CN117771265A - Small molecular RNA for delaying mesenchymal stem cell aging and application thereof - Google Patents
Small molecular RNA for delaying mesenchymal stem cell aging and application thereof Download PDFInfo
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Abstract
The invention relates to a small molecular RNA for delaying mesenchymal stem cell aging and application thereof. The small molecular RNA is tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4, and the nucleotide sequence is shown as SEQ ID NO. 1-3. The invention discovers that the tRF-1:32-Gly-GCC-1, the tRF-1:32-Glu-CTC-1-M2 and the tRF-1:29-Gly-CCC-1-M4 have the function of delaying the aging of mesenchymal stem cells for the first time, and the verification experiments of the invention show that the small molecular RNAs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4) can improve the proliferation capacity of the aging mesenchymal stem cells, reduce the active oxygen level of the aging mesenchymal stem cells, further delay the aging of the mesenchymal stem cells, can be used for preparing medicaments for treating related diseases caused by the aging of the mesenchymal stem cells, and provide a new method and a new idea for the clinical treatment and development of medicaments for the related diseases of the aging of the mesenchymal stem cells.
Description
Technical Field
The invention relates to a small molecular RNA for delaying mesenchymal stem cell aging and application thereof, belonging to the technical field of biomedicine.
Background
Aging refers to a phenomenon that an imbalance of homeostasis and a decrease of stress level in the body occur as the organism ages, causing a decrease of functions of various organs of the body, and finally causing irreversible deterioration and death of the organism. Cell aging is one of the causes of aging of organisms, is an important cause of aging-related diseases, and is also becoming an emerging medical and socioeconomic threat. Cell aging is a permanent and irreversible cell cycle arrest caused by various stresses, resulting in impaired functions of cell proliferation, migration, paracrine, etc. Prolonged exposure to risk factors such as smoking, high sugar, hypertension, hyperlipidemia, etc. can accelerate cell aging. Senescent cells are generally identified by a number of features, such as the overexpression of β -galactosidase (SA- β -Gal) in senescent cells, a common marker for senescent cells. In addition, P21 is highly expressed in a variety of stimulus-induced senescent cells. Many senescence-inducing factors cause damage to the genome of cells, leading to the appearance of DNA damage lesions and DNA damage response mediated signal transduction, where detection of DNA damage response marker molecules such as gamma-H2 AX is also a method of identifying senescent cells.
Mesenchymal stem cells (mesenchymal stem cells, MSCs) are progenitor cells with self-renewal and multipotent differentiation potential that can be committed to induce differentiation into various tissue cells, such as adipocytes, muscle cells, endothelial cells, vascular smooth muscle cells, osteoblasts and chondrocytes, and tissue cells of other germ layers. MSCs have low immunogenicity, immunoregulatory activity, inflammatory chemotaxis, homing ability, good multidirectional differentiation potential, good proliferation ability and other biological functions, are important seed cells for regenerative medicine, and are widely applied to clinical stem cell transplantation for treating various diseases, such as endocrine diseases, cardiovascular diseases, nervous system diseases, immune system diseases, bone system diseases (poor fracture healing, degenerative arthritis, multiple sclerosis) and the like. However, MSCs age with changes in biological characteristics due to aging, risk factor stimulation (extracellular environment and various physicochemical stimuli), or in vitro large number of replicative passaging amplifications. Therefore, searching for a method for effectively delaying MSCs has important significance for treating various clinical diseases.
MSCs are widely available and exist in various tissues such as bone marrow, periosteum, dental pulp, fat, placenta, muscle, hair follicle, umbilical cord, etc. Among them, self-renewal and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (bone marrow mesenchymal stem cells, BMMSCs) are important mechanisms for maintaining bone tissue homeostasis and fracture repair, and have great potential application value in bone repair and bone regeneration due to their wide sources and easy availability. The accumulation of aging BMMSCs directly affects bone remodeling and bone homeostasis, which causes significant reduction in bone marrow renewal activity, new bone formation rate and fracture repair rate, and causes bone tissue degenerative diseases (such as senile osteoporosis) such as delayed bone healing, bone mass loss and osteoarthritis; in addition, BMMSCs can play an important role in heart disease. BMMSCs are capable of reducing myocardial fibrosis, stimulating angiogenesis, endogenous stimulation of proliferation and differentiation of cardiac stem cells. BMMSCs exert paracrine effects to promote angiogenesis by secreting proteins or exosomes associated with angiogenesis. The decreased replicative capacity of senescent BMMSCs and abnormal changes in cellular function affect the regenerative potential of the cardiovascular system. Thus, aging of BMMSCs is closely related to the occurrence of various age-related degenerative diseases, and searching for a method for delaying BMMSCs is critical for the treatment of degenerative diseases.
Compared with other medicines, the small molecular RNAs generally play a role in genes or expression levels thereof, have higher specificity and targeting property, have short half-life, small side effect and high safety, and the advantages make the small molecular RNAs become new pets for clinical medicine research, and raise the hot trend of the small molecular RNAs in the field of biological pharmacy. Small molecule RNAs drugs mainly include micrornas (microRNA, miRNAs), small interfering RNAs (Small interfering RNAs, siRNAs), transfer RNA-derived fragments (transfer RNAs-derived fragments, tRFs), and the like. tRs (transfer RNA-derived fragments, tRs) are an emerging class of non-coding small fragment RNAs of less than 40 bases in length derived from transfer RNAs (tRNAs) that are cleaved by tRNA enzymes and Angiogenin (ANG) under special conditions such as hypoxia stress. Along with the development of high-throughput sequencing technology, more and more evidence shows that the tFs play an important role in the key processes of stem cell self-renewal, differentiation, proliferation and the like mainly by means of regulating gene expression through interaction with proteins, regulating gene expression through targeting, regulating protein level through influencing a translation process and the like, and are closely related to the occurrence and development of various diseases such as cancers, virus infection, metabolic diseases, neurodegenerative diseases and the like.
At present, research on small molecule RNA drugs, especially tFs, is still in the primary stage, and research on the effect of tFs on cell senescence delay, especially on mesenchymal stem cell senescence delay is still very lacking. Therefore, there is an urgent need to explore the role of small-molecule RNA drugs (especially tRFs) in delaying mesenchymal stem cell senescence, and simultaneously provide novel, effective and safe small-molecule RNA drugs for the treatment of senescence-associated diseases.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides small molecular RNA for delaying the aging of mesenchymal stem cells and application thereof.
The technical scheme of the invention is as follows:
a small molecular RNA for delaying mesenchymal stem cell aging, wherein the small molecular RNA is tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4;
the nucleotide sequence of the tRF-1:32-Gly-GCC-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the tRF-1:32-Glu-CTC-1-M2 is shown in SEQ ID NO. 2;
the nucleotide sequence of the tRF-1:29-Gly-CCC-1-M4 is shown as SEQ ID NO. 3.
According to the present invention, the mesenchymal stem cells are preferably derived from mice, humans, rabbits, and the like.
Further preferably, the mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta mesenchymal stem cells, gingiva mesenchymal stem cells or muscle mesenchymal stem cells.
Preferably, according to the present invention, the mesenchymal stem cell senescence is natural senescence, stress senescence or senescence caused by in vitro mass replication and passaging expansion. The natural aging refers to aging caused by intrinsic factors of mesenchymal stem cells; the stress aging refers to aging of mesenchymal stem cells caused by stimulation of dangerous factors such as light stimulation (ultraviolet rays), electric stimulation (ionizing radiation) or drug stimulation (doxorubicin, hydrogen peroxide, RO3306, nutlin-3 a).
According to the invention, the small molecular RNA for delaying the aging of the mesenchymal stem cells can delay the aging of the mesenchymal stem cells, improve the proliferation capacity of the aged mesenchymal stem cells and reduce the active oxygen level of the aged mesenchymal stem cells.
Application of the small molecular RNA in preparing a medicament for treating mesenchymal stem cell aging-related diseases.
According to a preferred embodiment of the present invention, the mesenchymal stem cell aging-related disease is senile osteoporosis caused by bone marrow mesenchymal stem cell aging.
According to the invention, preferably, the medicament for treating mesenchymal stem cell aging-related diseases comprises one or more than two of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4;
the nucleotide sequence of the tRF-1:32-Gly-GCC-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the tRF-1:32-Glu-CTC-1-M2 is shown in SEQ ID NO. 2;
the nucleotide sequence of the tRF-1:29-Gly-CCC-1-M4 is shown as SEQ ID NO. 3.
The application of the small molecular RNA in preparing an anti-aging culture medium for mesenchymal stem cell culture or passage.
Application of the small molecular RNA in preparing a reagent for improving the biological function of mesenchymal stem cells.
According to the invention, preferably, the medicament for treating mesenchymal stem cell aging-related diseases further comprises a pharmaceutically acceptable carrier.
According to the invention, the medicament for treating the mesenchymal stem cell aging-related diseases is preferably in the form of injection, capsule, tablet, oral preparation or microcapsule preparation.
The beneficial effects are that:
1. the invention discovers that the tRF-1:32-Gly-GCC-1, the tRF-1:32-Glu-CTC-1-M2 and the tRF-1:29-Gly-CCC-1-M4 have the function of delaying the aging of mesenchymal stem cells for the first time, and the verification experiments of the invention show that the small molecular RNAs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4) can improve the proliferation capacity of the aging mesenchymal stem cells, reduce the active oxygen level of the aging mesenchymal stem cells, delay the aging of the mesenchymal stem cells, can be used for preparing medicaments for treating related diseases caused by the aging of the mesenchymal stem cells, and provide a new method and a new idea for the clinical treatment and development of medicaments for the related diseases of the aging of the mesenchymal stem cells.
2. The application of the small molecular RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 in delaying mesenchymal stem cell aging is disclosed for the first time. The small molecular RNA medicine has the advantages of simple preparation method and easily obtained raw materials, and can realize engineering, large-scale and standardized preparation and production. Compared with the traditional medicines, the small molecular RNAs generally play a role in genes or expression levels thereof, have higher specificity and targeting property, and have high activity, short half-life, small side effect, high safety, low cost, sufficient control strategies such as sterility/microorganism/impurity and the like, perfect preparation process and quality system and long duration of treatment effect; in addition, compared with other medicines, the method for designing the small molecular RNA medicine is simple, and the corresponding intervention RNA can be designed on the basis of the pathogenic gene sequence, so that the screening time of target spots can be saved, any genes can be targeted, the off-target effect can be predicted, the disease treatment risk can be reduced, and the accurate treatment of the disease is possible.
3. The tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 provided by the invention have the function of delaying the aging of mesenchymal stem cells, can be applied to the culture and replicative passage expansion of the mesenchymal stem cells, can improve the cell proliferation characteristics, reduce the active oxygen level and delay the aging of the mesenchymal stem cells of different generations, and provides a new idea for the subsequent preparation of a culture medium or culture medicament of the mesenchymal stem cells.
Drawings
FIG. 1 is a graph showing the results of a small molecule RNA drug (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4) with significant delay in aging of BMMSCs in aged mice;
in fig. 1, a is a graph (400×) of the β -galactosidase staining results of aged mouse BMMSCs; b is a statistical plot of the results of beta-galactosidase staining of aged mouse BMMSCs.
FIG. 2 is a graph showing the results of improving the proliferation potency of BMMSCs in aged mice by small molecule RNA drugs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4), specifically, the results of detecting the proliferation potency of BMMSCs in aged mice by cell proliferation experiments.
FIG. 3 is a graph showing the results of small molecule RNA drugs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4) reducing BMMSCs Reactive Oxygen Species (ROS) in geriatric mice;
in fig. 3, a is a graph of the results of flow cytometry detection of ROS levels in aged mouse BMMSCs; b is a statistical plot of ROS levels detected in aged mouse BMMSCs by flow cytometry.
FIG. 4 is a graph showing the results of small molecule RNA drugs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4) with significant delay in doxorubicin-induced aging of BMMSCs;
in fig. 4, a is a graph of the result of beta-galactosidase staining (400×); b is a statistical plot of the results of beta-galactosidase staining.
FIG. 5 is a graph showing the results of small molecule RNA drugs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4) for improving the proliferation potency of doxorubicin-induced aged BMMSCs, specifically, the results of cell proliferation assay for the proliferation potency of doxorubicin-induced aged BMMSCs.
FIG. 6 is a graph showing the results of small molecule RNA drugs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4) reducing doxorubicin-induced Reactive Oxygen Species (ROS) in aging BMMSCs;
in fig. 6, a is a graph of the results of flow cytometry detection of ROS levels in BMMSCs; b is a statistical plot of flow cell count of BMMSCs detecting ROS levels in BMMSCs.
FIG. 7 is a graph showing the results of small molecule RNA drugs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4) with significant delay in doxorubicin-induced senescence in human BMMSCs;
in fig. 7, a is a graph (400×) of the β -galactosidase staining results of human BMMSCs; b is a statistical plot of the results of beta-galactosidase staining of human BMMSCs.
FIG. 8 is a graph showing the results of small RNA drugs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4) with the effect of treating senile osteoporosis, specifically a reconstructed image of the microCT scan of the femur of each group of mice; the upper graph and the lower graph in the graph are respectively reconstructed graphs of upper and lower angles of the same section at the same position.
Detailed Description
The technical scheme of the present invention will be further described with reference to examples, but the scope of the present invention is not limited thereto. The reagents and materials referred to in the examples are all commercially available products unless otherwise specified.
Wherein, the mouse bone marrow mesenchymal stem cells are obtained from primary culture of mice, and the human BMMSCs are purchased from Siro biosciences Inc.
The nucleotide sequence of tRF-1:32-Gly-GCC-1 in the small molecule RNA drug (tFs) is shown as SEQ ID NO. 1; the nucleotide sequence of the tRF-1:32-Glu-CTC-1-M2 is shown as SEQ ID NO. 2; the nucleotide sequence of tRF-1:29-Gly-CCC-1-M4 is shown in SEQ ID NO. 3. Then entrusting the engineering biological technology limited company to synthesize the tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 according to the specific nucleotide sequence by a chemical method.
The experimental C57BL/6J mice were purchased from Experimental animal technologies Inc. of Lewa, beijing, and approved by the ethical committee of Shandong university college of medicine.
The siRNA-NC referred to in the following examples contained two single strands, the sequences of which are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.
Example 1
Bone marrow-derived mesenchymal stem cells (BMMSCs) of aged (18 months old) C57BL/6J mice are isolated and extracted, three small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4 are respectively given to the aged mice by a cell transfection method, and the effect of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 on the aging of the aged mice BMMSCs is detected.
The specific implementation process is as follows:
(1) Isolation and extraction of bone marrow derived mesenchymal Stem cells (BMMSCs) from aged mice
Taking elderly (18 month old) C57BL/6J mice, separating tibia and femur of the mice, removing surrounding tissue, exposing bone marrow cavity, collecting whole bone marrow, centrifuging at 1000 r/min for 5 min, inoculating into cell culture flask, culturing with F12 culture solution (Hyclone Co., U.S.) containing 10% fetal bovine serum (BI, USA) and 1% green-streptomycin (Biyundian, china), placing into 37deg.C, and 5% CO 2 Culturing in a cell incubator (Thermo, U.S.) designated as Aged micro; at the same time, BMMSCs of young (3 month old) C57BL/6J mice were isolated and cultured in the same manner as control group BMMSCs (Young mice);
(2) Cell transfection
When the BMMSCs of the primary cultured old mice are passaged to 3-5 generations, carrying out cell transfection by using a special transfection reagent (Hantao) for RNAFit RNA and a serum-free Opti-MEM culture medium (Thermo), and respectively transfecting tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 into the BMMSCs of the old mice to obtain 3 groups of experimental groups of BMMSCs respectively transfected with 3 small molecular RNAs, wherein the final concentrations of the tRF-1:32-Gly-GCC-1, the tRF-1:32-Glu-CTC-1-M2 and the tRF-1:29-Gly-CCC-1-M4 are all 100 nM; meanwhile, the Aged mice+NC group is a negative control group, and the BMMSCs of the old mice are given the siRNA-NC of the same dosage according to the same method; after transfection of 6 h, fresh culture medium containing 10% serum was replaced, and after transfection of 48 h, beta-galactosidase (SA-beta-Gal) staining was performed;
(3) Beta-galactosidase (SA-beta-Gal) staining
Taking the BMMSCs of the aged mice after the transfection culture in the step (2) and the BMMSCs of the control group in the step (1), discarding the cell culture solution, washing for 1 time by using PBS buffer solution, respectively adding the beta-galactosidase staining fixative (Biyun Tian biotechnology Co., ltd.) of 1mL, and fixing for 15 minutes at room temperature; after discarding the cell fixative, the cells were washed 3 times with PBS buffer, and 1mL of beta-galactosidase staining working solution (Biyun biotechnology Co., ltd.) was added again and incubated overnight at 37 ℃. The next day, the aging condition of the cells is observed under an optical microscope, and the photographing and analysis are carried out, and the result is shown in fig. 1;
as can be seen from the results of the beta-galactosidase staining in fig. 1, the proportion of beta-galactosidase staining positive cells in BMMSCs (Aged mice+nc) was significantly increased in Aged mice in the negative control group compared to young mice BMMSCs (Young mice) in the control group. Compared with the BMMSCs (Aged mice+NC) of the Aged mice in the negative control group, the proportion of beta-galactosidase staining positive cells in the BMMSCs of the Aged mice treated by the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 is obviously reduced, namely the aging of the BMMSCs of the Aged mice can be obviously reversed by the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4.
The results of this example demonstrate that the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the invention have the effect of significantly delaying the aging of BMMSCs of aged mice.
Example 2
BMMSCs of aged (18 months old) C57BL/6J mice are isolated and extracted, three small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4 are respectively given to the aged mice by a cell transfection method, and the influence of the tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 on the proliferation capacity of the aged mice BMMSCs is detected.
The specific implementation process is as follows:
(1) BMMSCs from aged (18 months of age) and young (3 months of age) C57BL/6J mice were isolated and extracted as described in example 1 to give control BMMSCs (Young mice) and aged mice BMMSCs (Aged mice);
(2) Cell proliferation assay
Taking the BMMSCs of the control group and the BMMSCs of the aged mice in the step (1) to 2 multiplied by 10 3 The density of/mL was inoculated on DMEM (source culture organism, china) medium in 96 well plates (NEST, china), respectively; cell transfection was performed on aged mouse BMMSCs using tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 as described in example 1, to obtain 3 groups of 3 small RNA-transfected experimental BMMSCs, respectively; at the time of transfection culture 48 h, cell counting reagent (Cell Counting Kit-8, CCK-8) was added and incubated at 37℃under light-shielding conditions 2 h; meanwhile, the Aged mice+NC group is a negative control group, and the BMMSCs of the old mice are given the siRNA-NC of the same dosage according to the same method; finally, an enzyme-labeled instrument (TECAN, switzerland) is used for detecting the absorbance (OD) value at the wavelength of 450 nm on the BMMSCs treated by the above method, statistics are carried out, and the effect of a small molecule RNA drug (tFs) on the proliferation capacity of the BMMSCs of the aged mice is analyzed, and the result is shown in fig. 2.
As can be seen from the cell proliferation experimental results of fig. 2, the proliferation capacity of BMMSCs (agent+nc) of the Aged mice of the negative control group was significantly reduced compared to the young mice BMMSCs (Young mice) of the control group; compared with the BMMSCs (Agedmice+NC) of the Aged mice in the negative control group, the proliferation capacity of the BMMSCs of the Aged mice can be remarkably improved after the treatment of the small-molecule RNA medicaments such as tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4.
The results of this example demonstrate that the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 have the effect of remarkably improving the proliferation of BMMSCs of aged mice, can improve the proliferation capacity of mesenchymal stem cells, can be applied to the culture and replicative passage expansion of mesenchymal stem cells, and are used for preparing culture mediums or culture drugs of the mesenchymal stem cells.
Example 3
BMMSCs of aged (18 months old) C57BL/6J mice are isolated and extracted, three small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4 are respectively given to the aged mice by a cell transfection method, and the effect of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 on the active oxygen (ROS) level of the BMMSCs of the aged mice is detected.
The specific implementation process is as follows:
(1) BMMSCs from aged (18 months of age) and young (3 months of age) C57BL/6J mice were isolated and extracted as described in example 1 to give control BMMSCs (Young mice) and aged mice BMMSCs (Aged mice);
(2) Flow cytometry
Taking the BMMSCs of the control group and the BMMSCs of the aged mice in the step (1) to 1 multiplied by 10 5 The density of/mL was inoculated onto DMEM (source organism, china) medium in 6-well plates (NEST, china) respectively, and cell transfection was performed on aged mouse BMMSCs using tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 as described in example 1, to obtain 3 groups of BMMSCs each transfected with 3 small molecule RNAs; meanwhile, the Aged mice+NC group is a negative control group, and the BMMSCs of the old mice are given the siRNA-NC of the same dosage according to the same method; after 48 h transfection cultures, the above-mentioned differently treated BMMSCs were subjected to flow cytometry to detect MSCs Reactive Oxygen Species (ROS); 2, 7-dichlorofluorescein diacetate (DCFH-DA) was diluted 1:1000 with serum-free medium to a final concentration of 10. Mu.M according to the Reactive Oxygen Species (ROS) kit (Biyun Biotechnology Co., ltd.); removing cell culture solution, adding 1mL diluted DCFH-DA, incubating in cell culture box at 37deg.C for 20 minAnd (3) a clock. The cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells, and immediately after collecting the cells, they were examined by flow cytometry, and the results are shown in fig. 3.
As can be seen from the results of the flow cytometry experiments in fig. 3, the level of Reactive Oxygen Species (ROS) in BMMSCs (Aged mice+nc) was significantly increased in Aged mice of the negative control group compared to young mice BMMSCs (Young mice) of the control group; compared with the BMMSCs (Agedmice+NC) of the Aged mice in the negative control group, the active oxygen (ROS) level in the BMMSCs of the Aged mice can be obviously reduced after the treatment of the small molecular RNA medicaments such as tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4.
The results of this example demonstrate that the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the present invention have the effect of significantly reducing the levels of BMMSCs Reactive Oxygen Species (ROS) in aged mice.
Example 4
BMMSCs of young (3 month old) C57BL/6J mice are isolated and extracted, a BMMSCs aging model is constructed in vitro by using Doxorubicin (DOX), three small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 or tRF-1:29-Gly-CCC-1-M4 are respectively given to the BMMSCs aging cell model for treatment, and the influence of the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 on the BMMSCs aging model is detected.
The specific implementation process is as follows:
(1) BMMSCs from young (3 month old) C57BL/6J mice were isolated and extracted as described in example 1;
(2) In vitro construction of BMMSCs aging model by doxorubicin
Primary culturing BMMSCs of young mice, constructing a BMMSCs aging model by using doxorubicin induction when the BMMSCs are passaged to 3-5 generations, and culturing the BMMSCs after the doxorubicin action concentration is 100 nM and 24 h to obtain the BMMSCs aging model induced by the doxorubicin;
(3) Young mouse BMMSCs and doxorubicin-induced BMSCs senescence models were cultured, and cell transfection was performed using RNA specific transfection reagent RNAFit (Hantao) and serum-free Opti-MEM medium (Thermo), and tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 were transfected into BMMSCs senescence models, respectively, so that the final concentrations of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 were 100 nM; meanwhile, 100 nM DOX+NC group is a negative control group, and the BMMSCs aging model is given the siRNA-NC with the same dosage according to the same method; after transfection and culture of 6 h, fresh culture solution containing 10% of serum is replaced, and after transfection of 48 h, a BMMSCs aging model transfected with 3 small molecule RNAs and a BMMSCs aging model transfected with siRNA-NC are obtained; untreated BMMSCs (not induced with doxorubicin and not transfected) were used as a blank (CTL);
(4) Beta-galactosidase (SA-beta-Gal) staining was performed on the BMMSCs senescence model transfected with tFs, the BMMSCs senescence model transfected with siRNA-NC, and untreated BMMSCs as described in example 1, and the results are shown in FIG. 4.
As can be seen from the results of the β -galactosidase staining in fig. 4, the proportion of β -galactosidase-stained positive cells in the BMMSCs in the negative control group (100 nM dox+nc) was significantly increased compared to the blank group (CTL), i.e., the BMMSCs in the negative control group were senescent. Compared with the BMMSCs in the negative control group, the proportion of beta-galactosidase staining positive cells is obviously reduced after the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 are treated, namely the stress aging of the BMMSCs caused by the application of doxorubicin can be obviously reversed by the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4.
The results of this example demonstrate that the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the invention have the effect of significantly delaying the aging of BMMSCs.
Example 5
BMMSCs of young (3 month old) C57BL/6J mice are isolated, a BMMSCs aging model is constructed in vitro by using Doxorubicin (DOX), three small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the BMMSCs aging model are respectively given to the BMMSCs aging model for treatment, and the influence of the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 on the proliferation capacity of the aging BMMSCs is detected.
(1) BMMSCs from young (3 month old) C57BL/6J mice were isolated and extracted as described in example 1;
(2) Cell proliferation assay
Taking BMMSCs obtained in the step (1) to 2 multiplied by 10 3 Density of/mL was inoculated on DMEM (source organism, china) medium in 96-well plates (new, china), doxorubicin was administered as described in example 4 steps (2) and (3), and 3 small-molecule RNAs were administered for cell transfection to obtain a tRFs-transfected BMMSCs senescence model and a siRNA-NC-transfected BMMSCs senescence model; untreated BMMSCs (not induced with doxorubicin and not transfected) were used as a blank (CTL); at 48 and h, CCK-8 reagent was added to the above-mentioned different BMMSCs, incubated at 37℃in the absence of light for 2 h, absorbance (OD) values at 450 nm wavelength were measured using a microplate reader (TECAN, switzerland) and counted, and the effect of small molecule RNA drugs (tFs) on the proliferation capacity of senescent BMMSCs was analyzed, and the results are shown in FIG. 5.
As can be seen from the cell proliferation assay results of fig. 5, the proliferation potency of BMMSCs was significantly reduced in the negative control group (100 nM dox+nc) compared to the blank group (CTL); compared with the BMMSCs in the negative control group, the proliferation capacity of the aging BMMSCs can be remarkably improved after the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 are treated.
The results of this example demonstrate that the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the invention have the effect of significantly improving the proliferation capacity of aging BMMSCs.
Example 6
BMMSCs of young (3 month old) C57BL/6J mice are isolated, a BMMSCs aging model is constructed in vitro by using Doxorubicin (DOX), three small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the BMMSCs aging model are respectively given to the BMMSCs for treatment, and the effects of the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 on the Reactive Oxygen Species (ROS) in the aging BMMSCs are detected.
(1) BMMSCs from young (3 month old) C57BL/6J mice were isolated and extracted as described in example 1;
(2) Flow cytometry
Taking the transfected and cultured BMMSCs obtained in the step (1) to 1 multiplied by 10 5 Density of/mL was inoculated on DMEM (source organism, china) medium in 6-well plates (new, china), doxorubicin was administered as described in example 4 steps (2) and (3), and 3 small-molecule RNAs were administered for cell transfection to obtain a tRFs-transfected BMMSCs senescence model and a siRNA-NC-transfected BMMSCs senescence model; untreated BMMSCs (not induced with doxorubicin and not transfected) were used as a blank (CTL); after 48 to h cell transfection culture, flow cytometry was performed on the different treated BMMSCs to detect MSCs Reactive Oxygen Species (ROS); the final concentration was 10 μmol/liter by diluting DCFH-DA with serum-free medium at 1:1000 according to the Reactive Oxygen Species (ROS) kit (bi yun biotechnology limited) instructions; the cell culture medium was removed, 1mL diluted DCFH-DA was added, and the cells were incubated in a 37℃incubator for 20 minutes. The cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells, and immediately after collecting the cells, they were examined by flow cytometry, and the results are shown in fig. 6.
As can be seen from the flow cytometry experimental results of fig. 6, the active oxygen level in the BMMSCs of the negative control group (100 nM DOX) was significantly increased compared to the blank group (CTL); compared with the BMMSCs in the negative control group, the active oxygen level in the aging BMMSCs can be obviously reduced after the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 are treated.
The results of this example demonstrate that the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the present invention have the effect of significantly reducing the active oxygen levels in aging BMMSCs.
Example 7
Culturing human BMMSCs, constructing a human BMMSCs aging model in vitro by using Doxorubicin (DOX), respectively giving three small molecule RNA drugs of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 or tRF-1:29-Gly-CCC-1-M4 to the human BMMSCs aging model, treating, and detecting the influence of the small molecule RNA drugs of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 on the human BMMSCs aging model.
The specific implementation process is as follows:
(1) Human BMMSCs were purchased from Siro Biotech Inc. (Suzhou)
(2) In vitro construction of human BMMSCs aging model by doxorubicin
Culturing human BMMSCs, constructing a human BMMSCs aging model by using doxorubicin induction, enabling the action concentration of the doxorubicin to be 100 nM, and culturing for 24 h to obtain the human BMMSCs aging model induced by the doxorubicin;
(3) Cell transfection was performed on normal human BMMSCs and doxorubicin-induced human BMSCs senescence models using RNA specific transfection reagents RNAFit (Hantao) and serum-free Opti-MEM medium (Thermo), and tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 were transfected into human BMMSCs senescence models, respectively, so that the final concentrations of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 were 100 nM; meanwhile, 100 nM DOX+NC group is a negative control group, and is that the human BMMSCs aging model is given the siRNA-NC of the same dosage according to the same method; after transfection and culture of 6 h, fresh culture solution containing 10% of serum is replaced, and after transfection of 48 h, a human BMMSCs aging model transfected with 3 small molecule RNAs and a human BMMSCs aging model transfected with siRNA-NC are obtained; untreated human BMMSCs (not induced with doxorubicin and not transfected) were used as a blank group (CTL);
(4) Beta-galactosidase (SA-beta-Gal) staining was performed on the human BMMSCs senescence model transfected with tFs, the human BMMSCs senescence model transfected with siRNA-NC, and untreated human BMMSCs as described in example 1, and the results are shown in FIG. 7.
As can be seen from the results of the β -galactosidase staining in fig. 7, the proportion of β -galactosidase-stained positive cells in the negative control (100 nM dox+nc) human BMMSCs was significantly increased, i.e., the human BMMSCs of the negative control were senescent, compared to the blank (CTL). Compared with the human BMMSCs in the negative control group, the proportion of beta-galactosidase staining positive cells is obviously reduced after the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 are treated, namely the stress aging of the human BMMSCs caused by the induction of doxorubicin can be obviously reversed by the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4.
The results of this example demonstrate that the small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 of the invention have the effect of significantly delaying the aging of human BMMSCs.
Example 8
Young (3 months of age) mice and old (18 months of age) mice were prepared, and the old (18 months of age) mice were given 3 small molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 or tRF-1:29-Gly-CCC-1-M4 for treatment as follows:
(1) Selecting 3 month-old male C57BL/6J mice (Young mice) and 18 month-old male C57BL/6J mice (Aged mice), and carrying out adaptive breeding for 1 week;
(2) The lipid nanoparticles are used for respectively preparing small-molecule RNA drugs tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 into nano delivery systems for loading the small-molecule RNA drugs; the Aged mice+NC group mice are Aged male C57BL/6J mice, and siRNA-NC treatment is given;
the method comprises the following steps: respectively placing 3 kinds of small molecular RNAs and PEG-PGu (polyethylene glycol-guanidino polymer) in a pipe A and a pipe B of a microfluidic device according to a mass ratio of 1:20, regulating the flow rate to ensure that the small molecular RNAs and the PEG-PGu are fully and uniformly mixed, and standing for 15 minutes after the uniform mixing to form electrostatic interaction force to obtain primary nano particles a; PLGA (polylactic acid-glycolic acid copolymer) was then mixed with PEG-PGu in a mass ratio of 1:1 are respectively placed in an A pipe (PLGA solution) and a B pipe (primary nanoparticle a suspension) of a microfluidic device, are gently mixed at a slow flow rate, are placed for 15 minutes after being mixed uniformly, and form electrostatic interaction force to respectively obtain nanoparticle suspension tFs@PPNPs loaded with tRF-1:32-Gly-GCC-1, nanoparticle suspension tFs@PPNPs loaded with tRF-1:32-Glu-CTC-1-M2 and nanoparticle suspension tFs@PPNPs loaded with tRF-1:29-Gly-CCC-1-M4;
simultaneously preparing a nanoparticle suspension loaded with the siRNA-NC by the siRNA-NC according to the same method;
(3) Treating the 3 nanoparticle suspensions tFs@PPNPs prepared in the step (2) to Aged male C57BL/6J mice (Aged mice) by administering 0.02 mug/g of the 3 nanoparticle suspensions tFs@PPNPs prepared in the step (2) respectively according to the weight of the mice through tail intravenous injection, continuously injecting for 4 weeks according to the frequency of 1 time every day, and administering the same dose of the nanoparticle suspension loaded with siRNA-NC to the Aged mice+NC group according to the same method to serve as a negative control group (NC);
(4) The mice treated in the above way were anesthetized by intraperitoneal injection of 3.6% chloral hydrate (10 mL/kg), bilateral femur of the mice was taken, and distal cancellous bone and cortical bone near the middle section of the bilateral femur were scanned and three-dimensionally reconstructed by using microCT to detect bone loss of the mice, and the results are shown in fig. 8.
As can be seen from the three-dimensional reconstruction results of the microCT shown in fig. 8, compared with Young mice (Young mice), the Aged mice (Aged mice) showed a significant reduction in the number and volume of bone trabeculae, i.e., bone loss phenomenon, indicating that the Aged osteoporosis mouse model was successfully obtained (since the aging of bone marrow mesenchymal stem cells is the main cause of Aged osteoporosis, we succeeded in obtaining the disease model related to the aging of bone marrow mesenchymal stem cells: aged osteoporosis model). Compared with Aged mice (Aged mice), the number and the volume of bone trabeculae of the Aged mice are obviously increased by +3 small molecular RNAs of the Aged mice given with the small molecular RNA drugs, which indicates that the bone loss phenomenon of the Aged osteoporosis mice can be effectively improved by the 3 small molecular RNAs (tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4).
The results of the example show that the small molecular RNA medicaments tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2 and tRF-1:29-Gly-CCC-1-M4 can treat senile osteoporosis by delaying the aging of mesenchymal stem cells, and all the 3 small molecular RNAs have remarkable effect of treating senile osteoporosis.
The examples are given for the purpose of illustrating preferred embodiments of the invention and are not to be construed as limiting the invention, but are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (9)
1. The application of the small molecular RNA in preparing the medicine for treating the mesenchymal stem cell senescence-related diseases is characterized in that the small molecular RNA is tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4;
the nucleotide sequence of the tRF-1:32-Gly-GCC-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the tRF-1:32-Glu-CTC-1-M2 is shown in SEQ ID NO. 2;
the nucleotide sequence of the tRF-1:29-Gly-CCC-1-M4 is shown as SEQ ID NO. 3.
2. The use of small molecule RNA according to claim 1 for the preparation of a medicament for the treatment of diseases related to mesenchymal stem cell aging, wherein the mesenchymal stem cells are of murine, human and rabbit origin.
3. The use of small molecule RNA of claim 1 in the preparation of a medicament for the treatment of a disease associated with aging of mesenchymal stem cells, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells.
4. The use of small molecule RNA according to claim 1 for the preparation of a medicament for the treatment of diseases related to mesenchymal stem cell aging, wherein the medicament for the treatment of diseases related to mesenchymal stem cell aging is capable of delaying mesenchymal stem cell aging, increasing proliferation capacity of aged mesenchymal stem cells, and reducing active oxygen level of aged mesenchymal stem cells.
5. The use of small molecule RNA according to claim 1 for the preparation of a medicament for the treatment of diseases associated with mesenchymal stem cell senescence, wherein said mesenchymal stem cell senescence is natural senescence, stress senescence or senescence caused by mass replication and passaging in vitro.
6. The use of small molecule RNA of claim 1 for the preparation of a medicament for the treatment of a mesenchymal stem cell senescence-associated disease, wherein the mesenchymal stem cell senescence-associated disease is senile osteoporosis caused by bone marrow mesenchymal stem cell senescence.
7. The use of small molecule RNA of claim 1 in the preparation of a medicament for the treatment of a mesenchymal stem cell senescence-associated disease, comprising a combination of one or more of tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4.
8. The application of the small molecular RNA in preparing an anti-aging culture medium for mesenchymal stem cell culture or passage is characterized in that the small molecular RNA is tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4;
the nucleotide sequence of the tRF-1:32-Gly-GCC-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the tRF-1:32-Glu-CTC-1-M2 is shown in SEQ ID NO. 2;
the nucleotide sequence of the tRF-1:29-Gly-CCC-1-M4 is shown as SEQ ID NO. 3.
9. The application of small molecular RNA in preparing a reagent for improving proliferation capacity of mesenchymal stem cells, delaying aging of the mesenchymal stem cells and reducing active oxygen level of the mesenchymal stem cells is characterized in that the small molecular RNA is tRF-1:32-Gly-GCC-1, tRF-1:32-Glu-CTC-1-M2, tRF-1:29-Gly-CCC-1-M4;
the nucleotide sequence of the tRF-1:32-Gly-GCC-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the tRF-1:32-Glu-CTC-1-M2 is shown in SEQ ID NO. 2;
the nucleotide sequence of the tRF-1:29-Gly-CCC-1-M4 is shown as SEQ ID NO. 3.
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