CN-121992037-A - Method for quantitatively detecting lacrimal gland organoid epithelial cells by double fluorescence and application

CN121992037ACN 121992037 ACN121992037 ACN 121992037ACN-121992037-A

Abstract

The invention discloses a method for quantitatively detecting lacrimal gland organoids and application thereof, which comprises the following steps of providing a lacrimal gland organoids of a mouse, adding a recombinant adeno-associated virus combination into the organoids, wherein the combination comprises a first AAV vector containing a promoter for specifically driving the expression of acinar epithelial cells and a first fluorescent protein coding sequence, and a second AAV vector containing a promoter for specifically driving the expression of myoepithelial cells and a second fluorescent protein coding sequence.

Inventors

SUN LIJUAN
XIE XIAOHANG
WU JIAO
Dou Guorui
GUI YU
HE YANRU
QIN YIFEI
ZHANG XINYI
HUO FEI
YANG ZIWEI

Assignees

中国人民解放军空军军医大学

Dates

Publication Date: 20260508
Application Date: 20260205

Claims (7)

1. A method for quantitatively detecting lacrimal organoids epithelial cells by double fluorescence, comprising the steps of: (1) Providing a mouse lacrimal organoid; (2) Adding a double fluorescent carrier into the lacrimal gland organoids of the mice, and continuing culturing to express the fluorescent protein; (3) Detecting and quantifying the signal of the first fluorescent protein and the signal of the second fluorescent protein, thereby characterizing the number or ratio of acinar and myoepithelial cells; the double fluorescent vector comprises a first AAV vector and a second AAV vector; a first AAV vector comprising a promoter that specifically drives expression of acinar epithelial cells and a first fluorescent protein coding sequence; a second AAV vector comprising a promoter that specifically drives expression of a myoepithelial cell and a second fluorescent protein coding sequence; The promoter for specifically driving the expression of the acinar epithelial cells is an AQP5 promoter or a functional fragment thereof; the specific promoter for driving the expression of the myoepithelial cells is a MYL9 promoter or a functional fragment thereof; The first fluorescent protein is GFP and the second fluorescent protein is mCherry.
2. The method of claim 1, wherein the first AAV vector employs adeno-associated virus a serotype AAV9; the second AAV vector employs AAV5, an adeno-associated virus serotype B.
3. The method for quantitative detection of lacrimal organoids epithelial cells according to claim 1 or 2, wherein the step (2) specifically comprises: Preparing mixed virus liquid for infection, namely mixing a first AAV vector and a second AAV vector according to a volume of 1:1, and placing the mixed virus liquid on ice for light-proof preservation to ensure that the virus titer is stable; Transferring the second-generation lacrimal gland organoids into a 12-well plate, slowly adding a mouse lacrimal gland organoid amplification culture medium containing a double-fluorescence carrier along the wall of the well after the second-generation lacrimal gland organoids recover to grow for 48 hours, ensuring that the double-fluorescence carrier is uniformly distributed around the organoids, and placing the organoids in a 37 ℃ and 5% CO 2 incubator for incubation.
4. The method for the quantitative detection of lacrimal organoids epithelial cells according to claim 1 or 2, wherein the step (3) specifically comprises: The method comprises the steps of verifying infection efficiency and imaging, namely verifying and imaging by using a high content screening system or a confocal microscope, enabling the environment of an imaging cavity to be consistent with that of a cell incubator, arranging a fluorescent imaging module, stably placing a culture plate on an objective table of a living cell imaging system for fixing, searching a target visual field under an open field, detecting and adjusting an imaging position under a fluorescent imaging condition, setting a time interval to be 5-10 min/frame, and collecting the culture plate for more than 2 hours.
5. Use of the method for the quantitative detection of lacrimal organoids epithelial cells by double fluorescence according to any of claims 1-4, for screening a medicament for treating lacrimal gland injury.
6. The use according to claim 5, characterized in that it comprises: (1) Constructing a lacrimal gland organoid of the damage model; (2) Quantitatively detecting acinar epithelial cells and myoepithelial cells in the lacrimal organoid by using the method for quantitatively detecting lacrimal organoid epithelial cells by using the double fluorescence of any one of claims 1 to 4, and detecting the number or proportion of cells in a damaged state; (3) Adding a drug to be tested into the lacrimal gland organoids of the injury model; (4) Again, if the number or proportion of cells is rising, the drug is judged to have therapeutic potential.
7. A kit, characterized in that the method for quantitatively detecting the epithelial cells of lacrimal gland organoids by using the double fluorescence according to any one of claims 1-4 is used for quantitatively detecting acinar epithelial cells and myoepithelial cells in lacrimal gland organoids.

Description

Method for quantitatively detecting lacrimal gland organoid epithelial cells by double fluorescence and application Technical Field The invention belongs to the technical field of cell biology, and particularly relates to a method for quantitatively detecting lacrimal gland organoids epithelial cells by double fluorescence and application thereof. Background The lacrimal gland hypofunction is a chronic disease which is caused by dry eye surface due to insufficient lacrimal secretion, and is mainly represented by symptoms such as dry eyes, foreign body sensation, burning sensation, blurred vision and the like, such as dry eyes, senile lacrimal gland atrophy, dry eyes, radiation/toxic lacrimal gland injury and the like when the lacrimal gland is involved in autoimmune diseases. Under physiological conditions, the lacrimal gland functions to secrete tears to maintain ocular surface wetting and immune homeostasis. Lacrimal glands are composed mainly of acinar cells, ductal cells, and myoepithelial cells, with acinar cells accounting for 80% of the total. Acinar cells are arranged in a lobular arrangement around the central lumen with the cell apices closely connected, and this structure allows for unidirectional secretion of water, electrolytes, proteins and mucins, transport from the base to the top. Acinar cells synthesize, store and secrete water, electrolytes and mucins under neural and hormonal stimulation. The cells of the catheter are polarized by the tight junctions at the tip, assuming the transport task of tears. The myoepithelial cells are distributed between the acinar and ductal cells and the basal lamina, interconnected by gap junctions and desmosomes, the contractile function of which aids in the drainage of fluid from the acinar and ductal cells. Damage to lacrimal gland epithelial cells is the central pathological basis leading to hypofunction of the lacrimal gland. When the lacrimal gland is severely damaged (e.g., after irradiation) or affected by autoimmune diseases (e.g., sjogren's syndrome), the lacrimal gland is infiltrated by lymphocytes and other immune cells, resulting in the loss of acinar cells, ductal cells, and myoepithelial cells, which in turn, cause decreased tear secretion. As in lacrimal glands in dry eye patients associated with sjogren's syndrome, it is observed that the nuclear chromatin of acinar cells appears to be uniform in appearance, lacks heterochromatin or differentiation of euchromatin, and the endoplasmic reticulum, mitochondria and golgi apparatus are significantly reduced in the cytoplasm, and the myoepithelial cells lose fusiform projections. In the lacrimal glands of the Sjogren syndrome mouse model (NOD and MRL/MpJ-Fas lpr), the number of myoepithelial cells is reduced and the expression of contractile proteins, such as alpha-smooth muscle actin and calreducing proteins, is significantly reduced. AQP5 (aquaporin 5) is a key marker of the secretion function of lacrimal gland acinar cells, participates in the secretion process of tears, can maintain permeability of lacrimal gland epithelial cells to water molecules and guarantee basal secretion of tears, and MYL9 (myoglobin light chain 9) is an important marker of the contraction function of lacrimal gland myoepithelial cells, is closely related to the contraction activity of myoepithelial cells, and can assist acinar to empty secretion (tears) by regulating the contraction force of myoepithelial cells. In a mouse model of various diseases caused by hypofunction of the lacrimal gland, reduced AQP5 expression was observed and abnormal translocation from the cell membrane into the cytoplasm occurred, indicating reduced function of the lacrimal gland acinar cell water channel, leading to reduced lacrimal secretion. The decrease in MYL9 expression indicates that myoepithelial cell contractility is reduced and tear drainage efficiency is limited. The functional state and damage degree of lacrimal gland epithelial cells (acinar cells and myoepithelial cells) can be effectively evaluated by monitoring the expression level of the two specific markers. In the prior art, organoid damage is measured usually by qPCR (sample destruction), immunofluorescent staining (complicated operation, inability to dynamically observe), or ELISA to measure supernatant (only function, inability to reflect cell survival number), and failure to dynamically observe in real time. There is a lack of a method for simultaneously, real-time, and specific labeling and quantification of two key cells in vivo (in a three-dimensional organoid environment). Disclosure of Invention The invention provides a method for quantitatively detecting lacrimal gland organoids and application thereof, which solves the technical problem that the prior art cannot synchronously, dynamically and specifically track the functional states of acinus and myoepithelium in a living body three-dimensional organoid. In order to solve the problems, the technical scheme adopted