42 results about "C2C12" patented technology

The present invention provides methods of improving the osteogenic and / or chondrogenic activity of a bone matrix, e.g., a dermineralized bone matrix (DBM), by exposing the bone matrix to one or more treatments or conditions. In preferred embodiments the bone matrix is derived from human bone. The treatment or condition may alter the structure of the bone matrix and / or cleave one or more specific proteins. Cleavage may generate peptides or protein fragments that have osteoinductive, osteogenic, or chondrogenic activity. Preferred treatments include collagenase and various other proteases. The invention further provides improved bone and cartilage matrix compositions that have been prepared according to the inventive methods and methods of treatment using the compositions. The invention further provides methods of preparing, testing, and using the improved bone matrix compositions. Ona assay comprises exposing relatively undifferentiated mesenchymal cells to a bone matrix composition and measuring expression of a marker characteristic of osteoblast or chondrocyte lineage(s). Increased expression of the marker relative to the level of the marker in cells that have been exposed to a control matrix (e.g., an inactivated or untreated matrix) indicates that the treatment or condition increased the osteogenic and / or chondrogenic activity of the bone matrix. Suitable cells include C2C12 cells. A suitable marker is alkaline phosphatase. The inventive methods increase the osteogenic and / or chondrogenic activity of human DBM when tested using this assay system.

The invention belongs to the field of environmental-stimulus-response-type medicines, and particularly relates to a sol system used for preparing pH-sensitive double-network hydrogel, the hydrogel andapplication. The sol system is prepared from gelatin methacryloyl, oxidized alginate, a photoinitiator, gentamicin and mesoporous silica nano particles loaded with phenamil medicinal molecules, the sol system is transformed from sol to gel fast under ultraviolet irradiation, thus the pH-sensitive double-network hydrogel can be obtained, and the hydrogel is excellent in physical and chemical property and good in biocompatibility, can effectively control release of an antibacterial medicine GS and an osteogenic differentiation promoting medicine phenamil, prevent early infection of a graft, induce C2C12 cell osteogenic differentiation, and promote endogenous bone generation. Based on the character that the sol system is transformed from the sol to the gel fast under ultraviolet irradiation,in practical application, in-situ gelation can be conducted by means of minimally-invasive injection, and the requirements of complex orbital bone defect repair can be met, so that the sol system used for preparing the pH-sensitive double-network hydrogel has important values in orbit repair and reconstruction.

The present invention provides methods of improving the osteogenic and / or chondrogenic activity of a bone matrix, e.g., a dermineralized bone matrix (DBM), by exposing the bone matrix to one or more treatments or conditions. In preferred embodiments the bone matrix is derived from human bone. The treatment or condition may alter the structure of the bone matrix and / or cleave one or more specific proteins. Cleavage may generate peptides or protein fragments that have osteoinductive, osteogenic, or chondrogenic activity. Preferred treatments include collagenase and various other proteases. The invention further provides improved bone and cartilage matrix compositions that have been prepared according to the inventive methods and methods of treatment using the compositions. The invention further provides methods of preparing, testing, and using the improved bone matrix compositions. On a assay comprises exposing relatively undifferentiated mesenchymal cells to a bone matrix composition and measuring expression of a marker characteristic of osteoblast or chondrocyte lineage(s). Increased expression of the marker relative to the level of the marker in cells that have been exposed to a control matrix (e.g., an inactivated or untreated matrix) indicates that the treatment or condition increased the osteogenic and / or chondrogenic activity of the bone matrix. Suitable cells include C2C12 cells. A suitable marker is alkaline phosphatase. The inventive methods increase the osteogenic and / or chondrogenic activity of human DBM when tested using this assay system.

The invention discloses a method for preparing a bionic skeletal muscle composite tissue through multi-channel extrusion 3D biological printing. The preparation method comprises the following steps: preparing bone scaffold bionic bio-ink, periosteum bionic bio-ink, sarcolemma bionic bio-ink and muscle bionic bio-ink; respectively mixing MSCs and C2C12 with the corresponding bionic bio-inks; and printing and forming a bionic bone, bionic periosteum, bionic sarcolemma and bionic muscle four-layer composite tissue engineering scaffold by using a multi-channel extrusion 3D biological printer. Themethod for preparing the bionic skeletal muscle composite tissue through multi-channel extrusion 3D biological printing can minimize fibrosis during traumatic skeletal muscle injury recovery; the bionic skeletal muscle composite tissue prepared through multi-channel extrusion 3D biological printing can replace structures and functions of bones and skeletal muscles at the same time, and supports proliferation and differentiation of myoblasts and osteoblasts; and an implant is easy to customize by utilizing a 3D biological printing technology, so that the implant is suitable for any defect shape.

The present invention relates to establishing method of active cell strain for quantitative determination of bone morphogenetic protein (BMP). By means of the transgenic technology of molecular biology, partial promoter of BMP action target molecular osteocalcium together with leuciferinase report gene is transfered to myogenic cell C2C12, monoclonal cell strain to express leuciferinase report gene stably is obtained through screening and the reaction of the cell strains to BMP action is determined. Strain with high reaction strength to BMP stimulation and sensitive determination index is finally sorted. Specificity and stability test proves the stability and reliability of the cell strain and multiple BMP sample testing result shows that the action of BMP is correlated with leuciferinase activity linearly and directly. The cell strain of the present invention is suitable for quantitative determination of BMP activity.

The invention discloses an application of tyrosol in preparing a drug for treating a diabetic complication diabetic foot and an accelerant for expressing and secreting an angiogenic factor. The effective component of the accelerant is tyrosol and the accelerant promotes expression and secretion of the angiogenic factor of a skeletal muscle cell. The angiogenic factor comprises PDG-BB and VEGF-A. The accelerant promotes expression and secretion of the angiogenic factor of the skeletal muscle cell in a high glucose condition. An accelerant for proliferating and migrating a skeletal muscle cell C2C12 is characterized in that the effective component of the accelerant is tyrosol. The accelerant promotes proliferation and migration of the skeletal muscle cell C2C12 in the high glucose condition.

The present application discloses roles for miR-93 in treating hypoxia and ischemia. Endothelial cells (HUVEC) and myocytes (C2C12) expressed miR-93 and up-regulated miR-93 in response to hypoxia and serum starvation. Over-expression of miR-93 in HUVECs promoted cell proliferation, prevented hypoxia-induced apoptosis, and enhanced endothelial cell tube formation. miR-93 knockdown in HUVECs resulted in increased hypoxia-induced apoptosis and decreased tube formation. Over-expression or knockdown of miR-93 in myocytes resulted in reduced or increased hypoxia-induced apoptosis, respectively. Down-regulation of miR-93 in C57BL / 6 mice with antagomiR resulted in attenuated perfusion recovery (% non-ischemic leg at day-21: Scramble 85.22.9 vs. AntagomiR-93 67.96). Over-expression of miR-93 in BALB / C mice improved perfusion recovery (% non-ischemic leg at day 21: PremiR-93 757.5 vs. Scramble 59.62.5). The present invention encompasses the use of miR-93 and regulation of miR-93 to treat and prevent hypoxia, ischemia, and other injuries, diseases, disorders, and conditions associated with ischemia.

The invention provides a method for promoting GLUT4 gene expression in a muscle cell, relates to a method for promoting GLUT4 gene expression, aims to build a method for effectively improving GLUT4 gene expression, and particularly relates to a method for promoting GLUT4 gene expression in the muscle cell to promote glucose absorption of the muscle cell. The method comprises the following steps: firstly, performing induction and differentiation culture on a muscle precursor cell (C2C12) so as to enable the muscle precursor cell to be cultivated to a mature muscle cell, then adding 4-PBA with the concentration of 1-10 mol / L into a culture medium, culturing for 12-24 h under the condition that the temperature is 37 DEG C and the volume concentration of CO2 is 5%, and recycling the cell; then detecting GLUT4 gene expression of the muscle cell and glucose absorption change of the muscle cell. The method provided by the invention is applied to the field of scientific research and clinical application for reducing diabetes hyperglycemia.

The present invention belongs to the field of pig molecule genetics, and specifically relates to cloning identification of a pig muscle-specific expression gene integrin beta 1 binding protein 2 (ITGB1BP2) promoter, and an application thereof. The steps of the present invention comprise: extracting total RNA of different tissues of a large white pig, and carrying out fluorescence quantitative PCR to determine specific expression in muscle tissue. According to the present invention, the total DNA of the muscle tissue of the large white pig is adopted as a template to design primers; PCR amplification is performed to obtain a 1442 bp sequence of flank on 5' terminal of pig ITGB1BP2 gene; luciferase reporter gene plasmids of 15 ITGB1BP2 promoter deletion fragments with different lengths are constructed; and C2C12 cells are transfected with the plasmids, activity of the promoter of each deletion fragment is detected, and the pig ITGB1BP2 gene promoter is identified. The nucleotide sequence of the pig ITGB1BP2 gene promoter of the present invention is represented by SEQ ID NO:1. With the present invention, import elements and means are provided for pig genetic improvement and transgene, and muscle tissue specificity and stability of transgene expression can be strengthened.

The invention discloses an application of geniposide in alleviating skeletal muscle fibrosis, and belongs to the field of natural product treatment. C2C12 myoblasts are stimulated by the geniposide, and a result indicates that the geniposide can significantly alleviate the fibrosis degree of fibrocytes. Furthermore, by establishing a mice model with contusion-induced skeletal muscle injury, a geniposide standard product is intraperitoneally injected for treating mice, and the geniposide is proved to play an anti-fibrosis role in damaged skeletal muscles. In addition, the fibrosis degree of theC2C12 myoblasts stimulated by TGF-Beta and Smad 4 is increased significantly, but the fibrosis level is decreased significantly after the C2C12 myoblasts are treated with the geniposide as compared with that of untreated C2C12 myoblasts. Therefore, the action mechanism of the geniposide in alleviating skeletal muscle fibrosis can be closely related to a TGF-beta / Smad4 signaling pathway.

The invention provides application of a transcription factor SP1 in regulation and control of pig RTL 1 gene expression. According to the invention, RTL1 is used as a research object, recombinant plasmids of pig RTL1 gene promoter dual-luciferase reporter gene are constructed, and a core promoter region of RTL1 is found; then the interaction between a transcription factor SP1 and the core promoterregion of RTL1 is verified; subsequently, an SP1 overexpression vector and synthetic small interfering RNA are constructed to detect the influence of SP1 on RTL1; and a mutant fluorescent expressionvector SP1-mut of a transcription factor SP1 binding site is also constructed to detect luciferase activity after transfecting PK15 cells and C2C12 cells. The invention confirms for the first time theinfluence of the transcription factor SP1 on regulation and control of transcription of an RTL1 gene, brings deeper cognition for regulation and control of expression of RTL1, and has a good application prospect when the transcription factor SP1 is applied to improvement of meat quality traits and research on a molecular regulation mechanism of muscle development of livestock.

The invention discloses a bovin CMYA1 muscle specific expression core promoter and a preparation method thereof. The preparation method comprises the following steps: firstly cloning a full-length fragment 1155bp of the bovin CMYA1 muscle specific expression core promoter, building a pEGFP-1-CMYA1 promoter carrier, and transfecting mice C2C12 myoblast, bovine skeletal muscle satellite cells and bovine fibroblast to prove the muscle specificity expression of the carrier; then, carrying out the deficiency research on CMYA1 promoter, totally designing 8 deletion fragments of the CMYA1 promoter, respectively building deletion fragment carriers of pGL3-basic-CMYA1 promoters, and transfecting C2C12 cells; and finally verifying, thereby obtaining the CMYA1 core promoter of which the gene sequence is SQ2. The CMYA1 promoter is specifically expressed in muscle cells, and the promotion activity is high; moreover, such the characteristic can be utilized by people to make a research on the development and differentiation of muscles.

The invention provides application of cryptotanshinone in preparing a medicine for preventing and treating cachexia skeletal muscle atrophy. The medicine comprises an active component of cryptotanshinone and further comprises pharmaceutically acceptable auxiliary materials. Cryptotanshinone is capable of alleviating C2C12 myotube atrophy, and in addition, remarkably reducing expression of proteinsp-STAT3, MuRF1 and Atrogin-1 in muscles to inhibit degradation of skeletal muscle proteins, and novel ideas are provided for preparing medicines for treating and preventing cachexia skeletal muscle atrophy.

The invention discloses a differentiation promoting reagent YYQ1 capable of obviously promoting differentiation of mouse C2C12 myoblasts through external addition, and belongs to the technical field of cell biology. The differentiation promoting reagent YYQ1 is characterized in that: the component of the differentiation promoting reagent YYQ1 is 10[muM] of retinol, and after the differentiation promoting reagent YYQ1 is added into C2C12 cells cultured in vitro, good differentiation efficiency can be obtained, differentiation of the C2C12 cells is promoted, and finally the aim of remarkably promoting differentiation of mouse C2C12 myoblasts is finally achieved after external addition. The differentiation promoting reagent YYQ1 provides a basis for promoting muscle differentiation of animals, the retinol is used for promoting muscle differentiation, and a high practical application value is achieved. The differentiation promoting reagent YYQ1 has the advantages of simple manufacture, strong operability, low cost and obvious effect.

The invention discloses an application of a common fenugreek seed extract and an extraction method thereof. The tests prove that a fenugreek ethanol extract can promote GLUT4 to transpose to a membrane by activating an AMPK signal path; finally, the glucose uptake capability of C2C12 cells is improved; therefore, the common fenugreek seed extract can be used for preparing products for improving the glucose uptake capacity of skeletal muscle cells, AMPK agonists and products with a hypoglycemic effect, can be further used for preparing products for treating and / or preventing obesity, insulin resistance and diabetes mellitus, contributes to research and development of a new generation of antidiabetic drugs taking AMPK as a new target, and brings good news to human health.

The invention relates to a method for constructing a stable-expression SpCas9 protein cell line and application. The construction method comprises the following steps of: packaging and purifying lentivirus, namely preparing HEK 293T cells, transfecting packaging plasmids, performing transfection culture, collecting cell culture supernatant, and concentrating virus liquid; and infecting cells with the obtained concentrated virus, namely screening drug concentration, infecting cells with lentivirus, screening drugs, and selecting monoclone for identification. By adopting the method for constructing the stable-expression SpCas9 protein cell line, a mouse myoblast C2C12 stable-expression SpCas9 protein cell line and a mouse embryonic stem cell E14 stable-expression SpCas9 protein cell line are obtained, SpCas9 protein can be stably expressed, and the cell line can serve as a rapid verification tool, is used for performing rapid functional verification on designed SgRNA so as to detect whether SgRNA design is successful, and can serve as a functional gene screening system for subsequent large-scale functional gene screening.

The method of the invention provides for producing a heterologous protein in mammalian host cells having nucleic acid encoding Hepatitis B X protein and the heterologous protein, by growing mammalian host cells selected from the group consisting of HKB11, CHO, BHK21, C2C12, and HEK293 cells, by growing mammalian host cells in non-adherent suspension culture, or by growing mammalian host cells which contain nucleic acid providing exogenous X-box Binding Protein, XBP1s. The conditions should be such that HBx, exogenous XBP1s if present, and the heterologous protein are expressed by the mammalian cells. The invention includes compositions for carrying out the method.

The invention provides a method for promoting GLUT4 gene expression in a muscle cell, relates to a method for promoting GLUT4 gene expression, aims to build a method for effectively improving GLUT4 gene expression, and particularly relates to a method for promoting GLUT4 gene expression in the muscle cell to promote glucose absorption of the muscle cell. The method comprises the following steps: firstly, performing induction and differentiation culture on a muscle precursor cell (C2C12) so as to enable the muscle precursor cell to be cultivated to a mature muscle cell, then adding 4-PBA with the concentration of 1-10 mol / L into a culture medium, culturing for 12-24 h under the condition that the temperature is 37 DEG C and the volume concentration of CO2 is 5%, and recycling the cell; then detecting GLUT4 gene expression of the muscle cell and glucose absorption change of the muscle cell. The method provided by the invention is applied to the field of scientific research and clinical application for reducing diabetes hyperglycemia.

The embodiment of the invention provides an application of rhamnose monosulfate and a derivative thereof in resisting skeletal muscle atrophy. The rhamnose monosulfate or the rhamnose monosulfate derivative is applied to a medicine for resisting skeletal muscle atrophy, or the rhamnose monosulfate or the rhamnose monosulfate derivative is applied to a functional food for resisting skeletal muscleatrophy. The rhamnose monosulfate is used for inhibiting differentiation inhibition of TNF-alpha on skeletal muscle cells C2C12, promoting generation of myosin MHC and myoblast myogenin of C2C12, andpromoting conversion of skeletal muscle myocytes into muscle fibers.

The research is different from other previous researches, and the action mechanism of the astragalus flavone extract for treating the type 2 diabetes mellitus is discussed by applying a biological method. Skeletal muscle cells are taken as experimental subjects, an experimental group for blocking a P13 / Akt signal channel is constructed, then the astragalus flavone extract is given, liver cell related carbohydrate gene expression is analyzed, and the cellular mechanism of the astragalus flavone extract for treating type 2 diabetes mellitus is discussed. At present, the research is not reported at home and abroad. Therefore, a novel treatment method is provided by the astragalus flavone extract. By using the method, lots of expenditures for purchasing db / db mice and double-gene knockout mice can be saved. According to the research, the protein expression level of an insulin receptor substrate P-IRS-1 is measured, then the influence of the astragalus flavone extract on the insulin sensitivity of mouse myoblasts (C2C12) is detected, and a theoretical basis is provided for treatment of type 2 diabetes mellitus.

The invention discloses a bionic skeletal muscle composite tissue prepared by multi-channel extrusion 3D bioprinting. The preparation method comprises the following steps: preparing bone scaffold bionic bioink, periosteum bionic bioink, myofibrillar membrane bionic bioink, and muscle bionic bioink; Mix MSCs and C2C12 with the corresponding bionic bio-ink; use a multi-channel extrusion 3D bioprinter to print and form a four-layer composite tissue engineering scaffold of bionic bone, bionic periosteum, bionic sarcolemma, and bionic muscle. The bionic skeletal muscle composite tissue prepared by multi-channel extrusion 3D bioprinting of the present invention can minimize fibrosis during the recovery of traumatic skeletal muscle injury; the bionic skeletal muscle composite tissue prepared by multi-channel extrusion 3D bioprinting can simultaneously replace The structure and function of bone and skeletal muscle, supporting the proliferation and differentiation of myoblasts and osteoblasts; and using 3D bioprinting technology to make implants easy to customize to adapt to any defect shape.

The invention relates to malic acid phosphate ester and applications of the malic acid phosphate ester in inhibition of calcium ion deposition diseases, wherein the structure of the malic acid phosphate ester is represented by the following formula. The synthesis steps comprise: carrying out a stirring reaction of malic acid and benzyl alcohol for 6 h at a temperature of 80-130 DEG C, carrying out pressure reducing distillation at a temperature of 175-185 DEG C to remove the unreacted benzyl alcohol, carrying out recrystallization with ethanol to obtain dibenzyl malate, dissolving the dibenzyl malate in dichloromethane under N2 protection, adding triethylamine, adopting 4-dimethylaminopyridine as a catalyst, stirring for 0.5 h in a 0 DEG C ice bath, slowly adding diphenyl chlorophosphate in a dropwise manner, carrying out a stirring reaction for 0.5 h, carrying out a room temperature for 12 h, carrying out pressure reducing distillation, carrying out recrystallization with ethanol to obtain dibenzyl phosphate ester dibenzyl malate, adding the dibenzyl phosphate ester dibenzyl malate and platinum oxide to distilled water, carrying out a catalysis reaction for 20-24 h, extracting, and carrying out low temperature freeze-drying to obtain the product. According to the present invention, with the malic acid phosphate ester, the mouse myoblast C2C12 calcification can be well inhibited, and the concentration of the calcium ions in the human skeletal muscle cell HSK can be effectively reduced.

The present invention belongs to the field of pig molecule genetics, and specifically relates to cloning identification of a pig muscle-specific expression gene integrin beta 1 binding protein 2 (ITGB1BP2) promoter, and an application thereof. The steps of the present invention comprise: extracting total RNA of different tissues of a large white pig, and carrying out fluorescence quantitative PCR to determine specific expression in muscle tissue. According to the present invention, the total DNA of the muscle tissue of the large white pig is adopted as a template to design primers; PCR amplification is performed to obtain a 1442 bp sequence of flank on 5' terminal of pig ITGB1BP2 gene; luciferase reporter gene plasmids of 15 ITGB1BP2 promoter deletion fragments with different lengths are constructed; and C2C12 cells are transfected with the plasmids, activity of the promoter of each deletion fragment is detected, and the pig ITGB1BP2 gene promoter is identified. The nucleotide sequence of the pig ITGB1BP2 gene promoter of the present invention is represented by SEQ ID NO:1. With the present invention, import elements and means are provided for pig genetic improvement and transgene, and muscle tissue specificity and stability of transgene expression can be strengthened.

The invention belongs to differentiation culture of myoblasts, and relates to a differentiation culture method of C2C12 myoblasts. Separated mouse myoblasts sub-cultured more than five times are used as objects, a growth medium and a differentiation medium are optimized through steps of cell proliferation culture, differentiation culture and the like, and over 85% of the myoblasts sub-cultured more than five times can differentiate myotubes, thus effectively improving the utilization ratio of the separated primary myoblasts.

The invention discloses novel application of DHA as a feed additive in regulation and control of muscle fiber types. According to the novel application of DHA as a feed additive, DHA is added in high-fat diet to increase mRNA m6A demethylase FTO protein expression in skeletal muscle tissue of a mouse, so the total mRNA m6A modification level in the skeletal muscle of the mouse is reduced. Under the condition of the high-fat diet, the quantity of mitochondria in the skeletal muscle of the mouse is increased, the oxidative phosphorylation gene expression of the skeletal muscle of the mouse is promoted, the glycolysis gene expression is reduced, conversion from the muscle fiber type of the skeletal muscle to slow muscle fiber is promoted, and the exercise tolerance of the mouse is improved. The addition amount of DHA in the high-fat diet is 500 mg / kg of body weight / day. DHA promotes expression of m6A demethylase FTO in a C2C12 cell line and reduces the level of m6A. DHA does not contain substances harmful to animals or human bodies, is small in addition amount, can be directly added into an animal feed, food or drugs, has the advantages of being safe, effective, free of toxic and side effects and the like, and has good popularization and application prospects in the application as the feed additive to improve the quality of animal meat.

The invention discloses a CHO cellar strain of high-effective expressive exogenous rhBMP2, which is characterized by the following: adopting CHO-dhfr- in the dihydrofolic acid reductase flaw-typed Chinese hamster ovary cell as host cell; infecting exogenous hBMP2 plasmid; adopting secretory expressive product to induce myoblast system C2C12 to display the activity to stimulate sclerotomal cell; obtaining single-clone strain of CHO (rhBMP-2) with the content of BMP2 at 7.83ug / 24híñ106; fitting for clinic treatment.

The invention provides the application of transcription factor SP1 in regulating the expression of pig RTL1 gene. The present invention takes RTL1 as the research object, by constructing the recombinant plasmid of porcine RTL1 gene promoter dual luciferase reporter gene, and finding the core promoter region of RTL1; and verifying the interaction between the transcription factor SP1 and the RTL1 core promoter region; Then, the SP1 overexpression vector and synthetic small interfering RNA were constructed to detect the effect of SP1 on RTL1; the transcription factor SP1 binding site mutation fluorescent expression vector SP1-mut was also constructed to detect luciferase activity after transfection into PK15 cells and C2C12 cells. The invention confirms the influence of transcription factor SP1 on RTL1 gene transcriptional regulation for the first time, and brings a deeper level of cognition to the expression regulation of RTL1. It is applied to the improvement of livestock meat quality traits and the research on the molecular regulation mechanism of muscle development, and has good application prospect.