阅读说明：本技术 一种基于胚胎干细胞的抗衰老策略 (Anti-aging strategy based on embryonic stem cells ) 是由 刘娜 于璐 李宗金 于 2021-08-02 设计创作，主要内容包括：本发明是一种基于胚胎干细胞分泌的因子来延缓机体衰老的技术手段。胚胎干细胞具有自我更新能力和多向分化潜能,可以分化为机体几乎所有类型的细胞,可在体外无限传代,是一种不会发生衰老的细胞,因此胚胎干细胞在临床替代治疗及逆转衰老中具有极其重要的地位。但是胚胎干细胞的免疫排斥、伦理道德及致瘤性等问题限制了其在临床上的应用。外泌体是一种由细胞天然分泌的磷脂双分子层包裹的物质,富含核酸和蛋白质成分,可介导胚胎干细胞的部分作用与功能,并且外泌体成功地规避了上述免疫排斥、伦理及安全性等问题,是胚胎干细胞的良好替代产品。近年来,干细胞分泌的外泌体越来越多的应用于再生医学领域,但是胚胎干细胞来源的外泌体在抵御衰老中的作用还未见报道。本发明利用胚胎干细胞分泌的外泌体可显著延缓小鼠细胞的衰老,是一种很好的利用胚胎干细胞抵御衰老的策略。(The invention relates to a technical means for delaying the aging of an organism based on factors secreted by embryonic stem cells. The embryonic stem cell has self-renewal capacity and multidirectional differentiation potential, can be differentiated into almost all types of cells of an organism, can be subjected to unlimited passage in vitro, and is a cell which cannot cause senescence, so the embryonic stem cell has extremely important positions in clinical replacement therapy and reversal senescence. However, the problems of immunological rejection, ethical morality and tumorigenicity of the embryonic stem cells limit the clinical application of the embryonic stem cells. The exosome is a substance wrapped by phospholipid bilayers naturally secreted by cells, is rich in nucleic acid and protein components, can mediate partial functions and functions of the embryonic stem cells, successfully avoids the problems of immunological rejection, ethics, safety and the like, and is a good substitute product of the embryonic stem cells. In recent years, exosomes secreted by stem cells are increasingly used in the field of regenerative medicine, but the role of exosomes derived from embryonic stem cells in resisting aging has not been reported. The invention can obviously delay the aging of mouse cells by using exosomes secreted by the embryonic stem cells, and is a good strategy for resisting aging by using the embryonic stem cells.)

1. The effective component of the treatment means is exosome from stem cells, which is a paracrine component separated and extracted from cell supernatant cultured in vitro, carries various bioactive molecules such as protein, RNA and the like in donor cells, and has the function of treating injury.

2. Exosome according to claim 1, characterized in that: the exosomes are derived from embryonic stem cells.

3. A means for delivering exosomes and delaying senescence in a subject by treating senescent mouse cells with exosomes according to claim 1 to inhibit the appearance of a phenotype associated with cellular senescence.

4. The senescent mouse cell according to claim 3, characterized in that: the cells are replicative senescent mouse embryonic fibroblasts resulting from serial passages.

5. The technical means of claim 3, wherein the technology can delay the aging of mouse cells through exosomes, and comprises the steps of improving cell morphology, inhibiting the expression of aging-related proteins, reducing the positive rate of gamma-H2 AX, and promoting cell proliferation.

6. The use of claim 3 for delaying aging in the body, wherein aging in the body comprises cellular aging, immunosenescence, aging in mouse individuals, and the like.

Technical Field

The invention relates to a technical means for delaying cell aging by using exosomes secreted by embryonic stem cells, belonging to the technical field of tissue engineering and new medicines.

Background

An embryonic stem cell is a pluripotent cell isolated from a mammalian blastocyst. Embryonic stem cells are initially derived from mice, and embryonic stem cell lines or similar embryonic stem cell lines are subsequently isolated from other rodents, domesticated animal species, and non-human primates. Embryonic stem cells have high self-renewal capacity and multi-differentiation capacity, and can be induced to differentiate into various types of cells under certain conditions. The research reports that the traditional Chinese medicine composition has obvious therapeutic effects on treating neurodegenerative diseases, diabetes, repairing necrotic cardiac muscle and liver regeneration and the like. However, the use of embryonic stem cells in clinical therapy is limited due to immune rejection and ethical issues. Thus, scientists are looking for alternative methods of embryonic stem cells to develop the superior characteristics of embryonic stem cells. Several alternative therapies have been reported in recent years, such as disease treatment or tissue repair using therapeutic clones, parthenogenetic embryonic stem cells, and induced pluripotent stem cells. However, their tumorigenic potential still limits the clinical application of pluripotent stem cells.

Exosomes are vesicles encapsulated by phospholipid bilayers, produced by endosomal membrane invagination, with an average diameter of about 100nm, and contain RNA, lipids and proteins. The action of exosomes depends on the delivery of their contents to recipient cells, thereby altering the biological processes of the target cell. The transport and transport of exosomes affects various physiological functions of their target cells and plays an important role in intracellular and intercellular communication, including the regulation of biological processes such as immune response, cell proliferation, cell migration, angiogenesis, cancer progression, etc. Exosomes can also remove unwanted components from cells to maintain cellular homeostasis and are associated with radioactive tissue damage, cardiovascular disease, viral pathogenicity, central nervous system related diseases, and cancer progression. Exosomes are secreted by a variety of cells and can promote or limit disease progression. Thus, based on the intrinsic properties of the exosomes described above, exosomes may have more potential uses in the prevention and treatment of many diseases.

Many studies have reported that embryonic stem cells also secrete exosomes, and that this embryonic stem cell-derived exosome has been shown to play an important role in a variety of disease recovery models. The exosome derived from the embryonic stem cell can promote angiogenesis and cardiac muscle cell proliferation by transferring miR-294, so that cardiac function repair after myocardial infarction is promoted. In recent years, research shows that exosomes secreted by various cells have the effect of delaying body aging, exosomes derived from human adult light fibroblasts can improve aging-related tissue damage, exosomes derived from mesenchymal stem cells can promote skin regeneration by improving aging of skin fibroblasts, and research reports that exosomes derived from embryonic stem cells can also improve aging-related phenotype of aging mesenchymal stem cells and promote the effectiveness of the exosomes in treating skin damage.

And exosomes successfully circumvent the problems faced by embryonic stem cell therapy described above. First, exosomes derived from embryonic stem cells can avoid ethical problems, and since exosomes are derived from embryonic stem cell supernatant, and embryonic stem cells can be immortalized in vitro, exosomes derived from embryonic stem cells can be isolated continuously without destroying new embryos. Second, compared to cell therapy, extracellular vesicles are more stable, more convenient to store, and easier to manage and control. Third, exosomes derived from embryonic stem cells are less tumorigenic than embryonic stem cells. Therefore, the exosome derived from the embryonic stem cell is considered as a good substitute of the embryonic stem cell in cell therapy, avoids the defects of the embryonic stem cell in clinical application, and greatly exerts the excellent characteristics of the embryonic stem cell.

Aging is a necessary rule of life process, is a process of degenerative change of the functions of each tissue and organ of the organism along with the age, and is characterized in that the functions of the organism are reduced along with the time due to the accumulation of the biological process change in the organism. Aging in humans is accompanied by a gradual accumulation of cognitive and physical disorders and an increased risk of developing a variety of diseases, including cancer, diabetes, cardiovascular, musculoskeletal and neurodegenerative diseases. The occurrence of age-related aging diseases has a serious impact on the quality of life and ultimately increases the risk of death. With the increasing population of the elderly, China is gradually stepping into the aging society of the population at present. In the face of the coming of an aging society, how to realize healthy aging of the aged people becomes a problem which is urgently needed to be solved at present.

The mouse embryonic fibroblast is a mouse embryonic cell derived from the 13 th day of gestation, and is a feeder layer cell which can be used for stem cell culture, can secrete a plurality of important growth factors which are helpful for maintaining the pluripotency of the stem cells, and provides a cell matrix for the growth of the stem cells. However, mouse embryonic fibroblasts are aged quickly in the process of continuous passage, and show the typical characteristics of aged cells such as cell cycle arrest, slow cell proliferation, cell volume reduction and the like when passages reach 7 th generation.

Disclosure of Invention

The invention relates to a technical means for resisting aging by using exosomes secreted by embryonic stem cells.

The invention utilizes exosome from embryonic stem cells to treat replicative senescence mouse cells, thereby delaying senescence of the mouse cells.

The invention can effectively inhibit the expression of senescence-associated genes and proteins in replicative senescence mouse cells.

The invention can effectively delay the aging of mouse cells, improve the cell morphology, promote the cell proliferation and reduce the degree of cell oxidative damage.

The invention takes the mouse embryonic fibroblasts with continuous passage and replicative senescence as a model and explores the anti-senescence effect of exosomes derived from embryonic stem cells.

Drawings

FIG. 1 is a representation of exosomes, which are embryonic stem cell-derived exosomes.

FIG. 2 shows that exosomes derived from embryonic stem cells can be internalized by mouse embryonic fibroblasts to function.

FIG. 3 shows that the expression level of senescence-associated genes and proteins is significantly reduced when exosomes derived from embryonic stem cells are used for treating senescent P7 mouse embryonic fibroblasts.

FIG. 4 shows that exosomes derived from embryonic stem cells are used for treating aged P7 mouse embryonic fibroblasts to improve the cell proliferation capacity and reduce the DNA damage degree.

Detailed Description

In the following examples, unless otherwise specified, all methods used are conventional and all reagents used are commercially available.

Example 1, the present invention provides a method for extracting exosomes derived from embryonic stem cells.

Cell basal medium and serum were purchased from Hyclone; reagents such as diabody and pancreatin are purchased from Gibco; cell culture consumables were purchased from NEST.

The serum required by cell culture for extracting the exosome is fetal bovine serum without exosome, and the processing steps are as follows: fetal bovine serum was placed in an ultracentrifuge tube, after trimming, 120000g was centrifuged at 4 ℃ for 2 hours, after which the supernatant was taken out on an ultraclean bench, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm needle filter and stored in a refrigerator at-80 ℃ for further use.

The procedures of cell recovery, subculture, cryopreservation and other cell biological experimental operations are described in animal cell culture (sixth edition).

Collecting the culture supernatant of the embryonic stem cells containing the exosomes: when the embryonic stem cells are just recovered, culturing the embryonic stem cells by using a culture medium prepared by normal serum, when the confluence degree of the cells reaches 80%, absorbing the culture medium, gently washing the cells twice by using PBS, after the cells are subcultured, continuously culturing the cells for 24 hours by using the culture medium prepared by 15% of the serum without exosome, collecting cell culture supernatant into a 50ml centrifuge tube, wherein the supernatant is rich in exosomes from the embryonic stem cells.

Separating and extracting exosome by an ultracentrifugation method: centrifuging the cell culture supernatant obtained in the step at 4 ℃ at 500g for 10min, taking the supernatant, and removing cell debris; secondly, centrifuging the obtained supernatant for 30min at 4 ℃ at 2000g, taking the supernatant, and removing large cell debris such as apoptotic bodies; thirdly, filtering the obtained supernatant by using a 0.22 mu m needle filter to remove the microvesicles with the diameter more than 200 nm; placing the filtered supernatant into an ultracentrifuge tube, centrifuging at 4 ℃ for 2h at 120000g, discarding the supernatant, adding a proper amount of PBS (phosphate buffer solution) to resuspend the sediment at the bottom of the tube, subpackaging and storing at-80 ℃.

Example 2, the present invention provides a method for identifying exosomes derived from embryonic stem cells.

1. The exosome marker proteins Alix, TSG101 and CD63 were detected by Western Blot (fig. 1).

1) Protein sample preparation: according to the PMSF: RIPA ═ 1: 100, adding the protein lysate into the exosome precipitate obtained after ultracentrifugation to lyse the exosome. The sample was lysed on ice for 30min, shaken on a vortex shaker every 5min to ensure sufficient lysis, centrifuged at 12000rpm for 15min at 4 ℃ after 30min, and the supernatant was transferred to a new EP tube; measuring the protein concentration of the exosome by using a BCA method, adding a 5 xSDS loading buffer solution into the rest protein solution, boiling in boiling water for 5-10min for denaturation, immediately putting the sample into liquid nitrogen for quick-cooling after the denaturation is finished, and storing in a refrigerator at-80 ℃ for later use.

2) Preparing glue: carefully cleaning a glass plate used in a Western Blot experiment by using a sponge, washing the glass plate by using a detergent, washing the glass plate by using tap water, washing the glass plate by using ultrapure water for three times, and cleaning a clamp, the sponge, a glue preparation bottle, an electrophoresis tank, a film transfer tank and the like which are used subsequently. And (3) mounting the clean and dried glass plate on a glue making frame, enabling the bottom edges to be tightly fitted, and checking leakage by using distilled water. Preparing 10% separating gel solution according to the separating gel formula, mixing well, adding into a glass plate fixed with a clamp in advance by a 5ml pipette at one time, taking care to avoid air bubbles, adding the separating gel about 1.5cm away from the upper edge, adding water slightly for liquid sealing, and avoiding breaking the gel surface. After 40min, a clear boundary line is formed between the visible water and the visible glue, the separation glue is solidified, and the water of the seal separation glue is completely absorbed by absorbent paper. Preparing 5% concentrated glue solution according to the formula, adding 1.5ml concentrated glue solution to the upper part of the separation glue, immediately inserting a comb, and using after the concentrated glue is solidified.

3) Polyacrylamide gel electrophoresis: placing the prepared rubber plate into an electrophoresis tank, paying attention to the inner side of the short glass plate, adding a freshly prepared 1x Running buffer between the two glass plates, pulling out a comb, carrying out protein sample loading according to the principle of equal mass (20-50 ug), calculating the volume of the sample loading according to the measured concentration, aligning the total volume of the sample loading to 20ul by using 1xSDS, adding a protein sample into a sample loading hole, adding an electrophoresis solution to the mark position of the electrophoresis tank, covering the cover of the electrophoresis tank, paying attention to the direction of the positive electrode and the negative electrode to be consistent, Running the concentrated glue by using 80V voltage, stopping electrophoresis when bromophenol blue runs to the separation glue, when protein marker is separated, regulating the voltage to 110V, and stopping electrophoresis until the bromophenol blue is close to the bottom of the glass plate, wherein the time for the whole electrophoresis is 1.5 hours.

4) Film transfer: preparing a precooled membrane Transfer solution (1x Transfer buffer) containing 20% methanol in advance, soaking a membrane Transfer clamp, sponge and filter paper in a precooled membrane Transfer buffer solution, carefully prying off a rubber plate, slightly removing concentrated gel along a boundary line, placing separation gel after electrophoresis on one side of a black clamping plate, shearing a PVDF membrane with a proper size, placing the PVDF membrane in methanol for activating for 60s, placing the PVDF membrane on the gel, removing bubbles between the gel and the membrane, slightly covering the filter paper and the sponge on the membrane, sequentially clamping the membrane Transfer clamp according to the negative pole (black) -sponge-filter paper-gel-PVDF membrane-filter paper-sponge-membrane Transfer clamp positive pole (white) of the membrane Transfer clamp, placing the PVDF membrane in a membrane Transfer tank, adding the membrane Transfer solution, placing the whole membrane Transfer tank in a foam box filled with ice, and carrying out membrane Transfer at a constant pressure of 100V for 2h under ice bath.

5) And (3) sealing: the PVDF membrane after the membrane transfer is taken out, placed in 5% skimmed milk (confining liquid) with the protein side facing upwards, and horizontally shaken at 60rpm and sealed at room temperature for 2 h.

6) Antibody incubation: first-antibody incubation: diluting the primary antibody with a blocking solution according to the instruction (Alix, 1:2000 dilution; TSG101, 1:2000 dilution; CD63, 1:2000 dilution), sucking 2ml of the primary antibody, placing the primary antibody in an antibody incubation box, shearing a membrane by a control protein Marker, placing the membrane in the corresponding primary antibody, and incubating overnight at 4 ℃; and secondly, incubation with secondary antibody: the strips were placed in TBST on a horizontal shaker for 3 washes at 120rpm for 10min each, then the corresponding species of secondary antibody was added, incubated on a horizontal shaker at 60rpm for 2h at room temperature, and washed 3 times with TBST for 10min each after incubation.

7) Exposure: mixing the luminescent liquid A and the luminescent liquid B according to the proportion of 1:1 to prepare working liquid, and exposing by an exposure instrument.

2. Characterization of exosome morphology and particle size

The morphology of exosomes was observed using Transmission Electron Microscopy (TEM). The samples were deposited on a copper grid covered with a carbon film, dried at room temperature for 2min, and after removing excess liquid with a filter, the samples were negatively stained with 2% uranium acetate for 30 s. After the sample was air-dried for 60min, it was imaged by transmission electron microscopy. Nanoparticle Tracking Analysis (NTA) was used to determine the particle size of exosomes.

Example 3, the present invention provides a method for modeling mouse embryonic fibroblast senescence.

1. Extraction of Primary MEF cells

1) Taking a pregnant 13.5d female mouse, killing the female mouse by breaking the neck, and exposing the uterus under a sterile condition. The whole uterus was removed, washed 3 times with PBS and discarded for surface residual blood.

2) The uterus is cut along the mesenteric side, the embryo with the fetal membrane is taken out, the embryo is fully washed, and the surface red blood cells are discarded.

3) The fetal membranes were torn with a small forceps, the fetal mice were removed and washed 3 times with PBS.

4) The embryonic heads, viscera and limbs were removed and the torso was washed 3 times with PBS in another dish containing PBS.

5) Shearing the trunk of the rat into 1mm with sterile scissors³The following fragments were aspirated into a centrifuge tube.

6) Adding 5-10ml pancreatin, and placing the centrifuge tube containing the mouse trunk fragment in an incubator for digestion for 10-20 min.

7) Taking out the centrifuge tube, repeatedly blowing, adding sufficient culture medium to terminate digestion, and centrifuging at 1000rpm for 5 min.

8) And (4) discarding the supernatant, adding a proper amount of culture solution, repeatedly blowing and beating, and resuspending the cells.

9) Cells were split into T75, and one primary cell of T75 was typically made per 2 embryos.

10) Culturing in a saturated humidity incubator at 37 deg.C and 5% CO2, and culturing at a ratio of 1:3-1:6 when the cells are overlapped and fully cover the whole culture bottle for 5-7 days.

2. Establishment of MEF cell senescence model by continuous passage

The replicative senescence characteristic of MEF cells is utilized, and the obtained primary MEF cells are continuously subcultured until senescence occurs by a cell subculture method. The expression changes of Ki67, p21 and p53 proteins are used for detecting the aging state of cells.

Example 4, the present invention provides a technique for delaying the senescence of mouse embryonic fibroblasts using exosomes.

Aged mouse embryonic fibroblasts induced by serial passages were treated with exosomes derived from embryonic stem cells obtained by ultrarapid separation, and aged mouse embryonic fibroblasts were treated with exosomes (0, 50, 100, 150, 200ug/ml) at different concentrations for 48h or 96 h. The CCK-8 is used for detecting the proliferation speed change of cells under different treatment conditions, the Q-PCR is used for detecting the expression quantity change of senescence-related genes in the cells under different treatment conditions, and the detection results of various senescence-related indexes show that the exosome with the concentration of 100ug/ml from the embryonic stem cell can delay the senescence of mouse embryonic fibroblasts after being treated for 96 hours.