CN114853869B

CN114853869B - Alkaline fibroblast growth factor substitute, composition and application thereof

Info

Publication number: CN114853869B
Application number: CN202210422847.3A
Authority: CN
Inventors: 蔡娜
Original assignee: Hubei Cellway Biotechnology Co ltd
Current assignee: Hubei Cellway Biotechnology Co ltd
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2023-12-26
Anticipated expiration: 2039-12-10
Also published as: CN112940100A; CN114853869A; CN112940100B

Abstract

The invention provides an alkaline fibroblast growth factor substitute, which comprises the amino acid sequence shown in SEQ ID NO:2-4, and one or more nucleotide sequences in the sequence. The basic fibroblast growth factor substitute has stable structure and property and can perform similar biological functions of the basic fibroblast growth factor. The invention also provides a pharmaceutical composition containing the basic fibroblast growth factor substitute or the pharmaceutically acceptable salt thereof, and application of the pharmaceutical composition in preparing a medicament for wound healing or in an agent for regulating cell proliferation, differentiation or apoptosis.

Description

Alkaline fibroblast growth factor substitute, composition and application thereof

Technical Field

The invention relates to the field of medical biology, in particular to a nucleic acid aptamer, and particularly relates to an alkaline fibroblast growth factor substitute, a composition and application thereof.

Background

Basic fibroblast growth factor (basic fibroblast growth fator, bFGF) is a member of the fibroblast growth factor family. bFGF is a polypeptide growth factor widely existing in various tissues in vivo, has obvious proliferation promoting effect on cells derived from mesoderm and neuroectoderm, and plays an important role in regulating the processes of embryo development, angiogenesis, bone repair and wound repair of organisms. bFGF has also received extensive attention and research in terms of its characteristic biological activity pleiotropic.

However, bFGF is a protein product, which is unstable in properties and is easily decomposed by enzymes, and the application of bFGF and other protein products to clinical application is easy to generate immunogenicity, which also greatly limits the application of bFGF. Meanwhile, in the process of industrially preparing bFGF, as the bFGF belongs to protein products, the quality control requirements are very strict, and the existing bFGF or variants thereof have high preparation cost and uneven quality. Therefore, it is of great importance to develop a product with basic fibroblast growth factor related biological functions.

Disclosure of Invention

In order to provide a product which has low immunogenicity, stable property and low cost and can realize the related biological functions of the basic fibroblast growth factor, the invention adopts the following specific scheme:

a basic fibroblast growth factor replacement (bFGF replacement) comprising any one or more of the following three nucleotide sequences:

(1) And the sequence of SEQ ID NO:1-4, one or more nucleotide sequences having more than 60% homology to any of the sequences;

(2) And the sequence of SEQ ID NO:1-4, one or more nucleotide sequences that hybridize to any one of the sequences;

(3) The SEQ ID NO: 1-4.

Preferably, the nucleotide sequence comprises SEQ ID NO:1-4, which is capable of autonomous folding to form a secondary structure and a tertiary structure.

In the present invention, the SEQ ID NO:1-4 refers to any one of SEQ ID NOs: 1 to SEQ ID NO:4, the sequence of any one of SEQ ID NOs: 1-4 comprises SEQ ID NO: 1.SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, the SEQ ID NO: the sequences shown in 1-4 are shown in Table 1.

Table 1.Seq ID NO:1-4

In one embodiment of the invention, the basic fibroblast growth factor surrogate is SEQ ID NO:1, and a nucleotide sequence shown in the specification; or the basic fibroblast growth factor replacement is SEQ ID NO:2, a nucleotide sequence shown in seq id no; or the basic fibroblast growth factor replacement is SEQ ID NO:3, a nucleotide sequence shown in figure 3; or the basic fibroblast growth factor replacement is SEQ ID NO:4, and a nucleotide sequence shown in seq id no.

The basic fibroblast growth factor substitute can automatically curl and fold under the condition of no complementary strand, so that a specific G4 chain body special structure is formed; and is similar to the spatial structure of basic fibroblast growth factor (bFGF) protein, therefore, the basic fibroblast growth factor surrogate is capable of performing multiple biological functions similar to bFGF; can promote proliferation of endothelial cells, promote neovascularization, repair damaged endothelial cells, and promote proliferation and differentiation of chondrocytes. By adding complementary strand, the nature and function of the basic fibroblast growth factor can be regulated, and the adjustable activation and inhibition of the biological function can be realized.

Optionally, at least one nucleotide molecule in the nucleotide sequence is modified with a functional group, a nanomaterial and/or a proteinaceous material. For example, the functional groups, nanomaterials and/or proteinaceous materials may be, but are not limited to, modified at the bases of the nucleotide molecules.

In the present invention, the functional group may be used to effect the thioation, methylation, amination or esterification of the base of the nucleotide molecule or of the phosphate molecule. For example, functional groups may be modified at, but are not limited to, the C-5 position of a pyrimidine base or the C-8 position of a purine base. The functional group may be, but is not limited to being, at least one substituent selected from at least one of C1-C18 alkyl, C1-C18 alkenyl, C3-C6 cycloalkyl, phenyl, aryl, heteroaryl, and C4-6 heterocyclyl.

Alternatively, the functional group may also be a fluorescent group.

Optionally, the nanomaterial comprises one or more of magnetic particles, nanomaterials, biotin, or a nano-high molecular polymer. The nanometer high molecular polymer comprises monomethoxy polyethylene glycol.

Optionally, the proteinaceous material comprises one or more of a polypeptide and an enzyme.

The basic fibroblast growth factor substitute has stable structure, can perform the biological function of bFGF and replace bFGF protein; has dual effects of adjustable activation and inhibition. Functional groups, nano materials and/or protein materials decorated on the basic fibroblast growth factor substitute can further improve the functional properties of the basic fibroblast growth factor substitute, for example, the stability of the basic fibroblast growth factor substitute structure is effectively improved through methylation of bases and the like, and the probability of sequence degradation is reduced; for example, by labeling the fluorescent groups, it is possible to facilitate visual investigation of the mechanism by which the basic fibroblast growth factor surrogate functions on the cells; for example, the nanometer high molecular polymer can realize targeted proliferation or apoptosis of cells, etc.; alternatively, the basic fibroblast growth factor replacement may be modified to incorporate a polypeptide or enzyme, such as a transmembrane peptide or proteolytic enzyme, and the like.

In the present invention, the secondary structure or tertiary structure change formed by the autonomous folding of the basic fibroblast growth factor surrogate has an important effect on the biological function of the basic fibroblast growth factor (bFGF) protein, and therefore, the selection of the nucleotide sequence in the basic fibroblast growth factor surrogate cannot affect the structure formed by the basic fibroblast growth factor surrogate.

The invention provides a pharmaceutical composition comprising an alkaline fibroblast growth factor surrogate or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition can be used for preparing medicines for wound healing, can effectively promote endothelial cell proliferation, and is beneficial to promoting neovascularization and repairing damaged endothelial cells.

Optionally, the basic fibroblast growth factor surrogate is in the form of a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium salt, potassium salt, aluminum salt, lithium salt, zinc salt, calcium salt, magnesium salt, barium salt, ammonium salt, trimethylamine salt, tetramethylammonium salt, diethylamine salt, triethylamine salt, isopropylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, cyclohexylamine salt, dicyclohexylamine salt, and pyridine salt, picoline salt, 2, 6-lutidine salt, caffeine salt, procaine salt, choline salt, betaine salt, cocoa salt, purine salt, piperazine salt, piperidine salt, N-ethylpiperidine salt, polyamine resin salt, benzamine penicillin salt, hydrochloride, hydrobromide salt, sulfate salt, sodium hydroxide nitrate, phosphate, formate, acetate, glycolate, propionate, 2-hydroxypropionate, malonate, trifluoroacetate, methanesulfonate, ethanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, benzoate, phenylacetate, alginate, anthranilate, camphorite, maleate, tartrate, citrate, succinate, mandelate, fumarate, malate, oxalate, salicylate, glucuronate, galacturonate, citrate, aspartate, glutamate, cinnamate, or a combination thereof.

In the present invention, the term "pharmaceutically acceptable" refers to compounds and compositions which can be administered to a mammal without undue toxicity, without adverse side effects.

Optionally, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier or excipient.

Alternatively, the carrier may include, but is not limited to, one or more of hydrogels, liposomes, cellulose, and calcium phosphate.

Alternatively, the excipient comprises a solution of water, saline, ringer's solution, saccharide, ethanol, mannitol, sorbitol, polyethylene glycol, phosphate, acetate, gelatin, collagen, carbomer vegetable oil, and the like. The saccharide includes one or more of glucose, sucrose, dextran and mannose.

Further, the excipient may also include a preservative, a stabilizer, an antioxidant, an antimicrobial agent, or a buffer. For example, the excipient may be Butyl Hydroxy Anisole (BHA), dibutyl hydroxy toluene (BHT), citric acid, ascorbic acid, or tetracycline.

Alternatively, the basic fibroblast growth factor surrogate or pharmaceutically acceptable salt thereof is used as a single active ingredient or in combination with other pharmaceutically acceptable active ingredients to prepare the pharmaceutical composition. The other active agents include pharmaceutically acceptable bactericidal or analgesic ingredients such as p-aminobenzenesulfonamide, chitosan, and the like. The other active agent may also be one or both of MET (receptor tyrosine kinase) aptamer or VEGF (vascular endothelial growth factor) aptamer.

In a specific embodiment of the invention, the pharmaceutical composition comprises the active ingredients of the basic fibroblast growth factor surrogate or a pharmaceutically acceptable salt thereof, MET aptamer, and VEGF aptamer.

In the present invention, the effective dose of the pharmaceutical composition for treatment and the dose to be administered to a patient are adjusted according to the age, sex and weight of the patient, and the progress of the disease.

The invention also provides an application of the pharmaceutical composition in preparing a medicament for wound healing or an application in a reagent for regulating cell proliferation, differentiation or apoptosis; the cells include at least one of chondrocytes or stem cells.

For example, the present invention provides an adjuvant drug for skin wound healing comprising a hydrogel matrix and a basic fibroblast growth factor surrogate dispersed within the hydrogel matrix. The auxiliary material medicine can be applied to the skin wound to effectively promote the healing of the skin wound. Alternatively, the invention also provides a culture medium additive for cell proliferation or inhibiting differentiation, the additive comprising a basic fibroblast growth factor surrogate. The media additive may be stored, but is not limited to, in the form of a lyophilized powder.

The invention also provides a preparation method of the alkaline fibroblast growth factor substitute, which comprises the following steps:

(1) Constructing a random library of single-stranded DNA (deoxyribonucleic acid), wherein the length of the library of the random sequence is about 20-100 bases, incubating the library and a receptor of a target molecule basic fibroblast growth factor, eluting unbound sequences, collecting specific binding sequences, repeatedly amplifying and screening the nucleic acid library of a single-stranded nucleotide sequence specifically bound with an antibody of the basic fibroblast growth factor from the random library, and carrying out high-throughput sequencing and analysis to obtain a nucleic acid sequence with higher enrichment degree, wherein the single-stranded nucleotide sequence contains a nucleotide sequence identical to SEQ ID NO:1-4, one or more nucleotide sequences having at least 95% sequence homology;

(2) Screening the nucleic acid library by adopting a high performance liquid chromatography, and collecting to obtain the alkaline fibroblast growth factor substitute through a post-treatment step.

(3) If the nucleic acid aptamer is produced in large quantities, the nucleic acid aptamer can be expressed according to SEQ ID NO:1-4, inputting the nucleotide sequence design of one or more of 1-4 into a solid phase synthesizer matched software, synthesizing a crude product through a solid phase, and purifying the crude product through an ion exchange column or high performance liquid chromatography.

Optionally, the post-treatment step comprises modifying at least one base in the single-stranded nucleotide sequence with a biological or chemical modification method, a functional group, a nanomaterial, and/or a proteinaceous material.

Optionally, the invention also provides a preparation method of the basic fibroblast growth factor substitute, which comprises the following steps: synthesizing a single-stranded nucleotide sequence comprising a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:1-4, and one or more nucleotide sequences in seq id no; and collecting and obtaining the alkaline fibroblast growth factor substitute through a post-treatment step.

For example, the basic fibroblast growth factor surrogate is synthesized using a DNA synthesizer.

In the preliminary implementation of the invention, thousands of DNA nucleic acid sequences can be obtained through preliminary screening and DNA sequencing, and how to find the most effective DNA sequence from a plurality of information is the key point and the difficulty of the work.

The biggest problem in the prior application of the aptamer is poor stability, and in the screening process, special attention is paid to the DNA sequence capable of forming the G4 surface body, so that the DNA nucleic acid sequence in the invention can form the G4 surface body structure of DNA in a solution, has high stability, and can play the purpose and the performance of promoting the cell growth for a long time in the clinical application range in future.

The alkaline fibroblast growth factor surrogate of the present invention may be stored, but is not limited to, in the form of lyophilized powder. Or stored in a low temperature environment. Further, the lyophilized powder of the basic fibroblast growth factor replacement is reconstituted with a buffer for use. The buffer solution comprises at least one of deionized water, PBS buffer solution, HEPES buffer solution, tris-HCl buffer solution, MES buffer solution and MOPS buffer solution. The buffer solution in the present invention may also be a solution that does not cause chemical or physical damage to the nucleotide sequence.

In the present invention, the basic fibroblast growth factor surrogate in the down-buffer is capable of autonomous folding to form secondary and tertiary structures. Since the basic fibroblast growth factor surrogate is adaptable to a variety of systems; therefore, the basic fibroblast growth factor substitute can realize autonomous folding so as to perform various biological functions such as cell proliferation promotion and differentiation inhibition, as long as the basic fibroblast growth factor substitute is a medium which satisfies the autonomous folding of nucleotide sequence molecules.

The invention has the following beneficial effects:

(1) The alkaline fibroblast growth factor substitute has stable structure, low cost and easy storage and transportation, can effectively promote the proliferation of endothelial cells, and is beneficial to promoting the formation of new blood vessels and repairing damaged endothelial cells; has dual effects of adjustable activation and inhibition; the alkaline fibroblast growth factor substitute has good biocompatibility and is not easy to generate immunogenicity.

(2) The pharmaceutical composition comprises an alkaline fibroblast growth factor substitute or pharmaceutically acceptable salt thereof, has stable properties of all components, and can be used for preparing a reagent for regulating and controlling cell proliferation, differentiation or apoptosis; can also be applied to the preparation of medicaments for healing skin wounds.

(3) The preparation method of the alkaline fibroblast growth factor substitute is simple to operate, green and environment-friendly, and can be used for mass production; the prepared basic fibroblast growth factor substitute has biological functions similar to bFGF, can replace bFGF, and has low preparation cost and high purity.

Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the embodiments of the invention.

Drawings

For a clearer description of the present invention, reference will be made to the following detailed description of embodiments taken in conjunction with the accompanying drawings.

FIG. 1 is SEQ ID NO:1, and the basic fibroblast growth factor substitute self-assembled folding structure represented by the sequence shown in the specification.

FIG. 2 is an imaging of a basic fibroblast growth factor surrogate to promote neural stem cell proliferation.

FIG. 3 is an image of a basic fibroblast growth factor surrogate inhibiting neural stem cell differentiation.

FIG. 4 is a graph of biocompatibility testing of culture solutions containing various concentrations of alkaline fibroblast growth factor substitutes against neural stem cells.

FIG. 5 is a graph showing cell proliferation performance of culture solutions containing various concentrations of basic fibroblast growth factor substitutes against neural stem cells.

Detailed Description

The following description is of the preferred embodiments of the present invention, and it should be noted that it will be apparent to those skilled in the art that modifications and variations can be made without departing from the principle of the invention, and these modifications and variations are also regarded as the scope of the invention.

Unless otherwise specified, the chemical reagents used in the embodiments of the present invention are all commercially available reagents.

In one embodiment of the invention, 4 alkaline fibroblast growth factor substitutes are synthesized, and the nucleotide sequences of the alkaline fibroblast growth factor substitutes are shown as SEQ ID NO: 1-4.

The sequence represented by SEQ ID NO:1, which is capable of spontaneously folding to form a secondary structure and a tertiary structure, and which is tested by a professional DNA structural analysis software, the structure of which is shown in fig. 1.

In a specific embodiment of the present invention, there is also provided a pharmaceutical composition comprising an alkaline fibroblast growth factor surrogate and a hydrogel dressing, wherein the alkaline fibroblast growth factor surrogate is SEQ ID NO:1, and a nucleotide sequence shown in the specification.

In order to evaluate the above-described basic fibroblast growth factor replacement effect of the present invention, the following effect examples were conducted.

Effect examples

The model selected by the invention is a neural stem cell model, the model is a typical fibroblast factor response model, the treatment of the factor is available, the cell phenotype is obvious, and the comparison effect of the fibroblast growth factor analogue and the protein factor can be well observed.

(1) Assessment of the cell proliferation promoting function of the basic fibroblast growth factor surrogate of the present invention

Taking a plurality of cell plates paved with nerve stem cells, respectively adding a certain content of basic fibroblast growth factor (bFGF) substitute, basic fibroblast growth factor (bFGF) and a normal cell culture solution without addition, wherein the cell plates added with the cell culture solution containing the bFGF substitute are taken as experimental groups, the cell plates added with the cell culture solution containing the bFGF are taken as positive control groups, the cell plates cultured without addition of the normal cell culture solution are taken as blank control groups, and under the same conditions except for the cell culture solution, detecting the cell proliferation condition in each cell plate after 10 days; wherein the basic fibroblast growth factor substitute is SEQ ID NO:1, and a nucleotide sequence shown in the specification.

As shown in fig. 2, the bFGF surrogate in the experimental group of the present invention promotes the suspension growth of neural stem cells into neural stem cell pellets, which can effectively promote the proliferation of neural stem cells, and the bFGF surrogate has an effect similar to that of bFGF on the market; whereas the cells of the control group had little increase and the number of cell proliferation was much lower than that of the experimental group.

(2) Evaluation of alkaline fibroblast growth factor surrogate for use in cell differentiation

Taking neural stem cell spheres with consistent sizes, respectively adding a certain content of basic fibroblast growth factor (bFGF substitute), basic fibroblast growth factor (bFGF) and a normal cell culture solution without addition, wherein a cell plate added with the cell culture solution containing the bFGF substitute is taken as an experimental group, a cell plate added with the cell culture solution containing the bFGF is taken as a positive control group, and a cell plate cultured without addition of the normal cell culture solution is taken as a blank control group; wherein the basic fibroblast growth factor substitute is SEQ ID NO:1, and detecting the cell differentiation condition by immunofluorescent staining after 10 days under the same conditions except for a cell culture solution, wherein the basic fibroblast growth factor substitute is shown in SEQ ID NO:1, and the result is shown in FIG. 3.

In immunofluorescence staining detection, DAPI (4', 6-diamidino-2-phenylindole), tuj-1 (tubulin) antibody and Nestin (Nestin) antibody are adopted to carry out immunofluorescence staining on each group of cells, and as can be seen from fig. 3, the bFGF surrogate in the experimental group of the invention can effectively maintain a marker (Nestin) of neural stem cells expressed by neural stem cells, and the effect of the bFGF surrogate is similar to that of bFGF on the market; whereas the cells of the blank group did not proliferate, gradually differentiated into neural cells and expressed the differentiation marker (Tuj-1). Therefore, the bFGF substitute of the present invention has the property of inhibiting the differentiation of stem cells (such as neural stem cells), and the efficacy thereof is similar to that of bFGF on the market.

(3) Assessment of basic fibroblast growth factor surrogate biocompatibility

Seeding neural Stem cells in Multi-well plates, the number of seeded cells in each well being 3×10 ⁴ Well, the concentration gradient of basic fibroblast growth factor surrogate was set in the complete medium system at 0, 20, 40, 200nM, cultured in a cell incubator at 37℃with 5% CO2, the complete medium system without and with bFGF protein was used as a control, and the number of cells was determined by CCK8 method on the first, third, seventh and tenth days, with basic fibroblast growth factor surrogate as shown in SEQ ID NO:1, and the result is shown in FIG. 4.

From the results of FIG. 4, it is shown that the basic fibroblast growth factor surrogate of the present invention appears non-toxic to cells; and has obvious growth promoting effect on nerve stem cells, and the alkaline fibroblast growth factor substitute is safe and reliable.

(4) Comparing the performance of each basic fibroblast growth factor surrogate and the comparison sequence in the direction of cell proliferation

Laying multiple multi-well plates, inoculating nerve stem cells into the multi-well plates, and inoculating 3×10 cells in each well ⁴ Hole, then the SEQ ID NO:1-4 was added to a complete medium system at a concentration of 20nm, and the complete medium system without the addition and addition of a control sequence (SEQ ID NO: GCGCCACTAAAGGAGAGCGCCATTCGAATAGGTGGGCG) was used as a control, and the cell number was measured by the CCK8 method after seven days in a cell incubator at 37℃with 5% CO2, and the results are shown in FIG. 5.

From the results shown in fig. 5, the present invention has the sequence shown in SEQ ID NO:1-4, respectively corresponding to ID1-4 experimental groups on cells, compared with a blank group and a random sequence control group, each experimental group has obvious growth promoting effect on the neural stem cells, and the control sequence group has almost no growth promoting effect on the neural stem cells.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

SEQUENCE LISTING

<110> Hunan Sai Orthovitamin technology Co., ltd

<120> an alkaline fibroblast growth factor substitute, and compositions and uses thereof

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 76

<212> DNA

<213> Synthesis

<400> 1

cggcggtctt tatggctggg gatggtgtgg gttcgccgcg gcggtcttta tggctgggga 60

tggtgtgggt tcgccg 76

<210> 2

<211> 72

<212> DNA

<213> Synthesis

<400> 2

cgcggtcttt atggctgggg atggtgtggg ttcgcgcgcg gtctttatgg ctggggatgg 60

tgtgggttcg cg 72

<210> 3

<211> 75

<212> DNA

<213> Synthesis

<400> 3

cgcggtcttt atggctgggg atggtgtggg ttcgcgagac gcggtcttta tggctgggga 60

tggtgtgggt tcgcg 75

<210> 4

<211> 79

<212> DNA

<213> Synthesis

<400> 4

cggcggtctt tatggctggg gatggtgtgg gttcgccgag acggcggtct ttatggctgg 60

ggatggtgtg ggttcgccg 79

Claims

1. An alkaline fibroblast growth factor replacement comprising a nucleic acid aptamer; the sequence of the nucleic acid aptamer is shown as SEQ ID NO. 2, and the nucleic acid aptamer can be automatically folded to form a secondary structure and a tertiary structure;

the nucleic acid aptamer has functional groups, nano materials and/or protein materials modified on at least one nucleotide molecule in the sequence of the nucleic acid aptamer.

2. The alkaline fibroblast growth factor surrogate of claim 1, wherein the nanomaterial comprises one or more of magnetic particles, nanomedicines, biotin, or a nano-high molecular polymer.

3. The alkaline fibroblast growth factor surrogate of claim 1, wherein the proteinaceous material comprises one or more of a polypeptide or an enzyme.

4. A pharmaceutical composition comprising the basic fibroblast growth factor surrogate according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition of claim 4, wherein the basic fibroblast growth factor surrogate or pharmaceutically acceptable salt thereof is used as a single active ingredient or in combination with other pharmaceutically acceptable active ingredients to prepare the pharmaceutical composition.

6. Use of a pharmaceutical composition according to claim 4 or 5 for the preparation of an agent for promoting proliferation or growth of neural stem cells.