CN114045263B

CN114045263B - Method for precisely separating rat KISS1 neurons

Info

Publication number: CN114045263B
Application number: CN202111136595.XA
Authority: CN
Inventors: 沈根宏; 沈益行; 胡延平; 周莎莎; 臧少莲; 董卓; 佘金成
Original assignee: Shanghai Kunmeng Biotechnology Co ltd
Current assignee: Shanghai Kunmeng Biotechnology Co ltd
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2023-09-26
Anticipated expiration: 2041-09-27
Also published as: CN114045263A

Abstract

The application discloses a method for precisely separating rat KISS1 neurons, which belongs to the technical field of biomedicine, and comprises the steps of specifically expressing genes corresponding to proteins on cell membranes of the KISS1 neurons and sorting positive cells with specific gene coding proteins in arciform nucleus areas in hypothalamus by adopting flow cytometry, namely sorting out the selected KISS1 neurons; the application provides a method for finding out a specific gene corresponding to a specific expression protein on a cell membrane of a KISS1 neuron to determine a specific gene Slc18a 3; the extraction method provided by the application does not need to breed transgenic rats in the implementation process, has low experimental cost and is suitable for popularization; the KISS1 neuron cells obtained by the extraction method provided by the application are living cells, nucleic acid and protein in the cells can not be damaged, and the extracted KISS1 neuron cells can be used for subsequent cells and molecular experiments.

Description

Method for precisely separating rat KISS1 neurons

Technical Field

The application relates to the technical field of biomedicine, in particular to a method for precisely separating rat KISS1 neurons;

background

Puberty is one of the most important stages in the human developmental process, activation of the puberty genital axis (hypothalamic-pituitary-gonadal axis, hypothalamic Pituitary gonadal axis, HPG), the dramatic increase in sex hormones, which causes great changes in tissue organ structure, physiology and psychology; pubertal dysplasia may be closely related to the occurrence of later growth, development, reproduction, endocrine, metabolism and psychophysiological diseases, and is one of the hot spots focused on reproductive endocrine research; gonadotropin releasing hormone (Gonadotropin-releasing hormone, gnRH) rhythmic secretion is a marker for initiation of pubertal development; at present, research is considered that the development and function of GnRH neurons are started and regulated by a plurality of regulating factors and intercellular signal transduction systems in the central nervous system; of these, kisspptin protein encoded by KISS1 metastasis suppressor (KISS 1) gene and corresponding receptor KISS1 receptor (also called GPR 54) are considered to be the most critical acting factors for regulating GnRH neuron secretion GnRH; the kit is characterized in that KISS1 neurons distributed at the hypothalamic arciform nucleus position synthesize kisspeptin proteins, and the proteins act on GnRH neurons through paracrine; gnRH neuron surface GPR54 receptor responds to kisspeptin, and further, Gαq/11 coupled K ion channels are activated to continuously depolarize GnRH neurons, so that the regulation and control effect on an HPG axis is exerted; therefore, the KISS1 neuron expressing and secreting the kisspeptin is one of research hotspots for exploring the hypothalamic neuroendocrine network triggering mechanism in adolescent maturity at present and is also one of important acting objects for researching and treating sexual dysplasia diseases.

Common animal models for studying pubertal development are Sprague Dawley strain rats in rodents (hereinafter SD rats); it is generally considered that the 25 th day (PND 25 for short herein), 35 th day (PND 35 for short herein) and 45 th day (PND 45 for short herein) after birth of the rat correspond to the juvenile, adolescent and sexual maturity periods of the human, respectively; the research on the cell and biochemical mechanism of HPG axis activation and silencing is carried out by researching hypothalamic tissues of SD rats at different growth and development time points and combining indexes such as lutein (luteinizing hormone, LH), follicle Stimulating Hormone (FSH) and the like in blood; to date, people generally dye important markers such as kisspeptin on the whole hypothalamus by Immunofluorescence (IF) or fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH), and focus on detection of kisspeptin and other proteins in the arciform nucleus region rich in KISS1 neurons; however, this approach does not isolate KISS1 neurons alone; many studies involve high throughput gene sequencing or chip, often only the approximate location of the arcuately shaped nucleus, and even the entire hypothalamus, can be detected, which undoubtedly brings other types of neurons into the research system, greatly affecting the end result; in order to solve the problem, a green fluorescent protein (Green Fluorescent Protein, GFP) or beta-galactosidase transgenic rat is constructed, and a common method is to insert a GFP or beta-galactosidase between a promoter of a KISS1 gene and the KISS1 gene, so that the KISS1 neuron expresses GFP or beta-galactosidase while expressing the KISS1 gene, and finally forms a fusion protein of kisspeptin, and then GFP or beta-galactosidase positive cells are separated, thereby achieving the purpose of obtaining the KISS1 neuron; however, the problems of transgenic rats mainly include three aspects, namely unstable expression, and research data from different laboratories show that GFP or beta galactosidase can appear in the hypothalamic non-arciform nucleus region to cause false positives, and the expression abundance is very low when the expression should be carried out; 2. the kisspptin-GFP fusion protein is likely to cause abnormal structure of N-terminal or C-terminal protein of kisspptin, resulting in functional inactivation; 3. the cost is high, the transgenic rats need to be continuously bred in a laboratory with animal houses for seed conservation, and at present, the transgenic rats are only owned by a few laboratories worldwide and are not popularized; thus, there is a lack of a method by which the KISS1 neurons can be isolated accurately, and the platforms, instrumentation and techniques required for such a method are acceptable to most laboratories.

Disclosure of Invention

The present application aims to provide a method for precisely isolating rat KISS1 neurons, which solves the problems set forth in the background art;

in order to achieve the above purpose, the present application provides the following technical solutions: a method for precisely isolating rat KISS1 neurons, the method comprising the steps of:

a method for precisely isolating rat KISS1 neurons, the method comprising the steps of:

step one, finding out genes corresponding to the specific expression proteins on the cell membrane of the KISS1 neuron;

s11, embedding the whole brain of the SD rat with an OCT embedding medium and then fully cooling;

s12, slicing the hypothalamus of the SD rat treated by the step S11;

s13, placing the slice in a capture area of a special slide glass for constructing the space transcriptome library, fixing and permeabilizing the slice to release all RNA in cells and combine the RNA with a corresponding capture probe, and synthesizing cDNA by taking the captured RNA as a template to prepare a sequencing library; performing second-generation high-throughput short-reading long-sequencing on the prepared sequencing library;

s14, clustering the points of the captured area in a dimension reduction manner according to the significance differences of all the detectable gene expression in each point set, selecting a point set with significant high expression of the KISS1, further clustering in a dimension reduction manner according to the significance differences of all the detectable gene expression in the point set, and selecting a subset with significant high expression of the KISS 1; screening out a cell membrane expression specific marker of which the Slc18a3 is taken as a significant high expression subset of the KISS1, wherein the Slc18a3 gene is a nucleotide SEQUENCE shown as SEQUENCE LISTING ID NO. 1;

step two, sorting KISS1 neuron cells in the arciform nucleus region in hypothalamus by adopting a flow cytometry according to the Slc18a3 protein, wherein the Slc18a3 protein is an amino acid SEQUENCE shown in SEQUENCE LISTING ID NO.3, and the specific steps are as follows:

s21, taking an arciform nucleus region in hypothalamus, processing the arciform nucleus region into single-cell suspension, and re-suspending cells to about 5x106 cells/mL in ice-cold phosphate buffer saline solution containing 10% fetal calf serum and 1% sodium azide;

s22, adding the resuspended cell fluid obtained in the S21 into the Slc18a3 protein antibody, and incubating in the absence of light;

s23, centrifuging the cell sap obtained in the S22 after light-shielding incubation, re-suspending in an ice phosphate buffer salt solution, and repeating the operation for 3 times;

s24, adding anti-rabbit FITC secondary antibody into the cell fluid obtained in the S23, and incubating in a dark place on ice;

s25, after centrifuging the cell fluid obtained in the S24, re-suspending in an ice phosphate buffer salt solution, and repeating the operation for 3 times;

s26, adding 7-amino actinomycin D into the cell fluid obtained in the S25, and incubating at normal temperature;

s27, adopting a flow type sorter to carry out flow type sorting.

As a preferable technical scheme of the application, in the step S12, the section is tissue with the chimney spacing of 2.52-2.92mm and the double ear spacing of 6.08-6.48 mm.

As a preferred technical scheme of the application, the specific gene screening method in the step S14 specifically comprises the following steps: the genes of which the expressed proteins are on the cell membrane of the KISS1 neurons are selected from genes of which the subset with the remarkably high expression of the KISS1 has remarkably high expression relative to the other three subsets are analyzed and found by means of bioinformatics.

As a preferred technical scheme of the application, the S27 medium-flow sorting method of the second step specifically comprises the steps of firstly removing adhesive cells, then removing 7-amino actinomycin D positive dead cells, finally removing fragments, simultaneously obtaining FSC large and FSC small two groups of cells which are neurons and glial cells respectively, and then sorting the two groups of cells to obtain specific protein positive glial cells, specific protein negative glial cells, specific protein positive neuron cells and specific protein negative neuron cells, wherein the specific protein positive neuron cells are target cells;

as a preferable technical scheme of the application, the cooling temperature in the first step is-80 ℃.

As a preferred embodiment of the present application, in the step S12, SD rat hypothalamus is placed in a frozen microtome at-20℃and the slice thickness is 10. Mu.m.

As a preferable technical scheme of the application, in the step S14, the points of the capturing area are clustered into 14 point sets after dimension reduction according to the significance degree of the whole gene expression, the point set with the significant high expression of the KISS1 is selected, and the point sets are further clustered into 4 subsets after dimension reduction according to the significance degree of the whole gene expression.

Compared with the prior art, the application has the beneficial effects that:

the application provides a method for extracting the living cells of the KISS1 neurons by specifically expressing the genes corresponding to the proteins on the cell membranes of the KISS1 neurons, and realizes the accurate separation of the KISS1 neurons on wild SD rats. Compared with the prior art, the extraction method provided by the application does not need to breed transgenic rats in the implementation process, has low experimental cost and is suitable for popularization; in addition, the KISS1 neuron cells obtained by the KISS1 neuron extraction method provided by the application are living cells, nucleic acid and protein in the cells and the inside of the cells are not damaged, and the extracted KISS1 neuron cells can be used for subsequent cells and molecular experiments.

Drawings

FIG. 1 is a schematic representation of the distribution of the 1-14 point set over the spatial location of brain slices of 25 day SD rats;

FIG. 2 is a schematic representation of the distribution of the 1-14 point set over the spatial location of brain slices of 35 day SD rats;

FIG. 3 is a schematic representation of the distribution of the 1-14 point set over the spatial location of brain slices of 45 day SD rats;

FIG. 4 is a plot of violin at levels expressed by KISS1 neurons in different sets of points;

FIG. 5 is a diagram showing t-SNE grouping among 4 sub-set samples obtained by dimension reduction of a ninth point set in the application;

FIG. 6 is a graph showing the ratio of free single cells removed by the PND25 rat arcuate core flow assay of the present application;

FIG. 7 is a graph showing the ratio of viable cells positive for 7-amino actinomycin D selected in the arcuate nuclear flow sorting of PND25 rats according to the present application;

FIG. 8 is a graph showing FSC/SSC results in a PND25 rat arcuate core flow assay according to the present application;

FIG. 9 is a graph showing further sorting results of FSC large cell populations in PND25 rat arcuate core flow sorting according to the present application;

FIG. 10 is a graph showing the ratio of free single cells removed by PND35 rat arcuate core flow sorting in accordance with the present application;

FIG. 11 is a graph showing the ratio of viable cells positive for 7-amino actinomycin D selected in the arcuate nuclear flow sorting of PND35 rats according to the present application;

FIG. 12 is a graph showing FSC/SSC results in a PND35 rat arcuate core flow assay according to the present application;

FIG. 13 is a graph showing further sorting results of FSC large cell populations in PND35 rat arcuate core flow sorting according to the present application;

FIG. 14 is a graph showing the ratio of free single cells removed by the PND45 rat arcuate core flow assay of the present application;

FIG. 15 is a graph showing the ratio of viable cells positive for 7-amino actinomycin D selected in the arcuate nuclear flow sorting of PND45 rats according to the present application;

FIG. 16 is a graph showing FSC/SSC results in a PND45 rat arcuate core flow assay of the present application;

FIG. 17 is a graph showing the further sorting results of FSC large cell populations in PND45 rat arcuate nuclear flow sorting according to the present application.

Detailed Description

In order that those skilled in the art will better understand the present application, a technical solution in the embodiments of the present application will be clearly and completely described in the following description with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments; all other embodiments, based on the embodiments of the application, which would be apparent to one of ordinary skill in the art without having made inventive work, are intended to be within the scope of the application;

it should be noted that, without conflict, the embodiments of the present application and features in the embodiments may be combined with each other, and the present application will be described in detail with reference to the embodiments.

Example 1

The application provides a method for precisely isolating rat KISS1 neurons, which is concretely as follows.

Finding out the specific expression protein on the cell membrane of the KISS1 neuron:

s1, taking 1 complete hypothalamus of SD rats of 25 days after birth (PND 25 for short), 35 days after birth (PND 35 for short) and 45 days after birth (PND 45 for short), placing the basal part of the whole brain at the bottom of a specimen holder, uniformly smearing with an OCT embedding medium (Thermo Fisher Scientific) until the whole brain is completely covered, and fully cooling at-80 degrees;

s2, placing the cooled hypothalamus in a-20-degree frozen microtome, cutting the hypothalamus vertically to the longitudinal split of the brain, cutting the coronal plane of the position of the anterior chimney with 2.52-2.92mm and the position of the double ears with 6.08-6.48mm, wherein the thickness of the slice is 10 mu m;

s3, placing the slices on a capturing area of a special glass slide for constructing a 10x Genomics space transcriptome library, taking three hypothalamic slices of rats with different ages, respectively placing the hypothalamic slices on different capturing areas of the same glass slide, wherein the size of each capturing area is 6.5x6.5mm, each capturing area comprises 5000 bar code marked points (barcoded Spots), the diameter of each point is 55 mu m, the center distance between adjacent points is 100 mu m, each point contains capturing probes, and all probes in each point have a unique barcode sequence; fixing and permeabilizing the section, releasing RNA in the cells, and binding to the corresponding capture probes; using the captured RNA as a template, synthesizing cDNA, and sequencing library preparation; and carrying out second-generation high-throughput short-reading long-sequencing on the prepared sequencing library. The number of high-quality spots of Space Ranger quantitative quality control of samples at different time points is distributed between 3894 and 4317, the average UMI number in each sPot is distributed between 26546 and 33158, the average base factor in each sPot is distributed between 4881 and 5312, and the average mitochondrial gene proportion in each sPot is distributed between 27.22 and 28.26 percent.

S4, clustering the point clusters of each capturing area in S3 into 14 point sets according to the significance difference of all the detectable gene expression in each point set, wherein the distribution schematic diagrams of different point sets on sample space slice positions are reflected in FIGS. 1-3, and each area in the diagrams is formed by a plurality of discontinuous point circles; the numbers marked in the figures are point set numbers corresponding to the positions, and fig. 1 is a schematic diagram of brain sections of 25-day SD rats; FIG. 2 is a schematic representation of brain sections of 35 day SD rats, and FIG. 3 is a schematic representation of brain sections of 45 day SD rats; as can be seen from fig. 1-3, the positions of the points in each set of points in the slice are substantially continuous. The result of the abundance of the expression of the KISS1 neurons in the 14-point set is shown in fig. 4, wherein the abundance of the expression of the KISS1 neurons in the ninth point set is the highest, and as can be seen from the combination of fig. 1-3, all points in the ninth point set are positioned in the arciform nucleus region in the hypothalamic section, which is the highest consistent with the content of the KISS1 neurons in the arciform nucleus region in the prior research result;

further dimension-reducing clustering the ninth point set into 4 subsets according to the significance differences of all detectable gene expression in each point set, and as a result, the numbers in the curve areas in fig. 5 correspond to the subset labels as shown by the areas marked by the four curve coils in fig. 5; the difference of the expression of the KISS1 genes in the 4 subsets is counted, and a subset with obviously high expression of the KISS1 is selected, wherein the concrete counting method is as follows: counting the differences in expression of KISS1 in each subset relative to the expression in the other three subsets; the specific calculation method is the ratio of the expression quantity of the KISS1 expressed in a certain subset to the average value of the expression quantities in the remaining three subsets; the P value represents the statistically significant value of the gene expression protein difference, represents the degree of dispersion of the value of the expression difference obtained from 3 samples, and is statistically significant when the P value is less than 0.05 according to the statistical knowledge; comparing the difference of the expression levels of the KISS1 genes of the 4 subsets, the expression of the KISS1 in the 1 # subset is obviously higher than that of the expression of the KISS1 in 1 # subsets of the other 3 subsets, and the P value is 3.58x10-24, which indicates that the KISS1 neurons are mainly concentrated in the 1 # subset on the 9 # point set; the KISS1 gene is the nucleotide SEQUENCE shown in SEQUENCE LISTING ID NO. 2;

genes in subset 1 were then screened for significantly higher expression than the other three subsets. The screening method is as follows, the expression difference of all genes in the subset 1 relative to the other three subsets is counted, and the specific calculation method is that the larger the ratio is, the larger the expression difference is, namely the more the genes can be used as specific genes of the subset. Finally, the Slc18a3 gene was selected. The Slc18a3 gene is the nucleotide SEQUENCE shown in SEQ LISTING ID NO. 1; the Slc18A3 protein is a member of subfamily A subfamily 3 of solute carrier 18, designated generally as solute carrier family 18 membrane A3, and is commonly designated as Slc18A3. The Slc18a3 protein is an amino acid SEQUENCE shown in SEQUENCE LISTING ID NO. 3; the Slc18a3 protein is a transmembrane protein responsible for the absorption and transport of amino acids, nucleotides, sugars, inorganic ions and drugs on the cell membrane. The expression difference of the Slc18a3 protein is 6, the P value is 5.10E-18, which indicates that the expression quantity of the Slc18a3 protein in subset 1 is 6 times of the average value of the expression quantity of other 3 subsets, so that the Slc18a3 is a specific gene positioned on the cell membrane of the KISS1 neuron; at P values less than 0.05, the fold difference in expression is statistically significant.

KISS1 neuronal cells highly expressing the Slc18a3 protein in the arciform nuclear region in the hypothalamus of PND25 SD rat were sorted using flow cytometry. The tissue at the hypothalamic arcuate nucleus site of 30 rats was treated with Neural Tissue Dissociation Kits (Meitian, 130-092-628) and gentle MACS TM Octo Dissociator with Heaters to a single cell suspension, and the single cell suspension was resuspended to about 5X10 cells in an ice phosphate buffered saline solution containing 10% fetal bovine serum, 1% sodium azide ⁶ cells/mL; then 30. Mu.L of Slc18a3 antibody (Abclonal, manufactured) was added and incubated for 30min on ice protected from light; centrifuging at 400g for 5min after incubation, then re-suspending in ice phosphate buffer salt solution, and repeating the centrifugation re-suspending operation for 3 times; then 15. Mu.L of anti-rabbit FITC secondary antibody (manufactured by Abcam) was added, and incubated on ice for 30min in the absence of light; centrifuging at 400g for 5min, then re-suspending in ice phosphate buffer salt solution, and repeating the centrifugation re-suspending operation for 3 times; adding 5 μl of 7-amino actinomycin D (manufactured by Biyun) and incubating at room temperature for 15min; flow separation by AriaIII flow sorter (manufactured by Becton, dickinson and Company) is carried out by first removing adherent cells by FSCA/H and gating to separate brain tissue into individual free cells, as shown in the inner region of the frame line in FIG. 6The proportion of isolated cells to the total number of cells was 70.88%; then removing 7-amino actinomycin D positive dead cells to obtain 7-amino actinomycin D positive living cells, and the result is shown in figure 7, wherein the 7-amino actinomycin D positive living cells account for 89.48% of the total number of free cells; then FSC/SSC (FSC is a general abbreviation of forward scale, which represents forward scattered light, the parameter is proportional to the size of cells; SSC is a abbreviation of side scale, which represents side scattered light, the parameter reflects the complexity of cells, such as how many particles are in the cell), debris is removed by gating, the cells are divided into two groups according to the FSC parameter, the group with large FSC parameter is called FSC large cell group, and the group with small FSC parameter is called FSC small cell group, so that two groups of cells, namely FSC large cell and FSC small cell, are obtained, and the result is shown in a box line area in FIG. 8, wherein the FSC large cell is neuron, accounts for 31.17% of the total number of viable cells positive for 7-amino actinomycin D, the FSC small cell is glial cell, and accounts for 31.74% of the total number of viable cells positive for 7-amino actinomycin D; the FSC large cell population was then sorted, as shown in FIG. 9 by the corresponding box line P5, where the total number of the FSC large cells was 27.42% by the number of the SLC8a3 positive neuronal cells, so the total number of the SLC8a3 positive neuronal cells was 5.4% by the total hypothalamic tooling and area.

Example 2

The specific expression proteins found on the membranes of KISS1 neurons in example 2 are the same as in example 1 and will not be described in detail in this example.

KiSS1 neuronal cells with the Slc18a3 protein in the arciform nuclear region in the hypothalamus of PND35 SD rat were sorted using flow cytometry. The tissue at the hypothalamic arcuate nucleus site of 30 rats was treated with Neural Tissue Dissociation Kits (Meitian, 130-092-628) and gentle MACS TM Octo Dissociator with Heaters to a single cell suspension, and the single cell suspension was resuspended to about 5X10 cells in an ice phosphate buffered saline solution containing 10% fetal bovine serum, 1% sodium azide ⁶ cells/mL; then 30. Mu.L of slc18a3 antibody (Abclonal, manufactured) was added and incubated for 30min on ice protected from light; centrifugation at 400g for 5min after incubation was completed, followed by incubation in ice phosphate bufferRe-suspending in the solution, and performing centrifugal re-suspending operation for 3 times; then 15. Mu.L of anti-rabbit FITC secondary antibody (manufactured by Abcam) was added, and incubated on ice for 30min in the absence of light; centrifuging at 400g for 5 minutes, then re-suspending in ice phosphate buffer salt solution, and performing centrifugal re-suspending operation for 3 times; adding 5 μl of 7-amino actinomycin D (manufactured by Biyun) and incubating at room temperature for 15min; the method comprises the steps of carrying out flow separation by an AriaIII flow separator (manufactured by Becton, dickinson and Company), wherein FSCA/H is firstly adopted, a gate is arranged to remove adhesion cells, and brain tissue is divided into individual free cells, and the result shows that the proportion of the free cells to the total number of cells is 63.25% in the inner area of a frame line in FIG. 10; then, dead cells positive for 7-amino actinomycin D were removed to obtain live cells positive for 7-amino actinomycin D, and the result is shown in FIG. 11, wherein the live cells positive for 7-amino actinomycin D account for 84.79% of the total number of free cells; then FSC/SSC (FSC is a general abbreviation of forward scale, which represents forward scattered light, the parameter is proportional to the size of cells; SSC is a abbreviation of side scale, which represents side scattered light, the parameter reflects the complexity of cells, such as how many particles are in the cell), debris is removed by gating, the cells are divided into two groups according to the FSC parameter, the group with large FSC parameter is called FSC large cell group, and the group with small FSC parameter is called FSC small cell group, so that two groups of cells, namely FSC large cell and FSC small cell, are obtained, and the result is shown in a box line area in FIG. 12, wherein the FSC large cell is neuron, accounts for 16.26% of the total number of viable cells positive for 7-amino actinomycin D, the FSC small cell is glial cell, and accounts for 47.85% of the total number of viable cells positive for 7-amino actinomycin D; the FSC large cell population was then sorted, as shown in the corresponding box line at P5 in FIG. 13, wherein the slc8a3 positive neuronal cells account for 35.35% of the total FSC large cells, so the proportion of slc8a3 positive neuronal cells to total hypothalamic tooling and area was 3.08%.

Example 3

The specific expression proteins found on the membranes of KISS1 neurons in example 3 are the same as in example 1 and will not be described in detail in this example.

PND45 SD sorting by flow cytometryKISS1 neuronal cells with the Slc18a3 protein in the arciform nucleus region in the hypothalamus of rats. Hypothalamic tissue on a slide was treated with Neural Tissue Dissociation Kits (Meitian, 130-092-628) and gentle MACS TM Octo Dissociator with Heaters tissues at the hypothalamic arciform nucleus site of the rat to a single cell suspension, and then the single cell suspension was resuspended to about 5X10 cells in an ice phosphate buffered saline solution containing 10% fetal bovine serum, 1% sodium azide ⁶ cells/mL; then 30. Mu.L of slc18a3 antibody (Abclonal, manufactured) was added and incubated for 30min on ice protected from light; centrifuging at 400g for 5min after incubation, then re-suspending in ice phosphate buffer salt solution, and performing centrifugal re-suspending operation for 3 times; then 15. Mu.L of anti-rabbit FITC secondary antibody (manufactured by Abcam) was added, and incubated on ice for 30min in the absence of light; centrifuging at 400g for 5 minutes, then re-suspending in ice phosphate buffer salt solution, and performing centrifugal re-suspending operation for 3 times; adding 5 μl of 7-amino actinomycin D (manufactured by Biyun) and incubating at room temperature for 15min; the method comprises the steps of carrying out flow separation by an AriaIII flow separator (manufactured by Becton, dickinson and ComPany), wherein FSCA/H is firstly adopted, a gate is arranged to remove adhesion cells, brain tissue is divided into individual free cells, and the result shows that the proportion of the free cells to the total number of cells is 62.15% as shown in the inner area of a frame line in FIG. 14; then removing 7-amino actinomycin D positive dead cells to obtain 7-amino actinomycin D positive living cells, and the result is shown in figure 15, wherein the 7-amino actinomycin D positive living cells account for 93.13% of the total number of free cells; then FSC/SSC (FSC is a general abbreviation of forward scale, which means forward scattered light, this parameter is proportional to the size of the cells; SSC is a abbreviation of side scale, which means side scattered light, this parameter reflects the complexity of the cells, such as how many particles are inside the cells), gating off debris, dividing the cells into two large and small groups according to FSC parameters, the large FSC parameter group called FSC large cell group, and the small FSC parameter group called FSC small cell group, obtaining FSC large and FSC small cell groups, the result is shown in the box line region of FIG. 16, wherein FSC large cells are neurons, accounting for 18.11% of the total number of 7-amino actinomycin D positive living cells, FSC small cells are glial cells, accounting for44.62% of the total number of 7-amino actinomycin D positive living cells; the FSC large cell population was then sorted, as shown in the corresponding box line of P5 in fig. 17, where the slc8a3 positive neuronal cells account for 47.94% of the total FSC large cells, so the proportion of slc8a3 positive neuronal cells to total hypothalamic tooling and area total cells was 5.02%.

The foregoing description is only a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art, who is within the scope of the present application, should make equivalent substitutions or modifications according to the technical solution of the present application and the inventive concept thereof, and should be covered by the scope of the present application.

Sequence listing

<110> Shanghai Kunlun Biotech Co., ltd

<120> a method for precisely isolating rat KiSS1 neurons

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ctgcttatag gcctgggcgt catgttcgcc tccacagtca tgtttgcctt tgcagaagac 1380

tatgccacgc tcttcgctgc gcgcagtcta caaggcctgg gctcggcctt cgcggacacg 1440

tctggcattg ccatgatcgc cgacaagtat cccgaggagc ctgagcgcag tcgtgccctg 1500

ggcgtggcgc tagcctttat tagctttgga agcctagtgg cgccaccgtt tgggggcatc 1560

ctctacgagt tcgcgggcaa gcgtgtaccc tttctagtgc tcgccgctgt gtcccttttc 1620

gacgcgctcc tgctcctggc ggtggctaag cccttctcgg ctgcggctcg ggcgcgagcc 1680

aacctgccgg tgggcacacc tatccatcgc ctcatgctag acccttacat cgctgtggta 1740

gccggcgcgc tcaccacttg taacattccc cttgcgttcc tcgagcccac catagccacg 1800

tggatgaagc acacaatggc cgcatccgag tgggagatgg gcatggtttg gctgccggct 1860

ttcgtgccac acgtgttagg cgtctacctc accgtgcgcc tggcggcgcg ttatccacac 1920

ctgcactggc tgtacggcgc tctcgggcta gcggtaattg gagtgagctc ttgcgtcgta 1980

cctgcctgtc gctcattcgc gccgttagtg gtctcgctct gcggactctg cttcggcatc 2040

gcgttagtgg acacagcgct cctacccacg ctcgcctttc tggtggacgt gcgccacgta 2100

tccgtctatg gcagtgtcta tgccatagct gacatctcct attctgtggc ctacgcgctc 2160

gggcccatag tggcaggcca catcgttcac tctcttggct ttgagcagct cagcctgggc 2220

atgggcctgg ccaacctgct ctacgcacca gtccttcttc ttttgcgcaa tgtaggcctc 2280

cttacacgct cgcgttcgga gcgcgatgtg ttgcttgatg aaccgccgca gggtctgtac 2340

gacgcggtgc gcctgcgtga ggtgcagggc aaggatggcg gcgaaccttg tagcccacct 2400

ggcccttttg acgggtgcga ggacgactac aactattact cccgcagcta gcagacccgc 2460

ttctcctcca ggccacctac ccgccccatt taggtcaaga tggtcattct gcaagagcac 2520

tgtccaactt tggcctgggg cccacctcct ctaatgaata ccctagcccc tcgcccgtcc 2580

tgaattcctt tgctggaatc ccttctccat gacccctccc agtctaggcc cctccccaaa 2640

cacactcgta ttcattgggg aaatggagca gggaggcaga agaagctgtt gggctcttgg 2700

cagaggtgaa gaggtgtgcg ggtgatcgcc aatcacctac tgagagcccc caaatagagt 2760

catgcatctg tttgtccttc ctgcggatct ttccagtgcc aaacttggtc tctgcactcc 2820

ggtgcctccg gcctgaatta ataaaccata tctatctgag ga 2862

<210> 2

<211> 5885

<212> DNA

<213> rat genus (Rattus norvegicus)

<400> 2

agatactttc tctgcattgt ttggggtaca atgagtctcc agctgcgagg gaggggtgtc 60

aagaggctag agatgatggg agagccagtg agcttatgag aacagccttg gatggtttct 120

gggggcgagc aagtcgctgg ctccagactt gagagccagg aggcgggggc cagagggagg 180

gagtggagcc ctgaggaggg tccgggaggg cagaggtggg attatgctgg gatacgtcac 240

ccatccagac ttcataaaag gggctgtgat cgggcagcca gatagaggaa gcccaggagc 300

cagaggctcc cctcagtgtg ctccaactac ccaagtggct cttctccctc cggccctcaa 360

gccaggaccc agccaaggta ggcacactgc ccacaggatt tgggactgcc ctgacccagg 420

gaggatctag aggagcaggc ctctaaccca aggccagttg aagcatcaag ggagaaggag 480

gcaaggaaag cagggatcca ggaatgcagt gatgaagacg ttgtcttgtg tcttgtgtgt 540

cgctgggtgt tctcagagcc cacttctgcc gcgtaaggag tggatgttgt aaagagactc 600

tttgtgggaa ctacctgaag ggagcttggc aagccctttg ggttcatggg ctcaccacgg 660

cttgtctgag cctttccccc caatataaaa ctaactatat acatatatat gctttgagca 720

acttgctaag aactgggggg ggggggggat accgctggaa ggagccatct tatgtcaaaa 780

accttctgat cacgcctaga atttctgttg tttactagat ctaaaagccc ctagagtgaa 840

aggaaccacc atgattgttc atggcagacc agataaacct cttccggttt cttacatact 900

gaaaggggga gagggggcgg gcatgttcct ttttgttttc taacatttca gttctcatgc 960

actgagcgct ctctagaata ggcaactgag agttattggg gtctggggta catgtggcat 1020

gtgtgttact tgtgggatgt gtgtggtatg tatgtgaggt gtttgtgctt cgtatgttat 1080

gacatttgtt tatcatttat ttttttctgc gcatgtgtgt aagcgcagag cacccagggg 1140

agtcagagga caactttctg gaggtggttt tctccctccg ccatgtgggt ccaaggggtt 1200

aaactgagat cgtcaagcgc ggtagctagc aagcacattt atctgctgag ccatcatctc 1260

accatgtcgg gtcattgtat gtagtgtgtt catatgtggt acgtgtgtat acgtggggta 1320

agggtgtgtc gtgagcaggc acagttctcc cttggtacac ttgaaaaatg ctgtccatga 1380

tagtgcagag tactagaggg atgagaggca gaagtctgga tcaccaacct ccccacctcc 1440

cctccctctc ctcccctccc cttcctccct ctccacctcc cctccccttc ctccctctcc 1500

acctcccctc ccctacctcc ctcaccacct tctctgttta gagaaaacac gaggtaccac 1560

ttacggagca cgctgacaca ttcctgccct cctcaggccc cgttggggta taacctggca 1620

tggcttcctc tccctataaa gcgctaactt tgtatccagc acctctatac ccagccctgc 1680

ctgccatcct ggaccccctg gcgtcatcgg tcctgcagat cccctgaggt ggcttctgag 1740

agccctcttc atctggtgca tgctggggtg ccagcaccag cccggaccct cccgtcagac 1800

tccccagctg ccatcagctg ttagcatgtg tctggtgtca ttttggaatg atctatttat 1860

acaagctgcc tgtgagtcaa ctagacatga gatcagaggg tccctgcacc ctcagcacct 1920

tgagatcagg gggtccctgc accctcagca ccccagcacc tacacagtac tacttgtatt 1980

gtcagtactt tcagcagtga cttcctaacc ctggtccttg tgatccagac cgtctactga 2040

ttttggtttc cactgtattt ctattcaagc ctcagggcat ctacagaaag tgcctggctt 2100

ttggaatgtc acagagtcat agtctgctag tcttggcagc catctcattg agcagctggg 2160

ggccaggcta accaatggtg cctggagatg ggtgtggtcc gcttatgtct cgtgggatga 2220

ggccagagct ggagcactga aactcaggat tcatgtgtga gtctggagtc aggtggcagg 2280

ggtaggactg gggacaaagt ggcaagatgc acggtgccga gagatcacgc catcatggct 2340

cacaaggata tctgtcctca ctcctgtcct tgaacagaag ccctgggttc cgcctgttgt 2400

gcctccagcc tcccagggca cttaatgcca tttgttggac agtttcaaag tgcttggtga 2460

cattttcaaa gggaaattgg acttgggagc tggagacgtg gggtggagtg gggagcagga 2520

gaggagaaag gtggagtggc cagatgaggg tcaggccaca cagctcctac ctcagcagtc 2580

aaactgatga ggccaattta gttcacaacc tcccagagcc cacgagaaca gaagtgactg 2640

acgggccaga ggagagaaag gctttccctg ccctgaatta taggcaataa gacagtttga 2700

tgatggtctc ccaggctgcg gagggctttg agaatgtgca agatgattgc ctctttcttc 2760

ctttttccct ggaagagaga agaaactggg tttctattat atcatgggga cagagctaga 2820

ttgaggaaga ttgggtgctg cgtaggagag cagagcacag gacccagcac ggggcttggt 2880

tccaggctcc caagagaata gttgaacctc agagcacact cctgcctgac cttaccaacc 2940

tccccctccc tgactactcc tgtgcatgac atttattctt cttcagcatc cctgccctgc 3000

aaaccttcca cccagagccc taccacggtc caaccaaggc ttctcagccc ttcccaccaa 3060

atcagaccct tcccattttc cctgccttct tccgtccctc agtcctgttt ccggtggcct 3120

tgtttggggc ttatccttct gatcatctct ggtcaccggg ctttctcctc tcccccaggc 3180

gctctctttg acctaggtag gctctggtga atactaactc tggcctggtc ttcattgcct 3240

ctcttcgtct cagcctctgg acaccctgtg gatctgcctc ttccagaatg atctcgctgg 3300

cttcttggca gctgctgctt ctcctctgtg tggcctcttt tggggagcca ctggcaaaaa 3360

tggcacctgt ggtgaaccct gaacccacag gtacacacca tcccggaaaa gggggcaaga 3420

gagtcagtgc acgtgagact cgctttattt gggctggaga gatggctcag tggttaagag 3480

cactggctgc tcttccagag gtcctgagtt caattcccag caaccacatg gtggctcaca 3540

accacctgta atgagattca atgccctttt ctggtgtgtc tgaagacaga gactcgctgt 3600

atttttcaga ctgtagccaa ggtaccgaca cgagttcagt ttctgcactt gagaaatact 3660

cagggctggc ccaggccctt ctgctgtgag caccgggagg ctcagttaga gtcaggggcc 3720

tgtcaaccct aactcctttg tgattttttt ctcctgccgt gctctgctct accccaagca 3780

cttgtcttta tggatgctgg aaaggagctt catgtgtaag ttacttttat ctgtgacctc 3840

agatcccgaa aggggacttg aaaacaaaac tcaaaaaatg tcaacacagc cgggcatggt 3900

ggcacaggcc tttaactcca gcactcagaa ggtagaagca ggtagatctc ttgagttcga 3960

cgtcagtcta gtccaggaca tccagggctc cgttacacag agaaattgtc tcaaacaaac 4020

aaacaaacaa acaaacaaac ccaatacagc aacaacctca tcatccacac agaccttaat 4080

gcccgtcaag ctacctcacg gattcactgg gggctttgat gaactggaga aactaaagcc 4140

caaagaggca aagtgctccc cagaggtgct cagtactgac aaatcccaac aggtgccagg 4200

gtacttccaa gagcatcgtg gcttctaagt cagggtgtgg gtttgaactt tccttagctg 4260

ttctgtttct gtacatatta gagcttgctt tagcctttct gcacctcaga acttaggcat 4320

agaatgcagg atacatcctg caaatttgac accatcatac ataagcaaag caacgggaac 4380

aggggcccaa acactctcca atagggtcac catccccaga taagaataac cccatcactg 4440

tgcagaaaca ctggctactg gccctttccg gctgcacagg ttatcctgat ctgaggttcg 4500

tgtgtagaac taagatcttt tctcccacac cagctatcca gagaaggtag gaggggcctc 4560

ccagactatt attattaatc ttggaccaca gccacactca tctccagagt gtgccagggt 4620

atggaggagc caacacccac tcacaccacc gttccttttt actaatctct gtccataatt 4680

gactagacgc attgtgtact cagagctact tgccctgttt tactggaaaa agagaaagag 4740

agagagagac agaatttaat gagccttgtc tatgaagtgt tttcagagtg agccttctta 4800

gcctccaaca agaagggaag gccttgcacc tcgcctggga gagctgtacc tgctaaggca 4860

atctagatag tgtctggcgt cacattgcgt cttgatactg tgtattaggg attgcagttg 4920

tcctcagaga ccagagtcag gtcacaggtg catgggggca caacagcctt ggctccactt 4980

tttttttatt tctgttactc caagtttttc attagctatt gcttaatgag cacgtattga 5040

ggccatacta tgtgcagggg tactgtttca agcattctta gagatggaga aatgaatccc 5100

ctccgtcccc ccctcccccg gaggctgctg gaccgttcca actcccaaca cagacactca 5160

actgaaggat caggcagaga gcccagaaga gggctagagc gagttccccc tctataccct 5220

cctttttttc tcttgcatgt tgtttgcgtc tcagaggagg tagttttata aacgtattta 5280

gtagaattct ggcgagcact tgaaagtaaa gctatggaag gagggaggag caaacagcca 5340

agccacccgg actaagcagg atcctggctc cacacagagg tcagagcagt atttctgagg 5400

ttgaggaccc agggccagca tcggaagggg agggaagtct gagctggtgt ccaaagcaag 5460

ccttgctgtc ccagcctcac tcctctgtac tctgtcctag gccaacagtc cggaccccag 5520

gaactcgtta atgcctggca aaagggcccg cggtatgcag agagcaagcc tggggctgca 5580

ggactgcgcg ctcgccgaac atcgccatgc ccgccggtgg agaaccccac ggggcaccag 5640

cggcccccgt gtgccacccg cagtcgcctg atccctgcgc cccgcggatc ggtgctggtg 5700

cagcgcgaga aggacatgtc agcctacaac tggaactcct ttggcctgcg ctacggcagg 5760

aggcaggtgg cgcgggcggc acggggctga gtgcagggtt gcaggtggat tgcagcccac 5820

cccatggcag aggccaaggc agggagcttc tagacttgtg caataaaagc aatgctgtcg 5880

cctta 5885

<210> 3

<211> 530

<212> PRT

<213> rat genus (Rattus norvegicus)

<400> 3

Met Glu Pro Thr Ala Pro Thr Gly Gln Ala Arg Ala Ala Ala Thr Lys

1 5 10 15

Leu Ser Glu Ala Val Gly Ala Ala Leu Gln Glu Pro Gln Arg Gln Arg

20 25 30

Arg Leu Val Leu Val Ile Val Cys Val Ala Leu Leu Leu Asp Asn Met

35 40 45

Leu Tyr Met Val Ile Val Pro Ile Val Pro Asp Tyr Ile Ala His Met

50 55 60

Arg Gly Gly Ser Glu Gly Pro Thr Leu Val Ser Glu Val Trp Glu Pro

65 70 75 80

Thr Leu Pro Pro Pro Thr Leu Ala Asn Ala Ser Ala Tyr Leu Ala Asn

85 90 95

Thr Ser Ala Ser Pro Thr Ala Ala Gly Ser Ala Arg Ser Ile Leu Arg

100 105 110

Pro Arg Tyr Pro Thr Glu Ser Glu Asp Val Lys Ile Gly Val Leu Phe

115 120 125

Ala Ser Lys Ala Ile Leu Gln Leu Leu Val Asn Pro Leu Ser Gly Pro

130 135 140

Phe Ile Asp Arg Met Ser Tyr Asp Val Pro Leu Leu Ile Gly Leu Gly

145 150 155 160

Val Met Phe Ala Ser Thr Val Met Phe Ala Phe Ala Glu Asp Tyr Ala

165 170 175

Thr Leu Phe Ala Ala Arg Ser Leu Gln Gly Leu Gly Ser Ala Phe Ala

180 185 190

Asp Thr Ser Gly Ile Ala Met Ile Ala Asp Lys Tyr Pro Glu Glu Pro

195 200 205

Glu Arg Ser Arg Ala Leu Gly Val Ala Leu Ala Phe Ile Ser Phe Gly

210 215 220

Ser Leu Val Ala Pro Pro Phe Gly Gly Ile Leu Tyr Glu Phe Ala Gly

225 230 235 240

Lys Arg Val Pro Phe Leu Val Leu Ala Ala Val Ser Leu Phe Asp Ala

245 250 255

Leu Leu Leu Leu Ala Val Ala Lys Pro Phe Ser Ala Ala Ala Arg Ala

260 265 270

Arg Ala Asn Leu Pro Val Gly Thr Pro Ile His Arg Leu Met Leu Asp

275 280 285

Pro Tyr Ile Ala Val Val Ala Gly Ala Leu Thr Thr Cys Asn Ile Pro

290 295 300

Leu Ala Phe Leu Glu Pro Thr Ile Ala Thr Trp Met Lys His Thr Met

305 310 315 320

Ala Ala Ser Glu Trp Glu Met Gly Met Val Trp Leu Pro Ala Phe Val

325 330 335

Pro His Val Leu Gly Val Tyr Leu Thr Val Arg Leu Ala Ala Arg Tyr

340 345 350

Pro His Leu Gln Trp Leu Tyr Gly Ala Leu Gly Leu Ala Val Ile Gly

355 360 365

Val Ser Ser Cys Val Val Pro Ala Cys Arg Ser Phe Ala Pro Leu Val

370 375 380

Val Ser Leu Cys Gly Leu Cys Phe Gly Ile Ala Leu Val Asp Thr Ala

385 390 395 400

Leu Leu Pro Thr Leu Ala Phe Leu Val Asp Val Arg His Val Ser Val

405 410 415

Tyr Gly Ser Val Tyr Ala Ile Ala Asp Ile Ser Tyr Ser Val Ala Tyr

420 425 430

Ala Leu Gly Pro Ile Val Ala Gly His Ile Val His Ser Leu Gly Phe

435 440 445

Glu Gln Leu Ser Leu Gly Met Gly Leu Ala Asn Leu Leu Tyr Ala Pro

450 455 460

Val Leu Leu Leu Leu Arg Asn Val Gly Leu Leu Thr Arg Ser Arg Ser

465 470 475 480

Glu Arg Asp Val Leu Leu Asp Glu Pro Pro Gln Gly Leu Tyr Asp Ala

485 490 495

Val Arg Leu Arg Glu Val Gln Gly Lys Asp Gly Gly Glu Pro Cys Ser

500 505 510

Pro Pro Gly Pro Phe Asp Gly Cys Glu Asp Asp Tyr Asn Tyr Tyr Ser

515 520 525

Arg Ser

530

<210> 4

<211> 130

<212> PRT

<213> rat genus (Rattus norvegicus)

<400> 4

Met Ile Ser Leu Ala Ser Trp Gln Leu Leu Leu Leu Leu Cys Val Ala

1 5 10 15

Ser Phe Gly Glu Pro Leu Ala Lys Met Ala Pro Val Val Asn Pro Glu

20 25 30

Pro Thr Gly Gln Gln Ser Gly Pro Gln Glu Leu Val Asn Ala Trp Gln

35 40 45

Lys Gly Pro Arg Tyr Ala Glu Ser Lys Pro Gly Ala Ala Gly Leu Arg

50 55 60

Ala Arg Arg Thr Ser Pro Cys Pro Pro Val Glu Asn Pro Thr Gly His

65 70 75 80

Gln Arg Pro Pro Cys Ala Thr Arg Ser Arg Leu Ile Pro Ala Pro Arg

85 90 95

Gly Ser Val Leu Val Gln Arg Glu Lys Asp Met Ser Ala Tyr Asn Trp

100 105 110

Asn Ser Phe Gly Leu Arg Tyr Gly Arg Arg Gln Val Ala Arg Ala Ala

115 120 125

Arg Gly

130

Claims

1. A method for precisely isolating rat KISS1 neurons, the method comprising the steps of: step one, determining the specificity expression protein on the cell membrane of the KISS1 neuron; s11, embedding the whole brain of the SD rat with an OCT embedding medium and then fully cooling; s12, slicing the hypothalamus of the SD rat treated by the step S11; s13, placing the slice in a capture area of a special slide glass for constructing the space transcriptome library, fixing and permeabilizing the slice to release all RNA in cells and combine the RNA with a corresponding capture probe, and synthesizing cDNA by taking the captured RNA as a template to prepare a sequencing library; performing second-generation high-throughput short-reading long-sequencing on the prepared sequencing library; s14, clustering the points of the captured area in a dimension reduction manner according to the significance differences of all the detectable gene expression in each point set, selecting a point set with significant high expression of the KISS1, further clustering in a dimension reduction manner according to the significance differences of all the detectable gene expression in the point set, and selecting a subset with significant high expression of the KISS 1; screening out a cell membrane expression specific marker of which the Slc18a3 is taken as a significant high expression subset of the KISS1, wherein the Slc18a3 gene is a nucleotide SEQUENCE shown as SEQUENCE LISTING ID NO. 1; step two, sorting KISS1 neuron cells in the arciform nucleus region in hypothalamus by adopting a flow cytometry according to the Slc18a3 protein, wherein the Slc18a3 protein is an amino acid SEQUENCE shown in SEQUENCE LISTING ID NO.3, and the specific steps are as follows: s21, taking an arciform nucleus region in hypothalamus, processing the arciform nucleus region into single-cell suspension, and re-suspending cells to 5x106 cells/mL in ice-cold phosphate buffer saline solution containing 10% fetal calf serum and 1% sodium azide; s22, adding the resuspended cell fluid obtained in the S21 into the Slc18a3 protein antibody, and incubating in the absence of light; s23, centrifuging the cell sap obtained in the S22 after light-shielding incubation, re-suspending in an ice phosphate buffer salt solution, and repeating the operation for 3 times; s24, adding anti-rabbit FITC secondary antibody into the cell fluid obtained in the S23, and incubating in a dark place on ice; s25, after centrifuging the cell fluid obtained in the S24, re-suspending in an ice phosphate buffer salt solution, and repeating the operation for 3 times; s26, adding 7-amino actinomycin D into the cell fluid obtained in the S25, and incubating at normal temperature; s27, adopting a flow type sorter to carry out flow type sorting.

2. The method for precisely isolating rat KISS1 neurons according to claim 1, wherein the step S12 is performed with tissue having a pre-chimney spacing of 2.52-2.92mm and a binaural spacing of 6.08-6.48 mm.

3. The method for precisely isolating rat KISS1 neurons according to claim 1, wherein the specific gene screening method in step S14 is as follows: the genes of which the expressed proteins are on the cell membrane of the KISS1 neurons are selected from genes of which the subset with the remarkably high expression of the KISS1 has remarkably high expression relative to the other three subsets are analyzed and found by means of bioinformatics.

4. The method for precisely isolating rat KISS1 neurons according to claim 1, wherein the step S27 of the second method is specifically to remove adherent cells first, then remove dead cells positive for 7-amino actinomycin D, finally remove fragments, and obtain two groups of cells, namely neurons and glial cells, simultaneously, FSC large and FSC small, and then sort the two groups of cells to obtain specific protein positive glial cells, specific protein negative glial cells, specific protein positive neuronal cells and specific protein negative neuronal cells, wherein the specific protein positive neuronal cells are target cells.

5. A method for precisely isolating rat KISS1 neurons according to claim 1, characterized in that the cooling temperature in step one is-80 ℃.

6. A method for precisely isolating rat KISS1 neurons according to claim 1, wherein the SD rat hypothalamus is placed in a frozen microtome at-20 ℃ in step S12, with a slice thickness of 10 μm.

7. The method for precisely isolating rat KISS1 neurons according to claim 1, wherein in step S14, the points of the capturing region are clustered into 14 point sets according to the significance level of the whole gene expression, the point set with significantly high expression of KISS1 is selected, and the point set is further clustered into 4 subsets according to the significance level of the whole gene expression.