CN114657281A - Endogenous virus as marker of aging degree and application of endogenous virus as aging intervention target - Google Patents
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Abstract
The invention discloses a marker for identifying the aging degree of cells, tissues and individuals and application thereof as a target for aging intervention. The invention firstly discloses a marker of endogenous virus HERVK as human mesenchymal stem cells and application thereof. Secondly, further proves that the endogenous viruses HERVK, MMTV and HERVW are used as markers of aging degrees of cells, tissues and individuals in other cell types, mouse, non-human primate and human tissues and blood, and the application of the markers. The invention finally discloses application of HERVK inhibition or HERVK neutralization antibody as a target for aging intervention.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to an endogenous virus as a marker of aging degree and application thereof.
Background
Aging is the biggest risk factor of human diseases, and with the increasing aging pressure of the population in China, the prevention and treatment of senile diseases become a scientific problem which needs to be broken through urgently. Aging, a slowly progressing physiological decline of organs, is one of the most risk factors for many chronic diseases. However, little is currently known about the epigenetic and molecular changes of aging. Therefore, the identification of novel biomarkers that are capable of reflecting the degree of aging at the cellular, tissue and body level, provides an entry point for the development of interventions to delay aging, and is an important goal in the aging field and in geriatric medicine. Scientists have long sought aging biomarkers that meet the following conditions: 1) they can be used to monitor the aging process; 2) they can be tested repeatedly in a non-invasive manner. Several biomarkers of senescence have been identified, such as DNA methylation, telomere length, gene expression products, metabolomic profiles, and senescence-associated secreted phenotype (SASP) factors. Due to the complexity of the aging process, there are still few molecular biomarkers that can reflect determinants of aging-related molecular changes. Thus, there is a great need to further discover complex intrinsic biomarkers that can reflect determinants of aging-related molecular changes.
Previous studies have focused primarily on protein-coding regions of the genome, and no study is known as to whether non-protein-coding regions of the genome can serve as senescence markers, and we have therefore turned our attention to reverse transcription elements of the genome. Endogenous Retroviruses (ERVs), which are "dark materials" of the genome, belong to the LTR family of the reverse transcription elements and are thought to be present in the genome of almost all animals due to the legacy elements produced after the archaea infects germ cells of host progenitors and invades the host genome. Expression of Endogenous Retroviruses (ERVs) is under strict epigenetic regulation. HERVK (HML-2), as the youngest group among all Human Endogenous Retroviruses (HERV), is capable of encoding all viral proteins and producing virus-like particles. Under normal physiological conditions, transcription of HERVK is inhibited, whereas in certain pathological conditions such as cancer, autoimmune and neurodegenerative diseases HERVK is activated, it is not clear whether HERVK can exert a similar effect in age-related epigenetic changes.
Disclosure of Invention
The invention aims to solve the technical problem of how to identify endogenous viruses as markers of aging degree and application of the endogenous viruses as targets for aging intervention.
In order to solve the technical problems, the invention firstly provides the application of endogenous viruses as markers of aging degree;
the above-mentioned degree of aging includes aging at the cellular, organ and individual level.
The cells include adult stem cells including mesenchymal stem cells and neural stem cells, and cells derived from the stem cells by differentiation include at least one of adipose cells, osteogenic cells, chondrocytes, nerves, microglia, and the like, and terminally differentiated endothelial cells, fibroblasts, and the like.
The organ includes at least one of lung, liver, skin, muscle, brain, spleen, kidney, blood (blood plasma, blood serum, and peripheral blood mononuclear cells), etc.
The individual level includes at least one of humans, non-human primates, and rodents (mice, rats).
The above endogenous viruses include at least one of human endogenous viruses HERV, including HERVK and HERVW, and a homologous virus of HERVK in mice (MMTV).
The invention further provides a detection means and a kit of endogenous viruses as markers of aging degree.
The above-mentioned degree of aging includes aging at the cellular, organ and individual level.
The cells include adult stem cells including mesenchymal stem cells and neural stem cells, and cells derived from the stem cells by differentiation include at least one of adipose cells, osteogenic cells, chondrocytes, nerves, microglia, and the like, and terminally differentiated endothelial cells, fibroblasts, and the like.
The organ includes at least one of lung, liver, skin, muscle, brain, spleen, kidney, blood (blood plasma, blood serum, and peripheral blood mononuclear cells), etc.
The individual level includes at least one of humans, non-human primates, and rodents (mice, rats).
The above endogenous viruses include at least one of human endogenous viruses HERV, including HERVK and HERVW, and a homologous virus of HERVK in mice (MMTV).
The above detection means include RT-qPCR primer pair and RNA-FISH probe for detecting RNA level of endogenous virus (including HERVK, HERVW and MMTV), specific antibody of virus protein level, and immune colloidal gold electron microscope technology for detecting HERVK virus particle.
The above-mentioned kit comprises a substance for detecting the expression of endogenous viruses including HERVK, HERVW and MMTV.
The invention further provides a detection means and a kit for releasing the endogenous virus outside cells or in blood as a marker of aging degree.
The cells include adult stem cells including mesenchymal stem cells and neural stem cells, and cells derived from the stem cells by differentiation include at least one of adipose cells, osteogenic cells, chondrocytes, nerves, microglia, and the like, and terminally differentiated endothelial cells, fibroblasts, and the like.
The individual level includes at least one of humans, non-human primates, and rodents (mice, rats).
The above detection means include an experimental method for extracting free viral RNA or protein or virus particles in a culture medium or blood, a primer pair for digital PCR for detecting viral RNA, and antibodies specific to viral proteins and virus particles.
The above-mentioned kit comprises a substance for detecting the expression of endogenous viruses including HERVK, HERVW and MMTV.
Use of an endogenous virus as a marker for detecting the degree of ageing in blood.
The application of the endogenous virus or endogenous virus gene substance in the sample in preparing the product for detecting the aging degree of the mammal.
The sample is blood.
Use of an endogenous virus for the manufacture of a product for promoting or inhibiting senescence.
The application of endogenous virus, substance for inhibiting endogenous virus or silencing or knocking out endogenous virus gene in preparing products for promoting or inhibiting aging or premature senility.
The endogenous virus used in the method comprises human endogenous virus HERV.
The above applications include at least one of HERVK, HERVW and MMTV.
The MMTV is a virus homologous to herpk in mice.
Use of an endogenous virus as a target for intervention in the degree of senescence.
The application comprises a step of combining a substance capable of specifically binding to and inhibiting the activity of an endogenous virus, and the endogenous virus, wherein the endogenous virus comprises at least one of HERVK, HERVW and MMTV.
The substance capable of specifically binding with the endogenous virus is a monoclonal or polyclonal neutralizing antibody against the endogenous virus; or the substance specifically combined with the endogenous virus is a specific inhibitory small molecule compound of the endogenous virus; or the substance specifically combined with the endogenous virus is a specific primer pair for knocking down or knocking out the anti-endogenous virus.
The marker of the invention has important function in detecting the aging degree on the cell, organ and individual level, provides important molecular indication in human aging degree evaluation and disease diagnosis, and has important application value.
In order to solve the technical problems, the invention also provides application of endogenous viruses as targets for interfering with aging degree.
The above-mentioned degree of aging includes aging at the cellular, organ and individual level.
The cells include adult stem cells including mesenchymal stem cells and neural stem cells, and cells derived from the stem cells by differentiation include at least one of adipose cells, osteogenic cells, chondrocytes, nerves, microglia, and the like, and terminally differentiated endothelial cells, fibroblasts, and the like.
The organ includes at least one of lung, liver, skin, muscle, brain, spleen, kidney, blood (blood plasma, blood serum and peripheral blood mononuclear cells), etc.
The individual level includes at least one of humans, non-human primates, and rodents (mice, rats).
The above endogenous viruses include at least one of human endogenous viruses HERV, including HERVK and HERVW, and a homologous virus of HERVK in mice (MMTV).
Such intervention applications include substances that specifically bind to and inhibit the activity of endogenous viruses, including HERVK, HERVW and MMTV. The substance specifically binding to endogenous viruses (including HERVK, HERVW and MMTV) as described can be a monoclonal or polyclonal neutralizing antibody against endogenous viruses (including HERVK, HERVW and MMTV); the substance that specifically binds to endogenous viruses (including HERVK, HERVW and MMTV) can be a specific inhibitory small molecule compound of the endogenous viruses (including HERVK, HERVW and MMTV); the substance that specifically binds to endogenous viruses (including HERVK, HERVW and MMTV) can be a specific primer pair that knocks down or knocks out against endogenous viruses (including HERVK, HERVW and MMTV).
In the above application, the product is a reagent or a kit.
The target intervention means of the invention provides important theoretical basis for delaying senility and treating diseases related to senility.
The invention has the beneficial effects that: the invention discloses a marker for identifying the aging degree of cells, tissues and individuals and application thereof as an aging intervention target. The invention firstly discloses a marker of endogenous virus HERVK as human mesenchymal stem cells and application thereof. Secondly, further proves that the endogenous viruses HERVK, MMTV and HERVW are used as markers of aging degrees of cells, tissues and individuals in other cell types, mouse, non-human primate and human tissues and blood, and the application of the markers. The invention finally discloses application of HERVK inhibition or HERVK neutralization antibody as a target for aging intervention.
Drawings
FIG. 1 is a graph of upregulation of expression levels of human endogenous virus HERVK in senescent mesenchymal stem cells, wherein RNA-seq analysis increased HERVK levels in senescent mesenchymal stem cells; RT-qPCR verifies that HERVK RNA level is increased in the aging mesenchymal stem cells; RNA-FISH verifies that HERVK RNA level is increased in the mesenchymal stem cells of presenility (C) and replication (D), and the right figure is a statistical result; E-F: western blot confirmed elevated herpk viral protein levels in early senescence (E) and replicative (F) mesenchymal stem cells, statistical results are shown in the right panel; G-H: immunofluorescence confirms the elevated levels of HERVK virus protein in the early senescence (G) and replicative (H) mesenchymal stem cells, and statistics are shown in the right panel.
Fig. 2 is a diagram of the formation of virions by the human endogenous virus herpk in senescent mesenchymal stem cells, wherein a-B: the ordinary electron microscope shows that the number of similar virus particles is increased in the early senescence (A) and replicative (B) mesenchymal stem cells, and the right figure is a statistical result; C-D: colloidal gold immunolabeling electron microscopy showed an increase in the number of HERVK virions in the presenile (C) and replicative (D) mesenchymal stem cells, with statistics on the right panel. E: and (4) carrying out colloidal gold immune labeling on a negative control.
Fig. 3 is a graph of the up-regulation of the expression level of human endogenous virus herdk in senescent fibroblasts and the formation of viral particles, wherein a: immunoblotting verified elevated HERVK viral protein levels in senescent fibroblasts, and the right panel is statistical results; b: immunofluorescence verifies that HERVK virus protein level is increased in aged fibroblasts, and the right figure shows a statistical result; c: the increase in the number of similar virus particles in senescent fibroblasts was confirmed by ordinary electron microscopy, and the right panel is statistical.
Fig. 4 is a graph of the release of herpk viral particles into culture media by senescent mesenchymal stem cells, wherein a: digital PCR shows that HERVK RNA level in the culture medium of the presenility mesenchymal stem cells is increased, and the right attached figure is a statistical result; B-C: ELISA results showed elevated herpk protein levels in the culture media of presenile disease (B) and replicative senescence mesenchymal stem cells; D-E: common electrons microscopically show the presence of similar viral particles outside the mesenchymal stem cells of senilism (D) and replicative senescence (E); F-G: colloidal gold immunolabeling electrons microscopically indicate the presence of herpk viral particles outside mesenchymal stem cells of premature aging (F) and replicative senescence (G).
FIG. 5 shows the up-regulation of MMTV expression of the human endogenous virus HERVK analogue in the liver of aged mice, and shows the increase of MMTV level in the liver tissue of aged mice as verified by RT-qPCR using two primer pairs of MMTV.
Figure 6 is an upregulation of human endogenous virus HERVW expression in premature and naturally aging cynomolgus monkeys, wherein a: wild type and progeria HGPS cynomolgus monkey model; b: the western blot shows that HERVW virus protein level is increased in lung tissues of the early senescence HGPS cynomolgus monkey, and the right figure is a statistical result; c: immunohistochemistry showed increased HERVW signals in the lungs, liver and skin of the early-senescence HGPS cynomolgus monkey, and the right panel is the statistical result; d: young and old cynomolgus models; e: western blot showing elevated HERVW viral protein levels in the lung tissue of aged cynomolgus monkeys, statistical results are shown in the right panel; f: immunohistochemistry showed increased HERVW signals in the lungs, liver and skin of aged cynomolgus monkeys, and the right panel is statistical.
FIG. 7 is a graph of elevated HERVK levels in skin and blood of an elderly person, where A is a model of a young and elderly person; b: immunohistochemistry showed increased herpk signals in aged human skin, statistical results in the right panel; c: ELISA showed elevated levels of herpk viral protein in serum of elderly persons.
Fig. 8 is a graph showing that inhibition or activation of herpk may retard or accelerate senescence of mesenchymal stem cells, wherein a: schematic representation of activation or knockdown of herdk affecting mesenchymal stem senescence; b: western blot showing activation of endogenous herdk in wild type mesenchymal stem cells increased viral protein expression, statistical results in the right panel; c: SA-gal experiment shows that HERVK expression is activated to promote cell aging, and the right figure shows statistical results; d: immunoblotting shows that knocking down HERVK in the mesenchymal stem cells of the presenility disease reduces the expression of virus protein, and the right figure is a statistical result; e: SA-gal experiment shows that knocking down HERVK delays the senility of mesenchymal stem cells of presenility, and the right figure shows the statistical result.
Figure 9 is a graph of the ability of neutralizing herdk in aged cell culture media with antibodies to delay aging of aged media-induced cells, where a: HERVK schematic of the neutralization of senescence media with antibodies; b: western blot shows that antibodies specific for herpk can pull down herpk in senescent media; c: SA-gal experiments show that neutralization of HERVK in aging medium by neutralizing antibody can delay aging of young cells caused by aging medium, and statistical results are shown in the right figure.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The cynomolgus monkey experiments in the following examples were performed based on the ethical treatment criteria for non-human primates and have been approved by the ethical institute of animals of the Chinese academy of sciences.
The human sample experiments in the following examples were conducted based on the "ethical treatment criteria for human samples" and were approved by the ethical examination of the first subsidiary hospital of the Beijing cooperative medical college and Kunming medical college.
1. The cell lines used in the present invention are as follows:
mesenchymal stem cells carrying human childhood senilism gene mutation are childhood senilism carrying pathogenic gene LMNA mutation in Chinese patent (application of oltipraz in reversing human childhood senilism senescence phenotype, CN 105769869B).
The mesenchymal stem cell carrying human adult premature senility syndrome gene mutation is a mesenchymal stem cell with WRN function loss in Chinese patent (pluripotent stem cell carrying human adult premature senility syndrome gene mutation and a preparation method, ZL201510137846.4), and the public can obtain the biological material from the applicant.
Wild-type mesenchymal stem cells prepared according to the references Wu, Z.et. differential stem cell imaging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome, protein & cell 9,333-350, doi:10.1007/s13238-018-0517-8 (2018). The method comprises the following specific steps:
differentiation and culture of mesenchymal stem cells: human embryonic stem cells human H9(WiCell Research) were differentiated into Embryoid Bodies (EBs) for 3 days, and EBs were inoculated onto 6 plates coated with matrigel (matrigel Invitrogen), and cultured in the above mesenchymal stem cell differentiation medium for 2 weeks until fibrous cells appeared. After passage once again, mesenchymal stem cells are sorted by flow cytometry: and selecting a cell population with positive CD73, CD90 and CD105 as the high-purity human mesenchymal stem cells. And (3) culturing the purified mesenchymal stem cells, and carrying out subculture by using the mesenchymal stem cell culture medium for 45 days for one generation. One well in a 6-well plate was seeded at 1 × 105 cell density for each passage.
Wherein, the fluorescence labeling antibody used for flow cytometry sorting mesenchymal stem cells: the anti-human cell surface recognition molecule CD90 antibody labeled by fluorescein FITC has the cargo number 555595; fluorescein FITC labeled isotype control antibody cat # 555742. The cargo number of the fluorescein PE labeled anti-human cell surface recognition molecule CD73 antibody is 550257; fluorescein PE labeled isotype control antibody is available under the cat # 555749. The goods number of the fluorescein APC marked anti-human cell surface recognition molecule CD105 antibody is 17-1057-42; the isotype control antibody labeled by fluorescein APC was assigned with the accession number 555-751, and all the antibodies were BD Biosciences products. The product is preserved by the inventor at the institute of biophysics of the Chinese academy of sciences, and is publicly available from the applicant.
Fibroblasts were purchased from LONZA.
2. The cell culture media used in the present invention are as follows:
(1) mesenchymal stem cell culture medium: 89% (v/v% by volume) of α MEM + GlutaMAX (product of Invitrogen, cat. No. 12571071), 10% (v/v% by volume) of fetal bovine serum (product of Gibco, cat. No. 10100-147), 1% (v/v% by volume) of penicillin/streptomycin (product of Invitrogen, cat. No. 15070-063), and 1ng/ml of recombinant human fibroblast growth factor (JPCFGF 2).
(2) Human fibroblast culture medium: 87% (v/v) DMEM (Hyclone product), 10% (v/v) fetal bovine serum (Gibco product, cat. No. 10100-147), 1% v/v) nonessential amino acids (Invitrogen product, cat. No. 11140-050), 1% (v/v) GlutaMAX (Invitrogen product, cat. No. 35050-061), 1% (v/v) penicillin/streptomycin (Invitrogen product, cat. No. 15070-063).
3. The biological materials used for packaging lentiviruses in the present invention are as follows: lentiviral packaging vectors were psPAX2(Addgene, cat #12260) and pMD2G (Addgene, cat # 12259).
4. In the following examples, the antibodies used were as follows:
murine anti-HERVK-Env was purchased from Austral Biologicals under the accession number HERM-1811-5;
rabbit anti-ERVW-1 was purchased from Abcam under cat number ab 234850;
rabbit anti-pho-NF-. kappa.Bp 65(Ser536) was purchased from Cell Signaling technology under # 3033S;
Alexa
568Donkey Anti-Rabbit IgG(H+L)，Alexa
568Donkey Anti-Mouse IgG(H+L)，Alexa
488Donkey Anti-Mouse IgG (H + L) was purchased from Invitrogen and the accession numbers are A10042, A-10037, A21202;
HRP-jointed Goat-anti-Mouse (H + L), HRP-jointed Goat-anti-Rabbit (H + L) from China fir bridge, with the product numbers of 5332 and 2497; rabbit antibodies DES and FOXO3 were purchased from Cell signaling technology under the cat nos. ZB2305 and ZB2301, respectively;
5. in the following examples, the reagents and kits used were as follows:
the protein quantitative determination kit is purchased from Beijing ancient China Changsheng biotechnology limited company, and has a product number of BCA 02;
PVDF membranes, available from Millipore under the trade designation IPVH 00010;
SuperSignal West Femto Maximum Sensitivity Substrate available from Thermo Fisher Scientific under the product number 34096;
protein A/G sepharose magnetic beads from Sun Cruz Biotechnology under the cat # sc-2003;
GoScript Reverse Transcription System, available from Promega, cat # A5003;
THUNDERBIRD qPCR Mix, purchased from TOYOBO under the accession number QPS-101T;
QIAamp viral RNA mini Kit, purchased from Qiagen, cat # 52904;
DNase I using TURBO DNA-free Kit, purchased from Invitrogen, cat # AM 1907;
QX200ddPCR EvaGreen Supermix，QX200TMdroplet Generation Oil for EvaGreen, available from Bio-Rad Laboratories, Inc., having code numbers 1864034, 1864005, respectively;
human HERVK-7 p22.1Provirus antibiotic Env polypredin (ERVK6) ELISA kit available from CUSABIO under the cat number CBS-EL007812 HU;
hoechst 33342, purchased from Invitrogen under cat number H3570;
DAB Staining Kit, purchased from China fir Jinqiao, with a product number ZLI-9018;
RNA FISH probe HERVK Alexa Fluor 488, available from Thermo Fisher Scientific, having the accession number VA 10-3264982;
6. in the following examples, the instrumentation and analysis software used is as follows
ChemiDoc XRS system and Image Lab software, available from Bio-Rad Laboratories, Inc., for chemiluminescence imaging;
CFX384 Real-Time System, available from Bio-Rad Laboratories, Inc., for RT-qPCR;
QX200 droplet generator，PX1TMPCR Plate Sealer, DG 8cartridge, QX200 droplet reader, available from Bio-Rad Laboratories, Inc., for digital PCR;
enzyme reader Synergy H1, purchased from BioTek, for protein nucleic acid and ELISA absorbance determination;
leica SP5 focal microscope or Leica Aperio CS2 for immunofluorescence or immunohistochemical imaging;
microtome, available from Leica EM UC6, for electron microscopy cryosectioning;
a TEM Spirit120kV projection electron microscope (FEI Tecnai Spirit120kV) for electron microscope observation;
image J was used for data statistics and analyzed with GraghPad Prism8 statistical software, # P <0.05, # P <0.01, # P < 0.001.
7. In the following examples, the SA-. beta. -gal staining procedure for detecting cellular senescence was as follows:
SA-beta-Gal (senescent-associated beta-galactosidase) is a hydrolase in lysosomes with enhanced activity in senescent cells, which enzyme cleaves it and renders the cells blue when the substrate X-Gal is added. SA-beta-gal is therefore also a fast agentThe 'gold standard' of cell aging is rapidly and rapidly detected. The specific method comprises the following steps: 1) at 1X 105Density of wells mesenchymal precursor cells were seeded into one well of a gelatin (sigma) -coated 6-well plate and stained on day 2. 2) After completion of step 1), the cells were fixed with a fixative (2% (volume percent, v/v) formaldehyde + 0.2% (volume percent, v/v) glutaraldehyde + 97.8% (volume percent, v/v) PBS) for 4 minutes (which must not exceed 5 minutes), and washed 2 times with PBS. 3) Step 2) after completion 2mL of staining solution (40mM citrate/sodium phosphate buffer, 5mM K4[ Fe (CN) 6) was added per well]、5mM K3[Fe(CN)6]150mM NaCl, 2mM MgCl2, 1mg/mL X-gal), incubated overnight at 37 ℃ in a bacterial incubator protected from light. 4) And 3) after the step 3) is finished, washing for 2 times by PBS, observing under an inverted microscope, and taking a picture.
8. In the following examples, the names and nucleotide sequences of primers or probes used are shown in Table 1 below.
Example 1, aged mesenchymal stem cells expression level of human endogenous virus herpk is up-regulated.
Acquisition of replicative senescence wild-type and senescent mesenchymal stem cells
Culturing and passaging wild type mesenchymal stem cells in a mesenchymal stem cell culture medium according to a conventional mesenchymal stem cell culture method to obtain replicative wild type aged mesenchymal stem cells, taking the wild type mesenchymal stem cells passaged to the 3 rd or 4 th generation as early generation replicative wild type aged mesenchymal stem cells (early generation wild type or early generation, represented by EP in the drawing), taking the wild type mesenchymal stem cells passaged to the 9 th to 12 th generation as late generation replicative wild type aged mesenchymal stem cells (late generation wild type or late generation, represented by LP in the drawing),
the acquisition of the mesenchymal stem cells (HGPS for short) for the premature senility of children is as follows; human presenility mesenchymal stem cells (abbreviated as WS) are as described above. Wild-type mesenchymal stem cells which are passaged to the 8 th or 9 th generation corresponding to the children senilism mesenchymal stem cells (HGPS) and the adult senilism mesenchymal stem cells are taken as wild-type aged mesenchymal stem cells (wild type, WT in the attached drawing).
Second, RNA extraction and RNA sequencing (RNA-seq) and RNA-qPCR detection
RNA extraction
RNA was extracted from early-generation wild type (EP: P3, showing passage to 3 rd generation), late-generation wild type (LP: P9, showing passage to 9 th generation), wild type, childhood senilism mesenchymal stem cell (hereinafter abbreviated as HGPS), and adult senilism mesenchymal stem cell (hereinafter abbreviated as WS) according to the following methods, respectively: the cells were collected, rinsed with PBS, added to 1ml Trizol, and placed in a 10-well dish at room temperature to allow for sufficient lysis. Add 200. mu.L chloroform to each 1ml Trizol, mix well, stand at room temperature for 15min, centrifuge at 4 ℃ 15000g for 15 min. Taking the upper layer water phase to a zero centrifugal tube, adding 0.5ml isopropanol, mixing uniformly, standing at room temperature for 10min, and centrifuging at 4 ℃ for 10min at 10000 g. The supernatant was discarded, RNA was allowed to settle to the bottom of the tube, 1ml of pre-cooled 75% ethanol was added, the RNA pellet was floated by gentle shaking, and centrifuged at 8000g for 5min at 4 ℃. The supernatant was discarded as much as possible, air-dried at room temperature, and the RNA sample was dissolved in 30. mu.L of DEPC-treated water. The concentration was measured with microplate reader Synergy H1.
RNA sequencing and analysis: and (3) respectively taking the RNA of each cell extracted in the step (1) and sending the RNA to Novogold bioinformatics technology Limited company for library construction preparation, quality control and high-throughput sequencing. In terms of the pooling architecture, high throughput sequencing was performed according to the manufacturer's instructions using the NEBNext super RNA pool pre-base kit (NEB) and on the HiSeq X Ten platform. TrimGalore (version 0.4.5) (Babraham bioinformatics) (https:// github. com/FelixKrueger/TrimGalore) was trimmed to obtain end pairs and mapped to the human (homo sapiens) hg19 reference genome from the UCSC genome browser database using hisat2 (version 2.0.4). Then, reads mapping HTSeq to each gene (version 0.11.0) were calculated using high quality mapping reads (mapping quality scores over 20 points).
The results are shown in FIG. 1A, LTR: long terminal repeat (long terminal repeat); DNA: a DNA transposon; LINE: interspersed with repetitive sequences (LINE). By analyzing the total reverse transcription components that are up-regulated in senescent mesenchymal stem cells, LTR, DNA and LINE1 families were found to be up-regulated (left panel), and LTR was further analyzed to find that ERV1, ERVK and the like families were in the upper body in senescent cells (right panel).
2. Reverse transcription
Using the GoScript reverse transcription system, the RNA of each cell extracted in step 1 was taken as a template (2.5. mu.g), and primers (Random Primer 1. mu.L, oligo (dT))15 Primer 1. mu.L) and nuclease-free water to 10. mu.L, and performing pre-denaturation at 70 ℃ for 5min, taking out and placing on ice. Configuring RT-Mix (nuclease-free water 1.6 mu L, GoScript 5X Reaction Buffer 4 mu L, MgCl)22 μ L, PCR Nucleotide Mix 1 μ L, RNase inhibitor 0.4 μ L and GoScript reverse transcriptase 1 μ L), 10 μ L per sample was added. Setting a reverse transcription program: incubating at 25 deg.C for 5min, at 42 deg.C for 60min, at 70 deg.C for 15min, and cooling at 4 deg.C.
3. Instant polymerase chain reaction (qPCR)
Respectively taking each reverse transcription product obtained in the step 2 as a template, taking a primer pair (HERVK-Env, HERVK-Gag, HERVK-PolP21 or LAP2b) in the table 1 as a primer, and carrying out qPCR detection, wherein the specific steps are as follows: the obtained reverse transcription product was diluted 10 times, qPCR-Mix (template 2.1. mu.L, primer set (10. mu.M) 0.4. mu.L, THUNDERBIRD qPCR Mix 2.5. mu.L) was prepared according to the following reaction System, and detection was performed using CFX384 Real-Time System. The reaction conditions were as follows: pre-denaturation at 95 ℃ for 10min, PCR amplification: 95 ℃ 15sec, 60 ℃ 1min, 40 cycles, dissolution profile: 95 ℃ for 15sec, 60 ℃ for 1min, 95 ℃ for 15 sec.
The results are shown in fig. 1B, wherein hMPC represents human mesenchymal stem cells; EP represents the early generation; LP represents late generation; WT means a wild type; HGPS indicates premature senility in children; WS represents premature aging in adults; HERVK-Env/HERVK-pol/HERVK-gag the primer pair names for different regions of HERVK; p21Cip1And LAP2(TMOP), senescence-associated genes. RT-qPCR showed an upregulation of HERVK RNA level expression in the presenile mesenchymal stem cells (HGPS and WS) compared to wild type, while simultaneouslyHERVK RNA levels were elevated in later generations (LP: P9) of wild-type mesenchymal stem cells compared to earlier generations (EP: P3) of wild-type.
Three, RNA fluorescence in situ hybridization (RNA-FISH) detection
By using
HERVK-Env RNA levels in mesenchymal stem cells (WT, HGPS, WS, and replicative senescence wild-type early (EP: P4, passage to passage 4), late (LP: P12, passage to passage 12) were examined by HERVK RNA fluorescence in situ hybridization of the ViewRNA ISH Cell Assay Kit and probe HERVK Alexa Fluor 488.
The RNA fluorescence in situ hybridization comprises the following specific steps:
1. the above various mesenchymal stem cell slides were washed 2 times with PBS buffer, fixed with freshly prepared fresh 4% formaldehyde solution (1.08ml 37% formaldehyde stock and 8.92ml PBS) for 30min at room temperature, and washed three times with PBS buffer.
2. Add 400. mu.L of detegent Solution QC for 5min at room temperature and wash twice with PBS buffer.
3. 400 μ L of the Working Protease Solution (Protease QS diluted 1: 2000 with PBS) was added, digested for 10min at room temperature, and washed three times with PBS buffer.
4. mu.L of the HERVK Working Probe Set Solution (diluted with 396. mu.L of Probe Set dilution QF (pre-warmed at 40 ℃) 4. mu.L of Probe HERVK Alexa Fluor 488) was added, incubated in the hybridization oven at 40 ℃ for 3hr, washed 3 times with Wash Buffer for 2min each.
5. mu.L of the Working PreAmplifier Mix Solution (16. mu.L PreAmplifier Mix diluted with 384. mu.L Amplifier Diluent QF (pre-warmed at 40 ℃) was added to the pre-amplified signal, incubated in a hybridization oven at 40 ℃ for 30min and washed 3 times with Wash Buffer for 2min each.
6. mu.L of the amplification signal of the Working Amplifier Mix Solution (16. mu.L of the Amplifier Mix diluted with 384. mu.L of the Amplifier dilution QF (pre-warmed at 40 ℃) was added, incubated in a hybridization oven at 40 ℃ for 30min and washed 3 times with Wash Buffer, 2min each.
7. Add 400. mu.L of the Working Label Probe Mix Solution (diluted with 384. mu.L of Label Probe dilution QF (pre-warmed at 40 ℃) in 16. mu.L of Label Probe Mix), protected from light, incubated in the hybridization oven at 40 ℃ for 30min, washed 3 times with Wash Buffer, 2min for the first time and 10min for the second time.
8. Add 400. mu.L of Working DAPI Solution (4. mu.L of 100X DAPI diluted with 396. mu.L of PBS), avoid light, incubate at room temperature for 1min, Wash 1 time with Wash Buffer.
9. By using
Fade resistant mounting media was mounted and images were obtained using a confocal laser scanning microscope (Leica TCSSP5 ii).
The detection result of the RNA fluorescence in situ hybridization is shown as C and D in figure 1, and the HERVK probe in the figure C has green fluorescence; DNA, genome staining Hoechst and blue fluorescence; WT: a wild type; HGPS: premature senility of children; WS: premature senility syndrome of adults; and (3) on the right: and counting the green fluorescence intensity of the HERVK probe. RS hMPC in panel D replicative senescent mesenchymal stem cells. Early generation of EP early passage; and (3) LP: late passage late generation. And on the right: the HERVK probe green fluorescence intensity was counted. It can be seen from fig. 1C that the herpk RNA fluorescence intensity of the senilis mesenchymal stem cells (HGPS and WS) was increased compared to the wild type; meanwhile, as shown in FIG. 1D, HERVK RNA fluorescence intensity of the wild type (LP: P12) mesenchymal stem cells of later generation (representative picture in the left figure and statistical result in the right figure) is increased compared with the wild type of earlier generation (EP: P4).
Fourthly, detection by Western blot
The HERVK-Env protein expression levels in the mesenchymal stem cells (WT, HGPS, WS, and replicative senescence wild-type early generation (EP: P4, representing passage to passage 4) and late generation (LP: P12, representing passage to passage 12) were counted.
Mesenchymal stem cells (WT, HGPS, WS, and replicative senescence wild-type early (EP: P4, which indicates passage to 4 th passage) and late (LP: P12, which indicates passage to 12 th passage) were placed in a 1% SDS solution, and sufficiently lysed using a tissue grinder, heated to 105 ℃ for 10min to sufficiently denature the protein, and then Western blot (with the GAPDH expression level as a reference) was performed on the same amount of protein to obtain HERVK-Env protein expression levels in the mesenchymal stem cells (WT, HGPS, WS, and replicative senescence wild-type early (EP: P4, which indicates passage to 4 th passage) and late (LP: P12, which indicates passage to 12 th passage), respectively.
The Western blot comprises the following specific steps:
1. an appropriate amount of concentrated protein loading buffer (Dingguo) was added to an equivalent amount of protein sample.
After the step 1 is completed, the prepared protein sample is loaded into SDS-PAGE gel holes for protein electrophoresis experiments.
2. Electrophoresis was stopped when bromophenol blue in protein loading buffer reached near the bottom end of the gel.
3. Protein membrane transfer was performed using a membrane transfer apparatus for 2 hours.
4. Blocking was performed immediately with skim milk powder at room temperature for two hours.
5. Primary antibody incubations were performed with the herpk-Env antibody and the reference protein antibody GAPDH, respectively, at 4 ℃, overnight.
6. Incubations were performed with the corresponding resistant protein secondary antibody at room temperature for 1 hour. Development was carried out immediately after washing.
The result of the Western blot experiment is shown in E and F in figure 1, wherein hMPC is human mesenchymal stem cells in figure E; WT: a wild type; HGPS: premature senility of children; WS: premature senility syndrome of adults; HERVK-Env HERVK coat protein; p16INK4aAnd LAP2(TMOP), senescence-associated proteins; GAPDH: internal reference protein; the left side is a representative picture, and the right side is the statistical HERVK-Env protein expression level; RS hMPC in panel F replicative senescent mesenchymal stem cells. Early generation of EP early passage; and (3) LP: late passage late generation; HERVK-Env HERVK coat protein; p16INK4aAnd LAP2(TMOP), senescence-associated proteins; GAPDH: an internal reference protein; representative pictures are shown on the left, and HERVK-Env protein expression levels are counted on the right. E in FIG. 1 shows that HERVK protein level expression of HGPS and WS mesenchymal stem cells is up-regulated compared to wild type, while HERVK protein level of wild type mesenchymal stem cells of later generations (EP: P12) is increased compared to wild type of earlier generations (EP: P4) as described in FIG. 1-F.
Fifth, immunofluorescence assay
Immunofluorescence assays were performed on mesenchymal stem cells (WT, HGPS, WS, and replicative senescence wild-type early (EP: P4, for passage to passage 4), late (LP: P12, for passage to passage 12), respectively, and slides of mesenchymal stem cells were counted on average, each containing three slides, each slide taking at least 100 cells for quantification.
1. Mesenchymal stem cells (WT, HGPS, WS, and replicative senescence wild-type early (EP: P4, for passage to passage 4) and late (LP: P12, for passage to passage 12) were slide-fixed with 4% (m/v) paraformaldehyde solution at room temperature for 30min, washed twice with PBS buffer, permeabilized with 0.4% (v/v) TritonX-100 diluted with PBS buffer for 25min, and washed twice with PBS buffer.
2. Blocking with 10% donkey serum blocking buffer diluted in PBS buffer for 1h at room temperature and staining with primary anti-HERVK-Env antibody overnight at 4 ℃.
3. The primary antibody was washed 3 times with PBS buffer for 10min each, incubated with the corresponding secondary antibody at room temperature for 1h, and the secondary antibody was washed off 3 times with PBS buffer for 10min each.
4. The staining was carried out by Hoechst 33342, washed with PBS buffer, and then washed with PBS buffer
Fade resistant mounting media was mounted and images were obtained using a confocal laser scanning microscope (Leica TCS SP5 ii).
The immunofluorescence detection result is shown in G and H in figure 1, wherein in the figure G, hMPC is human mesenchymal stem cells; HERVK-Env protein is marked by green fluorescence; DNA, genome staining Hoechst and blue fluorescence; WT: a wild type; HGPS: premature senility of children; WS: premature senility of adults; the left panel is a representative picture and the right panel is a statistical HERVK-Env green fluorescence intensity. In Panel H, HERVK-Env protein was labeled with green fluorescence; DNA, genome staining Hoechst and blue fluorescence; RS hMPC is replication aging mesenchymal stem cell. Early generation of EP early passage; and (3) LP: late passage late generation; and on the right: and counting the HERVK-Env green fluorescence intensity. The HERVK-Env protein fluorescence intensity of the mesenchymal stem cells of the premature senility (HGPS and WS) is increased compared with the wild type, and the HERVK-Env protein fluorescence intensity of the mesenchymal stem cells of the later generation (EP: P12) is increased compared with the wild type of the early generation (EP: P4) as shown in figure 1-H.
According to the RNA-seq data analysis of the children early senescence mesenchymal stem cell HGPS, the adult early senescence mesenchymal stem cell WS and the replicative wild type aged mesenchymal stem cell (the replicative wild type aged mesenchymal stem cell is obtained by continuous passage of the wild type mesenchymal stem cell, and comprises a wild type, a premature generation and a late generation), the endogenous virus family including HERVK is found to be up-regulated in the children early senescence mesenchymal stem cell HGPS, the adult early senescence mesenchymal stem cell WS and the replicative aged mesenchymal stem cell. Expression of HERVK RNA levels was found to be upregulated in senescent cells using RT-qPCR and RNA fluorescence in situ hybridization (RNA-FISH) experiments. Meanwhile, the HERVK expressed viral protein level is increased through immunoblotting and immunofluorescence experiments. The HERVK can be used as a marker of mesenchymal stem cell aging.
Example 2 aged mesenchymal stem cells human endogenous virus herdk forms viral particles.
As mentioned above herpk viral RNA and protein expression is upregulated in senescent cells, the number of similar viral particles was found to increase in the presenility and replicative senescence mesenchymal stem cells using ordinary electron microscopy. The HERVK-Env antibody is used as an immune colloidal gold electron microscope marker, and the virus formed by the mesenchymal stem cells of the premature senility and the replicative senescence is found to be HERVK virus particles, and the number of the virus in the senescent cells is increased. Further illustrates that the endogenous virus HERVK can be used as a marker of mesenchymal stem cell senescence.
First, ordinary projection electron microscope detection
Similar viral particles in the wild-type early (EP: P4, representing passage to passage 4) and late (LP: P12, representing passage to passage 12) generations of (WT, HGPS, WS, and replicative senescence were counted using a common projection electron microscope, at least 50 cells per cell were counted for counting.
1. After the cells were washed with PBS, the cell pellet was fixed with 2.5% (vol/vol) glutaraldehyde diluted with Phosphate Buffer (PB) (0.1M, pH7.4) at 4 ℃ overnight.
2. After four washes with PBS buffer, cells were fixed with 1% (wt/vol) osmium tetroxide (PBS dilution) at 4 ℃ for 2 hr.
3. Dehydration was performed by ethanol gradient (30%, 50%, 70%, 80%, 90%, 100%, 7min each time in sequence) and finally twice in pure acetone for 10min each time.
4. The samples were immersed in a gradient mixture (mixing ratio 3: 1, 1: 3 in order) of acetone and SPI-PON812 resin (16.2g SPI-PON812, 10g DDSA, and 8.9g NMA), and then in the pure resin. Finally, the cells were embedded in pure resin with 1.5% BDMA and polymerized at 45 ℃ for 12hr and 60 ℃ for 48 hr.
5. Ultrathin sections (70nm thick) were sectioned with a microtome (Leica EM UC6), double stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (FEI Tecnai Spirit120 kV).
The detection results of the ordinary projection electron microscope are shown in A and B in figure 2, in the A, hMPC is human mesenchymal stem cells; WT: a wild type; HGPS: premature senility of children; WS: premature senility syndrome of adults; circled is the structure of the virus-like particle, the right panel is an enlarged view circled, rightmost: counting the number of virus particle-like structures per cell in each cell type; in panel B, RS hMPC is a replicating senescent mesenchymal stem cell. Early generation of EP early passage; and (3) LP: late passage late generation. Circled is a virus particle-like structure, the right panel is a circled enlarged view, right-most: the number of virus particle-like structures in each cell type was counted. As shown in FIG. 2A, the number of similar virions was increased in the mesenchymal stem cells of the premature senility (HGPS and WS) compared with the wild type, while as shown in FIG. 2B, the number of similar virions was increased in the mesenchymal stem cells of the late generation (EP: P12) compared with the wild type of the early generation (EP: P4).
Second, immune colloidal gold electron microscope detection
HERVK virus particles were labeled by using HERVK-Env antibody as an immune colloidal gold electron microscope, HERVK virus particles in mesenchymal stem cells (WT, HGPS, WS, and wild type early generation (EP: P4, which indicates passage to 4 th generation) and late generation (LP: P12, which indicates passage to 12 th generation) were counted, and at least 50 cells were counted for each cell for counting, and the immune colloidal gold electron microscope was specifically performed as follows:
1. cells seeded in a 35mm petri dish (CORNING) were previously fixed with 50% 4% formaldehyde + 50% pre-warmed medium in a cell culture chamber for 10 minutes and fixed with 4% formaldehyde for 1hr at room temperature and overnight at 4 ℃, and then changed to 1% formaldehyde.
2. Samples were dehydrated in ethanol at 30% (4 ℃,10 min), 50% (4 ℃,10 min), 50% (-20 ℃, 30min), 70% (-20 ℃, 30min), 90% (-20 ℃, 45min), 100% (-20 ℃, 20min, three times), respectively.
3. Then, the mixture was polymerized with 25% LR Gold resin (-20 ℃,60 min), 50% LR Gold resin (-20 ℃,60 min), 75% LR Gold resin (-20 ℃,60 min), 100% LRgold resin (-20 ℃, overnight) and LR Gold resin containing initiator (-20 ℃,60 min) for 24hr under UV at-20 ℃ by using Leica AFS2, by filling the mixture in capsules containing LR Gold initiator.
4. 70nm sections were cut using Leica EM UC7 and then picked up with a nickel grid with Formvar/carbon film.
5. Sections were blocked with 2% BSA (Jackson ImmunoResearch) for 1hr at room temperature.
6. Anti-HERVK-env (Austral biologicals) antibody was diluted 1:10 and incubated with sections for 2hr, followed by washing 5 times with 0.1% precooled Gelatin in PBS buffer at room temperature for 2 min.
7. Sections were incubated with 6nm colloidal gold labeled anti-mouse secondary antibody (Jackson ImmunoResearch) for 1h, washed with PBS buffer for 2min and 5 times.
8. Then fixed with 1% glutaraldehyde (diluted with ddH 2O). After washing with ddH2O, sections were stained with 2% uranyl acetate for 30min at room temperature and then imaged using TEMSpirit120 kV.
The result of the immune colloidal gold electron microscope is shown in C and D in figure 2, wherein, hMPC is human mesenchymal stem cells in the figure C; WT: a wild type; HGPS: premature senility of children; WS: premature senility syndrome of adults; circled is the structure of a similar viral particle that can be tagged with the HERVK-Env protein, the right panel is a circled enlarged view, right-most: counting the number of virus-like particle structures that can be tagged with HERVK-Env protein per cell in each cell type; RS hMPC in panel D replicative senescent mesenchymal stem cells. EP of the early generation; and (3) LP: and (4) late generation. Circled is the structure of a similar viral particle that can be tagged with HERVK-Env protein, the right panel is an enlarged view circled, right-most: the number of structures of similar virus particles that can be labeled with HERVK-Env protein in each cell type was counted. The HERVK virion number of the mesenchymal stem cells of the early senescence (HGPS and WS) was increased compared to the wild type as shown in FIG. 2C, while the HERVK virion number of the wild type mesenchymal stem cells of the late generation (EP: P12) was increased compared to the wild type of the early generation (EP: P4) as shown in FIG. 2D. FIG. E is a graph showing the staining results of a negative control with colloidal gold immunolabeling without HERVK-Env antibody.
Example 2 thus further demonstrates that the endogenous virus herpk can serve as a marker for mesenchymal stem cell senescence.
Example 3, senescent fibroblasts herdk are up-regulated and form viral particles.
Fibroblasts were purchased from LONZA under the culture conditions described above. Culturing and passaging the fibroblasts in a fibroblast culture medium according to a conventional fibroblast culture method to obtain replicative senescence fibroblasts, taking the fibroblasts passaged to 11 th or 12 th generation as early generation replicative senescence fibroblasts (called early generation for short, and denoted by EP in the attached drawing), and taking the fibroblasts passaged to 22 nd to 24 th generation as late generation replicative senescence fibroblasts (called late generation for short, and denoted by LP in the attached drawing).
The specific western blotting steps are as described above, and the experimental results are shown in 3A, wherein RS Fibroblast is replicative senescent Fibroblast; early generation of EP early passage; and (3) LP: late passage late generation; HERVK-Env HERVK coat protein; p16INK4aAnd LAP2(TMOP), senescence-associated protein; GAPDH: an internal reference protein; and on the right: and (5) counting the expression level of HERVK-Env protein. Western blot shows that expression of herpk-Env protein is up-regulated in late-generation fibroblasts compared to early-generation fibroblasts.
Immunofluorescence the specific procedures were as above, and the experimental results are shown in 3B, wherein RS Fibroplast is replicative senescent fibroblasts; early generation of EP early passage; and (3) LP: late passage late generation; HERVK-Env protein is marked by green fluorescence; DNA, genome staining Hoechst and blue fluorescence; and on the right: and counting the HERVK-Env green fluorescence intensity. Immunofluorescence showed an increase in HERVK-Env fluorescence intensity in later-passages as compared to earlier-passages.
The specific steps of the ordinary electron microscope are as described above, and the experimental result is shown in 3C, wherein RS Fibroplast is replicative senescent Fibroblast; early generation of EP early passage; and (3) LP: late passage late generation; circled is a virus particle-like structure, the right panel is a circled enlarged view, right-most: the number of virus particle-like structures in each cell type was counted. Conventional electron microscopy increases the number of similar viral particles in later-generation fibroblasts compared to earlier-generation fibroblasts.
Example 4, aged mesenchymal stem cells release herpk viral particles into the culture medium.
The existence of more HERVKRNA in the culture medium of the premature mesenchymal stem cells is detected by digital PCR, the existence of more HERVK-Env viral proteins in the culture medium of different mesenchymal stem cells is detected by using a Human HERVK-7 p22.1provirus antibiotic Env polyprotein (ERVK6) ELISA kit, and HERVK viral particles exist outside partially aged cells through the discovery of the ordinary electron microscope and an immune colloidal gold electron microscope, so that the HERVK level and the number of the viral particles in the aged mesenchymal stem cells are increased, the extracellular ERVK level and the number of the viral particles are also increased, and the endogenous HERVK can be used as a marker for detecting the aging of the mesenchymal stem cells.
Preparation of a culture medium: the senilism childhood mesenchymal stem cells, adult senilism mesenchymal stem cells, wild-type mesenchymal stem cells, early generation wild-type mesenchymal stem cells (EP: P4), and late generation wild-type mesenchymal stem cells (LP: P12) in example 1 were cultured in a vesicle serum-free medium (as configured below) for two days according to a conventional culture method, and then the cells were isolated to obtain a medium for senilism childhood mesenchymal stem cells (HGPS-CM), a medium for adult senilism mesenchymal stem cells (WS-CM), a medium for wild-type mesenchymal stem cells (WT-CM), a medium for early generation wild-type mesenchymal stem cells (EP-CM), and a medium for late generation wild-type mesenchymal stem cells (LP-CM), respectively. The resulting culture media were tested as follows.
One, digital PCR detection
The method utilizes digital PCR to count HERVKRNA in a culture medium of the mesenchymal stem cells of the premature senility symptoms of the children, a culture medium of the mesenchymal stem cells of the adult premature senility symptoms and a culture medium of the wild mesenchymal stem cells, and each cell has three biological repetitions.
The digital PCR detection comprises the following specific steps:
1. obtaining the culture Medium
(1) Preparation of vesicle-free serum medium: serum was centrifuged at 100,000g for 16hr at 4 ℃ to remove vesicles from the serum. The medium was prepared with vesicle-removed serum.
(2) The cells were cultured with vesicle-free medium for 48hr, the medium was collected, and the cells were counted. The culture medium for respectively obtaining the senescence according to the method comprises a culture medium (HGPS-CM) for obtaining the mesenchymal stem cells of the premature senility symptoms of children, a culture medium (WS-CM) for the mesenchymal stem cells of the adult premature senility symptoms, a culture medium (WT-CM) for the wild mesenchymal stem cells of the early generation, a culture medium (EP-CM) for the wild mesenchymal stem cells of the late generation and a culture medium (LP-CM) for the wild mesenchymal stem cells of the late generation.
(3) The same number of cells from which the culture medium was derived was counted for the extraction of RNA from vesicles released from the cells. The medium was centrifuged at 300g for 10min to remove cell debris, 15,000g at 4 ℃ for 20min and filtered through a 0.2 μm filter (Pohl) and ultracentrifuged at 110,000g for 2 hr. The vesicles were resuspended with the same volume of PBS and subjected to RNA extraction or western blotting.
2. Extraction of RNA from culture Medium
(1) RNA in vesicles in the medium was extracted using QIAamp viral RNA mini kit. Firstly, according to the number of samples, an AVL buffer solution and a carrier RNA-AVE buffer solution mixture are prepared. And adding 560. mu.L of the mixture of AVL buffer and carrier RNA-AVE buffer into 140. mu.L of the extracted medium vesicles, mixing uniformly, and standing at room temperature for 10 min. 560. mu.L of ethanol was added and mixed well. And taking 630 mu L of the mixture into a QIAamp Mini centrifugal column, centrifuging at 8000rpm for 1min, discarding the collecting tube, then replacing the collecting tube with a new collecting tube, adding the rest 630 mu L of the mixture into the QIAamp Mini centrifugal column, repeating the step, discarding the collecting tube, and then replacing the collecting tube with the new collecting tube. Add 500. mu.L Buffer AW1 to QIAamp Mini spin column, centrifuge at 8000rpm for 1min, discard the collection tube and replace it with a new collection tube. Add 500. mu.L Buffer AW2 to QIAamp Mini spin column, centrifuge for 3min at 14000 rpm. And (3) removing the collection tube, replacing a new Eppendorf collection tube, adding 40 mu L of Buffer AVE, standing at room temperature for 1min, and centrifuging at 8000rpm for 1min to elute the RNA in the vesicles in the culture medium.
(2) Excess DNA was removed using DNase I using TURBO DNA-free kit. mu.L of purified viral RNA was digested at 37 ℃ for 30min by adding 2. mu.L of DNase I and 2.4. mu.L of reaction buffer. DNase was inactivated and removed by addition of 2.4. mu.L of the inactivation reagent provided in the kit and centrifuged at 10,000g for 1.5 min. The supernatant was taken and heated at 70 ℃ for 10min to eliminate any residual DNase activity.
3. Digital PCR
(1) The purified viral RNA was reverse transcribed into cDNA using the GoScript reverse transcription system as described above.
(2) The power supply behind the QX200 Droplet Reader is turned on, and the computer and QuantaSoft software are turned on after preheating for at least 30 min.
(3) Preparing a 20-mu-L dye method quantitative reaction system: the template cDNA was 5. mu.L, the primer set (10. mu.M) was 0.4. mu.L, the 2XQX200 ddPCR EvaGreen Supermix was 10. mu.L, and nuclease-free water was 4.6. mu.L.
(4) A new DG 8cartridge was placed in the holder and 20. mu.L of the sample reaction was added to the middle row of 8 wells of DG8 cartridges, and the liquid was slowly decanted to avoid the introduction of air bubbles.
(5) 70 μ L of droplet generation Oil (DG Oil) was added to the bottom row of 8 wells of DG 8cartridge, and there were no empty wells.
(6) Gently placing the above holder in a microdroplet generator, covering with a rubber pad (gasket), and starting to generate microdroplets
(7) Carefully aspirate 40. mu.L down to 96-well plates slowly with a pipette tip.
(8) After transferring the oil drop and accomplishing in getting into 96 orifice plates, the one side (the bright face of reflection) that has the red line mark with the membrane is upwards placed on 96 orifice plates and is fixed, seals the membrane to it with the PX1 heat-seal appearance that preheats, and the operation procedure is: 180 ℃ for 6 s.
(7) After the membranes were sealed, amplification was completed on a 96-well PCR instrument. Reaction program, enzyme activation, 95 ℃,10 min; denaturation at 94 ℃ for 30 sec; annealing and extending, 60 ℃, 1min, and 40 cycles; inactivating enzyme at 98 deg.C for 10min, and keeping the temperature rising and lowering speed at2 deg.C/s.
(8) The 96-well plate was placed in a QX200 Droplet Reader to detect the signal.
The digital PCR results are shown in FIG. 4-A, where the ordinate: amplifying chromosomes; the abscissa: the number of events; ddH 2O: negative control; cDNA: a positive control; WT-CM which is a culture medium derived from wild-type mesenchymal stem cells; HGPS-CM: a culture medium derived from mesenchymal stem cells of the premature senility syndrome of the children; WS-CM derived from the culture medium of adult presenility mesenchymal stem cells; RNA levels of the culture species herdk of different cells were counted on the right. The culture medium of the same number of premature aging (HGPS and WS) mesenchymal stem cells contained more herpk viral RNA than the wild type.
Second, ELISA kit detection
The HERVK-Env virus protein levels in a culture medium of a childhood early senescence mesenchymal stem cell, a culture medium of an adult early senescence mesenchymal stem cell, a wild-type mesenchymal stem cell culture medium, a culture medium of an early generation wild-type mesenchymal stem cell and a culture medium of a late generation wild-type mesenchymal stem cell are counted by using a Human HERVK-7 p22.1Provirus antibiotic Env polypredein (ERVK6) ELISA kit, and each cell is subjected to three biological repetitions.
The HERVK-Env ELISA detection method comprises the following specific steps:
1. concentrated culture medium
Five media from the same number of sources were obtained as described above. The medium was concentrated using a Centricon Plus-70(Millipore) concentration tube. The tube was first activated by adding 70ml of ddH2O to the tube, and centrifuged at 4000g for 5min at 4 ℃. Adding the filtered culture medium into a concentration tube, and centrifuging at 4000g and 4 deg.C for 1 hr. The concentrated sample was collected by inverting the tube at 4000g and centrifuging at 4 ℃ for 5 min.
HERVK-Env ELISA assay
(1) mu.L of the concentrated medium was added to Human HERVK-7 p22.1Provirus antibiotic Env polypredin (ERVK6) ELISA plates and incubated at 37 ℃ for 2 hr.
(2) Add 100. mu.L Biotin-antibody, incubate 1hr at 37 ℃.
(3) Add 200. mu.L Washing Buffer for Washing 2min each time, 3 times in total.
(4) 100 μ L of HRP-avidin was added and incubated at 37 ℃ for 30 min.
(5) Add 200. mu.L Washing Buffer for Washing 2min each time, 5 times total Washing.
(6) Add 90. mu.L of TMB substrate and incubate for 30min at 37 ℃ in the dark.
(7) Adding 50 μ L of stop solution, and measuring absorbance at 450nm with microplate reader Synergy H1.
The results of the ELISA experiments of Human HERVK-7 p22.1Provirus antibiotic Env polypredin (ERVK6) are shown in FIGS. 4B and C, wherein WT-CM in FIG. B is a culture medium derived from wild-type mesenchymal stem cells; HGPS-CM: a culture medium derived from mesenchymal stem cells of the presenility of children; WS-CM derived from the culture medium of adult presenility mesenchymal stem cells; performing ELISA statistics on HERVK-Env protein levels of culture media from different cell sources; RS hMPC in panel C is replicative senescent mesenchymal stem cells. The EP-CM is a culture medium of early passaged conditioned medium early-generation wild mesenchymal stem cells; LP-CM: a culture medium of late passageconditioned medium late-generation wild-type mesenchymal stem cells. And (3) carrying out ELISA statistics on the HERVK-Env protein level of the culture medium of the replicative senescence early-late generation wild-type mesenchymal stem cells. FIG. 4B shows that the culture medium of the same number of mesenchymal stem cells of premature senility (HGPS and WS) contains more HERVK-Env viral proteins than the wild type, and as shown in FIG. 4-C, the culture medium of the same number of mesenchymal stem cells of late generation (LP: P12) contains more HERVK-Env viral proteins than the wild type of early generation (EP: P4).
Third, detection by electron microscope
Similar virus particles in the culture medium of the senilism mesenchymal stem cells of children, the culture medium of the adult senilism mesenchymal stem cells, the culture medium of the wild-type mesenchymal stem cells, the culture medium of the early generation wild-type mesenchymal stem cells and the culture medium of the late generation wild-type mesenchymal stem cells were counted according to the methods of the ordinary electron microscope and the immune colloidal gold electron microscope in example 2. Common electron microscope is shown in D and E in FIG. 4, wherein, in D, hMPC is human mesenchymal stem cell; WT: a wild type; HGPS: premature senility of children; WS: premature senility syndrome of adults; circled is the secretion of virus particle-like structures outside the cell, and the right panel is an enlarged circled view; RS hMPC in panel E replicative senescent mesenchymal stem cells. Early generation of EP early passage; and (3) LP: late passage late generation. Circled is secretion of the structure of the analogous viral particle outside the cell, the right panel is circled enlarged showing the analogous viral particle near or outside the cell membrane in partially senescent (HGPS and WS, fig. 4C) and replicative (late P12 wild-type, fig. 4E) senescent mesenchymal stem cells; the same immune colloidal gold electron microscope as F and G in figure 4, wherein, hMPC is human mesenchymal stem cell in figure F; WT: a wild type; HGPS: premature senility of children; WS: premature senility of adults; circled is the secretion of the structure of the virus-like particle that can be tagged with HERVK-Env protein outside the cell, the right panel is circled in an enlarged view; RShMPC in Panel G, replicating senescent mesenchymal stem cells. Early generation of EP early passage; and (3) LP: late passage late generation. Circled is the secretion of the structure of the virus-like particle that can be tagged with HERVK-Env protein outside the cell, the right panel is circled in an enlarged view. HERVK virions were shown to be located near the cell membrane or outside the cell in partially senescent (HGPS and WS, FIG. 4F) and replicative (late generation P12 wild type, FIG. 4G) senescent mesenchymal stem cells.
Thus example 3 further demonstrates that endogenous virus HERVK can be released extracellularly and can serve as a marker of mesenchymal stem cell senescence.
Example 5, premature aging and naturally aging mice in human endogenous virus HERVW expression up-regulated.
The mice used in the present invention were products of sbeft (beijing) biotechnology limited, and were housed in animal welfare facilities of the chinese centers for disease prevention and control, which had a 12-hour day-night cycle at room temperature of 23 ℃ and had free access to food and water. All animal experiments were approved by the animal welfare committee of the institute of animal research, academy of sciences, china. The level of herpk in mice was tested in the livers of mice 2 months (5), 8 months (5) and 24 months (5) old for the homologous virus MMTV.
RNA extraction and RT-qPCR detailed procedures were as described above, and the results are shown in FIG. 5, wherein MMTV: HERVK is a homologous virus in mouse species; MMTV-1: MMTV primer pair No. 1; MMTV-2: MMTV primer pair No. 2; the abscissa is 2 months, 8 months and 24 months sized mouse liver tissue. The results showed that the expression of MMTV was up-regulated in aged mice (24 months old) compared to young mice (2 months or 8 months old).
Example 6 expression of human endogenous virus HERVW in premature and naturally aging cynomolgus monkeys is upregulated.
The level of the endogenous virus HERVW was detected in the lung, liver and skin of wild-type and early-aging HGPS cynomolgus monkeys using a laboratory-established model of early-aging HGPS cynomolgus monkeys (the model preparation process can be referred to in Wang, F.et al., Generation of a Hutchinson-Gilford progeria syndrome monkey model by base edition. protein & cell, doi:10.1007/s13238-020-00740-8(2020)).
Ordinary cynomolgus monkeys of the same age are used as wild cynomolgus monkeys, and the premature senility HGPS cynomolgus monkeys are prepared by the method in Wang, F.et al.Generation of a Hutchinson-Gilford progeria syndrome monkey by base edition, protein & cell, doi:10.1007/s 13238-020-; wild type cynomolgus monkeys and early-aging HGPS cynomolgus monkeys are fed in the same way according to the general feeding mode of the cynomolgus monkeys until the age is 6-12 months old (since the early-aging HGPS cynomolgus monkeys die in the first year), 2 wild type cynomolgus monkeys and 2 early-aging HGPS cynomolgus monkeys are taken, and the level of endogenous virus HERVW is detected in the lung, the liver and the skin of the cynomolgus monkeys. Simultaneously, 8 wild-type cynomolgus monkeys (young cynomolgus monkeys) of 4-6 years old and 8 naturally aged 18-21 year old wild-type cynomolgus monkeys (old cynomolgus monkeys) were selected and tested for the level of endogenous virus HERVW in the lungs, liver and skin. The specific test method is as follows:
first, detection by Western blot
And (4) counting the expression quantity of HERVW-Env protein in the lung of the wild type HGPS, the premature senility HGPS and the young cynomolgus monkey and the old cynomolgus monkey.
Respectively taking 8 wild type cynomolgus monkeys (young cynomolgus monkeys) of 4-6 years old, 8 naturally aged 18-21 years old, 1 wild type cynomolgus monkeys of 6-12 months old and 1 premature senility HGPS cynomolgus monkeys, placing the tissue blocks in 1% SDS solution, fully cracking the tissue blocks by using a tissue grinder, and heating to 105 ℃ for 10min to fully denature proteins. Then taking equivalent protein to carry out Western blot (taking GAPDH expression as reference, the concrete method is the same as above) respectively, and carrying out HERVW-Env protein expression quantity in the mesenchymal stem cells of the premature senility and the replicative senescence.
The results of the Western blot experiments are shown in B and E in FIG. 6, wherein WT: a wild-type cynomolgus monkey; HGPS: premature senility syndrome cynomolgus monkey; lung is Lung; ERVW-Env is a virus homologous to HERVK in monkey species; P-P65 inflammation-related protein; GAPDH: internal reference; right side: counting the protein level of ERVW-Env in wild type and presenility cynomolgus monkey lung species; young in fig. E: young cynomolgus monkeys; old: old cynomolgus monkey; lung is Lung; ERVW-Env is a virus homologous to HERVK in monkey species; P-P65 inflammation-related protein; GAPDH: internal reference; right side: and (4) counting the protein level of ERVW-Env in the lung of the young and old cynomolgus monkey. As shown in fig. 6B, the premature senility HGPS cynomolgus monkey lung HERVW-Env protein level expression is increased compared to the wild type cynomolgus monkey, while as shown in fig. 6E, the old cynomolgus monkey lung HERVW-Env protein level expression is increased compared to the young cynomolgus monkey (the left figure in the figure is a representative picture, and the right figure is a statistical result).
Second, immunohistochemical detection
Respectively taking lung tissues, liver tissues and skins of a young 4-6 year-old wild type cynomolgus monkey (young cynomolgus monkey) and a naturally aged 18-21 year-old wild type cynomolgus monkey (old cynomolgus monkey) and a 6-12 month old wild type cynomolgus monkey and a premature senility disease HGPS (HGPS) cynomolgus monkey to prepare paraffin slides, preparing 2 slides of each of the 6-12 month old wild type cynomolgus monkey and the premature senility disease HGPS cynomolgus monkey in each group, repeatedly preparing 4 slides of each of the wild type cynomolgus monkey and the premature senility cynomolgus monkey, and preparing 2 slides of each of the young cynomolgus monkey and the old cynomolgus monkey in each group by 8 slides of each of the young cynomolgus monkey and the old cynomolgus monkey.
The ERVW-Env signal intensity in each of the above products was statistically counted by immunohistochemistry.
The immunohistochemical method comprises the following specific steps:
1. dewaxing and hydrating: slicing paraffin into fresh xylene, soaking for 10min for 3 times; removing excessive liquid, soaking in 100% anhydrous ethanol for 5min for 2 times; soaking in 95% ethanol for 5 min; soaking in 90% ethanol for 5 min; soaking in 80% ethanol for 5 min; soaking in 75% ethanol for 5 min; soaking in 50% ethanol for 5 min; ddH2O, soak for 5min,2 times.
2. Antigen retrieval: placing in antigen repairing solution, and heating with microwave oven for 3min, 3 times, each time at an interval of 1 min.
3. Blocking endogenous peroxidase: adding appropriate amount of endogenous peroxidase blocker, incubating at room temperature for 15min, and washing with PBS for 5min for 3 times.
4. Then 0.4% (v/v) Triton X-100 diluted with PBS buffer was permeabilized for 1hr, and washed twice with PBS buffer.
5. Blocking with 10% donkey serum blocking buffer diluted in PBS buffer for 1h at room temperature and staining with primary anti-HERVW-Env antibody overnight at 4 ℃.
6. The primary antibody was washed 3 times with PBS buffer for 10min each time. Adding appropriate amount of enhancer, incubating at room temperature for 30min, and washing with PBS for 10min for 3 times.
7. The cells were incubated with the corresponding secondary antibodies for 1h at room temperature, and the secondary antibodies were washed off 3 times with PBS buffer for 10min each time.
8. DAB color development: mixing the prepared DAB chromogenic working solution (1 drop (50 mu L) of concentrated DAB solution with 1ml of DAB substrate uniformly, adding a proper amount of freshly prepared DAB chromogenic working solution, incubating at room temperature for 5-10min, and observing the chromogenic result under a microscope.
9. Counterdyeing: washing with tap water, incubating with hematoxylin staining solution for 5min, differentiating, washing with reverse blue for 10 s.
10. Dehydrating, transparent and sealing.
Immunohistochemistry results are shown in fig. 6, C and F, where WT: a wild-type cynomolgus monkey; HGPS: premature senility syndrome cynomolgus monkey; ERVW-Env immunohistochemistry color is brown; DNA is developed into blue by hematoxylin; lung, liver and skin were analyzed separately; right side: immunohistochemical signal intensity of ERVW-Env in wild type and presenility cynomolgus monkey lung, liver and skin species; young in fig. F: young cynomolgus monkeys; old: old cynomolgus monkey; ERVW-Env immunohistochemistry color is brown; DNA is developed into blue by hematoxylin; lung, liver and skin were analyzed separately; right side: ERVW-Env immunohistochemical signal intensities in lung, liver and skin species of young and old cynomolgus monkeys. As shown in figure 6C, the HERVW-Env signal was elevated in the lungs, liver and skin of the premature aging HGPS cynomolgus monkey compared to the wild type cynomolgus monkey. Also, as shown in fig. 6F, the lung, liver and skin HERVW-Env signals were elevated in older cynomolgus monkeys compared to younger cynomolgus monkeys.
Western blot showed elevated HERVW-Env protein levels in the lungs of early senescence HGPS cynomolgus monkeys compared to wild type. Immunohistochemistry results showed that the HERVW-Env protein signal intensity was increased in the lungs, liver and skin of the premature aging HGPS cynomolgus monkey compared to the wild type. Similarly, the level of HERVW-Env protein in the lungs of naturally aged young (4-6 years old, n ═ 8) and aged (18-21 years old, n ═ 8) cynomolgus monkeys was examined by western blotting and found to be significantly higher than that of young cynomolgus monkeys. The results of immunohistochemistry showed that HERVW-Env protein signal intensity was increased in the lungs, liver and skin of old cynomolgus monkeys compared to young cynomolgus monkeys. Indicating that expression of endogenous virus can be used as a marker for aging in non-human primates.
Example 7 elevated HERVK levels in skin and blood of elderly people
The HERVK-Env protein level in human skin tissues was measured by immunohistochemistry on the upper eyelid skin group of young (20-28 years old, n-3 years old (55-76 years old, n-3 years old) and aged (55-76 years old, n-3 years old) persons, and the results showed that the HERVK-Env protein level was increased in the skin tissues of the elderly persons as compared with the young persons. Levels of HERVK-Env protein in young (18-25 years old, n 30) and old (71-90 years old, n 30) human serum were measured using the HERVK-Env ELISA kit and showed elevated levels of HERVK-Env protein in the serum of old people. Indicating that endogenous viruses may serve as markers of human aging.
The immunohistochemical detection and HERVK-Env ELISA detection specific steps are the same as above.
The immunohistochemical assay results are shown in fig. 7B and C, where Young: a young; old: the elderly; HERVK-Env immunohistochemistry was brown; DNA is developed into blue by hematoxylin; the skin was analyzed separately; right side: immunohistochemical signal intensity of HERVW-Env in skin species of young and old people; young in panel C: a young; old: the elderly; the young and old human serum HERVK-Env protein levels were analyzed by ELISA. The results show that the signal of HERVK-Env virus protein is increased in skin tissues of the elderly compared with the skin of young people (in the figure, the left side figure is a representative picture, and the right side figure is a statistical result); HERVK-Env ELISA assay results As shown in FIG. 7-C, HERVK-Env viral protein levels were elevated in serum from old versus young human.
Example 8 inhibition or activation of herdk may delay or accelerate senescence of mesenchymal stem cells.
HERVK is knocked down in the mesenchymal stem cells of the presenility HGPS and WS, and the results show that the knocking down of HERVK can delay the aging of the mesenchymal stem cells of the presenility. The CRISPR activation system is utilized in early-generation wild mesenchymal stem cells to activate endogenous HERVK expression, and the aging of the mesenchymal stem cells is accelerated. The HERVK level is increased to be the driving force of the mesenchymal stem cells, and can be used as a target point for delaying the aging of the mesenchymal stem cells.
Preparation of recombinant lentivirus for knocking down human HERVK and activating HERVK gene
1. shRNA for knockdown of herdk and sgRNA sequence design for activation of herdk:
shRNAs designed according to HERVK for knocking down HERVK are shHERVK-1 (corresponding to a sequence 1: CCTGAACATCCAGAATTAT in a sequence table) and shHERVK-2: (corresponds to the sequence 2: ACATTATTAGATTCCATTGC in the sequence table); the sgRNA sequence sgHERVK for activating HERVK was designed according to HERVK (sequence 3: GATAGGGAAAAACCGCCTTAGGG in the corresponding sequence listing).
Synthesizing the oligonucleotide sequence of the HERVK-targeted shRNA in a company (ThermoFisher), annealing to obtain a double-chain shHERVK-1 containing the sequence, connecting the double-chain shHERVK-1 to a lentiviral vector framework obtained by cutting pLVTHM (Addgene product, #12247) by ClaI/MluI (NEB) through T4 ligase (NEB), marking the obtained recombinant vector with correct sequence as a recombinant vector pLVTHM-shHERVK-1, replacing the sequence between ClaI and MluI of the recognition site of the pLVTHM with the sequence of shHERVK-1 by the recombinant vector pLVTHM-shHERVK-1, and keeping other sequences unchanged.
Synthesizing the oligonucleotide sequence of the HERVK-targeted shRNA in a company (Thermo Fisher), annealing to obtain a double-chain shHERVK-2 containing the sequence, connecting the double-chain shHERVK-2 to a lentiviral vector framework obtained by cutting pLVTHM (Addgene product, #12247) by ClaI/MluI (NEB) through T4 ligase (NEB), marking the obtained recombinant vector with correct sequence as a recombinant vector pLVTHM-shHERVK-2, replacing the sequence between ClaI and MluI of the recognition site of the pLVTHM with the sequence of shHERVK-2 through the recombinant vector pLVTHM-shHERVK-2, and keeping other sequences unchanged.
According to the method, sgRNA activating HERVK is annealed to obtain double-stranded sgHERVK containing the sequences, T4 ligase (NEB) is connected to the vector framework of into lenti-SAM v2 (adddge #75112) (Addgene product, #52961) cut by Fastdigest _ Esp3I (NEB), and the obtained recombinant vector with correct sequences is marked as recombinant vector SAM-sgHERVK which is a recombinant vector that is inserted into the sequence of the sgHERVK at the position of Esp3I recognition site of the into lenti-SAM v2 vector and keeps other sequences unchanged.
shRNA control shControl and endogenously activated control sgNTC were constructed as described above, and the sequence information can be found in Hu, H.et al.ZKSCAN3 polynucleotides cellular sensitivity by labeling heterologous nucleic acid research 48,6001-6018, doi:10.1093/nar/gka 425 (2020). Wherein the reference plasmid of shRNA for knocking down HERVK is shControl, and the plasmid skeleton and the preparation method thereof are as described in the recombinant vectors shHERVK-1 and shHERVK-2, so as to obtain the recombinant vector shControl; sgNTC is a control plasmid of sgRNA of endogenously activated HERVK, the plasmid framework and the preparation method thereof are described in the recombinant vector sgHERVK, and the recombinant vector sgNTC is obtained.
2. Packaging recombinant lentiviruses knocked-down HERVK and activated HERVK gene
The method comprises the following steps of co-transfecting 293T cells with knockdown HERVK recombinant plasmids shHERVK-1 and shHERVK-2 or endogenous activation HERVK recombinant plasmids sgHERVK (corresponding to the recombinant vectors pLVTHM-shHERVK-1, pLVTHM-shHERVK-2 and SAM-sgHERVK) and lentivirus packaging vectors psPAX2(Addgene product, #12260) and pMD2G (Addgene product, #12259) to obtain shHERVK-1 virus solution, shHERVK-2 virus solution and sgHERVK virus solution, wherein the method comprises the following steps:
(1) lentiviral plasmids shHERVK-1, shHERVK-2 or sgHERVK, lentiviral packaging vectors psPAX2 and pMD2G were co-transfected into 293T cells (the ratio is 110 cm dish 293T cell: 9. mu.g lentiviral plasmid, 6. mu.g psPAX2 and 3. mu.g pMD2G) by using Lipo3000 transfection kit (ThermoFisher) and cultured for 8 hours.
(2) After 6hr of transfection, the medium was replaced with fresh 293T cell medium, and the culture was continued for 48 to 54 hours.
(3) The supernatant was collected, filtered through a 0.22 μm filter membrane, and the filtrate was collected.
(4) Taking the filtrate, centrifuging at 4 ℃ and 19400rpm for 2.15 hours, removing the supernatant, and carrying out heavy suspension precipitation by using a mesenchymal stem cell culture medium to obtain the virus liquid containing the knocked-down HERVK lentiviral plasmid shHERVK-1, shHERVK-2 or endogenously activated HERVK lentiviral plasmid sgHERVK recombinant lentivirus, namely shHERVK-1 virus liquid, shHERVK-2 virus liquid and sgHERVK virus liquid for short.
(5) The virus solutions shControl and sgNTC for control were prepared in the same manner as described above. Co-transfecting a HERVK-knocked-down control recombinant plasmid shControl or an endogenously activated HERVK control recombinant plasmid sgNTC with lentiviral packaging vectors psPAX2(Addgene product, #12260) and pMD2G (Addgene product, #12259) to obtain shControl virus solution and sgNTC virus solution
Secondly, the lentivirus infects the mesenchymal stem cells.
The early senescence HGPS and WS mesenchymal stem cells are taken as test cells to respectively infect control shGl2 and shHERVK viruses (comprising shHERVK-1 virus solution and shHERVK-2 virus solution); wild-type mesenchymal stem cells were used as test cells to infect sgNTC and sgherpk viruses, which were used as controls. The specific method comprises the following steps: mu.L of lentivirus solution and 2. mu.L of LPolybrene were added to culture wells (one well of 6-well plates) seeded with passage 4 WS mesenchymal precursor cells. The liquid is changed the next day, and then the culture is normally carried out and subcultured.
After lentivirus infection, the resulting cells were serially passaged to passage 3. Cells were collected, tested for HERVK protein knockdown and activation efficiency using Western blotting, and stained for the cellular senescence marker SA- β -gal. The specific procedures for Western blotting and SA- β -gal detection were the same as above.
Experimental results as shown in fig. 8B, fig. 8B is an immunoblot showing that activation of endogenous herdk in wild-type mesenchymal stem cells increases viral protein expression, wherein the left figure is a representative picture and the right figure is a statistical result, wherein WT mpc: wild-type mesenchymal stem cells; CRISPR-activation, namely utilizing a CRISPR endogenous activation system; sgNTC: a control sgRNA that does not target any sequence; sgHERVK: a sgRNA targeting herpk; kda is protein size unit; GAPDH: internal reference. As can be seen in fig. 8B, the immunoblot showed that shherkk can reduce the herkk-Env viral protein level.
The SA-beta-gal result is shown in FIG. 8C, and FIG. 8C is a schematic diagram of SA-beta-gal experiment showing that cell senescence is promoted after HERVK expression is activated, wherein CRISPR-activation is realized by using CRISPR endogenous activation system; sgNTC: a control sgRNA that does not target any sequence; sgHERVK: a sgRNA targeting herpk; the left panel is a representative picture and the right panel is the SA- β -gal statistics related to senescence using the CRISPR endogenous activation system. From figure 8C, it can be seen that knocking down herpk delays senescence of the mesenchymal stem cells of the premature aging disorders (HGPS and WS).
The immunoblotting results are shown in fig. 8D, and fig. 8D is a graph showing that reduction of viral protein expression by reducing herpk in the mesenchymal stem cells of premature senility by immunoblotting, wherein the left side of the graph is a representative graph, and the right side of the graph is a statistical result, wherein C HGPS or WS hMPC is the mesenchymal stem cell of premature senility or adult, shControl (without this mark, whether it should be shControl in the graph): a control shRNA without knock-down of any human gene sequence; shHERVK: shRNA knock-down of herdk; kda is the protein size unit; GAPDH: internal reference; protein expression statistics using shRNA to knock down herpk-Env are on the right. From the results in fig. 8D, it can be seen that sgherpk can activate the protein level of endogenous herpk-Env.
The SA-beta-gal results are shown in FIG. 8E, FIG. 8E is SA-beta-gal experiments showing that knocking-down HERVK delays the aging of the mesenchymal stem cells of presenility, the left drawing in the figure is a representative picture, and the right drawing in the figure is a statistical result, wherein CHGPS or WS hMPC, the mesenchymal stem cells of presenility of children or adults, shControl (without the mark, whether it should be shControl in the figure): a control shRNA without knock-down of any human gene sequence; shHERVK: shRNA knock-down of herdk; on the right are age-related SA- β -gal statistics using shRNA knock-down of HERVK-Env. It can be seen from fig. 8E that activation of herpk promotes senescence of wild-type mesenchymal stem cells.
The above example 6 shows that HERVK activation is one of the driving forces for causing cell senescence, and reducing HERVK level can be used as one of the technical means for delaying cell senescence.
Example 9 neutralization of HERVK in aged cell culture media with antibodies can delay aging of aged media-induced cells.
The aged cells were released into the medium with the HERVK-Env antibody and neutralized, and then young cells were cultured with the control (IgG) -treated and HERVK-depleted aged cell-derived medium, and the degree of aging was observed.
Antibody immune neutralization detection
The HERVK-Env antibody is used for releasing aged cells into HERVK in a culture medium to be neutralized, and the specific steps are as follows:
1. the Protein A/G sepharose beads were washed 3 times with PBS, at 4 ℃ for 5 min.
2. The culture medium for childhood senilism mesenchymal stem cells (HGPS-CM), the culture medium for adult senilism mesenchymal stem cells (WS-CM), and the culture medium for late-generation wild-type mesenchymal stem cells (LP-CM) prepared in example 3 were incubated with Protein A/G sepharose magnetic beads at 4 ℃ for 2-3hr, respectively.
3. Removing the magnetic beads by centrifugation, mixing the antibody (HERVK-Env or IgG) and the culture medium in a ratio of 1:1000 (volume ratio), adding Protein A/G sepharose magnetic beads, and incubating overnight at 4 ℃.
4. The culture medium obtained by centrifugation at 1000g at 4 ℃ was used for culturing early-generation (EP: P4) wild-type mesenchymal stem cells. After washing the magnetic beads for 3 times with PBS, the magnetic beads are used for western blot to detect whether HERVK-Env antibodies can pull down HERVK in a culture medium derived from aged cells.
Western blot results as shown in fig. 9B, fig. 9B is a western blot showing that herdk-specific antibodies can pull down herdk in senescent media, where IP: performing immunoprecipitation; IgG: control, no binding to any protein; HERVK-Env: HERVK-Env antibodies; HGPS-CM: a culture medium derived from mesenchymal stem cells of the premature senility syndrome of the children; WS-CM derived from the culture medium of adult presenility mesenchymal stem cells; RS-CM derived from the culture medium of late-generation wild mesenchymal stem cells; these three belong to the aging medium; IB: HERVK-gag proteins together with HERVK-Env, in principle both proteins can be pulled down simultaneously. It can be seen from the figure that HERVK-Env and HERVK-gag viral proteins were not detected in the magnetic beads bound with the control antibody IgG, while HERVK-Env and HERVK-gag viral proteins were detected in the magnetic beads bound with the HERVK-Env antibody, indicating that the HERVK-Env antibody can pull down HERVK in the senescent cell-derived medium.
Second, culturing young cells in conditional aged medium
And (3) performing control treatment, mixing the culture medium from which the HERVK is removed and the mesenchymal stem cell culture medium according to a ratio of 1:1, culturing early-generation wild cells, adding 10ml of prepared culture medium and 10 mu L of polybrene to cells cultured on a10 cm culture dish, and detecting the aging degree of the cells by using SA-beta-gal after culturing for 2 generations.
The results of SA- β -gal staining are shown in FIG. 9C, which is a graph showing that SA- β -gal staining experiments show that neutralization of HERVK in senescence medium by neutralizing antibody can delay senescence of young cells caused by senescence medium, and the right graph is a statistical result, wherein IgG: control, no binding to any protein; HERVK-Env: HERVK-Env antibodies; HGPS-CM: a culture medium derived from mesenchymal stem cells of the premature senility syndrome of the children; WS-CM derived from the culture medium of adult presenility mesenchymal stem cells; RS-CM derived from the culture medium of late-generation wild mesenchymal stem cells; these three belong to the aging medium; right side: SA- β -gal statistics no treatment (three-no group), treatment with senescence medium (middle second group), treatment with IgG control treated senescence medium (middle third group) and treatment with herpk-Env antibody herpk-Env were removed from the treatment group (last fourth group). The results in the figure show that the aging of young cells can be caused by culturing young cells in a medium of old cells, and the removal of HERVK from the medium derived from old cells by using the HERVK-Env antibody can delay the aging degree of cells, which indicates that the preparation of HERVK-Env neutralizing antibody can be used as one of the technical methods for delaying the aging degree of cells.
Immunoblot detection of HERVK-Env antibodies HERVK in senescent cell-derived media was pulled down. The media derived from senescent cells depleted of HERVK can retard the extent to which senescent media causes cellular senescence compared to control-treated media derived from senescent cells. The neutralizing antibody of HERVK can be used as one of the technical means for delaying senility.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
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Claims (10)
1. Use of an endogenous virus as a marker of the degree of senescence.
2. Use of an expression product of an endogenous virus as a marker for the degree of senescence.
3. The use according to claim 2, wherein the expression product of the endogenous virus is a primer pair and a probe for detecting the RNA level of the endogenous virus, an antibody specific for a viral protein or a kit.
4. Use of an endogenous virus as a marker for detecting the degree of ageing in blood.
5. The use of a substance for detecting an endogenous virus or endogenous viral gene in a sample for the manufacture of a product for detecting the degree of senescence in a mammal.
6. Use of an endogenous virus for the manufacture of a product for promoting or inhibiting senescence.
7. The application of endogenous virus, substance for inhibiting endogenous virus or silencing or knocking out endogenous virus gene in preparing products for promoting or inhibiting aging or premature senility.
8. Use of an endogenous virus as a target for intervention in the degree of senescence.
9. The use according to claim 8, comprising a step of binding a substance that specifically binds to an endogenous virus and inhibits the activity thereof to the endogenous virus; the substance capable of specifically binding with the endogenous virus is a monoclonal or polyclonal neutralizing antibody against the endogenous virus; or the substance specifically combined with the endogenous virus is a specific inhibitory small molecule compound of the endogenous virus; or the substance specifically combined with the endogenous virus is a specific primer pair for knocking down or knocking out the anti-endogenous virus.
10. The use according to any one of claims 1 to 9, wherein the endogenous virus comprises at least one of human endogenous viruses HERV, HERVK, HERVW and HERVK, and the homologous virus MMTV in mice.
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| EP21909481.0A EP4163379A1 (en) | 2020-12-22 | 2021-12-22 | Method for identifying and/or regulating senescence |
| PCT/CN2021/140550 WO2022135486A1 (en) | 2020-12-22 | 2021-12-22 | Method for identifying and/or regulating senescence |
| CN202180048695.9A CN116157529A (en) | 2020-12-22 | 2021-12-22 | Methods for identifying and/or modulating aging |
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