Method for inhibiting senescence of tendon cells through spongy

Abstract

The invention relates to a method for inhibiting senescence of cells, in particular to a method for inhibiting senescence of tendon cells through spongy. The method comprises the following steps: collecting a tendon sample, carrying out in-vitro isolated culture through a block implanting method, and respectively carrying out marker tracking on tendon cells in the sample and RNA molecules in the cells; according to the gene expression level of the tendon cells, in combination with the expression levels of RNA molecules of the corresponding tendon cells in different growth periods, finding out RNA molecules related to the gene expression level change of the tendon cells; screening to obtain RNA molecules strongly related to tendon cell senescence change, and determining a sponge expression vector targeting the strongly related RNA molecules; the sponge expression vector is loaded into the tendon cells, and expression of strongly correlated RNA molecules is negatively regulated so as to inhibit senescence of the tendon cells. In the invention, the senescence of the tendon cells can be effectively inhibited by spongizing the strongly correlated RNA molecules and negatively regulating the expression of the strongly correlated RNA molecules.

Description

Technical Field

[0001] The present invention relates to a method for inhibiting cell aging, in particular to a method for inhibiting tendon cell aging through spongiosis. Background Art

[0002] Cell aging refers to the process in which cells' proliferation and differentiation abilities and physiological functions gradually decline over time as they perform their life activities. The physiological manifestations of cell aging are functional decline and low metabolism. As cell aging progresses, the body's metabolism slows down, causing metabolic diseases such as diabetes and arteriosclerosis, as well as muscle atrophy and osteoporosis.

[0003] For example, CN115404210B involves a method for inhibiting the aging of umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells are treated with a Dermaseptin-TA1 polypeptide at a concentration higher than 100 μg / ml, which effectively improves the proliferation ability of the umbilical cord mesenchymal stem cells and promotes the expression of cell proliferation-related proteins in the umbilical cord mesenchymal stem cells; at the same time, it can reduce the aging of the umbilical cord mesenchymal stem cells.

[0004] Tendon cells are the basic functional units of tendons. They synthesize and secrete extracellular matrices such as collagen to maintain the metabolism of tendon tissue. As we age, tendons gradually age. Aging of tenocytes can lead to age-dependent tendon diseases and destroy the ability of human tendons to repair and replace.

[0005] In order to inhibit the aging of tenocytes and thus maintain the repair and replacement capabilities of tendons, a method of inhibiting the aging of tenocytes by spongiosis is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for inhibiting the aging of tendon cells by spongiosis, so as to solve the problems raised in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides a method for inhibiting the aging of tendon cells by spongiosis, comprising the following steps: S1. After collecting tendon samples, they are isolated and cultured in vitro using the explant method, and the RNA molecules in the tendon cells and cells in the samples are labeled and tracked; S2. According to the gene expression level of the tenocytes, combined with the expression level of the RNA molecules at different growth stages of the corresponding tenocytes, the RNA molecules related to the changes in the gene expression level of the tenocytes are found; S3. Screening RNA molecules that are strongly correlated with the aging changes of tendon cells, and determining the sponge expression vector targeting the strongly correlated RNA molecules; S4. Load the sponge expression vector into tendon cells to negatively regulate the expression of strongly related RNA molecules and inhibit the aging of tendon cells.

[0008] As a further improvement of the present technical solution, in S1, the tendon samples are equally divided and placed in multiple culture media to allow them to grow naturally by adhering to the wall, and all RNA molecules in the sample tendon cells are incorporated into the coding gene library.

[0009] As a further improvement of the present technical solution, in S1, after in vitro isolation and culture, multiple tendon cells in the sample are divided into multiple cell data groups, and multiple tendon cells in each cell data group are arranged in order of life cycle, and the gene expression levels of the tendon cells and RNA molecules in one of the cell data groups are detected by RNA sequencing.

[0010] As a further improvement of the technical solution, in S2, the specific steps are as follows: S2.1. Label RNA molecules that appear throughout the life cycle of tenocytes and screen out RNA molecules in the coding gene library that are insensitive to changes in tenocyte gene expression levels; S2.2. Combine the expression levels of the remaining RNA molecules in the coding gene library with the gene expression levels of tendon cells at different growth stages to form associated data and establish a gene database.

[0011] As a further improvement of the present technical solution, in S2.1, the insensitive RNA molecule is an RNA molecule whose expression level fluctuates within 5% of the entire life cycle of the tendon cells based on the average expression level during the entire life cycle of the tendon cells.

[0012] As a further improvement of the present technical solution, in S3, based on the change in expression level of RNA molecules when the expression level of tendon cell genes changes in the gene database, the RNA molecules that are strongly correlated with the change in expression level of tendon cell genes are determined through a correlation algorithm.

[0013] As a further improvement of this technical solution, the correlation algorithm is as follows:

[0014] In the formula, is the gene expression level of tenocytes, is the average gene expression level of tenocytes, is the RNA molecule expression level, is the average expression level of RNA molecules, for and The correlation of The value range is less than 1 and greater than -1. when When the absolute value of is greater than 0 and less than 0.6, and Weakly correlated; when When the absolute value of is greater than 0.6 and less than 1, and Strongly correlated.

[0015] As a further improvement of the present technical solution, in S3, after obtaining the strongly correlated RNA molecule, an inhibitor targeting the strongly correlated RNA molecule is searched in the coding gene library as a sponge expression vector, and the sponge expression vector is proliferated in vitro.

[0016] As a further improvement of the present technical solution, in S4, the sponge expression vector is loaded into the tendon cells via lipid nanoparticles.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In the method for inhibiting the aging of tendon cells by spongiosis, a plurality of tendon cells are first arranged in order of their life cycle, and then changes in the intracellular gene expression levels and RNA molecule expression levels during the maturation and aging stages of the tendon cells are detected by RNA sequencing, and RNA molecules that are strongly correlated with changes in tendon cell gene expression are obtained by the above-mentioned correlation algorithm, and then a sponge expression vector targeting the strongly correlated RNA molecule is determined, and after the sponge expression vector is proliferated and cultured in vitro, the sponge expression vector is introduced into the tendon cells to inhibit the expression of the spongiosis-related RNA molecules, thereby inhibiting the aging of the tendon cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the accompanying drawings in the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] See also Figure 1 As shown, the purpose of this embodiment is to provide a method for inhibiting tendon cell aging by spongiosis, comprising the following steps: S1. It is preferred to use tendon samples collected from the operating room, and divide the tendon samples into multiple culture media, and culture them in vitro using the explant method. The tendon cells and RNA molecules in the cells in the sample are labeled and tracked. During culture, they are allowed to grow naturally on the wall, and all RNA molecules in the tendon cells of the sample are included in the coding gene library. The explant method can provide a scaffold for the growth and differentiation of tendon cells, while allowing tendon cells to migrate out from the edge of the tendon sample tissue block to form a new cell population. This method does not require a complex enzymatic digestion process, so it is relatively simple and helps to maintain the original morphology and function of the cells.

[0021] Since RNA sequencing will kill cells, multiple tendon cells in the sample will be divided into multiple cell data groups after in vitro isolation and culture. In each cell data group, multiple tendon cells will be arranged in order of their life cycle, and RNA sequencing will be used to detect the gene expression levels of the tendon cells and RNA molecules in one of the cell data groups. The specific operation is to divide the surviving tendon cells in the sample into a normal observation group, an experimental intervention group, and an effect verification group. In each group, the tendon cells will be arranged in the order of cell proliferation, maturation, and aging. Then RNA sequencing will be performed on the experimental intervention group to obtain the numerical values ​​of the tendon cell gene expression levels and RNA molecule expression levels throughout the tendon cell life cycle.

[0022] S2. According to the gene expression level of the tenocytes, combined with the expression level of the RNA molecules in the different growth stages of the corresponding tenocytes, the RNA molecules related to the changes in the gene expression level of the tenocytes are found. The specific steps are as follows: S2.1. Mark the RNA molecules that appear throughout the life cycle of tenocytes, and screen out the RNA molecules in the coding gene library that are insensitive to changes in the expression levels of tenocyte genes. Insensitive RNA molecules are RNA molecules whose expression levels fluctuate by no more than 5% during the life cycle of tenocytes based on the average expression level during the life cycle of tenocytes.

[0023] S2.2. Combine the expression levels of the remaining RNA molecules in the coding gene library with the gene expression levels of tendon cells at different growth stages to form associated data and establish a gene database.

[0024] S3. According to the changes in the expression of RNA molecules when the expression of tendon cell genes changes in the gene database, the RNA molecules that are strongly correlated with the changes in the expression of tendon cell genes are determined by a correlation algorithm, the strongly correlated RNA molecules are screened, and the sponge expression vector targeting the strongly correlated RNA molecules is determined. The correlation algorithm is as follows:

[0025] In the formula, is the gene expression level of tenocytes, is the average gene expression level of tenocytes, is the RNA molecule expression level, is the average expression level of RNA molecules, for and The correlation of The value range is less than 1 and greater than -1. when When the absolute value of is greater than 0 and less than 0.6, and Weakly correlated; when When the absolute value of is greater than 0.6 and less than 1, and is strongly correlated; After obtaining the strongly related RNA molecule, an inhibitor targeting the strongly related RNA molecule is searched in the coding gene library as a sponge expression vector, and the sponge expression vector is proliferated in vitro.

[0026] S4. The sponge expression vector is loaded into the tendon cells through lipid nanoparticles, negatively regulating the expression of strongly related RNA molecules to inhibit the aging of tendon cells.

[0027] The sponging principle is that certain specific long non-coding RNA (lncRNA) or pseudogene transcripts contain multiple miRNA response elements (MRES). The MRES on the sponged miRNA is complementary to the seed sequence on the miRNA, which can adsorb and isolate a large number of miRNAs, forming a stable double-stranded structure, preventing the binding of miRNA to its target mRNA, thereby relieving the inhibitory effect of miRNA on its target mRNA. Since the miRNA is isolated, the target mRNA originally inhibited by the miRNA is released, and its expression level increases.

[0028] In the implementation process of the present invention, the obtained tendon samples are first divided into three groups, namely, a normal observation group, an experimental intervention group and an effect verification group, on the premise that the tissue shape can be maintained. Each group is isolated and cultured by the explant method, wherein a plurality of tendon cells in the experimental intervention group are arranged in order of the life cycle, and then the changes in the intracellular gene expression levels and the RNA molecule expression levels during the maturation and aging stages of the tendon cells are detected by RNA sequencing. The RNA molecules such as microRNA-199a-5p that are strongly correlated with the changes in the gene expression levels of the tendon cells are obtained by the above-mentioned correlation algorithm, and then the sponge expression vector targeting the strongly correlated RNA molecule (microRNA-199a-5p) is determined to be circPVT1. After the sponge expression vector is proliferated and cultured in vitro, the sponge expression vector is introduced into the tendon cells of the effect verification group to sponge the strongly correlated RNA molecules to inhibit expression, and then compared with the normal observation group.

[0029] Experimental methods: After completing the in vitro proliferation of the sponge expression vector according to the above-mentioned method flow, the rats aged 18-24 months were divided into a normal observation group and an effect verification group. The sponge expression vector was injected into the tendon cells of the effect verification group, and senescence-associated-β-galactosidase (SA-B-Gal) staining and telomerase activity detection were used. Western blot or reverse transcription quantitative polymerase chain reaction (RTqPCR) were used to detect gene expression levels. Finally, an in vivo model was established, and tendon tissue was used for hematoxylin and eosin (HE) and Masson's trichrome staining, as well as immunohistochemical analysis of Co1·I and TNMD.

[0030] Results: Compared with the tendon cells of the rats in the normal observation group, the cell senescence of the rats in the effect verification group was lower, and a stronger tendon differentiation ability was retained. That is, the present invention can effectively inhibit the aging of tendon cells by sponging strongly correlated RNA molecules and negatively regulating the expression of strongly correlated RNA molecules.

[0031] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for inhibiting tenocyte aging by spongiosis, characterized in that: The following steps are involved: S1. After collecting tendon samples, they are isolated and cultured in vitro using the explant method, and the RNA molecules in the tendon cells and cells in the samples are labeled and tracked; S2. According to the gene expression level of the tenocytes, combined with the expression level of the RNA molecules at different growth stages of the corresponding tenocytes, the RNA molecules related to the changes in the gene expression level of the tenocytes are found; S3. Screening RNA molecules that are strongly correlated with the aging changes of tendon cells, and determining sponge expression vectors that target the strongly correlated RNA molecules; S4. Load the sponge expression vector into tendon cells to negatively regulate the expression of strongly related RNA molecules and inhibit the aging of tendon cells.

2. The method for inhibiting tenocyte aging by spongiosis according to claim 1, characterized in that: In the S1, the tendon samples are equally divided and placed in multiple culture media to allow them to grow naturally attached to the wall, and all RNA molecules in the sample tendon cells are incorporated into the coding gene library.

3. The method for inhibiting tenocyte aging by spongiosis according to claim 1, characterized in that: In S1, after in vitro isolation and culture, multiple tendon cells in the sample are divided into multiple cell data groups, and multiple tendon cells in each cell data group are arranged in order of life cycle. The gene expression levels of the tendon cells and RNA molecules in one of the cell data groups are detected by RNA sequencing.

4. The method for inhibiting tenocyte aging by spongiosis according to claim 1, characterized in that: In S2, the specific steps are as follows: S2.

1. Label RNA molecules that appear throughout the life cycle of tenocytes and screen out RNA molecules in the coding gene library that are insensitive to changes in tenocyte gene expression levels; S2.

2. Combine the expression levels of the remaining RNA molecules in the coding gene library with the gene expression levels of tendon cells at different growth stages to form associated data and establish a gene database.

5. The method for inhibiting tenocyte aging by spongiosis according to claim 4, characterized in that: In S2.1, the insensitive RNA molecule is an RNA molecule whose expression level fluctuates within 5% of the entire life cycle of the tendon cell, based on the average expression level during the entire life cycle of the tendon cell.

6. The method for inhibiting tenocyte aging by spongiosis according to claim 4, characterized in that: In S3, based on the change in the expression level of RNA molecules when the expression level of tendon cell genes changes in the gene database, the RNA molecules that are strongly correlated with the change in the expression level of tendon cell genes are determined through a correlation algorithm.

7. The method for inhibiting tenocyte aging by spongiosis according to claim 6, characterized in that: The correlation algorithm is as follows: In the formula, is the gene expression level of tenocytes, is the average gene expression level of tenocytes, is the RNA molecule expression level, is the average expression level of RNA molecules, for and The correlation of The value range is less than 1 and greater than -1. when When the absolute value of is greater than 0 and less than 0.6, and is weakly correlated; when When the absolute value of is greater than 0.6 and less than 1, and Strongly correlated.

8. The method for inhibiting tenocyte aging by spongiosis according to claim 2, characterized in that: In S3, after obtaining the strongly correlated RNA molecule, an inhibitor targeting the strongly correlated RNA molecule is searched in the coding gene library as a sponge expression vector, and the sponge expression vector is proliferated in vitro.

9. The method for inhibiting tenocyte aging by spongiosis according to claim 1, characterized in that: In S4, the sponge expression vector is loaded into the tendon cells via lipid nanoparticles.

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