CN116870198A - Method for regulating skeletal muscle injury repair - Google Patents
Method for regulating skeletal muscle injury repair Download PDFInfo
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- CN116870198A CN116870198A CN202311139530.XA CN202311139530A CN116870198A CN 116870198 A CN116870198 A CN 116870198A CN 202311139530 A CN202311139530 A CN 202311139530A CN 116870198 A CN116870198 A CN 116870198A
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Abstract
The invention belongs to the technical field of biology, and relates to a method for regulating skeletal muscle injury repair. The invention provides application of CLIC5 gene in preparing medicines for regulating skeletal muscle injury repair. The invention reveals for the first time that CLIC5 participates in the repair process after skeletal muscle injury, and skeletal muscle specific knockout of CLIC5 destroys the regeneration capacity of muscle; in contrast, skeletal muscle overexpression of CLIC5 accelerates the repair process following injury. The invention provides a new thought and a target for promoting the repair of human skeletal muscle after injury.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a CLIC5 gene in preparation of a medicament for regulating skeletal muscle injury repair.
Background
Skeletal muscle is the largest organ of the human body and occupies 35% of the body weight, playing a key role in supporting, exercising, metabolizing and the like. External force or aging can cause skeletal muscle injury, and reduce life quality of people. After the muscle is damaged, satellite cells activate, proliferate and differentiate, and fuse to form new muscle fibers or fuse with the original muscle fibers to promote muscle repair. Numerous cells and signaling molecules are involved in the repair process following muscle injury.
The family of intracellular chloride ion channels (Chloride intracellular channel, CLIC) includes six members (CLIC 1-6) that are found on the cytoplasm, cell membranes and various organelle membranes, and that play an important role in cardiovascular disease, cancer, neurodegenerative disease, and can be used as diagnostic markers for these diseases. CLIC5 is a member of the CLIC family, whose natural mutation would impair hearing and mouse balance. However, no report on the skeletal muscle repair ability of CLIC5 is currently available.
Disclosure of Invention
The invention aims to provide an application of CLIC5 gene in preparing medicines for regulating skeletal muscle injury repair. Skeletal muscle specific knockout of CLIC5 can disrupt the repair ability after muscle injury, whereas skeletal muscle overexpression of CLIC5 can significantly promote repair after muscle injury.
The invention provides application of CLIC5 gene in preparing medicines for regulating skeletal muscle injury repair.
The invention also provides application of the reagent for over-expressing the CLIC5 gene in preparing medicaments for promoting skeletal muscle injury repair.
Preferably, the promoting skeletal muscle injury repair comprises any one or more of the following: reducing necrotic muscle fiber; increasing the area of regenerated muscle fibers; the expression of Pax7 was increased.
The invention also provides application of the over-expressed CLIC5 gene in preparation of medicines for relieving skeletal muscle inflammation.
Preferably, the nucleotide sequence of the CLIC5 gene is shown as SEQ ID NO. 1.
The invention also provides a medicament for promoting skeletal muscle injury repair, which comprises a reagent for over-expressing the CLIC5 gene and pharmaceutically acceptable auxiliary materials.
Preferably, the auxiliary material comprises physiological saline.
Preferably, the agent that overexpresses the CLIC5 gene comprises a vector that overexpresses the CLIC5 gene.
Preferably, the vector over-expressing the CLIC5 gene comprises an adeno-associated viral vector over-expressing the CLIC5 gene.
The invention provides application of CLIC5 gene in preparing medicines for regulating skeletal muscle injury repair. The invention reveals for the first time that CLIC5 participates in the repair process after skeletal muscle injury, and skeletal muscle specific knockout of CLIC5 destroys the regeneration capacity of muscle; in contrast, skeletal muscle overexpression of CLIC5 accelerates the repair process following injury. The invention provides a new thought and a target for promoting the repair of human skeletal muscle after injury.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of the result of genotyping of offspring provided by the invention;
FIG. 2 is a graph showing the results of the mRNA expression level of CLIC5 provided by the present invention;
FIG. 3 is a graph showing the results of protein expression levels of CLIC5 provided by the present invention;
FIG. 4 is a graph of the result of detecting the protein expression level of CLIC5 by using the Western blot technology provided by the invention;
FIG. 5 is a graph of the results of tissue slice analysis provided by the present invention;
FIG. 6 is a graph of the results of tissue slice analysis provided by the present invention;
FIG. 7 is a graph showing the results of protein expression levels of Pax7 provided by the present invention.
Detailed Description
The invention provides application of CLIC5 gene in preparing medicines for regulating skeletal muscle injury repair. Skeletal muscle specific knockout of CLIC5 can disrupt the repair ability after muscle injury, whereas skeletal muscle overexpression of CLIC5 can significantly promote repair after muscle injury.
The invention also provides application of the reagent for over-expressing the CLIC5 gene in preparing medicaments for promoting skeletal muscle injury repair. In the present invention, the promotion of skeletal muscle injury repair preferably includes any one or two or more of the following: reducing necrotic muscle fiber; increasing the area of regenerated muscle fibers; the expression of Pax7 was increased. In the present invention, the over-expressed CLIC5 gene is preferably skeletal muscle CLIC5 gene. In the invention, the nucleotide sequence of the CLIC5 gene is shown as SEQ ID NO. 1. In the present invention, the agent for overexpressing the CLIC5 gene preferably includes a vector for overexpressing the CLIC5 gene. In the present invention, the vector overexpressing the CLIC5 gene preferably includes an adeno-associated viral vector overexpressing the CLIC5 gene. In the present invention, the adeno-associated drug vector preferably comprises AAV9. The construction method of the adeno-associated virus vector for over-expressing the CLIC5 gene is not particularly limited, and the construction is carried out by adopting a conventional method.
Knockout of the CLIC5 gene has an adverse effect on repair after skeletal muscle injury. Specifically, the knockout of CLIC5 later disrupts the regenerative capacity of the muscle. In the present invention, the knockout CLIC5 gene is preferably a specific knockout skeletal muscle CLIC5 gene. In the invention, the nucleotide sequence of the CLIC5 gene is shown as SEQ ID NO. 1. The method of the present invention is not particularly limited, and can be any gene knockout method known to those skilled in the art, such as a method of using Cre/LoxP recombinase system to perform the knockout of the CLIC5 gene. The present invention preferably constructs CLIC5 from Siro biotechnology Co., ltd loxp/loxp Transgenic mice. The source of Myf5-Cre transgenic mice of the invention is preferably Jackson laboratories (cat. J007893). The present invention preferably defines the exon 2 of the CLIC5 gene as a conditional knockout region. The invention preferably designs 2 sgrnas according to introns 1 and 2, the sgrnas sequences are as follows: sgRNA-1: CTTAGTATAGTAACAGCGGTTGG (SEQ ID NO. 2); sgRNA-2: GTAGATGTTACGACGAGGTAAGG (SEQ ID NO. 3). In the examples of the present invention, myf5-Cre transgenic mice and CLIC5 are preferred loxp/loxp The transgenic mice are mated, and the invention preferably detects the protein expression level of CLIC5 in the offspring tibialis anterior to obtain CLIC5 skeletal muscle specific knockout mice.
The invention also provides application of the over-expressed CLIC5 gene in preparation of medicines for relieving skeletal muscle inflammation. Skeletal muscle overexpression of CLIC5 significantly relieves the inflammatory response following muscle injury, promoting muscle repair.
The invention also provides a medicament for promoting skeletal muscle injury repair, which comprises a reagent for over-expressing the CLIC5 gene and pharmaceutically acceptable auxiliary materials. In the present invention, the auxiliary material includes physiological saline. In the present invention, the agent for overexpressing the CLIC5 gene preferably includes a vector for overexpressing the CLIC5 gene. In the present invention, the vector overexpressing the CLIC5 gene preferably includes an adeno-associated viral vector overexpressing the CLIC5 gene. In the present invention, the adeno-associated drug vector preferably comprises AAV9. The construction method of the adeno-associated virus vector for over-expressing the CLIC5 gene is not particularly limited, and the construction is carried out by adopting a conventional method.
For further explanation of the present invention, the application of the CLIC5 gene provided by the present invention in preparing a medicament for modulating skeletal muscle injury repair is described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Preparation of CLIC5 skeletal muscle-specific knockout mice
Construction of CLIC5 by Sitting Biotechnology Co., ltd loxp/loxp Transgenic mice. The CLIC5 gene is located on chromosome 17 of the mouse (NCBI Reference Sequence: NM-172621; ensembl: ENSMUSG00000023959), 6 exons in total, exon 2 being selected as the conditional knockout region. 2 sgrnas were designed based on introns 1 and 2, and the sgrnas were of the following sequence:
sgRNA-1：CTTAGTATAGTAACAGCGGTTGG（SEQ ID NO.2）；
sgRNA-2：GTAGATGTTACGACGAGGTAAGG（SEQ ID NO.3）；
myf5-Cre transgenic mice and CLIC5 loxp/loxp The transgenic mice are mated, the generated offspring are subjected to genotype identification by utilizing a rat tail genome, and the primer sequences are as follows:
loxp-F：CGGAGTCATCCTTTAGTGCCTTAC（SEQ ID NO.4）；
loxp-R：CTGCTCGGCTTCCTTGTCTAATA（SEQ ID NO.5）；
Cre-F：CATATTGGCAGAACGAAAACGC（SEQ ID NO.6）；
Cre-R：CCTGTTTCACTATCCAGGTTACGG（SEQ ID NO.7）；
PCR amplification System (10. Mu.L):
2 XPCR reaction reagents: 5 mu L
An upstream primer: 0.25 mu L
A downstream primer: 0.25 mu L
ddH 2 O：4.1μL
Rat tail lysate: 0.4 mu L
PCR reaction conditions:
1:95 ℃,5min,2:95 ℃,30s,3:60 ℃,30s,4:72 ℃,45s, 2-4: 35 cycles, 5:72 ℃,5min,6:4 ℃ and infinity.
The agarose gel electrophoresis results are shown in FIG. 1. As can be seen from FIG. 1, 2, 3, 11 are skeletal muscle specific knockout CLIC5 mice (CLIC 5 MKO ) Genotype is Myf5-Cre: CLIC5 loxp/loxp 8 is wild type mouse (WT), genotype is CLIC5 loxp/loxp . The CLIC5 primers were used to determine whether to achieve a knockout of CLIC5, the genes were derived from tibialis anterior, extensor digitorum longus and gastrocnemius, respectively, with the following sequences:
CLIC5-F：GGTGACTCAGACGCATCCAG（SEQ ID NO.8）；
CLIC5-R：AGGTGCCGTCGTAGCAGTTC（SEQ ID NO.9）；
qRT-PCR amplification System (10. Mu.L):
2×Taq PCR master mix：5μL
an upstream primer: 0.3 mu L
A downstream primer: 0.3 mu L
cDNA：4μL
ddH 2 O：0.4μL
qRT-PCR reaction conditions:
1:95 ℃,5min,2:95 ℃,15s,3:64 ℃,15s,4:72 ℃,15s, 2-4: 35 cycles, 5:4 ℃ and infinity.
The qRT-PCR results are shown in fig. 2, where the mRNA expression level of CLIC5 was significantly reduced in the muscle of CLIC5 skeletal muscle-specific knockout mice. Protein expression levels of CLIC5 in tibialis anterior were detected using Western blot techniques, as shown in fig. 3, the protein expression levels of CLIC5 were significantly reduced in the muscle of CLIC5 skeletal muscle-specific knockout mice. The above results indicate that specific knockout of CLIC5 in skeletal muscle is achieved.
In fig. 2: WT, wild-type; CLIC5 MKO CLIC5 skeletal muscle-specific knockout mice; in fig. 3: WT, wild-type; CLIC5 MKO CLIC5 skeletal muscle-specific knockout mice.
Example 2
Preparation of CLIC5 skeletal muscle overexpressing mice
Cloning the CLIC5 CDS region sequence into AAV9: CMV-MSC-3 XFlag-EF 1-ZsGreen-T2A-Puro plasmid (purchased from Hayota Biotechnology (Shanghai) Inc.), and subjected to a large number of processes such as extraction, packaging, concentration and purification of adeno-associated virus (AAV) vectors, and titer measurement, an AAV vector for over-expressing CLIC5 was obtained. The CDS region sequence of CLIC5 is as follows:
atgacggactcagcgacaactaatggggacgacagggaccccgagatcgagctcttcgtgaaggctgggatcgacggggaaagcattggcaactgtcccttctctcagcgtctcttcatgatcctctggctgaaaggagtcgtgttcaatgtcaccaccgtggatctgaaaagaaagccagccgatctacacaacctggctcctggcacgcacccgcccttcctgaccttcaatggggatgtgaagacagatgtcaacaagattgaagagttcctggaggagaccctaacccccgagaagtaccccaaactggctgcaaaacaccgggaatctaacaccgcgggcatcgacatcttctccaagttctcagcctacatcaaaaacaccaaacaacagaacaatgctgcccttgagagaggcttgacaaaggcgctgaggaagctggatgactacctaaacagccctctgccagaggagattgacaccaacacccacggggacgagaaggggtcccagcgcaagttcctggatggggatgagctgaccctggccgactgcaatctgctgcccaagctgcatgtggtcaagattgtggctaagaagtaccgaaactatgacatcccagctgagatgacaggcttgtggcgatacctcaagaatgcctatgcacgggacgagttcaccaacacctgtgcagctgacagtgagatcgagttggcctacgcagatgtcgccaggcgcctcagccgatcgtga（SEQ ID NO.1）；
c57BL/6J mice with the age of 8 weeks are used, 40-50 mu L of AAV vector or control vector which is used for over-expressing CLIC5 is injected into tibialis anterior muscle after anesthesia, and protein expression level of the CLIC5 is detected by Western blot technology after three weeks. As shown in fig. 4, injection of the AAV vector of CLIC5 in tibialis anterior significantly increased the protein expression level of CLIC5, indicating successful construction of CLIC5 skeletal muscle overexpressing mice.
Example 3
Effects of skeletal muscle knockout CLIC5 and overexpression of CLIC5 on repair of muscle injury
Skeletal muscle injury models were constructed using 8 week old CLIC5 skeletal muscle-specific knockout mice (prepared in example 1) and wild-type mice or mice in which skeletal muscle overexpresses CLIC5 (prepared in example 2) and mice injected with control AAV vectors. The specific operation is as follows: cardiotoxin (purchased from Latoxan corporation, cat No. L8102) at a concentration of 10 μm and a volume of 50 μl was injected into tibialis anterior muscle, and tibialis anterior muscle was collected on days 3, 7 and 14 after injection, stored in 4% paraformaldehyde or-80 ℃ refrigerator for tissue section analysis or Western blot. The tissue section analysis was performed as follows:
(1) The tibial anterior muscle samples of mice were fixed in 4% paraformaldehyde for more than 48 hours.
(2) Repairing tissue blocks, fully washing fully fixed tissues with PBS, and then dehydrating with gradient alcohol: treating with 50% ethanol for 1 hr; treating with 70% ethanol for 1 hr; treating with 80% ethanol for 1 hr; treating with 90% ethanol for 1 hr; 95% ethanol I for 45min; treating with 95% ethanol II for 45min;100% ethanol I for 30min;100% ethanol II for 30min.
(3) The tissue was thoroughly dehydrated and then subjected to transparency of xylene, xylene I treatment for 10min and xylene II treatment for 8min.
(4) And immersing the wax I and the wax II for 1h respectively after the wax I and the wax II are fully transparent, and packaging the wax blocks to finish the preparation of the wax blocks.
(5) The embedded paraffin block is placed in a refrigerator at the temperature of minus 20 ℃ for precooling, then 4 mu m of slices are carried out, the slices are spread in distilled water at the temperature of 43 ℃, the fully spread tissues are fished out by a glass slide and placed at the room temperature overnight, and finally the slices are baked in an oven at the temperature of 63 ℃ for 1h.
(6) Hydration: paraffin wax on the sections was first removed with xylene and twice xylene treatments were performed for 10min each. Removing xylene on the slice with gradient alcohol, and treating with 100% ethanol twice for 5min each time; treating with 95% ethanol twice for 5min each time; treating with 80% ethanol for 5min;70% ethanol treatment for 5min: distilled water was treated 3 times for 5min each.
(7) Hematoxylin staining: hematoxylin is used for dying cell nuclei, different dying times are selected according to the injury condition of tibialis anterior, normal muscles are dyed for 10min, and muscles on the 3 rd day and the 7 th day are dyed for 3min. After dyeing, the mixture is blued in tap water at 37 ℃ until the water is clear and transparent, and then distilled water is used for washing twice for 3min each time.
(8) Eosin staining: treating the hematoxylin marked pieces with 95% ethanol for 30s, coloring the pieces with eosin dye solution for 10s, and then placing the pieces into 95% ethanol I, II for 2min each; placing the slices in 100% ethanol I, II for 5min to completely remove water on the slices; finally, the flakes were placed in xylene I, II for 10min each: finally, the film is sealed by neutral quick-drying adhesive, and the film is placed in a fume hood overnight for collecting pictures.
The results of the tissue section analysis are shown in fig. 5 and 6. Early muscle fiber necrosis after muscle injury can cause severe inflammatory reactions and macrophage infiltration. In this example, on day 3 after injury, the wild type mouse tibialis anterior had developed newly formed muscle fibers, while CLIC5 MKO The mice had substantially no regeneration of muscle fibers. On day 14 after injury, wild type murine muscle fibers were tightly aligned, structurally sound, CLIC5 MKO The number of muscle fibers with large rat area is smaller. In contrast, skeletal muscle over-expression of CLIC5 caused the replacement of most of the inflammatory muscle fibers by neogenic muscle fibers on day 7 after muscle injury, a significant relief of inflammatory response and a significant increase in regeneration area.
Activation of satellite cells is a prerequisite for repair after muscle injury. Pax7 is a marker gene for satellite cells. Protein expression level of tibialis anterior Pax7 at day 3 after muscle injury was detected by Western blot technique. The results showed that skeletal muscle-specific knockout of CLIC5 significantly reduced the protein expression level of Pax7 (a and B in fig. 7), and that overexpression of CLIC5 significantly increased the protein expression level of Pax7 (C and D in fig. 7). From the results of fig. 5-7 of example 3, it can be seen that specific knockout of CLIC5 by skeletal muscle destroys the repair ability after muscle injury, whereas overexpression of CLIC5 by skeletal muscle significantly relieves the inflammatory response after muscle injury and promotes muscle repair.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (9)
- Application of CLIC5 gene in preparing medicine for regulating skeletal muscle injury repair.
- 2. Use of an agent that overexpresses the CLIC5 gene in the preparation of a medicament for promoting repair of skeletal muscle injuries.
- 3. The use of claim 2, wherein the promotion of skeletal muscle injury repair comprises any one or more of: reducing necrotic muscle fiber; increasing the area of regenerated muscle fibers; the expression of Pax7 was increased.
- 4. The application of over-expressed CLIC5 gene in preparing medicines for relieving skeletal muscle inflammation.
- 5. The use according to any one of claims 1 to 4, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID No. 1.
- 6. A medicament for promoting skeletal muscle injury repair, which is characterized by comprising an agent for over-expressing CLIC5 gene and pharmaceutically acceptable auxiliary materials.
- 7. The medicament according to claim 6, wherein the auxiliary material comprises physiological saline.
- 8. The medicament of claim 6, wherein the agent that overexpresses the CLIC5 gene comprises a vector that overexpresses the CLIC5 gene.
- 9. The medicament of claim 6, wherein the vector that overexpresses the CLIC5 gene comprises an adeno-associated viral vector that overexpresses the CLIC5 gene.
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