A specific single biomarker ndufa4l2 for detecting pericytes and applications thereof

Abstract

The application provides a specific single biomarker NADH dehydrogenase alpha subcomplex subunit 4-L2 (NDUFA4L2) for detecting pericytes. The biomarker is located in the mitochondria of pericytes. The application also provides application of NDUFA4L2 in pericyte identification. The specific single biomarker NDUFA4L2 provided by the application has a pericyte recognition accuracy of more than 90%, which is higher than the accuracy of pericyte recognition of the previously generally considered pericyte marker, and can be used as a novel single pericyte biomarker with higher specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell detection, in particular to a pericyte-specific single biomarker NDUFA4L2 and application thereof. BACKGROUND

[0002] Pericytes are structural cells widely distributed in the walls of microvessels throughout the body, and together with endothelial cells constitute the barrier of microvessels and interstitial space. Pericytes communicate with microvascular endothelial cells through cell junctions or paracrine signals, and play an important role in regulating microcirculation blood flow, microvascular permeability and angiogenesis. In addition, in tumor tissues, pericytes are proved to be functional cells that can mediate endothelial maturation and stability and reduce vascular leakage. The structural and functional abnormalities of pericytes are accompanied by microvascular lesions in various diseases, and the regulation of pericytes has become a research hotspot. Based on its morphology, pericytes are often confused with vascular smooth muscle cells (vSMC), fibroblasts, macrophages, and even epithelial cells. Although the same cell markers as vSMC are currently widely used, there is no specific antibody marker that can specifically identify pericytes. The cell markers commonly used cannot successfully distinguish pericytes from other vascular cells to a large extent. Therefore, as a compromise for pericyte identification, a comprehensive standard of location, morphology and gene / protein expression pattern is usually used to define or describe. Several recent reviews provide a list of molecular markers for pericytes. However, from a practical point of view, not all markers are useful. It is also important that there is no single completely pericyte-specific marker known, and all markers currently used are in their expression dynamics, and can be down-regulated together with developmental status, pathological response, in vitro culture, etc. Therefore, negative markers for endothelial cells and combinations of two or more pericyte markers become necessary.

[0003] Pericerocytes originate differently in different tissues and exhibit heterogeneity even within the same tissue. Pericerocytes can express a variety of biomarkers, but most lack specificity. In brain tissue, pericytes can be identified by labeling proteins such as alpha smooth muscle actin (α-SMA), pericyte aminopeptidase N, platelet-derived growth factor beta receptor (PDGFRβ), chondroitin sulfate proteoglycan NG2, and G protein signaling regulator 5 using immunostaining. Differences in biomarkers between pericytes and vascular smooth muscle cells in human brain tissue can be discovered using techniques such as brain tissue section staining and primary brain cell culture. Therefore, cell sorting can be performed based on differences in protein expression among pericytes. Furthermore, studies have also found certain differences in the expression of pericyte biomarkers between human and rodent brain tissues. Recent studies have shown that human heart pericytes (hHPs) express CD146, NG2, PDGFRβ, αSMA, and smooth muscle myosin heavy chain, but do not express CD31, CD34, CD144, and vWF. This allows hHPs to be distinguished from endothelial cells. At the same time, Chen et al. did not detect the expression of CD117 or Wt1 in hHPs, while pluripotent cardiac progenitor cells can express CD117 and epicardial progenitor cells can express Wt1. This also indicates that hHPs can be distinguished from cardiac progenitor cells and epicardial progenitor cells through the above markers.

[0004] To date, there is no single biomarker for pericyte identification; instead, a combination of methods is used, such as morphological analysis, in situ anatomical localization, antigen profiles co-expressed by two or more markers, and functional characteristics. Therefore, pericyte identification remains a challenge in pericyte research, a major obstacle to understanding its mechanism of action, and a difficult problem that future research needs to overcome. Summary of the Invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present invention provides a specific single biomarker for detecting pericytes and its application.

[0006] The pericyte-specific singlet biomarker provided by this invention is NADH dehydrogenase α subcomplex subunit 4-L2 (NDUFA4L2).

[0007] Furthermore, the biomarker is located in the mitochondria of pericytes.

[0008] Furthermore, the specificity refers to the AUC value of the biomarker NADH dehydrogenase α subcomplex subunit 4-L2 recognizing pericytes as greater than 0.9.

[0009] The application also provides application of NADH dehydrogenase alpha subcomplex subunit 4-L2 in pericyte identification, and the pericyte includes: brain microvascular pericyte, retinal microvascular pericyte, spinal cord microvascular pericyte, brain pericyte, heart pericyte, glioma pericyte or eye choroid capillary pericyte.

[0010] The application also provides application of NDUFA4L2 specific antibody in pericyte detection or separation identification.

[0011] The application also provides a pericyte tissue immunofluorescence detection kit for realizing the above-mentioned application, and the kit composition includes: NDUFA4L2 primary antibody working solution, immunofluorescence secondary antibody working solution, 10% donkey blocking serum, rabbit IgG isotype control antibody, DAPI-containing anti-fluorescence quencher and PBS buffer.

[0012] The application also provides a pericyte WB detection method for realizing the above-mentioned application, and the method includes the following steps:

[0013] 1) adding protein lysis solution to the cells to be detected; 2) electrophoresis concentration after protein denaturation; 3) protein electrophoresis to PVDF membrane, room temperature oscillation blocking; 4) incubating the PVDF membrane in the primary antibody NDUFA4L2 polyclonal antibody; 5) incubating the PVDF membrane in the HRP-labeled goat anti-rabbit IgG secondary antibody after washing the membrane; 6) developing using developing solution after washing the membrane; 7) the cells to be detected are pericytes if a protein band with a molecular weight of 10 kd can be observed.

[0014] The application also provides application of NDUFA4L2 qRT-PCR primer in pericyte detection or separation identification, and the primer is shown in SEQ.NO.1 and SEQ.NO.2.

[0015] The application also provides a pericyte qRT-PCR detection kit for realizing the above-mentioned application, and the kit composition includes: total RNA rapid extraction kit, qRT-PCR kit, forward and reverse primers: as shown in SEQ.NO.1 and SEQ.NO.2.

[0016] Pericytes are cells that exist along the interval of capillary (and post-capillary venules) walls. In the central nervous system, they are very important for vasculogenesis, maintenance of blood-brain barrier, regulation of immune cell entry into the central nervous system and control of brain blood flow. Pericytes also play a key role in pathology, for example, in ischemia, pericytes will contract capillaries, thereby preventing microcirculation reperfusion after stroke thrombolysis.

[0017] The application analyzes that NDUFA4L2 has better pericyte recognition specificity and higher expression abundance compared with other cells through single cell sequencing technology, and considers that NDUFA4L2 can be used as a new pericyte biomarker to identify pericyte after immunofluorescence staining combined with bioinformatics analysis.

[0018] At present, the specificity of the pericyte biomarker is moderate, and common pericyte biomarkers include PDGFRB, MCAM, NG2, ACTA2 and CD248, and usually a combination of markers is needed to identify pericyte. The current search literature does not mention that NDUFA4L2 can be used as a new pericyte marker.

[0019] Compared with the prior art, the application has the beneficial effects that:

[0020] 1. Single cell RNA sequencing analysis shows that the accuracy of NDUFA4L2 for pericyte recognition is as high as 90% or more, which is higher than the accuracy of the previously commonly believed pericyte marker for pericyte recognition, and can be used as a new single pericyte biomarker with high specificity.

[0021] 2. The immunofluorescence results show that the NDUFA4L2 antibody in the kit of the application can specifically recognize pericytes in glioma, and has co-localization characteristics with pericytes recognized by the pericyte antibody CD146, and is located outside the CD31 positive endothelial cells, which is consistent with the anatomical positioning of pericytes, indicating that the NDUFA4L2 antibody in the kit can specifically recognize pericytes in glioma.

[0022] 3. The kit and identification method provided by the application can quickly and simply identify pericytes. DETAILED DESCRIPTION

[0023] Figure 1 is a UMAP graph and a bubble graph showing the composition of single cells in a human glioma sample;

[0024] Among them Figure 1 A. is a UMAP graph showing the composition of single cells in a human glioma sample;

[0025] Figure 1 B. is a bubble graph showing single cell markers in a human glioma sample;

[0026] Figure 2 is a bubble graph showing the gene expression of common pericyte markers and NDUFA4L2 in various cells in glioma;

[0027] Figure 3 is a Umap graph showing the composition of pericyte subtypes in a human glioma sample;

[0028] Figure 4Figure 1 is a bubble plot showing the gene expression of common pericyte markers and NDUFA4L2 in each subtype of pericytes;

[0029] Figure 5 Figure 2 is a ROC curve analysis showing the specificity and sensitivity of common pericyte markers and NDUFA4L2 in predicting pericytes, AUC is the area under the ROC curve;

[0030] Figure 6 Figure 3 is a heatmap showing the spatial localization of common pericyte markers and NDUFA4L2 predicted in IVY database;

[0031] Figure 7 Figure 4 is a heatmap showing the correlation of expression of common pericyte markers and NDUFA4L2 in TCGA database;

[0032] Figure 8 Figure 5 is the expression of NDUFA4L2 in TCGA glioma database and its prognostic significance;

[0033] Figure 6 is a box plot showing the expression of NDUFA4L2 in different grades of glioma; Figure 8 A. is a box plot showing the expression of NDUFA4L2 in different grades of glioma;

[0034] Figure 8 B. is a univariate COX regression analysis and Kaplan-Meier survival analysis showing the prognostic significance of NDUFA4L2 gene expression in TCGA glioma database;

[0035] Figure 9 Figure 7 is a volcano plot showing the differential genes between high expression group and low expression group of NDUFA4L2 in TCGA glioma database;

[0036] Figure 10 Figure 8 is a bubble plot showing GSEA gene enrichment analysis and GO enrichment analysis of differential genes between high expression group and low expression group of NDUFA4L2 in TCGA glioma database;

[0037] Figure 9 is a bubble plot showing the results of gene enrichment analysis of H gene set in GSEA database; Figure 10 A. is a bubble plot showing the results of gene enrichment analysis of H gene set in GSEA database;

[0038] Figure 10 B. is the results of BP part in GO enrichment analysis;

[0039] Figure 11 Figure 10 is an immunofluorescence staining showing the protein expression of NDUFA4L2 in pericytes of human glioma tissue;

[0040] Figure 12 Figure 11 is a qRT-PCR showing the mRNA expression of NDUFA4L2 in pericytes of human glioma tissue;

[0041] Figure 13 This is a Western blot showing the protein expression of NDUFA4L2 in pericytes of human glioma tissue. Detailed Implementation

[0042] The method of the present invention will be described and illustrated in detail below with specific examples. This description is intended to explain the invention and not to limit its scope of protection.

[0043] Example 1: A tissue immunofluorescence kit for identifying glioma-derived pericytes containing a specific antibody of NDUF4AL2.

[0044] The kit consists of: NDUFA4L2 primary antibody working solution, immunofluorescence secondary antibody working solution, 10% donkey blocking serum, rabbit IgG isotype control antibody (isotyped with the primary antibody, trade name Rabbit IgG Control Polyclonal antibody), DAPI-containing antifluorescence quencher, and PBS buffer.

[0045] The specific steps are as follows:

[0046] 1) Remove frozen sections of glioma tissue from the -20℃ freezer and allow them to equilibrate to room temperature. Once the moisture on the sections has disappeared, place the sections in PBS (Zhongshan Jinqiao, ZLI-9062) for 5 minutes each time, for a total of 3 times.

[0047] 2) Circle the tissue with an immunohistochemical pen and add 10% donkey blocking serum (Solarbio, SL050) to the tissue. Incubate at 37°C.

[0048] Close for 30 minutes;

[0049] 3) Discard the blocking serum and add the prepared primary antibody working solution to the tissue. Incubate overnight at 4°C. The primary antibody is NDUFA4L2 polyclonal antibody (proteintech, 16480-1-AP, 1:100).

[0050] 4) On the second day, warm the sections at 37°C for 30 minutes, then wash them three times in PBS for 5 minutes each time;

[0051] 5) Add the prepared secondary antibody working solution to the tissue under light-protected conditions and incubate at 37°C for 30 minutes. The secondary antibody is donkey anti-rabbit AF488 fluorescent secondary antibody (Abcam, ab150073, 1:500).

[0052] 6) Wash the sections three times in PBS, 5 minutes each time;

[0053] 7) Mounting with anti-fluorescence quencher containing DAPI (Biotium, P0131), using confocal microscope (ZEISS, LSM900)

[0054] Observe the image.

[0055] 8) The fluorescent cells observed by the above-mentioned immunofluorescence method of the tissue are pericytes.

[0056] Example 2: Pericyte WB detection method using specific antibody of NDUF4AL2

[0057] The specific operation method is as follows:

[0058] 1) Collect the cell precipitate to be tested, add appropriate amount of RIPA protein lysate containing protease and phosphatase inhibitor to the cell precipitate, lyse for 20 minutes on ice, and shake every 10 minutes for 15 seconds on a shaker;

[0059] 2) Place the protein lysate in a low-temperature centrifuge that has been cooled to 4°C, centrifuge at 16000g for 20 minutes, and then absorb the supernatant, i.e., the lysed protein, and store it on ice;

[0060] 2) Measure the protein concentration of the lysed protein using the BCA method;

[0061] 3) Add 4x loading buffer to the protein to be tested at a ratio of 3:1, mix well, and then place it in a 95°C metal bath for denaturation for 10 minutes, and then store it at -80°C;

[0062] 4) Place the protein marker and the protein sample to be tested in the electrophoresis device in sequence, and perform protein electrophoresis experiment, with the initial running voltage set to 100V, and then adjust the voltage to 150V-180V after the protein sample runs out of the concentrated gel;

[0063] 5) After the running is completed, perform the electrotransfer operation, activate the PVDF membrane with methanol, and the sequence from the anode to the cathode is sponge, thin filter paper, PVDF membrane, electrophoresis gel, thin filter paper, and sponge, set the membrane transfer conditions to constant current 150-300mA and 90-150 minutes after removing the air bubbles completely with the roller;

[0064] 6) After the membrane transfer is completed, gently wash the residual transfer solution with PBST, and place the PVDF membrane in the blocking milk powder for 1 hour of room temperature shaking blocking;

[0065] 7) Gently wash the residual blocking milk powder with PBST, and incubate the primary antibody NDUFA4L2 polyclonal antibody (proteintech, 16480-1-AP, 1:1000) at 4°C overnight;

[0066] 8) Transfer the PVDF membrane into PBST for membrane washing, 5 minutes / time, for a total of 3 times;

[0067] 9) Transfer the PVDF membrane into the prepared secondary antibody, shake incubate at room temperature for 1 hour;

[0068] 10) Transfer the PVDF membrane into PBST, wash the membrane for 5 minutes / time, for 3 times, and develop using developing solution;

[0069] 11) If protein band with molecular weight of 10 kd can be observed, the tested cell is pericyte.

[0070] Example 3: A qRT-PCR kit for identifying pericyte derived from glioma

[0071] The kit composition comprises: total RNA rapid extraction kit, qRT-PCR kit, forward and reverse primers: as shown in SEQ. No. 1 and SEQ. No. 2.

[0072] The specific operation is as follows:

[0073] 1) Collect the tested cell precipitate, use the total RNA rapid extraction kit of Feijie Biological Company, extract RNA according to the requirements of the instruction manual, and conduct concentration determination;

[0074] 2) Use the qRT-PCR kit of Takara Company, add 2 μl of 5×PrimeScript RT Master Mix to 500 ng of RNA, supplement with DEPC water to 10 μl, and then conduct reverse transcription, the condition is 37℃ for 30 minutes for reverse transcription, 85℃ for 1 minute to terminate the reaction, and 4℃ for storage; TM

[0075] 3) The fluorescence quantitative PCR system is 10 μl, wherein 0.4 μl of forward and reverse primers (10 μmol / L), 5 μl of SYBR Green, 0.2 μl of cDNA, and 4.0 μl of DEPC water are added, the condition is 95℃ for 30 seconds, 95℃ for 5 seconds, 60℃ for 30 seconds for 40 cycles, and the melting curve is measured; the primer sequence is shown in SEQ. No. 1 and SEQ. No. 1.

[0076] 4) According to the expression of the internal reference molecule, the standardization processing is conducted, and the difference of the target gene is compared.

[0077] NDUFA4L2 F-CTGGGACAGAAAGAACAACCCG SEQ. NO. 1 NDUFA4L2 R-CAGCCTGGCTTAGAAGTCTGGC SEQ. NO. 2

[0078] 5) If it is observed that the expression of NDUF4L2 mRNA is significantly higher than that of known non-pericyte (such as tumor cells, etc.), the tested cell is identified as pericyte.

[0079] Test Example 1:

[0080] ​The single-cell sequencing data of 20 cases of adult diffuse glioma was pretreated and quality controlled by R language Seurat package, and different samples were integrated by harmony package. The first 2,000 genes with the highest dispersion were selected from each data set, and cells with less than 200 and more than 6,000 genes and more than 10% mitochondrial genes were filtered out. The results after harmony integration data were visualized by UMAP chart, and each cell type was annotated by using FindClusters and known markers (as shown in Figure 1 , for example). It was found that pericytes specifically expressed NDUFA4L2 (as shown in Figure 2 , for example) compared with other cells. Pericytes were extracted alone for cell subpopulation, and the expression abundance of NDUFA4L2 was high in all pericyte subgroups (as shown in Figure 3 , Figure 4 , for example).

[0081] Test Example 2:

[0082] The ROC curve was drawn and the AUC value was analyzed by R language pROC package to determine the sensitivity and specificity of each marker of pericytes and NDUFA4L2 in single-cell data. Among the common and new pericyte markers, the AUC value of PDGFRB, RGS5, HIGD1B and NDUF4AL2 for pericyte recognition can reach more than 0.9, indicating that their effectiveness for pericyte recognition is higher. (as shown in Figure 5 , for example)

[0083] Test Example 3:

[0084] The spatial distribution of pericyte markers and NDUFA4L2 in single-cell data was analyzed by IVY database. After the glioma gene expression profile downloaded from IVY database was standardized, data normalization and heat map visualization were performed by R language Pheatmap package, and the results showed that NDUFA4L2 and other pericyte markers were located in the vascular area. (as shown in Figure 6 , for example)

[0085] Test Example 4:

[0086] The correlation of NDUFA4L2 and other pericyte markers was analyzed by TCGA glioma database. After the glioma gene expression profile downloaded from TCGA glioma database was standardized, the pearson correlation coefficient was calculated and the correlation heat map was visualized by R language ggcorrplot package, and the results showed that NDUFA4L2 had high correlation with CD248, MCAM, ACTA2 and other pericyte markers.

[0087] (as shown in Figure 7 , for example)

[0088] Test Example 5:

[0089] The relationship between NDUFA4L2 and glioma grade was analyzed by TCGA glioma database, Wilcoxon test was used for non-parametric test of mean comparison between two groups, and R language ggplot2 package was used for box plot visualization. The results showed that NDUFA4L2 was expressed the highest in grade IV glioblastoma. The relationship between NDUFA4L2 and patient prognosis in TCGA glioma database was analyzed by R language survival package and survivalminer package, and the results were visualized by forestplot package. Single factor cox regression analysis showed that NDUFA4L2 expression was a risk factor and was significant. The best cut off value of KM survival analysis was selected by pROC package, and the difference in overall survival of patients between NDUFA4L2 high expression group and low expression group was compared. The results showed that NDUFA4L2 high expression was associated with short overall survival of patients. Figure 8

[0090] Test Example 6:

[0091] The differential genes and GSEA gene enrichment of NDUFA4L2 high expression group and low expression group were analyzed by TCGA glioma database. The TCGA glioma gene expression profile was divided into NDUFA4L2 high expression group and NDUFA4L2 low expression group by using R language TCGAbiolinks package with the median of NDUFA4L2 expression. The differential gene analysis of the two groups showed that the expression of pericyte markers (MCAM\CD248, etc.) was also high in NDUFA4L2 high expression group. The results were visualized by volcano plot using ggplot2 package (as shown in Figure 9 Figure 10

[0092] Test Example 7:

[0093] Immunofluorescence staining was performed on human glioma patient frozen sections, and it was found that the cells recognized by NDUFA4L2 antibody had obvious colocalization with pericyte marker CD146, and were outside the endothelial cell marker CD31, which was consistent with the localization of pericytes. The fluorescence results showed that NDUFA4L2 was located in mitochondria, and the staining was discontinuous and dotted (as shown in

[0094] The staining steps are as follows:

[0095] ​​​1. Take out the corresponding frozen section from the -20℃ refrigerator and equilibrate to room temperature. When the water vapor on the section disappears, place the section in PBS (Zhongshanjinqiao, ZLI-9062) for hydration, 5 minutes / time, a total of 3 times;

[0096] 2. Use an immunohistochemical pen to circle the tissue and add 10% donkey blocking serum (Solarbio, SL050) dropwise to the tissue, block at 37℃ for 30 minutes;

[0097] 3. Discard the blocking serum and add the prepared primary antibody working solution to the tissue, incubate overnight at 4℃. The primary antibody is CD146 monoclonal antibody (self-made, 1:100, patent application number 201611078356.2), CD31 polyclonal antibody (R&D Systems, AF3628, 1:200), NDUFA4L2 polyclonal antibody (proteintech, 16480-1-AP, 1:100)

[0098] 4. The next day, rewarm the section at 37℃ for 30 minutes, then place the section in PBS and wash 3 times, 5 minutes / time;

[0099] 5. Add the prepared secondary antibody working solution to the tissue under light-proof conditions, incubate at 37℃ for 30 minutes. The secondary antibody is donkey anti-mouse AF555 fluorescent secondary antibody (Abeam, ab150110, 1:500), donkey anti-rabbit AF488 fluorescent secondary antibody (Abeam, ab150073, 1:500), and donkey anti-goat AF647 fluorescent secondary antibody (Abeam, ab15013, 1:500)

[0100] 6. Place the section in PBS and wash 3 times, 5 minutes / time;

[0101] 7. Use DAPI-containing anti-fluorescence quencher (Bi Yun Tian, P0131) to mount the section, and use a confocal microscope (ZEISS, LSM900) to observe and take pictures.

[0102] Test Example 8:

[0103] qRT-PCR detection of pericytes and other non-pericytes found that the mRNA expression of pericyte markers (such as CD146\CD248, etc.) was consistent, and the expression of NDUFA4L2 mRNA in pericytes (about 20 cycles of cycle number) was significantly higher than that in other non-pericytes Figure 12 ), the specific operation is as follows:

[0104] 1) Collect the required cell precipitate, use the total RNA rapid extraction kit of Feijie Biological Company, extract RNA according to the requirements of the instruction manual, and conduct concentration determination;

[0105] 2) using Takara qRT-PCR kit, 2 μl 5x PrimeScript added to each 500 ng RNA, DEPC water to 10 μl, and then reverse transcription, conditions: 37 °C for 30 min, 85 °C for 1 min to terminate the reaction, 4 °C for storage; TM RTMaster Mix, DEPC water to 10 μl, and then reverse transcription, conditions: 37 °C for 30 min, 85 °C for 1 min to terminate the reaction, 4 °C for storage;

[0106] 3) The fluorescence quantitative PCR system is 10 μl, including 0.4 μl of forward and reverse primers (10 μmol / L), 5 μl of SYBR Green, 0.2 μl of cDNA, and 4.0 μl of DEPC water. The conditions are 95 °C for 30 seconds, 95 °C for 5 seconds, 60 °C for 30 seconds, 40 cycles, and determination of melting curve. The primer sequence is

[0107]

[0108]

[0109] 4) According to the expression of the internal reference molecule, the difference of the target gene is compared.

[0110] Test Example 9:

[0111] Western Blot detection was performed on pericytes and other non-pericytes, and it was found that the protein expression amount of NDUFA4L2 was consistent with the expression of pericyte markers (such as CD146, etc.). NDUFA4L2 protein was expressed in pericytes, but not in other non-pericytes Figure 13 ), and the specific operation is as follows:

[0112] 1) Collect the required cell precipitate, add appropriate amount of RIPA protein lysis buffer containing protease and phosphatase inhibitor to the cell precipitate, and lyse on ice for 20 minutes, and shake on a shaker every 10 minutes for 15 seconds;

[0113] 2) Put the protein lysis buffer into a low-temperature centrifuge that has been cooled to 4 °C, centrifuge at 16000g for 20 minutes, and then absorb the supernatant, i.e. the lysed protein, and store it on ice;

[0114] 3) The lysed protein is measured for protein concentration using the BCA method;

[0115] 4) Add 4x loading buffer to the protein to be tested at a ratio of 3:1, mix well, and then place in a 95 °C metal bath for denaturation for 10 minutes, and then store at negative 80 °C;

[0116] 5) In the electrophoresis device, the protein marker and the protein sample to be tested are sequentially loaded, and the protein electrophoresis experiment is performed, the initial running voltage is set to 100V, and after the protein sample is run out of the concentrated gel, the voltage is adjusted to 150V-180V;

[0117] 6) After the running gel is finished, the electrotransfer operation is performed, the methanol activates the PVDF membrane, the order from the positive electrode to the negative electrode is sponge, thin filter paper, PVDF membrane, electrophoresis gel, thin filter paper, sponge, and after the air bubbles are completely removed by the roller, the membrane transfer conditions are set to constant current 150-300mA, 90-150 minutes;

[0118] 7) After the membrane transfer is finished, the residual transfer solution is gently washed away with PBST, and the PVDF membrane is placed in the blocking milk powder and shaken at room temperature for 1 hour;

[0119] 8) The residual blocking milk powder is gently washed away with PBST, and the primary antibody NDUFA4L2 polyclonal antibody (proteintech, 16480-1-AP, 1:1000), CD146 monoclonal antibody (abcam, ab24577, 1:1000), and β-Tubulin antibody (cst, #2128s, 1:1000) are incubated, and the temperature is 4°C overnight;

[0120] 9) The PVDF membrane is transferred into PBST for washing, 5 minutes / time, for a total of 3 times;

[0121] 10) The PVDF membrane is transferred into the prepared secondary antibody, and shaken at room temperature for 1 hour;

[0122] 11) The PVDF membrane is transferred into PBST for washing, 5 minutes / time, for a total of 3 times, and the developing solution is used for development.

[0123] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. Use of NADH dehydrogenase alpha subcomplex subunit 4-L2 in the detection of pericytes, characterized in that, The NADH dehydrogenase alpha subcomplex subunit 4-L2 is located in the pericyte mitochondria, and the application comprises the following steps: 1) adding protein lysate to the cells to be tested; 2) electrophoresis concentration after protein denaturation; 3) transferring the protein of the concentrated gel to a PVDF membrane, blocking at room temperature; 4) transferring the PVDF membrane into the primary antibody NDUFA4L2 polyclonal antibody for incubation; 5) transferring the PVDF membrane into the HRP-labeled goat anti-rabbit IgG secondary antibody for incubation after washing the membrane; 6) developing using developing solution after washing the membrane; and 7) observing the protein band with a molecular weight of 10 kd, and the cells to be tested are pericytes.

2. Use according to claim 1, characterized in that, The pericytes include brain microvascular pericytes, retinal microvascular pericytes, spinal cord microvascular pericytes, brain-derived pericytes, heart-derived pericytes, glioma-derived pericytes or eye choroid capillary-derived pericytes.

3. The pericyte tissue immunofluorescence test kit for use according to claim 1, characterized in that, The kit composition comprises NDUFA4L2 primary antibody working solution, immunofluorescence secondary antibody working solution, 10% donkey blocking serum, rabbit IgG isotype control antibody, DAPI-containing anti-fluorescence quencher and PBS buffer.

Images