Protein marker for detecting or evaluating paracrine of human neural stem cells and application of protein marker

Abstract

The invention discloses a protein marker for detecting or evaluating human neural stem cell paracrine and application thereof, the protein marker is a combination of SERPINE2, NCAN, VCAN, PTN, NRP2, PTPRF, C1QL1 and LDLR.The screening method comprises the steps that total protein in a human neural stem cell sample is extracted for proteome analysis and reference genome transcriptome analysis; and carrying out data processing and comparison screening to obtain common protein with high protein abundance expression and a forward regulation function, and carrying out ELISA (enzyme-linked immunosorbent assay). The human neural stem cell paracrine is detected or evaluated by detecting the expression level of the protein marker, and the application value is high.

Description

Technical Field

[0001] The present invention belongs to the field of cell biotechnology, and specifically relates to a protein marker for detecting or evaluating paracrine secretion of human neural stem cells and its application. It should be noted that the present invention is a divisional case filed on the basis of the patent application with application number 202411597299.3. Background Art

[0002] Human neural stem cells are a type of cell with self-renewal and differentiation potential. Their application mechanism in diseases is mainly through cell replacement, repair, micro-regulation of paracrine in the microenvironment, and immune regulation. During the in vitro cell culture process, growth factors such as LIF, EGF, and FGF are usually added to adjust the balance to promote cell growth and development. So what specific substances do the cells secrete during this culture process? What role do they play? These questions have been puzzling researchers. At present, most people use VEGF, which has the function of vascular repair, as an indicator, but it is relatively simple.

[0003] In the existing technology, most researchers understand the paracrine effect and know that it has broad application prospects. However, most of the current screening technologies are based on mass spectrometry and reverse transcription analysis, which have complex mechanisms, take too long, and are time-consuming and labor-intensive. There are suitable methods for detection (enzyme-linked immunosorbent assay to detect secreted proteins), but there is no direction for selecting protein markers for detection, and there is no targeted combination of marker proteins as application standards to adapt to form a unified industry understanding. Summary of the invention

[0004] In order to overcome the above-mentioned deficiencies in the prior art, the main purpose of the present invention is to provide a protein marker for detecting or evaluating the paracrine secretion of human neural stem cells. Through proteomic analysis and transcriptome analysis with a reference genome, the analysis data obtained are used for comparative screening of secretory protein types, expression levels, and action pathway mechanisms, and proteins with high common protein abundance expression are screened out, and protein markers are screened by ELISA enzyme-linked immunosorbent assay.

[0005] Another object of the present invention is to provide an application of the protein marker in the preparation of a product for detecting or evaluating the paracrine secretion of human neural stem cells, for the detection or evaluation of the paracrine secretion of human neural stem cells, so as to understand the paracrine situation during the culture of human neural stem cells.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides a protein marker for detecting or evaluating paracrine secretion of human neural stem cells, which is one or more of SERPINE2, NCAN, VCAN, PTN, NRP2, PTPRF, C1QL1 and LDLR.

[0008] The present invention provides a protein marker for detecting or evaluating paracrine secretion of human neural stem cells, which is a combination of SERPINE2, NCAN, VCAN, PTN, NRP2, PTPRF, C1QL1 and LDLR.

[0009] Preferably, the human neural stem cells include, but are not limited to, primary neural stem cells and neural progenitor cells obtained from tissue sources, and neural stem cells or neural progenitor cells and cell lines thereof obtained by inducing embryonic stem cells or iPSCs.

[0010] The present invention also provides a method for screening the protein marker, comprising the following steps:

[0011] Step 1: Extract total protein from human neural stem cell culture supernatant samples and perform proteomic analysis;

[0012] Step 2: Take human neural stem cell samples and perform transcriptome analysis with a reference genome, including:

[0013] Step 3: Perform data processing and comparative screening on the analysis data obtained in steps 1 and 2, and screen out the proteins with the highest protein abundance expression as screening proteins;

[0014] Step 4: Perform ELISA enzyme-linked immunosorbent assay on the protein screened in step 3 to screen out protein markers.

[0015] Preferably, in step 1, the proteomic analysis includes: extracting total protein from a sample of the supernatant of a human neural stem cell culture, taking out a portion for protein concentration determination and SDS-PAGE detection, taking another portion for trypsin hydrolysis, and performing LC-MS / MS high-resolution mass spectrometry identification on the sample after desalting the hydrolyzed peptides; the LC-MS / MS high-resolution mass spectrometry identification uses DIA technology to collect mass spectral data of each sample, and performs spectrum matching, quantitative information extraction and subsequent statistical analysis.

[0016] Preferably, in step 2, the reference genome database is selected from one or more of NCBI_GI, egg NOG, KEGG, GO, EC, Ensembl and UniProt ID.

[0017] Preferably, in step 2, the reference genome transcriptome analysis includes: total RNA extraction, total RNA quality detection, mRNA purification, mRNA fragmentation, CDNA synthesis, PCR-enriched library fragments, library quality inspection and second-generation sequencing (NGS) sequencing of the human neural stem cell sample.

[0018] More preferably, in step 2, the mRNA fragmentation process comprises: enriching the mRNA with polyA structure in the total RNA by Oligo (dT) magnetic beads, and breaking the RNA into fragments of 200-400 bp in length by ion shearing;

[0019] The cDNA synthesis process includes: using RNA as a template, using 6-base random primers and reverse transcriptase to synthesize the first-chain cDNA, and using the first-chain cDNA as a template to synthesize the second-chain cDNA;

[0020] The process of PCR-enriched library fragments includes: using PCR amplification to enrich library fragments, and then selecting the library according to the fragment size, and the library size is 450 bp;

[0021] The library quality inspection process includes: performing quality inspection on the library by Agilent 2100 Bioanalyzer, and then detecting the total concentration of the library and the effective concentration of the library;

[0022] The NGS sequencing process includes: performing double-end (PE) sequencing on the library based on the Illumina HiSeq sequencing platform.

[0023] Preferably, in step 3, the data processing and comparative screening include secretory protein types, expression levels and action pathway mechanisms, and the process includes: data filtering, data quality assessment, alignment to genome, alignment result assessment, expression level calculation, expression difference analysis, and enrichment analysis of the analysis data obtained in steps 1 and 2 in sequence;

[0024] Wherein, the alignment to the genome includes cSNP analysis and transcript structure analysis, and the transcript structure analysis includes alternative splicing statistics, UTR analysis, and new transcription site prediction;

[0025] The expression difference analysis adopts cluster analysis, including two-way cluster analysis, PCA analysis and sample correlation analysis;

[0026] The enrichment analysis adopted GO enrichment analysis and KEGG Pathway analysis.

[0027] Preferably, in step 4, the screening protein in step 3 is subjected to ELISA enzyme-linked immunosorbent assay by double antibody sandwich ELISA method, and the process comprises: adding the test sample or standard to the microplate coated with anti-screening protein antibody and incubating, wherein the screening protein binds to the antibody fixed on the plate, and washing the plate to remove the unreacted test sample or standard; adding biotin-detection antibody for a second incubation, wherein the biotin-detection antibody binds to the screening protein already bound to the coated antibody, and the unbound components are washed away; adding HRP-streptavidin (SABC) for a third incubation, and then washing away the unbound components; adding TMB colorimetric substrate solution to the reaction well, wherein TMB is oxidized by horseradish peroxidase to form a blue complex; adding a reaction stop solution to terminate the reaction, wherein the color of the substrate solution changes from blue to yellow; measuring the absorption value of the complex, and drawing a standard curve based on the absorption value versus the concentration of the screening protein in the standard, and the concentration of the screening protein in the test sample is obtained by comparing the absorption value of the sample with the standard curve ... The standard curve was fitted with a logistic regression model, and the secretion amount of the screened protein in the test sample was calculated.

[0028] The present invention also provides the use of the protein marker in preparing a product for detecting or evaluating paracrine secretion of human neural stem cells.

[0029] The present invention also provides a kit for detecting or evaluating paracrine secretion of human neural stem cells, comprising a reagent for detecting the expression level of the protein marker.

[0030] The present invention also provides the use of a reagent for detecting the protein marker in preparing a product for detecting or evaluating paracrine secretion of human neural stem cells.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention combines gene detection, reverse transcriptase analysis, proteomic analysis, and immunoassay technology to screen out highly expressed proteins as protein markers for detecting or evaluating the paracrine secretion of human neural stem cells. By detecting the above protein markers present in human neural stem cells and their culture supernatants, the paracrine secretion of human neural stem cells can be evaluated, reflecting the biological efficacy of human neural stem cells in neural development, axonogenesis, synapse formation, etc., and has important cell biological value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the process of transcriptome analysis of human neural stem cell samples with reference genome in the embodiment.

[0034] Figure 2 This is a schematic diagram of the data processing and comparative screening process for human neural stem cell samples in the embodiment.

[0035] Figure 3 These are the standard curves of NCAN, LAMB2, MMP2, and SFRP1 in the examples.

[0036] Figure 4 These are the standard curves of RTN4, B4GAT1, NPY, and VCAN in the examples.

[0037] Figure 5 These are the standard curves of GPC2, CHL1, SERPINE2, and PTPRF in the examples.

[0038] Figure 6 These are the standard curves of PTN, LDLR, NRP2, and NLGN3 in the examples.

[0039] Figure 7 These are the standard curves of CNTN2, C1QL1, NLGN4X, and VLDLR in the examples.

[0040] Figure 8 The results of GO enrichment analysis and KEGG Pathway analysis in the examples are shown in FIG. 1 , wherein red indicates up-regulation and green indicates down-regulation.

[0041] Fig. 9 Schematic diagram of PPI network-gene expression level in the embodiment.

[0042] Fig.10 It is a volcano plot made for the difference comparison data in the embodiment.

[0043] Fig.11 This is an example of GO analysis of the differentially expressed proteins screened in the examples. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with embodiments.

[0045] In the following examples, multiple batches of human neural stem cell samples obtained by Anji Xiekang Biotechnology Co., Ltd. were first obtained by transcriptome detection and analysis, and a total of 20025 gene entries were obtained, and a total of 2738 protein gene entries were obtained using a protein spectrum expression matrix. Then, the protein spectrum expression matrix and the transcriptome expression matrix of the sample were analyzed in series, and the expression matrix of the products expressed in both test results was compared to obtain 2594 gene entries. Then, this matrix was compared with the human secretory protein gene list to obtain the secretory protein gene expression matrix of the sample, and a total of 771 gene entries were obtained. Finally, clusterProfiler software was used to perform GO enrichment analysis on this expression matrix, and a total of 631 entries of various functional pathways were obtained. From it, the gene pathways related to positive regulation of functions such as neural development, immune response, and angiogenesis were screened, which were 78, 26, and 21 related pathways, respectively, each containing 130, 58, and 77 genes. According to the secretory protein expression matrix, genes with high protein expression abundance were screened among the genes contained in the above pathways (human secretory protein gene data were obtained from the HPA (The human protein atlas) database (https: / / www.proteinatlas.org / ), with a total of 3947 gene entries).

[0046] The following embodiments propose a protein marker for detecting or evaluating paracrine secretion of human neural stem cells, which is one or more of NCAN, LAMB2, MMP2, SFRP1, RTN4, B4GAT1, NPY, VCAN, GPC2, CHL1, SERPINE2, PTPRF, PTN, LDLR, NRP2, NLGN3, CNTN2, C1QL1, NLGN4X, and VLDLR.

[0047] Among them, human neural stem cells can be neural stem cells or neural progenitor cells obtained by IPSC induction, as well as primary neural stem cells obtained from tissue sources.

[0048] The following examples provide a method for screening the above protein markers, and the steps are as follows:

[0049] 1. Proteomic Analysis

[0050] 1. Technical background: Data-independent acquisition (DIA) is one of the mass spectrometry acquisition technologies that has attracted much attention in recent years. Its principle is to perform a full scan of all parent ions and their fragmented daughter ions in each acquisition window at high speed and cyclically during mass spectrometry data acquisition, rather than screening according to the parent ion signal intensity and then re-scanning the secondary fragments. It has the advantages of high throughput, high resolution, high reproducibility, and accurate quantification. In addition, samples do not need to be graded and loaded onto the machine, which greatly shortens the detection time of each sample and is suitable for proteomics research with large sample sizes. However, since DIA obtains almost all ion information with no loss, the amount of data and its complexity are greatly increased, and the difficulty of data analysis is increased. The current mainstream data analysis is based on the spectral library constructed by DDA / DIA and the data generated by the experiment to identify and quantify the proteins in the sample.

[0051] 2. Experimental principle: After the extracted protein is hydrolyzed, the peptides are analyzed by mass spectrometry using liquid chromatography-mass spectrometry technology. By comparing the signal intensities of the corresponding peptides in different samples, the proteins corresponding to the peptides can be relatively quantified. The abundance (quantity) of each peptide is proportional to its peak area or signal intensity in the mass spectrum; and the retention time (RT) of the same peptide in different samples on liquid chromatography is consistent, so the information in the spectral library can be compared and then integrated into the corresponding protein to achieve quantitative analysis of the relative expression level of proteins in different samples.

[0052] 3. Experimental process: The test samples were divided into an experimental group and a control group. The experimental group consisted of 3 batches of culture supernatants of human neural stem cells that passed the test, and the control group consisted of 3 batches of blank culture media that were cultured synchronously with the experimental group.

[0053] First, extract the total protein in the sample, take out a part for protein concentration determination and SDS-PAGE detection, take another part for trypsin hydrolysis, desalt the hydrolyzed peptides, and perform LC-MS / MS high-resolution mass spectrometry identification on the sample. LC-MS / MS identification uses DIA technology to collect mass spectrometry data of each sample, perform spectrum matching, extract quantitative information, and perform subsequent statistical analysis.

[0054] The basic process of bioinformatics analysis is: search the database for qualitative and quantitative data, perform expression level analysis and functional analysis through quality assessment and preprocessing. Use multiple common databases to perform functional annotation analysis on the identified proteins, perform GO analysis, pathway analysis and interaction analysis on the screened differential proteins, and make volcano maps, expression pattern clustering heat maps and venn analysis for the differential comparison group data. Figure 8-11 shown.

[0055] 2. Transcriptome analysis with reference genome

[0056] 1. Technical background: Transcriptome sequencing technology uses high-throughput sequencing technology to perform sequencing analysis, reflecting the expression level of genes in the transcripts of the tested samples, studying gene function and gene structure from a holistic level, revealing specific biological processes and the molecular mechanisms of disease occurrence. It has been widely used in basic research, clinical diagnosis, and drug development.

[0057] 2. Experimental principle: Oligo (dT) magnetic beads are used to enrich mRNA with polyA structure in total RNA, and RNA is sheared into fragments of about 300bp in length by ion shearing. Fragments of 300bp in length are selected because the length of the linker is fixed. If the length of the sheared fragment is shorter, the proportion of linker sequences will be higher, thereby reducing the proportion of valid data; if the length of the sheared fragment is longer, it will be unfavorable for the generation of clusters during sequencing. Using RNA as a template, 6-base random primers and reverse transcriptase are used to synthesize the first chain of cDNA, and the first chain cDNA is used as a template for the synthesis of the second chain cDNA. After the library is constructed, PCR amplification is used to enrich the library fragments, and the library is selected according to the fragment size. The library size is 450bp. Then, the library is quality checked by Agilent 2100 Bioanalyzer, and the total concentration and effective concentration of the library are tested. Then, according to the effective concentration of the library and the amount of data required for the library, the libraries containing different Index sequences (different Indexes are added to each sample, and the offline data of each sample is finally distinguished according to the Index) are mixed in proportion, and the mixed library is uniformly diluted to 2nM and denatured by alkali to form a single-stranded library. After RNA extraction, purification, and library construction, the samples are subjected to double-end (PE) sequencing based on the Illumina HiSeq sequencing platform using the second-generation sequencing technology (NGS).

[0058] 3. Reference genome

[0059] Databases: NCBI_GI, egg NOG, KEGG, GO, EC, Ensembl, UniProtID.

[0060] 4. Test process

[0061] 4.1 Samples: The test samples are 3 batches of human neural stem cells that have passed the company's quality inspection.

[0062] 4.2 Sample processing: Figure 1As shown in the figure, human neural stem cell samples were first subjected to total RNA extraction, concentration detection, mRNA purification and fragmentation (mRNA with polyA structure in total RNA was enriched by Oligo (dT) magnetic beads, and RNA was broken into fragments of about 300 bp in length by ion shearing), CDNA synthesis (using RNA as a template, 6-base random primers and reverse transcriptase to synthesize the first-chain cDNA, and the first-chain cDNA was used as a template for the synthesis of the second-chain cDNA), PCR-enriched library fragments (PCR amplification was used to enrich library fragments, and library selection was performed based on fragment size, with a library size of 450 bp), then the library was quality checked by Agilent 2100 Bioanalyzer, and the total concentration and effective concentration of the library were detected. Finally, NGS sequencing technology was used to perform double-end (PE) sequencing on these libraries based on the Illumina HiSeq sequencing platform.

[0063] 4.3 Transcriptome analysis process with references: Figure 2 As shown, the raw data was first filtered (the samples were sequenced on the machine to obtain image files, which were converted by the software provided by the sequencing platform to generate FASTQ raw data. The sequencing data contained some low-quality reads with adapters. Cutadapt was used to remove the sequences with adapters at the 3' end and the reads with an average quality score lower than Q20). The filtered high-quality sequences were aligned to the human reference genome using HISAT2 software. The reference genome and annotation information used the GRCh38 version of the human reference genome provided by NCBI. According to the alignment results, feature Counts software was used to count the alignment results, obtain the transcriptome expression matrix, and calculate the expression of each gene. On this basis, the samples were further subjected to expression difference analysis, enrichment analysis, and cluster analysis. The reads on the alignment were spliced ​​to restore the transcript sequence.

[0064] Human secretory protein gene data was obtained from the HPA (The human protein atlas) database. The transcriptome expression matrix was compared with the reference data to obtain the secretory protein gene expression data, which was then compared with the protein abundance expression results obtained by protein spectrum sequencing to screen out genes that existed in both test results to eliminate experimental errors.

[0065] Through the above process, the secretory protein expression matrix of the sample was obtained. The data was subjected to GO enrichment analysis using clusterProfiler software, and the generated data was displayed in an excel table. After obtaining the excel table data, the gene pathways related to the positive regulation of neural development and neural regeneration, immune response, and angiogenesis were manually screened out. Based on the secretory protein expression matrix, genes with high protein expression abundance in the three aspects were screened out from the genes contained in the above pathways, and multiple representative proteins were selected according to gene function, and their secretion levels in human neural stem cells were detected by ELISA. Among them, examples of GO enrichment analysis samples are as follows: Figure 8 The specific instructions are as follows:

[0066] Background proteins are all proteins with this function in the species, and foreground proteins are all proteins with this function in the differential proteins. The hypergeometric distribution test is used to calculate the p-value representing whether the function set is significantly enriched in the foreground protein. The p-value is then corrected by Benjamini&Hochberg multiple tests to obtain the FDR:

[0067]

[0068] The hypergeometric distribution calculation formula is as follows:

[0069]

[0070] Based on the secretory protein expression matrix, gene pathways related to positive regulation of neural development, immune response, angiogenesis and other functions were screened, and genes with high abundance of secretory protein expression were screened. According to the gene function, multiple representative proteins were selected, and their secretion levels in human neural stem cells were detected by ELISA. The proteins are shown in Table 1, and the protein expression levels and gene expression levels are shown in Table 2.

[0071] Table 1

[0072]

[0073]

[0074] Table 2

[0075]

[0076]

[0077] 3. ELISA test

[0078] Test samples: The experimental group was the culture supernatant of freshly cultured human neural stem cells, and the control group was the blank culture medium of synchronous culture.

[0079] Method: Double antibody sandwich ELISA method was used. After adding samples or standards to the microplate coated with anti-screening protein antibody, the screening protein in the detection sample or standard was incubated with the antibody fixed on the plate, and the plate was washed to remove the unreacted sample or standard; biotin-detection antibody was added, and in the second step of incubation, the biotin-detection antibody was combined with the screening protein that had been bound to the coated antibody. After a certain period of incubation, the unbound components were washed away; HRP-streptavidin (SABC) was added for the third step of incubation. After a certain period of incubation, the unbound components were washed away again; TMB color substrate solution was added to the reaction well, and TMB was oxidized by horseradish peroxidase to form a blue complex; the reaction was terminated by adding the reaction stop solution, and the color of the substrate solution changed from blue to yellow. The absorption value of the complex was measured, and a standard curve was made from the absorption value to the concentration of the screening protein in the standard. The concentration of the screening protein in the detection sample was obtained by comparing the absorption value of the sample with the standard curve. The standard curve was fitted by a four-parameter logistic regression model and the secretion amount of the screened protein in the test sample was calculated.

[0080] Example 1

[0081] ELISA detection: A total of 20 proteins, including NCAN, LAMB2, MMP2, SFRP1, RTN4, B4GAT1, NPY, VCAN, GPC2, CHL1, SERPINE2, PTPRF, PTN, LDLR, NRP2, NLGN3, CNTN2, C1QL1, NLGN4X, and VLDLR, were selected for ELISA detection.

[0082] Methods: Double antibody sandwich ELISA method was used. A microplate coated with anti-(NCAN, LAMB2, MMP2, SFRP1, RTN4, B4GAT1, NPY, VCAN, GPC2, CHL1, SERPINE2, PTPRF, PTN, LDLR, NRP2, NLGN3, CNTN2, C1QL1, NLGN4X, VLDLR) antibodies is incubated with samples or standards, the proteins in the detection samples or standards are bound to the antibodies fixed on the plate, and the plates are washed to remove unreacted samples or standards; biotin-detection antibodies are added, and in the second step of incubation, the biotin-detection antibodies are bound to SFRP1 that has been bound to the coated antibodies, and after incubation for a certain period of time, the unbound components are washed away; SABC is added for the third step of incubation, and after incubation for a certain period of time, the unbound components are washed away again; TMB color substrate solution is added to the reaction well, and TMB is oxidized by horseradish peroxidase to form a blue complex; a reaction stop solution is added to stop the reaction, and the color of the substrate solution changes from blue to yellow. The absorption value of the complex is measured, and a standard curve is made from the absorption value versus the protein concentration in the standard. The protein concentration of the test sample is obtained by comparing the absorption value of the sample with the standard curve. The standard curve is fitted by a four-parameter regression model and the protein secretion amount in the test sample is calculated.

[0083] Test samples: The experimental group was the culture supernatant of freshly cultured human neural stem cells, and the control group was the blank culture medium of synchronous culture.

[0084] Example 2

[0085] Take secreted frizzled-related protein 1 (SFRP1) as an example. The SFRP1 gene encodes an inhibitor of the Wnt / β-catenin signal transduction pathway. The encoded product is a secreted glycoprotein that can compete with the Wnt protein receptor or directly bind to the Wnt protein, thereby blocking the Wnt signal transduction pathway and inhibiting abnormal cell division and proliferation. Methylation silencing of the SFRP1 gene promoter is associated with the occurrence and metastasis of various tumors. Inactivation of the SFRP1 gene exists in many tumors, and the SFRP1 gene is studied as a tumor suppressor gene.

[0086] 1. Preparation of standard solution is shown in Table 3.

[0087] Table 3

[0088] name source Volume (μL) Assay Diluent Volume (μL) Concentration (ng / mL) STD1 Lyophilized Standards / 1000 10 STD2 STD1 300 300 5 STD3 STD2 300 300 2.5 STD4 STD3 300 300 1.25 STD5 STD4 300 300 0.625 STD6 STD5 300 300 0.312 STD7 STD6 300 300 0.156 Blank NA NA 300 0.000

[0089] 2. Preparation of quality control samples is shown in Table 4.

[0090] Table 4

[0091] name source Volume (μL) Assay Diluent Volume (μL) Concentration (ng / mL) ULOQ Lyophilized Standards / 1000 10 HQC ULOQ 100 25 8 MQC HQC 100 300 2 LQC MQC 100 300 0.5 LLOQ LQC 100 220 0.156

[0092] 3. The test steps are shown in Table 5.

[0093] Table 5

[0094]

[0095] 4. Data analysis:

[0096] Standard curve parameters of SFRP1: y = 5.20961 + ((0.081555 -5.20961) / (1 + (x / 6.59661) ^ 1.08186)), R 2 : 1.000, as shown in Table 6, the standard curve is as follows Figure 3 shown.

[0097] Table 6

[0098]

[0099]

[0100] 5. Test results are shown in Table 7.

[0101] Table 7

[0102]

[0103] BQL: below the quantitative limit, not detected.

[0104] Example 3

[0105] Take human matrix metalloproteinase 2 (MMP-2) as an example. MMP-2 is a gene on chromosome 2. This gene belongs to the RENETON family of coding genes. It is an extracellular matrix protease in the metalloproteinase family. MMP-2 plays a role in tissue morphogenesis, growth, tissue repair and angiogenesis. It promotes tissue remodeling and repair by degrading proteins in the ECM. It is mainly involved in the hydrolysis activity of various extracellular matrix proteins such as collagen, cholinesterol and cis-2-ene carboxylase. It plays an important role in regulating important biological processes such as cell proliferation, migration and invasion in vitro and apoptosis, including various normal physiological processes such as embryonic development, morphogenesis, reproductive tissue absorption and tissue remodeling. The activity of MMP-2 is inhibited by a variety of external factors, including metalloproteinase inhibitors, proteins and sugar chains. Studies have shown that MMP-2 plays an important role in the occurrence and development of cardiovascular diseases, arthritis, tumors, diabetes and epidemic infections. MMPs are regulated at multiple levels, including mRNA expression levels, zymogen activation processes, and the inhibition of endogenous TIMPs.

[0106] 1. Preparation of standard solution is shown in Table 8.

[0107] Table 8

[0108] name source Volume (μL) Assay Diluent Volume (μL) Concentration (ng / mL) STD1 Lyophilized Standards / 1000 32 STD2 STD1 300 300 16 STD3 STD2 300 300 8 STD4 STD3 300 300 4 STD5 STD4 300 300 2 STD6 STD5 300 300 1 STD7 STD6 300 300 0.5 Blank NA NA 300 0

[0109] 2. Preparation of quality control samples is shown in Table 9.

[0110] Table 9

[0111] name source Volume (μL) Assay Diluent Volume (μL) Concentration (ng / mL) ULOQ Lyophilized Standards / 1000 32 HQC ULOQ 100 33 24 MQC HQC 100 140 10 LQC MQC 100 400 2 LLOQ LQC 100 300 0.5

[0112] 3. Test steps are shown in Table 10.

[0113] Table 10

[0114]

[0115]

[0116] 4. Data analysis:

[0117] Standard curve parameters of MMP-2: y = 4.30355 + ((0.135892 - 4.30355) / (1 + (x / 5.70074) ^ 1.19808)), R 2 : 0.999, as shown in Table 11, the standard curve of MMP-2 is as follows Figure 3 shown.

[0118] Table 11

[0119]

[0120]

[0121] Table 12

[0122]

[0123] BQL: below the quantitative limit, not detected.

[0124] Example 4

[0125] Take reticulin 4 (RTN-4) as an example. Rtn4 is a member of the reticulin family 4. RTN-4 is a myelin-associated endoplasmic reticulum protein that plays an important role in regulating ER structure, especially the bending and remodeling of ER membranes. RTN4 protein can form disulfide bonds through its conserved C-terminal reticulin homology domain (RHD) to affect the tubular structure of ER. Increased expression of RTN4 may reduce the production of Aβ, which may be of great significance in the pathological process of Alzheimer's disease (AD). RTN4 reduces the production of Aβ40 and Aβ42 by interacting with β-amyloid protein converting enzyme 1 (BACE1) and preventing it from approaching the substrate APP. RTN4 is involved in the secretion of neuroendocrine and is widely known as an inhibitor of axon regeneration in the nervous system. It has been identified as a neuronal growth inhibitor specific to the central nervous system. RTN4 participates in the inhibition of axon growth and regeneration in the central nervous system through its receptor NgR complex and limits the plasticity of the CNS. During neural development, it is mainly expressed by neurons, providing inhibitory signals for the migration and sprouting of endothelial cells in the central nervous system, thereby limiting vascular density and playing an important role in a variety of physiological and pathological processes.

[0126] 1. Preparation of standard solution, as shown in Table 13.

[0127] Table 13

[0128] name source Volume (μL) Assay Diluent Volume (μL) Concentration (ng / mL) STD1 Lyophilized Standards / 1000 10 STD2 STD1 300 300 5 STD3 STD2 300 300 2.5 STD4 STD3 300 300 1.25 STD5 STD4 300 300 0.625 STD6 STD5 300 300 0.312 STD7 STD6 300 300 0.156 Blank NA NA 300 0.000

[0129] 2. Preparation of quality control samples is shown in Table 14.

[0130] Table 14

[0131] name source Volume (μL) Assay Diluent Volume (μL) Concentration (ng / mL) ULOQ Lyophilized Standards / 1000 10 HQC ULOQ 100 25 8 MQC 2 HQC 100 300 2 MQC 1 MQC2 300 300 1 LQC MQC1 300 300 0.5 LLOQ LQC 100 220 0.156

[0132] 3. The test steps are shown in Table 15.

[0133] Table 15

[0134]

[0135]

[0136] 4. Data analysis:

[0137] Standard curve parameters of RTN-4: y = 5.54408 + ((0.0859118 - 5.54408) / (1 + (x / 10.3244) ^ 1.14641)), R 2 : 1.000, as shown in Table 16, the standard curve is as follows Figure 4 shown.

[0138] Table 16

[0139]

[0140]

[0141] 5. Test results are shown in Table 17.

[0142] Table 17

[0143]

[0144] BQL: below the quantitative limit, not detected.

[0145] Example 5-21

[0146] Similar to the method of Example 4, Examples 5-21 respectively use NCAN, LAMB2, B4GAT1, NPY, VCAN, GPC2, CHL1, SERPINE2, PTPRF, PTN, LDLR, NRP2, NLGN3, CNTN2, C1QL1, NLGN4X, and VLDLR as examples to prepare standard solutions and quality control samples and perform data analysis. The results are shown in Tables 18, 19, and Figure 3-7 shown.

[0147] Table 18

[0148]

[0149]

[0150] Table 19

[0151]

[0152]

[0153] After screening in the above embodiments and excluding the interference of the blank group, one or more of NCAN, LAMB2, MMP2, SFRP1, RTN4, B4GAT1, NPY, VCAN, GPC2, CHL1, SERPINE2, PTPRF, PTN, LDLR, NRP2, NLGN3, CNTN2, C1QL1, NLGN4X, and VLDLR were finally determined as protein markers for detecting or evaluating the paracrine secretion of human neural stem cells. By detecting the above protein markers present in human neural stem cells and their culture supernatants, the paracrine secretion of human neural stem cells can be evaluated, which reflects the biological efficacy of human neural stem cells in neural development, axonogenesis, synapse formation, etc., and has important cell biological value.

[0154] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the protection scope of the present invention.

Claims

1. A protein marker for detecting or evaluating paracrine secretion of human neural stem cells, characterized in that: The protein markers are a combination of SERPINE2, NCAN, VCAN, PTN, NRP2, PTPRF, C1QL1 and LDLR.

2. The protein marker for detecting or evaluating paracrine secretion of human neural stem cells according to claim 1, characterized in that: The human neural stem cells include, but are not limited to, primary neural stem cells and neural progenitor cells obtained from tissue sources, and neural stem cells or neural progenitor cells and their cell lines obtained by inducing embryonic stem cells or iPSCs.

3. Use of a reagent for detecting a protein marker in the preparation of a product for detecting or evaluating paracrine secretion of human neural stem cells, wherein the protein marker is as described in claim 1 or 2.

4. A kit for detecting or evaluating paracrine secretion of human neural stem cells, comprising a reagent for detecting the expression level of the protein marker according to claim 1 or 2.

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