Method for bifluorescence quantitative detection of epithelial cells of lacrimal gland type organs and application

Abstract

The invention discloses a method for bifluorescence quantitative detection of epithelial cells of a lacrimal gland organ and application. The method comprises the following steps: providing a mouse lacrimal gland organ; a recombinant adeno-associated virus combination is added to the organoid, the combination comprising: a first AAV vector comprising a promoter specifically driving the expression of alveolar epithelial cells and a first fluorescent protein coding sequence; the second AAV vector contains a promoter for specifically driving expression of myoepithelial cells and a second fluorescent protein coding sequence; according to the method, the two cells can be visually distinguished, and by calculating the fluorescence area or the number of the cells, the alveolar / muscular epithelial cell proportion is established as a damage index, so that high-throughput drug screening is realized.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically a method and application for quantitative detection of lacrimal gland organoid epithelial cells using dual fluorescence. Background Technology

[0002] Lacrimal gland hypofunction is a chronic disease characterized by insufficient tear secretion, leading to dryness of the ocular surface. Symptoms include dry eyes, foreign body sensation, burning sensation, and blurred vision. Causes include dry eye, age-related lacrimal gland atrophy, dry eye due to autoimmune diseases affecting the lacrimal glands, and radiation / toxic lacrimal gland damage. Under physiological conditions, the lacrimal gland's function is to secrete tears to maintain ocular surface moisture and immune homeostasis. The lacrimal gland is mainly composed of acinar cells, ductal cells, and myoepithelial cells, with acinar cells accounting for 80% of the total. Acinar cells are arranged in a lobular pattern around the central lumen, with tightly connected apical junctions. This structure allows for unidirectional secretion and transport of water, electrolytes, proteins, and mucins from base to tip. Acinar cells synthesize, store, and secrete water, electrolytes, and mucins under nerve and hormonal stimulation. Ductal cells, polarized through tight junctions at their apex, are responsible for tear transport. Myoepithelial cells are dispersed between the acinar and ductal cells and the basal layer, interconnected by gap junctions and desmosomes. Their contractile function helps drain fluid from the acinar and duct. Damage to lacrimal gland epithelial cells is the core pathological basis for lacrimal gland dysfunction. When the lacrimal gland is severely damaged (e.g., after radiation exposure) or affected by autoimmune diseases (e.g., Sjögren's syndrome), the lacrimal gland is infiltrated by lymphocytes and other immune cells, leading to the loss of acinar cells, ductal cells, and myoepithelial cells, thereby causing reduced tear secretion. For example, in the lacrimal glands of patients with Sjögren's syndrome-related dry eye, the nuclear chromatin of acinar cells appears homogeneous, lacking differentiation of heterochromatin or euchromatin; the endoplasmic reticulum, mitochondria, and Golgi apparatus in the cytoplasm are significantly reduced; and myoepithelial cells lose their spindle-shaped processes. In mouse models of Sjögren's syndrome (NOD and MRL / MpJ-Fas... lpr In the lacrimal gland, the number of myoepithelial cells is reduced, and the expression of contractile proteins (such as α-smooth muscle actin and calcium reduction protein) is significantly decreased.

[0003] AQP5 (aquaporin 5) is a key marker of the secretory function of lacrimal gland acinar cells, participating in the tear secretion process. It maintains the permeability of lacrimal gland epithelial cells to water molecules, ensuring the basal secretion volume of tears. MYL9 (myosin light chain 9) is an important marker of the contractile function of lacrimal gland myoepithelial cells, closely related to the contractile activity of myoepithelial cells. It can assist in the emptying of secretions (tears) by regulating the contractile force of myoepithelial cells. In mouse models of various diseases caused by lacrimal gland dysfunction, reduced AQP5 expression and abnormal translocation from the cell membrane to the cytoplasm were observed, indicating weakened aquaporin function of lacrimal gland acinar cells, leading to decreased tear secretion. Decreased MYL9 expression indicates weakened myoepithelial cell contractile ability and limited tear drainage efficiency. Monitoring the expression levels of these two specific markers can effectively assess the functional status and degree of damage of lacrimal gland epithelial cells (acinar cells and myoepithelial cells). In existing technologies, organoid damage is typically assessed using qPCR (for damaged samples), immunofluorescence staining (cumbersome procedures that cannot provide dynamic observation), or ELISA to measure the supernatant (which only reflects function and not the number of viable cells), and none of these methods allow for dynamic, real-time observation. There is a lack of a method that can simultaneously, in real-time, and specifically label and quantify these two key cell types in vivo (within the three-dimensional environment of organoids). Summary of the Invention

[0004] This invention provides a method and application for quantitative detection of epithelial cells in lacrimal gland organoids using dual fluorescence, which solves the technical problem that existing technologies cannot simultaneously, dynamically, and specifically track the functional status of acinar and myoepithelial cells in living three-dimensional organoids.

[0005] To solve the above problems, the technical solution adopted by the present invention includes:

[0006] A method for quantitative detection of lacrimal gland organoid epithelial cells using dual fluorescence includes the following steps: (1) Provide mouse lacrimal gland organoids; (2) Add a dual fluorescent carrier to the mouse lacrimal gland organoid; continue culturing to express the fluorescent protein; (3) Detect and quantify the signals of the first fluorescent protein and the second fluorescent protein to characterize the number or ratio of acinar epithelial cells and myoepithelial cells; The dual fluorescent carrier includes a first AAV carrier and a second AAV carrier; The first AAV vector contains a promoter that specifically drives expression in acinar epithelial cells and a first fluorescent protein coding sequence; The second AAV vector contains a promoter and a second fluorescent protein coding sequence that specifically drive myoepithelial cell expression; The promoter that specifically drives acinar epithelial cell expression is the AQP5 promoter or a functional fragment thereof; The promoter that specifically drives myoepithelial cell expression is the MYL9 promoter or a functional fragment thereof; The first fluorescent protein is GFP, and the second fluorescent protein is mCherry.

[0007] Optionally, the first AAV vector is adeno-associated virus A serotype AAV9; The second AAV vector uses adeno-associated virus serotype B AAV5.

[0008] Optionally, step (2) specifically includes: Preparation of mixed virus solution for infection: Mix the first AAV vector and the second AAV vector at a volume ratio of 1:1, store on ice in the dark, and ensure stable virus titer. The second-generation lacrimal gland organoids were transferred to 12-well plates. After 48 hours of recovery and growth, mouse lacrimal gland organoid amplification medium containing dual fluorescent vectors was slowly added along the well wall to ensure that the dual fluorescent vectors were evenly distributed around the organoids. The plates were then incubated at 37°C in a 5% CO2 incubator. Optionally, step (3) specifically includes: Verification of infection efficiency and imaging: Verification and imaging were performed using a high-content screening system or confocal microscope. The environment of the imaging chamber was made consistent with that of the cell culture incubator, and a fluorescence imaging module was set up. The culture plate was placed stably on the stage of the live cell imaging system and fixed. The target field of view was located under bright field, and the imaging position was detected and adjusted under fluorescence imaging conditions. The time interval was set to 5-10 min / frame, and the acquisition time was more than 2 hours.

[0009] The dual-fluorescence quantitative detection method for lacrimal gland organoid epithelial cells described in any one of the present invention can be used to screen drugs for the treatment of lacrimal gland injury.

[0010] Optional, including: (1) Constructing a lacrimal gland organoid model of injury; (2) Using the dual fluorescence quantitative detection method for lacrimal gland organoid epithelial cells as described in any one of claims 1-4, the acinar epithelial cells and myoepithelial cells in lacrimal gland organoids are quantitatively detected, and the number or proportion of cells under the state of injury is detected. (3) Add the test drug to the lacrimal gland organoid of the injury model; (4) If the number or proportion of cells increases again after retesting, the drug is deemed to have therapeutic potential.

[0011] A kit for quantitatively detecting acinar epithelial cells and myoepithelial cells in lacrimal organoids using the dual-fluorescence quantitative detection method for lacrimal organoid epithelial cells described in any one of the present invention.

[0012] Technical effects of the present invention: It can intuitively distinguish between two types of cells, and by calculating the fluorescence area or cell number, it can establish the "acini / myoepithelial cell ratio" as a damage indicator to achieve high-throughput drug screening. Attached Figure Description

[0013] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 Normal organoids + double AAVs (showing abundant green and red), green represents AQP5-labeled acinar epithelial cells, and red represents MYL9-labeled myoepithelial cells. Both are clearly structured and evenly distributed, with a scar bar of 100 µm. Figure 2 Damaged organoids (IFNγ) + double AAV (showing significant reduction in green and red), green represents AQP5-labeled acinar epithelial cells, and red represents MYL9-labeled myoepithelial cells. The green and red fluorescence signals are significantly weakened, and the ratio of acinar cells (GFP+) to myoepithelial cells (mCherry+) is significantly reduced, suggesting functional degeneration of the organoids. Scar bar = 100 µm; Figure 3 Damage and treatment with N-acetylcysteine ​​+ double AAV (showing color recovery), green represents AQP5-labeled acinar epithelial cells, and red represents MYL9-labeled myoepithelial cells. The fluorescence signal was significantly enhanced compared to the damaged group, the structural outline was clear, and the cells were arranged in an orderly manner, indicating that N-acetylcysteine ​​can effectively alleviate IFNγ-induced cell damage, promote the functional recovery of acinar and myoepithelial cells, and maintain organoid integrity. Scar bar = 100 µm; Figure 4 The fluorescence intensity of GFP and mCherry in the three groups of organoids was quantitatively analyzed using ImageJ software. The results showed that the fluorescence intensity of GFP and mCherry in the injury model group (IFNγ) was significantly lower than that in the control group (P < 0.01), while the fluorescence intensity of both increased significantly after the addition of N-acetylcysteine ​​(NAC) (P < 0.05). Detailed Implementation

[0014] Unless otherwise specified, the scientific and technical terms used in this document are intended for understanding by one of ordinary skill in the art. The invention will be further described below with reference to specific embodiments. It should be noted that the embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the invention.

[0015] I. Explanation of English Abbreviations in the Full Text: AQP5: Aquaporin 5; MYL9: Myosin light chain 9; AAV: Adeno-associated virus; AAV9: Adeno-associated virus type 9; AAV5: Adeno-associated virus type 5; GFP: Green fluorescent protein; mCherry: Cherry fluorescent protein.

[0016] Organoids are ideal three-dimensional models for mimicking the structure and function of the lacrimal gland. They can reproduce the polar arrangement and secretory function of lacrimal gland epithelial cells in vitro and support long-term culture and dynamic observation. In previous studies, the extent of organoid damage was usually assessed through destructive methods or static detection. For example, qPCR requires sample lysis, making continuous monitoring impossible; immunofluorescence staining can locate protein expression, but the procedure is cumbersome and cannot be used for dynamic observation; ELISA detection of secretory protein levels in the supernatant can only indirectly reflect functional status and cannot simultaneously assess cell viability and structural integrity. Therefore, existing methods are insufficient for real-time, dynamic, and multi-dimensional assessment of damage progression and functional changes without damaging organoids. By using viral labeling technology, fluorescent proteins driven by the AQP5 and MYL9 promoters can be labeled in organoids, enabling dual-color specific tracing of acinar cells and myoepithelial cells. This fluorescent reporter system, based on specific functional markers such as AQP5 and MYL9, can non-invasively reflect the functional integrity of acinar and myoepithelial cells in real time. Combined with live-cell imaging technology, it can continuously track the dynamic changes in cell polarity, water channel localization, and contractile activity of organoids under injury stimulation. Thus, without destroying the sample, it can accurately assess the decline process of its structure and function, providing a more efficient and reliable technical platform for mechanism research and drug screening of diseases such as lacrimal gland hypofunction.

[0017] Example 1: The dual fluorescent carrier in this embodiment is: Adeno-associated virus A (serotype AAV9): carries promoter AQP5 (specifically drives acinar cell expression of AQP5) + first fluorescent gene (GFP).

[0018] Adeno-associated virus B (serotype AAV5): carries the promoter MYL9 (specifically drives myoepithelial cell expression of MYL9) + second fluorescent gene (mCherry).

[0019] Materials preparation: AQP5 promoter was used to drive acini, and MYL9 promoter was used to drive myoepithelial cells.

[0020] To construct an AAV9 virus carrying a GFP fluorescent group and an AQP5 promoter, the rat AQP5 promoter (approximately 1.7-4.3 kb, containing enhancer elements), a GFP expression cassette (C-terminal fusion or self-cleaved peptide linker), and a WPRE / polyA regulatory sequence are integrated into the ITR space of the AAV9 shuttle plasmid through vector design. HEK293T cells are then co-transfected with these three plasmids to package the virus. After purification by iodixanol density gradient centrifugation, the titer (≥1×10⁻⁶) is determined by qPCR. 12 (vg / mL). Following the same procedure as above, construct an AAV5 virus carrying the mCherry fluorescent group and the MYL9 promoter.

[0021] Experimental steps: 1. According to Marie Bannier-Hélaouët [1] Mouse lacrimal gland organoids were constructed using methods such as [method name missing], and basal culture medium, amplification medium, and differentiation medium for mouse lacrimal gland organoids were prepared. The organoids were then passaged to the second generation in mouse lacrimal gland organoid proliferation medium. The specific operational steps are as follows: (1) Dissect and separate mouse lacrimal gland tissue: ① Prepare surgical tools, including scissors, forceps, surgical pads, etc.; euthanize the mice by inhaling O2 / CO2.

[0022] ② Place the euthanized mouse on an abdominal dissection pad and fix its limbs. Moisten the hair between the mouse's ears and on its forehead with 70% ethanol.

[0023] ③ Use dissecting scissors to make an incision between the two ears behind the skull, extending the incision from the forehead to the nose, and cut away the skin behind the ears to form two skin flaps; pull the skin flaps forcefully toward the nose until the lacrimal gland is exposed, and fix the skin flaps to the interlayer pad; use scissors to make a small incision above the lacrimal gland to fully expose the lacrimal gland, and then use forceps to remove the lacrimal gland.

[0024] ④ Place the mouse lacrimal gland in tissue culture medium PBS for further processing; process the lacrimal gland within 2–4 hours to limit cell death; if longer processing time is required, place the mouse lacrimal gland in biopsy collection medium.

[0025] (2) Construction of lacrimal gland organoids: ① Remove mouse lacrimal gland tissue from the culture medium and place it in a culture dish; use pre-wetted ophthalmic scissors to mince the tissue (<0.5 mm). 3The cells were resuspended in tissue digestion solution and transferred to 15 mL centrifuge tubes. The tubes were incubated in a 37°C water bath for 15 min. The tubes were periodically inverted and resuspended, and cell dissociation was monitored under a microscope to avoid over-digestion of tissue. When many single cells and small clumps were visible under the microscope, 10 mL of mouse lacrimal gland organoid basal culture medium (the formulation of which can be found in the literature: Bannier-Hélaouët, M., Geurts, MH, Korving, J., Begthel, H., & Clevers, H. (2023). Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids. Journal of visualized experiments : JoVE, (192), 10.3791 / 65040. https: / / doi.org / 10.3791 / 65040.) was added to stop dissociation. The cells were then rotated at 400 g for 5 min to form spheres.

[0026] ② Remove the supernatant and resuspend the particles in 10 mL of mouse lacrimal gland organoid basal culture medium, and wash repeatedly. Filter through a 70 µm filter to remove undigested large tissue fragments and remaining collagen fibers, and centrifuge at 400 g for 5 min.

[0027] ③ Remove the supernatant and resuspend the cell particles in 100 µL of cold matrix gel. Add approximately 2-3 droplets (about 20 µL each) to each well of a 12-well plate. Invert the plate and incubate at 37°C for 15 min to allow the matrix gel to solidify. After solidification, add 1 mL of mouse lacrimal gland organoid amplification medium to each well of the 12-well plate. Replace the medium every 2-3 days until the organoids reach a size of 300 µm, then passage.

[0028] 2. Preparation and Infection with Mixed Virus Solution: Based on preliminary experimental results, the two AAV viruses were mixed at a 1:1 volume ratio and stored on ice in the dark to ensure stable viral titers. Second-generation lacrimal gland organoids were transferred to new 12-well plates. After 48 hours of recovery, amplification medium containing the mixed virus solution was slowly added along the well walls, ensuring even distribution of the virus solution around the organoids. The plates were incubated at 37°C with 5% CO2 for 6 hours, followed by fresh amplification medium. Morphological changes in the organoids were observed daily. Once the organoids reached a uniform size of approximately 200–300 µm, amplification medium containing 10 μg / mL Hoechst 33342 cell staining solution was added, and imaging was performed after 30 minutes of staining.

[0029] 3. Verification of Infection Efficiency and Imaging: Verification and imaging were performed using a high-content screening system or confocal microscope. The instrument was pre-set to ensure the imaging chamber environment was consistent with the cell culture incubator (37℃, 5% CO2), and a fluorescence imaging module was set up. The culture plate was placed stably on the stage of the live-cell imaging system and secured with a special clamp to prevent sample displacement during imaging. The target field of view was located under bright field (4× / 10×), and the imaging position was detected and adjusted under fluorescence imaging conditions. The time interval was set to 5–10 min / frame, and the acquisition time was over 2 hours. Results showed that the organoids under the microscope were morphologically intact and structurally clear. AQP5, with green fluorescence, was expressed in lacrimal gland acinar epithelial cells, and MYL9, with red fluorescence, was expressed in lacrimal gland myoepithelial cells. The fluorescence intensity was uniform, with no obvious fragmentation. Figure 1 ).

[0030] Damage model and lacrimal gland recovery validation: Lactal gland organoids in good growth condition were used for group construction, divided into a control group, a damage model group (100 ng / mL IFNγ), and an intervention group (100 ng / mL IFNγ and 3 mM N-acetylcysteine). First, AAV virus infection was performed according to the experimental procedures described above. After confirming the infection efficiency, the culture was continued for 72 hours, followed by replacement with damage induction medium containing IFNγ. The intervention group received additional N-acetylcysteine. After 48 hours of continuous treatment, organoid morphology and activity were assessed in each group.

[0031] The results showed that the control group organoids were morphologically intact, structurally clear, and exhibited uniform fluorescence intensity without significant fragmentation. However, after treatment with 100 ng / mL IFNγ for 48 hours, a significant decrease in the proportion of acinar cells (GFP+) and myoepithelial cells (mCherry+) was observed, suggesting functional degeneration of the organoids. Figure 2In organoid culture media with added N-acetylcysteine, the decrease in GFP and mCherry fluorescence signals was not significant, suggesting that organoids maintained the stability of acinar cells under N-acetylcysteine ​​intervention. Figure 3 This indicates that the dual fluorescent labeling system can effectively reflect the protective effect of the drug on lacrimal gland organoid function. Figure 4 ).

[0032] Although the illustrative specific embodiments of the present invention have been described above to enable those skilled in the art to understand the invention, it should be understood that the invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of the present invention are protected.

Claims

1. A method for quantitative detection of lacrimal gland organoid epithelial cells using dual fluorescence, characterized in that, Includes the following steps: (1) Provide mouse lacrimal gland organoids; (2) Add a dual fluorescent carrier to the mouse lacrimal gland organoid; continue culturing to express the fluorescent protein; (3) Detect and quantify the signals of the first fluorescent protein and the second fluorescent protein to characterize the number or ratio of acinar epithelial cells and myoepithelial cells; The dual fluorescent carrier includes a first AAV carrier and a second AAV carrier; The first AAV vector contains a promoter that specifically drives expression in acinar epithelial cells and a first fluorescent protein coding sequence; The second AAV vector contains a promoter and a second fluorescent protein coding sequence that specifically drive myoepithelial cell expression; The promoter that specifically drives acinar epithelial cell expression is the AQP5 promoter or a functional fragment thereof; The promoter that specifically drives myoepithelial cell expression is the MYL9 promoter or a functional fragment thereof; The first fluorescent protein is GFP, and the second fluorescent protein is mCherry.

2. The method for quantitative detection of lacrimal gland organoid epithelial cells according to claim 1, characterized in that, The first AAV vector used is adeno-associated virus A serotype AAV9; The second AAV vector uses adeno-associated virus serotype B AAV5.

3. The method for quantitative detection of lacrimal gland organoid epithelial cells according to claim 1 or 2, characterized in that, Step (2) specifically includes: Preparation of mixed virus solution for infection: Mix the first AAV vector and the second AAV vector at a volume ratio of 1:1, store on ice in the dark, and ensure stable virus titer. The second-generation lacrimal gland organoids were transferred to 12-well plates. After 48 hours of recovery and growth, mouse lacrimal gland organoid amplification medium containing dual fluorescent vectors was slowly added along the well wall to ensure that the dual fluorescent vectors were evenly distributed around the organoids. The plates were then incubated in a 37°C, 5% CO2 incubator.

4. The method for quantitative detection of lacrimal gland organoid epithelial cells according to claim 1 or 2, characterized in that, Step (3) specifically includes: Verification of infection efficiency and imaging: Verification and imaging were performed using a high-content screening system or confocal microscope. The environment of the imaging chamber was made consistent with that of the cell culture incubator, and a fluorescence imaging module was set up. The culture plate was placed stably on the stage of the live cell imaging system and fixed. The target field of view was located under bright field, and the imaging position was detected and adjusted under fluorescence imaging conditions. The time interval was set to 5-10 min / frame, and the acquisition time was more than 2 hours.

5. The method for dual-fluorescence quantitative detection of lacrimal gland organoid epithelial cells according to any one of claims 1-4 is used for screening drugs for the treatment of lacrimal gland injury.

6. The application according to claim 5, characterized in that, include: (1) Constructing a lacrimal gland organoid model of injury; (2) Using the dual fluorescence quantitative detection method for lacrimal gland organoid epithelial cells as described in any one of claims 1-4, the acinar epithelial cells and myoepithelial cells in lacrimal gland organoids are quantitatively detected, and the number or proportion of cells under the state of injury is detected. (3) Add the test drug to the lacrimal gland organoid of the injury model; (4) If the number or proportion of cells increases again after retesting, the drug is deemed to have therapeutic potential.

7. A reagent kit, characterized in that, The dual-fluorescence quantitative detection method for lacrimal gland organoid epithelial cells according to any one of claims 1-4 is used to quantitatively detect acinar epithelial cells and myoepithelial cells in lacrimal gland organoids.

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