CN111471771B

CN111471771B - Marker for evaluating or assisting in evaluating identity and aging of islet cells

Info

Publication number: CN111471771B
Application number: CN202010472871.9A
Authority: CN
Inventors: 曲静; 刘光慧; 汤富酬; 张维绮; 李静宜; 郑宇轩; 颜鹏泽; 宋默识; 王思
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2022-03-22
Anticipated expiration: 2040-05-29
Also published as: CN111471771A

Abstract

The invention discloses a marker for evaluating or assisting in evaluating the identity of islet cells and aging. According to the invention, through constructing and analyzing a sequencing library of cynomolgus monkey islet cells and carrying out immunofluorescence detection, at least one of ERO1B and TMEM132B can be used as a marker to identify or assist in identifying the islet beta cells. HSP90B1 can be used as a marker to distinguish between senescent and young islet beta cells. The markers can be used for evaluating diseases related to islet aging such as diabetes, pancreatic cancer, islet presenility and the like. The invention has important application value.

Description

Marker for evaluating or assisting in evaluating identity and aging of islet cells

Technical Field

The invention relates to a marker for evaluating or assisting in evaluating the identity of islet cells and aging, belonging to the field of biomedicine.

Background

The pancreas is an important metabolic organ of a human body, is divided into an exocrine part and an endocrine part, and provides pancreatic juice rich in digestive enzymes and a plurality of blood sugar regulating hormones which are necessary for life activities for the human body respectively. Wherein the cells of the endocrine part (i.e. the pancreatic islets) are aged, which can cause the blood sugar regulation disorder of the body, the decrease of the glucose tolerance and even the development of diabetes. Diabetes is a kind of aging-related disease, the incidence rate of which rises with the increase of age, has become the third most serious disease that endangers human health, and is a health problem facing all over the world. The diabetic needs to strictly control diet, take a plurality of hypoglycemic drugs orally, and the serious patient needs to inject insulin, so that the life quality is greatly influenced. In addition, abnormal hyperglycemia in vivo causes metabolic disorders of tissues and organs such as retina, kidney, blood vessel, nervous system and the like, and causes a series of complications. The research on how to prevent and intervene diabetes has important social significance. There is increasing evidence that aging leads to increased islet mass, impaired turnover, and aberrant gene transcription. Senescent cells accumulate in the islets of the elderly and in type 2 diabetic patients. On the contrary, the elimination of aged islet cells improves the endocrine function of the pancreas and restores the blood sugar balance of the body. Therefore, the molecular mechanism of islet senescence is systematically explored, a key targeting factor is provided for delaying islet senescence, and a new treatment idea is provided for effectively preventing and intervening diabetes. Digging cell type markers and aging markers of islet cells, and providing important molecular indications for developing islet regeneration strategies and developing and evaluating islet aging intervention strategies.

Disclosure of Invention

The technical problem to be solved by the invention is how to detect the islet beta cells and how to distinguish the aged islet beta cells from the young islet beta cells.

In order to solve the above technical problems, the present invention provides the following applications:

1. use of any of the following substances for detecting expression of HSP90B1 gene:

x1, preparing a product for distinguishing or assisting in distinguishing aged islet beta cells from young islet beta cells;

x2, differentiating or aiding in differentiating aging islet beta cells from young islet beta cells;

2. the following applications of the substance for detecting the content of HSP90B1 protein are disclosed:

3. the application of a substance for distinguishing aged islet beta cells from young islet beta cells by taking HSP90B1 gene or HSP90B1 protein as a marker in preparing a product for distinguishing or assisting in distinguishing aged islet beta cells from young islet beta cells;

4. use of any of the following substances for detecting expression of ERO1B and/or TMEM132B genes:

x3, preparing a product for detecting or assisting in detecting the islet beta cells;

x4, detection or detection-assisted islet beta cells;

x5, preparing a product for screening or auxiliary screening of islet beta cells;

x6, screening or screening-assisted islet beta cells;

5. use of a substance for detecting ERO1B and/or TMEM132B protein content of any of the following:

x4, detection or detection-assisted islet beta cells;

x6, screening or screening-assisted islet beta cells;

6. the application of the substance for detecting the islet beta cells by using the ERO1B and/or TMEM132B gene as a marker comprises the following steps:

x4, detection or detection-assisted islet beta cells;

x6, screening or screening-assisted islet beta cells;

7. the application of the substance for detecting the islet beta cells by using ERO1B and/or TMEM132B protein as a marker comprises the following steps:

x4, detection or detection-assisted islet beta cells;

x6, screening or screening-assisted islet beta cells.

The invention also provides a substance for distinguishing or assisting in distinguishing aged islet beta cells from young islet beta cells, wherein the substance is used for detecting the expression of HSP90B1 gene or detecting the content of HSP90B1 protein.

The invention also provides a substance for detecting or assisting in detecting the islet beta cells, wherein the substance is used for detecting the expression of the ERO1B and/or TMEM132B gene, or is used for detecting the content of the ERO1B and/or TMEM132B protein.

The invention also provides any one of the following methods:

y1, a method for detecting whether a test cell is an islet beta cell, comprising (S1) or (S2):

(S1) detecting whether the cell to be detected contains a target protein, wherein the target protein is ERO1B and/or TMEM132B protein: if the target protein is contained, the cell to be detected is or is selected as an islet beta cell; if the target protein is not contained, the cell to be detected is or is selected as a non-islet beta cell;

(S2) detecting whether a target gene in the cell to be detected is expressed, wherein the target gene is ERO1B and/or TMEM132B gene: if the target gene is expressed, the cell to be detected is or is selected as an islet beta cell; if the target gene is not expressed, the cell to be detected is or is selected as a non-islet beta cell;

y2, a method of distinguishing between senescent and young islet beta cells, comprising (S3) or (S4):

(S3) detecting the content of a target protein in the cell to be detected, wherein the target protein is HSP90B1 protein: the age of islet beta cells with high target protein content is greater than or is candidate for greater than the age of islet beta cells with low target protein content; the age of the islet beta cells with low target protein content is less than or is less than the age of the islet beta cells with low target protein content; islet beta cells with equal target protein content are of equal age or are candidate for equal;

(S4) detecting the expression level of a target gene in the cell to be detected, wherein the target gene is HSP90B1 gene: the age of the islet beta cells with high target gene expression is greater than or is more than the age of the islet beta cells with low target gene expression; the age of the islet beta cells with low target gene expression level is smaller than or is less than the candidate age of the islet beta cells with low target gene expression level; islet beta cells with equal target gene expression levels are of equal age or are candidates for equal expression levels.

In the present invention, the substance for detecting the expression of the HSP90B1 gene may include a substance capable of specifically recognizing the HSP90B1 gene, such as a primer and/or a probe. The substance for measuring the content of the HSP90B1 protein may include a substance, such as an antibody, capable of specifically recognizing the HSP90B1 protein.

The substance for detecting the expression of ERO1B and/or TMEM132B gene may include a substance, such as a primer and/or a probe, capable of specifically recognizing the ERO1B and/or TMEM132B gene.

The substance for detecting the ERO1B and/or TMEM132B protein content may include a substance, such as an antibody, capable of specifically recognizing the ERO1B and/or TMEM132B protein.

The test sample of the product may be an islet tissue sample isolated from a mammal, a paraffin section thereof, or a frozen section thereof.

The test sample of the product may be islet cells. Islet cells refer to cells isolated from islet tissue.

The mammal is a cynomolgus monkey or a human.

When the mammal is a cynomolgus monkey, the old is 18-21 years old, and the young is 4-6 years old. The mammal is human, the old means 60-80 years old, and the young means 16-20 years old.

The inventor of the invention discovers that at least one of ERO1B and TMEM132B can be used as a marker to identify or assist in identifying the islet beta cells by constructing and analyzing a sequencing library of the cynomolgus monkey islet beta cells and carrying out immunofluorescence detection. HSP90B1 can be used as a marker to distinguish between senescent and young islet beta cells. The markers can be used for evaluating diseases related to islet aging such as diabetes, pancreatic cancer, islet presenility and the like. The invention has important application value.

Drawings

FIG. 1 is a schematic diagram of cynomolgus monkey islet single cell sequencing library construction and analysis.

FIG. 2 shows the expression levels of islet beta cell molecular markers in islet beta cells.

FIG. 3 is a graph showing the identification of the expression and localization of islet beta cell molecular markers in islet beta cells by immunostaining. The upper panel shows the results of immunohistochemical detection, and the lower panel shows the results of immunofluorescence detection.

FIG. 4 is a bioinformatics analysis of senescence-associated differentially expressed gene HSP90B1 of islet beta cells.

FIG. 5 shows the expression and localization of HSP90B1 as a molecular marker in aged islet beta cells by immunofluorescence. YF represents a young female individual, OF represents an old female individual, YM represents a young male individual, and OM represents an old male individual.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

ERCC RNA Spike-In Mix: life Technologies, cat # 4456740;

dNTP: fermentas, Cat number R0193;

nuclease-free water: ambion, cat # AM 9937;

murine antibodies to INS, Triton X-100, betaine and MgCl₂The products are Sigma-Aldrich products, and the product numbers are I2008, T9284, 61962 and M8266 in sequence; AMPure XP magnetic beads: beckman, cat # A63881;

Tissue-

OCT^TMa compound: sakura Finetek, cat # 4583;

fade resistant fixative media: vector Laboratories, Cat number H-1000;

bovine Serum Albumin (BSA): MP, cat # 0219989991;

propidium Iodid, RNase inhibitor, SuperScript II reverse transcriptase, 5 XSuperScript II one-strand synthesis buffer, Dithiothreitol (DTT), Dynabeads MyOne streptavidin C1, Alexa

488 donkey anti-rabbit IgG (H + L) secondary antibody, Alexa

488 donkey anti-rat IgG (H + L) secondary antibody and Alexa

568 donkey anti-mouse IgG (H + L) secondary antibodies are Invitrogen products, and the product numbers are P3566, AM2684, 18064-071, 65001, A21206, A-21208 and A10037 in sequence;

rabbit anti-ERO 1B: proteitech, a good number of 11261-2-AP; rabbit anti-TMEM 132B: novus Biologicals, cat # NBP 2-38259; rat anti-HSP 90B1 and Hoechst 33342 are Invitrogen products with the code of MA3-016 and H3570 respectively;

HBSS and FBS are Gibco products with the product numbers of 14025 and 10270-106 respectively;

accumax is Millipore product, and the product number is SCR 006;

hematoxylin staining solution, rabbit two-step method detection kit, mouse two-step method detection kit and DAB color development kit are China fir Jinqiao products, and the product numbers are ZLI-9610, PV-9001, PV-9002 and ZLI-9018 in sequence;

the 2 XKAPA HiFi HotStart ReadyMix and KAPA Hyper Prep library building kits are products of KAPA company, and the product numbers are KK2602 and KK8054 in sequence.

In the following examples, the names and nucleotide sequences of primers used to construct the cynomolgus monkey single cell sequencing library are shown in Table 1.

TABLE 1

Note: in the barcode primer, "XXXXXXXX" is an 8bp barcode sequence (i.e., barcode), "NNNNNNNN" is an 8bp random Unique Molecular Identifier (UMI) sequence, "T25" represents 25T; in the TSO primer, "rG" is riboguanosine and "+ G" is guanosine modified by Locked Nucleic Acid (LNA); in the biotin primer, "/biotin/" is the 5' -end of the primer subjected to biotin modification, and "/index/" is the library barcode sequence of 6 bp.

The cynomolgus monkey experiments in the following examples were performed based on the ethical treatment criteria for non-human primates and have been approved by the ethical institute of animals of the Chinese academy of sciences.

Example 1 obtaining of sequencing library for cynomolgus monkey Single cells (including sequencing library for cynomolgus monkey islet beta cells)

A schematic diagram of cynomolgus monkey single cell sequencing library construction and analysis is shown in FIG. 1.

First, obtaining a suspension of cynomolgus monkey cells

According to the average life span of the cynomolgus monkeys and the corresponding relationship with the age of human beings, 8 (4 female and 4 male) cynomolgus monkeys of 4-6 years old constitute a young group, and 8 (4 female and 4 male) cynomolgus monkeys of 18-21 years old constitute an old group. All cynomolgus monkeys were subjected to physical examination, including measurement of basic parameters (e.g., Body Mass Index (BMI)), detection of biochemical indicators (e.g., blood routine, urine routine), and ultrasound detection of important internal organs such as brain nuclear magnetism and heart, to confirm that none of the cynomolgus monkeys in the young and old groups had significant disease.

Obtaining a cell suspension of each cynomolgus monkey, and specifically comprising the following steps:

1. cynomolgus monkeys were anesthetized and perfused with normal saline, and then pancreatic tissue was isolated and immediately immersed in normal saline.

2. The pancreas was ligated with surgical thread at 1/3, perfused through the catheter with 20ml of 0.5mg/ml collagenase V (HBSS dilution) to engorge the pancreas, incubated in a thermostatted water bath at 37 ℃ for 35min, added with 25ml HBSS containing 10% FBS, stopped from digestion, centrifuged at 1000rpm at 4 ℃ for 1min, and the precipitate 1 was collected.

3. 10ml of HBSS containing 10% FBS was added to the precipitate 1, and the mixture was centrifuged at 1000rpm at 4 ℃ for 1min to collect the precipitate 2.

4. 10ml of HBSS was added to the pellet 2 for resuspension to obtain suspension 1.

5. Suspension 2 was obtained by taking suspensions in batches from 1 to 3.5cm petri dishes and picking up yellow islets under a microscope.

6. Adding accumax into suspension 2, incubating at 37 deg.C for 5-10min in a constant temperature water bath, adding 25ml HBSS containing 10% FBS, stopping digestion, centrifuging at 1000rpm for 5min, and collecting precipitate 3.

7. To pellet 3 was added 2ml of HBSS for resuspension to obtain suspension 3. Propidium Iodide (PI) was added to suspension 3 and Fluorescence Activated Cell Sorting (FACS) (BD FACSAria) was used^TMII) removing debris in the residual cell suspension to obtain a cell suspension; the cell suspension contains islet cells.

Secondly, construction of cynomolgus monkey islet beta cell sequencing library

Lysis buffer was prepared from 0.05. mu.L of RNase inhibitor at a concentration of 40U/. mu.L, 0.095. mu.L of 10% (volume percent) Triton X-100, 0.5. mu.L of an aqueous solution of dNTP at a concentration of 10mM, 1.105. mu.L of nuclease-free water, 0.1. mu.L of 1: 2000000 dilutions of ERCC RNA Spike-In Mix and 0.15. mu.L of 10. mu.M aqueous barcode primer solution.

Reverse transcription mixture: the reaction mixture was composed of 0.25. mu.L of 200U/. mu.L SuperScript II reverse transcriptase, 1. mu.L of 5 XSuperpcript II single-strand synthesis buffer, 0.125. mu.L RNase inhibitor, 0.25. mu.L RNase inhibitormu.L of 0.1M aqueous DTT solution, 1. mu.L of 5M aqueous betaine solution, and 0.03. mu.L of 1M MgCl₂Aqueous solution, 0.145. mu.L nuclease-free water and 0.05. mu.L aqueous TSO primer solution at a concentration of 100. mu.M.

PCR mixture: consisting of 6.25. mu.L of 2 XKAPA HiFi HotStart ReadyMix, 0.25. mu.L of a 10. mu.M aqueous pre-amplification primer solution, 0.75. mu.L of a 10. mu.M aqueous 3' P2 primer solution, and 0.25. mu.L of nuclease-free water.

The optimized STRT-seq single-cell transcriptome database building method is adopted to respectively perform cracking, reverse transcription and pre-amplification on islet cells of each cynomolgus monkey, respectively build a sequencing library of the islet cells of the cynomolgus monkey, and then perform sequencing on an Illumina X Ten platform. The method comprises the following specific steps:

1. taking the cell suspension of the first step, manually picking islet cells under a dissecting microscope and transferring to PBS medium drops of 0.1mg/100mL BSA.

2. The islet cells collected in step 1 were individually pipetted into Eppendorf tubes (200. mu.L) containing 2. mu.L lysis buffer via a pipette and lysed at 72 ℃ for 3 min.

3. After step 2 was completed, 2.85. mu.L of the reverse transcription mixture was added, mixed well, followed by incubation at 25 ℃ for 5min, 42 ℃ for 60min, 50 ℃ for 30min, and 70 ℃ for 10 min.

4. And (3) adding the system which completes the step 3 into an EP tube filled with 7.5 mu L of PCR mixture for PCR amplification, then combining PCR products marked with different barcodes, and purifying the combined DNA twice by using AMPure XP magnetic beads to obtain the combined DNA.

PCR reaction procedure: 3min at 95 ℃; 20s at 98 ℃, 30s at 65 ℃ and 5min at 72 ℃; 20s at 98 ℃, 15s at 67 ℃, 5min at 72 ℃ and 15 cycles; 5min at 72 ℃.

5. Preparing a biotin PCR reaction system, carrying out PCR amplification, and then purifying twice by using AMPure XP magnetic beads to obtain a biotin-labeled DNA fragment.

The biotin PCR reaction system was 50. mu.L, consisting of 25. mu.L of 2 XKAPA HiFi HotStart ReadyMix, 2. mu.L of 10. mu.M aqueous solution of the anchor primer, 2. mu.L of 10. mu.M aqueous solution of the biotin primer, 30-40ng of pooled DNA and nuclease-free water.

6. After the step 5 is completed, breaking the biotin-labeled DNA fragment to about 300bp by using an ultrasonication instrument, and purifying for 1 time by using AMPure XP magnetic beads; the biotin-labeled 3' ends were then enriched with Dynabeads MyOne streptavidin C1.

7. And (3) taking the DNA fragment enriched in the step 6, and performing terminal repair, dA tailing and adaptor connection according to the steps of the KAPA Hyper Prep library building kit instruction to obtain the DNA connected with the adaptor.

8. Preparing a PCR reaction system, and then carrying out PCR amplification to obtain a PCR amplification product.

The PCR reaction system was 50. mu.L, consisting of 25. mu.L of 2 XKAPA HiFi HotStart ReadyMix, Illumina QP2 primer, short universal primer, adaptor ligated DNA and nuclease free water. The concentrations of Illumina QP2 primer and short universal primer in the PCR reaction system were both 0.3. mu.M.

9. And (3) carrying out 150bp reading-length double-end sequencing on the PCR amplification product obtained in the step 10 by using Illumina HiSeq 4000 to obtain a cynomolgus monkey islet cell sequencing library.

Example 2 obtaining and detection of molecular markers of pancreatic islet cells

Firstly, analyzing biological information to obtain islet cell molecular markers

And comparing sequencing data after quality control by taking the cynomolgus monkey genome Macaca _ fascicularis _5.0(Ensemble) as a reference, counting unique comparison reading length, and splitting into different cell data, wherein the cells are islet alpha cells, islet beta cells and the like. Marking single molecules with the unique molecular sequence number, correcting amplification deviation, and obtaining the cynomolgus monkey islet single cell expression map. Searching for high-variation genes through a Seurat software package, performing nonlinear dimensionality reduction (t-SNE) on the genes in the tested cells based on the gene group, performing clustering through a FindClusters function of the Seurat software package and a FindAllMarkers function of the Seurat software package, and obtaining the expression condition in partial cells (see figure 2). INS is an islet beta cell marker. The results show that ERO1B and TMEM132B are specifically expressed in islet beta cells.

Second, immunofluorescence assay

1、The pancreas obtained in step 1 of example 1 was cut into 0.5X 0.5cm³The left and right small blocks are embedded in Tissue-

OCT^TMCompound (iii) and then frozen.

2. Frozen sections (about 10 μm) were placed on positively charged slides and stored at-80 ℃.

3. The slide is taken out, washed for 3 times by PBS buffer solution, and fixed for 30min at room temperature by 4g/100mL paraformaldehyde solution. Then 0.4% (v/v) Triton X-100 diluted in PBS buffer was permeabilized for 30min, followed by blocking with 10% donkey serum blocking buffer diluted in PBS buffer for 1h at room temperature and staining with primary antibody overnight at 4 ℃. The primary antibody is murine anti-INS or rabbit anti-ERO 1B.

4. After completion of step 3, the sections were taken, washed extensively with PBS buffer and incubated with the corresponding secondary antibody for 1h at room temperature.

5. After completion of step 4, the sections were stained with Hoechst 33342, washed 3 times with PBS buffer, and then washed with PBS buffer

The fade resistant mounting medium was mounted and images were obtained using a confocal laser scanning microscope (Leica TCS SP5 ii).

The results of partial immunofluorescence were shown in FIG. 3. The results show that ERO1B is specifically expressed in islet beta cells.

Third, immunohistochemical detection

1. The pancreas obtained in step one 1 of example 1 was fixed with 4g/100mL paraformaldehyde solution overnight at 4 ℃ and then washed thoroughly with PBS buffer, and then immersed in 30g/100mL aqueous sucrose solution, embedded in paraffin, and stored at room temperature.

2. Paraffin sections (about 5 μm) were placed on slides and stored at room temperature.

3. Soaking the slices according to the following steps, and dewaxing and rehydrating: xylene 2 times for 10 minutes each; 100% ethanol 2 times, each for 5 minutes; 70% ethanol for 5min each time for 1 time; 50% ethanol for 5min each time for 1 time; 30% ethanol for 5min each time for 1 time; PBS 2 times for 5min each

4. The sections were washed with PBS buffer and then placed in 10mM sodium citrate buffer at pH6.0 and heated with microwaves at 98 ℃ for 3 times (2 min each time) for antigen retrieval.

5. After completing step 4, cooling, adding endogenous peroxidase inhibitor (China fir gold bridge), and treating at room temperature for 10min to remove endogenous peroxidase activity.

6. Slides were washed 3 times with PBS buffer, permeabilized with 0.4% (v/v) Triton X-100 diluted in PBS buffer for 25min, then blocked with 10% donkey serum blocking buffer diluted in PBS buffer for 1h at room temperature and stained with primary antibody overnight at 4 ℃. The primary antibody comprises murine anti-INS, rabbit anti-ERO 1B or rabbit anti-TMEM 132B.

7. After completion of step 6, the sections were taken, washed extensively with PBS buffer and incubated with the corresponding secondary antibody for 1h at room temperature.

8. After step 7, the slices are taken, and the slice is firstly cut by a cutter with the weight ratio of 1: 20 developing solution was prepared, developed for 30s (mouse anti-INS) and 5min (rabbit anti-ERO 1B or rabbit anti-TMEM 132B), and immediately placed in PBS.

9. After completion of step 8, the sections were taken, stained with hematoxylin for 2-5min, rinsed 3-5 times with tap water, and then treated with 1% hydrochloric acid diluted with ethanol.

10. After step 9, taking the slices, and adding 70% ethanol for 1 time for 2 minutes each time; 100% ethanol 2 times, each for 2 minutes; xylene 2 times for 2 minutes each.

11. After completion of step 10, the sections were taken, mounted with neutral resin and images were collected using a microscope.

The results of the partial detection are shown in FIG. 3. The results show that ERO1B and TMEM132B are specifically expressed in islet beta cells.

The above results indicate that ERO1B (Endoplasmic Reticulum Oxidoreductase 1 Beta) and TMEM132B (Transmembrane Protein 132B) genes are islet cell molecular markers.

Example 3 obtaining and testing of molecular markers of aging pancreatic islet cells

Biological information analysis to obtain aging islet cell molecular marker

Based on young and old cynomolgus monkey islet cell sequencing library, the aging islet cell molecular marker (| log) is obtained through the FindMarkers function of the Seurat software package₂(fold change)|≥0.5，P<0.05, more than 25% of the total number of cells in the group expressing the gene in the corresponding age group). The results for HSP90B1 are shown in fig. 4. Wherein PC4 is the dimension with the largest differentiation degree of young and old beta cells obtained by a principal component analysis method, and the direction indicated by the abscissa arrow is the young-to-old direction. The results show that HSP90B1 is expressed in the beta cells of the aged pancreatic islets in an increased amount.

Second, immunofluorescence assay

On average, islet sections from 8 young and 8 old cynomolgus monkey individuals were counted, each containing 13-132 (on average 55.6) independent fields of islet beta cells for quantification.

The method of step two in example 2 was followed, replacing the primary antibody with rat anti-HSP 90B1, step (5) with step (5A), and none of the other steps were changed. The step (5A) is as follows: after the completion of step (4), the section was stained with Hoechst 33342, washed 3 times with PBS buffer, and then washed with

Fade resistant fixative media was fixed, images were obtained using the high content system Opera Phenix high-content imaging system (PerkinElmer, Massachusetts, USA), and the fluorescence intensity of HSP90B1 was measured and averaged.

The relative fluorescence intensity of HSP90B1 of β cells of islets of langerhans of other cynomolgus monkeys was calculated with the average fluorescence intensity of HSP90B1 of β cells in islets of young cynomolgus monkeys taken as 1.

The results of immunofluorescence detection and quantification are shown in FIG. 5.

The above results indicate that HSP90B1(Heat Shock Protein 90Beta Family Member 1) is a molecular marker for senescent islet Beta cells.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. Application of a substance for detecting HSP90B1 gene expression in preparing products for distinguishing or assisting in distinguishing aged islet B cells from young islet B cells.

2. Application of a substance for detecting the content of HSP90B1 protein in preparing a product for distinguishing or assisting in distinguishing aged islet B cells from young islet B cells.