CN114214363B - Anti-mesenchymal stem cell aging modification method and application thereof - Google Patents
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Abstract
The invention provides an anti-mesenchymal stem cell aging modification method and application thereof. The invention inhibits AMSC aging through the intervention modification of SINHCAF gene posttranscriptional level, and confirms the relieving and inhibiting effect of SINHCAF intervention modification on AMSC aging through the research means such as WB, PCR, CCK cell proliferation analysis, SA-beta-gal staining, cell and mitochondrial morphology observation at the cellular level; and experiments in vivo such as a mouse liver failure model prove that SINHCAF is used for intervening modification to improve the curative effect of the aging AMSC liver disease. The biological preparation for treating liver diseases provided by the invention has a shorter preparation period, and can eliminate adverse effects caused by the cell function damage related to the AMSC aging, thereby improving the application potential of the cell preparation in the clinical treatment of liver diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an anti-mesenchymal stem cell aging modification method and application thereof, wherein the anti-mesenchymal stem cell aging is modified by intervention SINHCAF to resist adipose-derived mesenchymal stem cell aging, so that the proliferation capacity of the adipose-derived mesenchymal stem cell is improved, and the application of SINHCAF intervention modified AMSC (AMSC-SINHCAF) in preparing a biological preparation for treating liver diseases is provided. .
Background
Mesenchymal stem cells (MESENCHYMAL STEM CELL, MSC) have been demonstrated to have powerful differentiation potential, self-renewal capacity, immunomodulatory effects, and targeted therapeutic functions. MSC cell transplantation has shown clear efficacy in the treatment of a variety of degenerative diseases and acute and chronic injuries of tissues. Animal level research shows that transplanted MSC can relieve liver inflammation of acute and chronic liver injury models of mice and promote liver cell regeneration. Clinical experimental study also shows that the infusion of MSC by patients with severe liver has good tolerance, can obviously improve liver function, reduce Child-Pugh and MELD scores, and reduce ascites and total mortality. Therefore, for major diseases for which no effective drugs and treatments exist clinically at present, such as liver failure, MSC transplantation is expected to be a promising new cell therapy strategy for supplementing or even replacing two main modes of current clinical liver failure treatment limited by plasma and liver source shortage, artificial liver system and liver transplantation. Thereby effectively improving the prognosis of the patients and reducing the fatality rate of the patients.
Although the role of MSC in regenerative medicine and liver disease treatment has been established, its efficacy has been limited. Many of these defects, which are poor proliferation, differentiation and homing capacity, may be associated with their cellular senescence. This is because in order to meet the amount of cells required for treatment, in vitro expansion is often required prior to MSC transplantation, which provides an opportunity for replication-induced cell senescence. In addition, MSCs obtained from elderly and metabolic disease patients also tend to have higher aging loads. Therefore, the method can relieve or even inhibit MSC aging, eliminate cell function damage related to aging, and is expected to remarkably improve the treatment effect after MSC transplantation.
Disclosure of Invention
The invention aims to provide an anti-mesenchymal stem cell aging modification method, in particular to an epigenetic modification method for up-regulating the expression level of SINHCAF (SIN 3A and HDAC-associated factor, SIN3A and HDAC related factors) in AMSC.
The invention is realized by the following technical scheme:
1. preparation of adipose-derived mesenchymal stem cells (AMSC):
Mouse AMSC (mAMSC) and adult AMSC (hAMSC) were isolated by conventional methods, and mAMSC and hAMSC were cultured and amplified using STEMCELLTM company mouse and human MesenCult TM amplification kit (cat# 05513, # 05411), respectively, and L-glutamine was added as a complete medium. AMSC proliferation was close to 75% fusion, and digestion and passage were performed.
Construction of sinhcaf modified AMSC:
construction of sinhcaf modified hAMSC:
hAMSC was infected with hSINHCAF lentiviral vector pLVX-IRES-ZsGreen 1-SINHCAF. hSINHCAF the lentiviral vector comprises a fragment of interest sequence shown in SEQ ID NO.1, and is characterized by comprising a targeting binding sequence for the 3' -UTR region of HSINHCAF MRNA. The modification is based on epigenetic regulation of SINHCAF expression. Epigenetic modification is used as reversible modification of gene functions without changing DNA sequences, and has higher safety than gene editing.
SEQ:NO.1：
5'-CGCCTCAGAGCCGCCCGCCGTTCCTTTTTCCTATGCATATACTTCTTTGAGGATCTGGCCTAAAGAGGTATAGGGCATGGGAAAACGGGGCGGTCGGGTCCTCCCCAGCGCAGTAGTCCAGAGGACACCTCCACTCCGTCTACCCAGTGTTTAGACTATCTGTTCAGGACTCCCAAATTGTACAGTAGTCTGCACATTGGTTAGGCTGGGCTGGGTTAGACCCTCGGCAGTAGTCAGTAGTGTACTTGGGCAGTAGTGTAGAGATTGGTTTGCCTGTTAATGAATTCAAACTAATCTCTACACTGCTGCCCAAGAGCCAGTAGTCCTGCCGGGGCTAAAGTGCTGACAGTGCAGATAGTGGTCCTCTCCGTGCTACCGCACTGTGGGTACTTGCTGCTCCAGCAGGGTAGCACTAAAGTGCTTATAGTGCAGGTAGTGTTTAGTTATCTACTGCATTATGAGCACTTAAAGTACTGCACAGTATTGGGGCCCTGGCTGGGATATCATCATATACTGTAAGTTTGCGATGAGACACTACAGTATAGATGATGTACTAGTCCGGGCACCCCCACAGTAT-3'
The generation 2 hAMSC cells are paved with 6 pore plates, cultured in a complete culture medium overnight, and are ready for use when the fusion degree reaches 50%. 200. Mu.l of Opti-MEM TM medium was then added, 50. Mu.l of HitransG infection-enhancing solution was added, followed by 10. Mu. lpLVX-IRES-ZsgGeen1-SINHCAF lentivirus (titre 10 8 TU/ml, MOI value=10) (MOI: multiplicity of infection), and after mixing, 1.75ml of complete medium was added and added together to hAMSC medium, and the cell state was observed for 8-12 hours, and if the cell state appeared bad, the solution was immediately changed, and if no change was made, the culture was continued. After 24 hours, the culture is changed into a complete culture medium containing 5 mug/ml polybrene (polybrene), the fluorescence condition of ZsGreen1 is observed by a fluorescence microscope after 3-4 days of screening culture, and polybrene can be removed when the fluorescence rate reaches nearly 100%. hAMSC (hAMSC-Ctrl) infected with empty lentivirus was used as a control.
Construction of sinhcaf modified mAMSC:
mAMSC was infected with mSINHCAF lentiviral vector pLVX-IRES-ZsGreen 1-SINHCAF. mSINHCAF the lentiviral vector comprises a fragment of interest sequence shown in SEQ ID NO.2, and is characterized by comprising a targeting binding sequence for the 3' -UTR region of MSINHCAF MRNA.
SEQ:NO.2：
5'-GTTCCTTTTTCCTATGCATATACTTCTTTGTGGATCTGGTCTAAAGAGGTATAGCGCATGGGAAGATGGAGCCAGTAGTGCCATCCCAGTGTTCAGACTACCTGTTCAGGAGGCTGGGACATGTACAGTAGTCTGCACATTGGTTAGGCCAGTAGTCCAGAGGATACCTCCACTCCGTCTACCCAGTGTTTAGACTACCTGTTCAGGACTCCCAAATTGTACAGTAGTCTGCACATTGGTTAGGCTGGGCTGGGTTAGACCCTCGGCAGTAGTCAGCATGTGGAACACTGTAAAGTGCTGTGTCACGGACACTGACTTTTCAAAGTCCAGTAAGTCAGTCTGCTTAGAGGTCAGTGGGCAATACTATGCTATGTTCTGGCCCCATGTTTACAGTATGGCTGGGATATCATCATATACTGTAAGTTTGTGATGAGACACTACAGTATAGATGATGTACTAGTCACAGTAT-3'
MSINHCAF lentivirus infects mAMSC and construction of mAMSC-SINHCAF is performed as before. Also, mAMSC (mAMSC-Ctrl) infected with empty lentivirus was used as a control.
Analysis of SINHCAF expression level in amsc-SINHCAF:
Protein expression levels of SINHCAF in native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells were detected by Western Blot. The method comprises the following specific steps:
(1) Cells were collected by digestion and centrifugation, and a suitable amount of RIPI lysate (priwill, cat# C1053) containing protease inhibitors was added to the cell pellet, and the mixture was thoroughly mixed, centrifuged at 12000g at 4℃for 5 minutes, and the supernatant was collected.
(2) Protein quantification was performed using BCA kit (Thermo, cat# 23228).
(3) Before loading, glycerin in each hole of 4-20% gradient concentration prefabricated glue (GenScript) is blown off, a cell protein sample is loaded according to 40 ug/empty protein amount, the glue is run at 140V for about 1 hour, and then an automatic film transfer machine is used for transferring films (GenScript, trans-Blot Turbo) for about 15 minutes.
(4) Blocking was performed in 5% BSA (Servicebio, cat# G5001-100G) for about 1 hour, then incubation of SINHCAF primary antibody (Novus, 1:1000 dilution) was performed overnight at 4 ℃.
(5) TBST washes the membrane 3 times for 10 minutes each time; the membrane was then washed 3 times with HRP-labeled anti-rabbit secondary antibody for 10 minutes each, followed by 1 hour incubation with TBST.
(6) Finally, ECL (BI, cat# 20-500-120) exposure was used. The strips were scanned using ChemiScope Western Blot imaging system (Clinx Science Instruments co., ltd) and then subjected to grey scale ratio analysis using Image J software (Rawak Software, inc.
The results show that the expression level of SINHCAF protein of the AMSC modified by SINHCAF intervention is obviously lower than that of the AMSC-Ctrl and natural AMSCs of the control group, and can be down-regulated to 15-50 percent.
4. Analysis of native AMSC, AMSC-Ctrl and AMSC-SINHCAF cell senescence phenotypes in an in vitro culture passage-induced cell replication senescence model:
Adult adipose tissue-derived natural hAMSC, hAMSC-Ctrl and hAMSC-SINHCAF cells were transferred to the 20 th generation, respectively; mouse fat-derived native mAMSC, mAMSC-Ctrl and mAMSC-SINHCAF cells were transferred to passage 8, respectively. As can be seen by observation under a light microscope, the natural hAMSC and hAMSC-Ctrl cells are obviously enlarged and flattened, the cell nucleus is enlarged, and cavitation occurs; the mitochondria in the natural hAMSC and hAMSC-Ctrl cells are obviously fused and increased under the electron microscope, and the characteristic of the aging cells is achieved. The hAMSC-SINHCAF cells can still maintain the long shuttle type characteristics of classical MSC cells, and the cell nucleus and the mitochondrial morphology are not obviously changed compared with cells in the early passage (such as the 2 nd generation cells). Likewise, mAMSC of mouse fat origin also performed similarly after in vitro culture passaging. The SINHCAF modification is shown to be effective in preventing AMSC cell senescence.
4.1. Further, the staining conditions of the natural AMSC, AMSC-Ctrl and AMSC-SINHCAF cells were compared by using a cell senescence beta-galactosidase (SA-beta-gal) staining kit (C0602, biyun Tian). The method comprises the following specific steps:
(1) Cells were transferred to 6-well plates for overnight incubation, the cell culture fluid was aspirated, washed 1 time with PBS or HBSS, and 1ml of staining fixative contained in the kit was added and fixed at room temperature for 15 minutes.
(2) The cell fixative was aspirated and the cells were washed 3 times for 3 minutes with PBS.
(3) The PBS was removed by pipetting, and 1 ml of staining fluid was added to each well. The formula of the working solution comprises the following steps: staining solution A (10. Mu.l) +staining solution B (10. Mu.l) +staining solution C (930. Mu.l) +X-Gal solution (50. Mu.l).
(4) After sealing the 6-well plate with a preservative film, the plate was incubated overnight at 37 ℃.
(5) The staining working solution was removed, 2 ml of PBS was added, and the mixture was observed under a common optical microscope.
The results show that SINHCAF intervening modification can significantly reduce the SA- β -gal blue positive rate of hAMSC and mAMSC.
4.2. The expression levels of p21 and p16 proteins in native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells were detected by Western Blot.
The steps of cell lysis, protein sample preparation, quantification, gel running, membrane transfer, antibody incubation, membrane washing, exposure, imaging, gray scale ratio analysis and the like are similar to the steps described above. One antibody was p21 primary antibody (Abcam, cat No. ab109199,1:1000 dilution) and one p16 primary antibody (Abcam, human cell sample ab108349, mouse cell sample ab211542,1:1000 dilution).
The results show that SINHCAF intervening modifications can significantly reduce the p21 and p16 protein expression levels of hAMSC and mAMSC.
The design idea of the invention is to construct SINHCAF intervening modified AMSC, thereby delaying or even inhibiting AMSC cell aging, eliminating cell function damage related to aging and improving cell expansion capability. The cell senescence includes, but is not limited to, replicative senescence caused by passage expansion, cell senescence induced by chemotherapeutics, toxins, oxidative stress, irradiation and the like, and senescence load of cells themselves derived from aged individuals or metabolic disease individuals.
It is another object of the present invention to provide the use of SINHCAF tamper-modified AMSC (AMSC-SINHCAF) for the preparation of a biologic for the treatment of liver disease.
The invention also discloses the effect and advantages of the AMSC-SINHCAF cell preparation in treating liver diseases, and the AMSC-SINHCAF cell preparation has any one or more of the following effects: compared with the natural AMSC with the same cell number, the AMSC-SINHCAF can more effectively exert the curative effects on liver diseases such as liver failure and the like; (2) reducing serum ALT, AST levels; (3) reducing liver inflammation and reducing the degree of necrosis of liver tissue; (4) correcting mitochondrial dysfunction of liver tissue cells.
Preferably, the liver disease includes, but is not limited to, drug or toxin induced acute liver injury, hepatitis, liver failure, liver fibrosis. The hepatitis includes viral hepatitis, drug hepatitis, steatohepatitis, alcoholic hepatitis and autoimmune hepatitis.
The preparation period of the main cell component AMSC-SINHCAF of the biological preparation for treating liver diseases provided by the invention can be reduced by more than 50% compared with that of the traditional cell component AMSC; and can eliminate the function damage of the aging-related AMSC cells, and improve the application potential of the AMSC preparation in clinical treatment of liver diseases.
The method may be in vivo. It is also possible to use it in vitro, for example only for scientific research.
The inventors have succeeded in establishing techniques for isolating cultured MSCs from adipose tissue in earlier studies. Compared with bone marrow-derived MSC, adipose-derived MSC (AMSC) has the advantages of more abundant sources, more convenient acquisition and higher amplification efficiency, thereby having good application prospect in clinic. However, we found that during both in vitro culture expansion of AMSCs (hAMCs) in mice (mAMSC) and humans, AMSCs showed significant senescence characteristics, such as increased SA- β -gal staining, increased intracellular p21 and p16 protein expression levels, and also significantly increased cell senescence-associated secretory phenotype (SASP) associated factors in cells and supernatants, with a decrease in cell proliferation rate, a significant increase and flattening of cells and vacuolization of nuclei. At the same time, the cell mitochondria also show increased fusion and up-regulated mitochondrial ROS levels. In addition, the treatment of mice liver injury and liver failure model is carried out by adopting AMSC with aging phenotype, and the curative effect is also obviously reduced, and the curative effect is obviously different from that of the non-aging (good-state) AMSC in various aspects of inhibiting liver inflammation, repairing liver injury, recovering liver function and the like, and even has no definite curative effect compared with a solvent control group. Thus, the present invention aims to provide a method for alleviating or even inhibiting AMSC aging by epigenetic modification, thereby increasing the therapeutic potential of AMSC in severe liver diseases such as liver failure.
The SINHCAF of the present invention intervenes in the modified AMSCs has the advantages of: (1) Compared with natural AMSC, the method has higher amplification efficiency and resists cell aging induced by various factors such as replication, chemotherapeutics, radiation and the like; (2) The cell senescence phenotype of the AMSC derived from adipose tissue of an elderly individual or a metabolic disease patient can be corrected, the treatment potential of the AMSC is improved, and feasibility is provided for autologous AMSC transplantation to treat senescence-associated degenerative diseases or metabolic diseases; (3) The AMSC and the liver disease treatment preparation or medicament prepared from the AMSC have better liver disease treatment effect.
Drawings
Fig. 1: SINHCAF protein and mRNA expression levels in AMSC cells: SINCGAF intervention modification reduced SINHCAF protein expression levels in mouse mAMSC and adult hAMSC cells without affecting SINHCAF mRNA levels. * P < 0.01, ns indicates no statistical difference.
Fig. 2: effect of SINHCAF intervention modification on AMSC senescence phenotype: SINCGAF intervention modification reduces the expression level of SASP-related molecules in AMSC of the replicative senescence model. SINCGAF intervention modification can reduce p21 and p16 protein expression levels in AMSC of replicative senescence model. SINCGAF intervention modification can reduce mitochondrial ROS levels in AMSCs replicating aging models. * P < 0.01, ns indicates no statistical difference.
Fig. 3: SINHCAF intervention modification corrects the senescent phenotype of aged mice AMSC: western Blot experiments show that the expression level of p21 and p16 proteins of oAMSC modified by SINHCAF intervention is obviously lower than that of a control group oAMSC-Ctrl and is similar to that of yAMSC cells. B. Cellular ROS level assays showed that the ROS level in oAMSC cells was significantly higher than yAMSC; whereas oAMSC-SINHCAF had significantly lower ROS levels than the control oAMSC-Ctrl. * P < 0.01; yAMSC AMSC of young 6-week-old mice; oAMSC: AMSC of old mice of 16 months old.
Fig. 4: SINHCAF intervention modifies the senescent phenotype of mice derived from AMSC that corrects metabolic disease: the SA-beta-gal staining result shows that the blue staining positive rate of mdAMSC cells is obviously higher than ncAMSC, and the blue staining positive rate of mdAMSC-SINHCAF cells is obviously lower than that of a control group mdAMSC-Ctrl; B. reactive Oxygen Species (ROS) assay showed that mdAMSC cells had ROS levels significantly higher than ncAMSC, while mdAMSC-SINHCAF cells had ROS levels significantly lower than control mdAMSC-Ctrl. * P < 0.01; mdAMSC mouse AMSC as metabolic disease model; ncAMSC: AMSC in normal mice of the same week age.
Detailed Description
The invention is further described with reference to the drawings and examples.
Example 1SINHCAF intervention modification affects SINHCAF protein expression levels in AMSC cells:
Protein expression levels of SINHCAF in native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells were detected by Western Blot. And detecting the mRNA expression level of SINHCAF in the above cells by PCR method.
As shown in FIG. 1, SINHCAF-modified mAMSC (mAMSC-SINHCAF) had significantly lower SINHCAF protein expression levels than control mAMSC-Ctrl and native mAMSC; likewise, SINHCAF-modified hAMSC (hAMSC-SINHCAF) also had significantly lower SINHCAF protein expression levels than control hAMSC-Ctrl and native hAMSC; however, none of the SINHCAF MRNA levels in hAMSC and mAMSC cells differed significantly after SINHCAF intervention modification, indicating that the modification was predominantly posttranscriptional at levels of translation affecting SINHCAF expression.
Example 2SINHCAF intervention modification against culture passage-induced AMSC cell senescence:
The native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells derived from adipose tissue of mice and adults are respectively transferred to the 8 th generation (mAMSC) and the 20 th generation (hAMSC), and a cell replicative aging model induced by culture passage is established.
The staining of native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells was compared using SA- β -gal staining. The staining of 5 fields was counted by light microscopy and statistically analyzed. The results show that the SA-beta-gal blue-dyeing positive rate of natural hAMSC reaches 39.5+/-6.4%, the blue-dyeing positive rate of hAMSC-Ctrl reaches 44.1+/-5.4%, and the blue-dyeing positive rate of hAMSC-SINHCAF cells is only 12.5+/-1.9%. The SA-beta-gal blue-dyeing positive rate of natural mAMSC reaches 67.9+/-8.8%, the blue-dyeing positive rate of mAMSC-Ctrl reaches 71.6+/-8.7%, and the blue-dyeing positive rate of mAMSC-SINHCAF cells is only 21.4+/-4.2%.
Expression levels of senescence-associated secretion phenotypes Mmp3, mmp13, PAI-1, MCP-1, TNF-alpha and IL-6 in native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells were analyzed by the relative quantitative Real-time PCR method (FIG. 2A). The results show that the SASP related molecule expression level of the SINHCAF intervention modified AMSC is significantly lower than that of the control AMSC-Ctrl and the native AMSC.
The expression levels of p21 and p16 proteins in native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells were detected by Western Blot. The results showed that the p21 and p16 protein expression levels of the SINHCAF-intervention modified AMSC were significantly lower than those of the control AMSC-Ctrl and native AMSC (fig. 2B).
Increased mitochondrial fusion and increased ROS levels are an important marker of MSC aging. Cell mitochondrial morphology was observed by electron microscopy and mitochondrial ROS levels were analyzed by MitoSOX TM Red staining (Thermofisher, M36008). The result shows that the mitochondrial fusion enhancement degree of the AMSC modified by SINHCAF intervention is obviously lower than that of the AMSC-Ctrl and the natural AMSC of the control group; furthermore, AMSC-SINHCAF cells, which also had significantly lower Mitosox TM red fluorescence levels than the control AMSC-Ctrl and native AMSC (FIG. 2C).
Example 3SINHCAF intervention modification against chemotherapy drug-induced AMSC senescence:
Cell senescence was induced using Doxorubicin (Doxorubicin, dox). AMSC was plated in 6-well plates overnight, treated with 100nM Dox for 48 hours, then cultured in normal medium for 6 days, and cells were collected and assayed for p21 and p16 protein expression levels in native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells by Western Blot.
Results show that SINHCAF intervention modification can significantly reduce the increase of p21 and p16 protein expression in AMSC cells induced by chemotherapeutic drugs. In addition, there is a similar phenomenon in the 5-FU induced cellular senescence model. The SINHCAF intervention modification has a protective effect on AMSC aging induced by various chemotherapeutics.
Example 4SINHCAF intervention modification against radiation-induced AMSC senescence:
And (3) establishing a radiation-induced hAMSC cell aging model by adopting 137Cs gamma radiation, and carrying out 5.0Gy radiation on cell passages. Collecting about 10 generations of cells, and detecting the expression level of p21 and p16 proteins in natural AMSC, AMSC-Ctrl and AMSC-SINHCAF cells by using a Western Blot method; the staining of native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells was compared using SA- β -gal staining.
The results show that SINHCAF intervening modification can significantly reduce the increase of p21 and p16 protein expression in AMSC cells induced by irradiation, and reduce SA-beta-gal blue staining positive caused by irradiation. The SINHCAF intervention modification is also indicated to have a protective effect on irradiation-induced AMSC senescence.
Example 5SINHCAF intervention modification against AMSC senescence caused by oxidative stress and the like:
Cell senescence was induced by H 2O2 or the like. AMSC was plated in 6-well plates, cultured overnight, treated with 50. Mu. M H 2O2 for 2 hours, then cultured with a common medium for 48 hours, and cells were collected and assayed for p21 and p16 protein expression levels in native AMSC, AMSC-Ctrl and AMSC-SINHCAF cells by Western blot. And the cellular ROS levels were analyzed using the bi yun tian Reactive Oxygen Species (ROS) detection kit.
The results show that SINHCAF intervention modification can significantly reduce the elevation of p21 and p16 protein expression and elevation of ROS levels in H 2O2 -induced AMSC cells. In addition, the phenomenon is similar to that in the model of aging of cell aging induced by D-galactose and the like. The SINHCAF intervention modification is also shown to have a protective effect on cell senescence induced by oxidative stress and the like.
Example 6SINHCAF intervention modification reverses the senescent phenotype of aged mouse AMSC:
The 16 month old mice AMSC (oAMSC) were infected with SINHCAF lentivirus to construct SINHCAF-intervention modified cells (oAMSC-SINHCAF), oAMSC cells (oAMSC-Ctrl) infected with empty lentiviral vectors were used as controls, and 6 week old young mice AMSC (yAMSC) were used as comparisons.
The cells were taken at passage 4 and stained with SA-. Beta. -gal to compare the staining of yAMSC, oAMSC, oAMSC-Ctrl and oAMSC-SINHCAF cells. The results show that the blue staining positive rate of yAMSC cells is significantly lower than oAMSC; however, oAMSC modified by SINHCAF was found to have a significantly lower blue dye positive rate than the control group oAMSC-Ctrl, similar to the levels in yAMSC cells.
The expression levels of p21 and p16 proteins in native yAMSC, oAMSC, oAMSC-Ctrl and oAMSC-SINHCAF cells were detected by Western blot. The results showed that p21 and p16 protein expression levels in yAMSC cells were significantly lower than oAMSC; whereas SINHCAF intervened oAMSC, the p21 and p16 protein expression levels were significantly lower than those of the control group oAMSC-Ctrl, similar to those in yAMSC cells (fig. 3A).
Analysis of the above cellular ROS levels using Reactive Oxygen Species (ROS) detection kits also showed that the ROS levels in oAMSC cells were significantly higher than yAMSC; whereas oAMSC-SINHCAF had significantly lower ROS levels than the control oAMSC-Ctrl (FIG. 3B).
Example 7SINHCAF intervention modification corrects the senescent phenotype of the metabolic disease mice derived AMSC:
Separating metabolic disease mice (mice with type 2 diabetes combined fatty liver model induced by high fat diet and STZ treatment, mdAMSC), culturing until generation 2, infecting SINHCAF lentivirus to construct cells (mdAMSC-SINHCAF) modified by SINHCAF intervention, and continuing subculturing until generation 4 by using mdAMSC cells (mdAMSC-Ctrl) infected with empty lentiviral vector as control; and the 4 th generation cells of AMSC (ncAMSC) derived from normal mice of the same week age were used as a comparison.
Then, as shown in example 6, the aging characteristics of the cells were analyzed by SA-. Beta. -gal staining, western Blot, reactive Oxygen Species (ROS) assay kit, respectively. The results show that the blue staining positive rate, the p21 and p16 expression levels and the ROS level of mdAMSC cells are all obviously higher than ncAMSC; the SINHCAF intervention modification can obviously correct the aging characteristics, and the characteristics are shown that the blue dyeing positive rate, the p21 and p16 expression level and the ROS level of mdAMSC-SINHCAF cells are all obviously lower than those of mdAMSC-Ctrl of a control group and are similar to those of ncAMSC cells (figure 4).
Example 8SINHCAF intervention modification improves AMSC cell expansion efficiency:
The proliferation of native hAMSC, hAMSC-Ctrl and hAMSC-SINHCAF cells was compared using the CCK8 method. The results show that the proliferation rate of hAMSC-SINHCAF cells is significantly higher than that of native hAMSC and hAMSC-Ctrl cells; especially in cells after 10 passages, the difference in proliferation rate of cells is more remarkable; after 20 generations, hAMSC-SINHCAF cells can increase their proliferation rate by more than 2.5 times compared with native hAMSC and hAMSC-Ctrl cells.
The passaging times of the native mAMSC, mAMSC-Ctrl and mAMSC-SINHCAF cells were further compared. After the natural mAMSC and mAMSC-Ctrl are respectively transmitted to the 9 th generation and the 8 th generation, the cell proliferation rate is obviously reduced, the cell enters a cell cycle arrest state, and the expansion is almost difficult; however, mAMSC-SINHCAF cells continued to expand by passage although their proliferation rate was reduced after 15 passages.
Example 9SINHCAF intervention modification can enhance the corrective effect of aging AMSC on liver disease mitochondrial abnormalities:
The intraperitoneal injection of APAP in C57 mice establishes a drug-induced acute liver injury (DILI) model. Immediately after molding, 5X 10 5 of yAMSC, oAMSC, oAMSC-Ctrl and oAMSC-SINHCAF cells were infused into the tail vein, and a corresponding volume of PBS was injected into the tail vein as a control group (vehicle). After 12h of modeling, mice were sacrificed and serum and liver tissue were collected for detection of liver function, liver pathology, mitochondrial dynamics related molecules, mitochondrial function, etc.
The results show that young mouse AMSC (yAMSC) has significantly better efficacy on the mouse DILI model than old mouse AMSC (oAMSC), and is expressed as: more effectively reducing serum ALT and AST levels; relieving liver inflammation and reducing liver tissue necrosis; correcting mitochondrial dynamics related molecules (e.g., MFN1, MFN2, OPA1, FIS1, and p-DRP1/DRP 1) levels; reduce the level of mitochondrial ROS, restore mitochondrial membrane potential and inhibit the opening of mitochondrial permeability transition pore.
SINHCAF intervention modification can remarkably improve the curative effect of oAMSC on liver failure mouse models, and oAMSC-SINHCAF can reduce serum ALT and AST levels more effectively than oAMSC-Ctrl; relieving liver inflammation and reducing liver tissue necrosis; correcting the expression level of the molecule related to mitochondrial dynamics. oAMSC-SINHCAF has therapeutic effect on liver failure equivalent to yAMSC and even better effect on correcting mitochondrial dysfunction.
In addition, SINHCAF intervention modification can enhance the correction effect of replicative senescence and metabolic senescence AMSC on liver disease mitochondrial abnormalities.
Example 10SINHCAF intervention modification can enhance the efficacy of aging AMSC on liver failure mice models:
C57 mice were intraperitoneally injected with LPS/GalN (10. Mu.g/kg LPS+500mg/kg D-GalN) to establish an acute liver failure model. Immediately after molding, 5X 10 5 of yAMSC, oAMSC, oAMSC-Ctrl and oAMSC-SINHCAF cells were infused into the tail vein, and a corresponding volume of PBS was injected into the tail vein as a control group (vehicle). After 6h of modeling, mice were sacrificed and serum, liver tissue, etc. were collected for liver function, liver pathology, inflammatory factor detection, etc. Serum ALT and AST were assayed using FUJIDRI-CHEM SLIDE GFP/ALT-PIII and GOT/AST-PIII kits, respectively, using DRI-CHEM 4000ie (FUJIFILM). Liver pathology was detected using HE. Serum inflammatory factors were detected using the corresponding ELISA kit.
The results show that the curative effect of young mouse AMSC (yAMSC) on acute liver failure of mice is significantly better than that of old mouse AMSC (oAMSC), and the results are shown as follows: more effectively reducing serum ALT and AST levels; relieving liver inflammation and reducing liver tissue necrosis; reduces serum IL-1 beta, IL-6 and TNF-alpha levels.
SINHCAF intervention modification can remarkably improve the curative effect of oAMSC on liver failure mouse models, and oAMSC-SINHCAF can reduce serum ALT and AST levels more effectively than oAMSC-Ctrl; relieving liver inflammation and reducing liver tissue necrosis; reduces serum IL-1 beta, IL-6 and TNF-alpha levels. oAMSC-SINHCAF has the same therapeutic effect on liver failure as yAMSC, and even has better effect on reducing the serum inflammatory factor level.
In addition, SINHCAF intervention modification has a remarkable improvement effect on the liver failure treatment effect of replicative senescence and metabolic senescence AMSC. The high algebra mAMSC or mdAMSC tail vein infusion can not effectively relieve liver injury in a LPS/GalN induced liver failure model, and reduce serum ALT, AST and inflammatory factor levels; compared with the same algebra mAMSC-Ctrl, mdAMSC-SINHCAF, the high algebra mAMSC-SINHCAF can obviously improve liver pathology and liver function, and has definite control effect on liver inflammation.
In mice acute and chronic liver injury models induced by ConA, APAP, CCl 4 and other factors, SINHCAF intervention modification is also shown to remarkably improve the curative effect of aging AMSC.
Sequence listing
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taggctgggc tgggttagac cctcggcagt agtcagcatg tggaacactg taaagtgctg 300
tgtcacggac actgactttt caaagtccag taagtcagtc tgcttagagg tcagtgggca 360
atactatgct atgttctggc cccatgttta cagtatggct gggatatcat catatactgt 420
aagtttgtga tgagacacta cagtatagat gatgtactag tcacagtat 469
Claims (2)
1. A method for anti-mesenchymal stem cell aging modification for non-disease treatment, which is characterized by comprising the following steps:
(1) Preparation of adipose-derived mesenchymal stem cells:
Separating mouse AMSC and adult AMSC according to conventional method, respectively adopting STEMCELL ™ mouse and human MesenCult ™ amplification kit, adding L-glutamine as complete culture medium to make culture amplification, and when AMSC proliferation is close to 75%, making digestion and passaging;
(2) Construction of SINHCAF modified AMSC:
Construction of sinhcaf modified hAMSC: infecting hAMSC with hSINHCAF lentiviral vector pLVX-IRES-ZsGreen1-SINHCAF, wherein the hSINCFAF lentiviral vector comprises a target fragment sequence shown as SEQ No.1 and is a targeting binding sequence comprising a 3' -UTR region of HSINHCAF MRNA;
Construction of sinhcaf modified mAMSC:
infection of mAMSC with mSINHCAF lentiviral vector pLVX-IRES-ZsGreen1-SINHCAF, the mSINCFAF lentiviral vector comprising the target fragment sequence shown in SEQ No.2, which is a targeting binding sequence comprising the 3' -UTR region of MSINHCAF MRNA; mAMSC infected with empty lentivirus was used as a control;
(3) Analysis of SINHCAF expression level in AMSC-SINHCAF:
Detecting protein expression levels of SINHCAF in natural AMSC, AMSC-Ctrl and AMSC-SINHCAF cells by adopting a Western Blot method; the method comprises the following specific steps:
(a) Digesting, centrifuging and collecting cells, and collecting supernatant;
(b) Carrying out protein quantification;
(c) Loading a cell protein sample, running gel, transferring a membrane, incubating a first antibody SINHCAF, incubating a second antibody labeled with HRP, and washing the membrane;
(d) The band is subjected to gray ratio analysis, and the result shows that the expression level of the SINHCAF protein of the AMSC modified by SINHCAF intervention is obviously lower than that of the AMSC-Ctrl and natural AMSCs of a control group, and can be adjusted to 15-50%;
(4) Analysis of native AMSC, AMSC-Ctrl and AMSC-SINHCAF cell senescence phenotypes in an in vitro culture passage-induced cell replication senescence model:
the staining conditions of the natural AMSC, AMSC-Ctrl and AMSC-SINHCAF cells are compared by using a cell senescence beta-galactosidase staining kit, and the expression levels of p21 and p16 proteins in the natural AMSC, AMSC-Ctrl and AMSC-SINHCAF cells are detected by using a Western Blot method, so that the expression levels of p21 and p16 proteins in hAMSC and mAMSC can be obviously reduced by SINHCAF intervention modification.
2. Use of SINHCAF of the modified AMSC provided by the method of claim 1 for the preparation of a biologic for treating liver disease, which is liver failure, drug or toxin induced acute liver injury, by constructing SINHCAF of the modified AMSC to delay or even inhibit AMSC cell senescence, eliminate cell function damage associated with senescence, and increase cell expansion capacity.
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