antibodies and EpCAM antibodies, and cells that bind to CD73 antibodies and EpCAM antibodies are separated to obtain type I alveolar cells.

8. A method for labeling type I alveolar cells, comprising: Lung cells are labeled with CD73 antibodies, and cells that bind to CD73 antibodies are type I alveolar cells.

9. A method for labeling type I alveolar cells, comprising: Lung cells were labeled using CD73 antibodies and EpCAM antibodies. Cells that bind to CD73 antibodies and EpCAM antibodies are type I alveolar cells.

10. The use according to any one of claims 2 to 5, or the method according to any one of claims 6 to 9, characterized in that: The lung cells are lung epithelial cells or whole lung cells.

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