CN116898988A - Skin reprogramming factor preparation, biological material and application - Google Patents
Skin reprogramming factor preparation, biological material and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0091—Purification or manufacturing processes for gene therapy compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Abstract
The skin reprogramming factor preparation, the biological material and the application provided by the embodiment of the application comprise 2-4 mole parts of mRNA nucleic acid molecule encoding Oct4 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding Sox2 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding Klf4 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding Glis1 transcription factor and 1 mole part of mRNA nucleic acid molecule encoding Lin28 transcription factor; after the skin reprogramming factor preparation is introduced into skin, the skin aging is reversed in situ, so that the expression of the skin aging-related genes is regulated down, the skin wrinkle eliminating effect is better, and meanwhile, the hair follicle-related genes are regulated up, so that the hair follicle proliferation and hair growth can be promoted.
Description
Technical Field
The application relates to the technical field of biological medicine, in particular to a skin reprogramming factor preparation, a biological material and application.
Background
Skin aging is manifested as: gradual loss of elasticity and reduced collagen content of the skin, leading to skin sagging and the formation of wrinkles and fine lines, especially in the forehead, corners of the eyes and neck of the face; at the same time, wrinkles are also created by muscle contraction caused by repetitive facial expressions.
Currently, botulinum toxin injections are currently the primary non-surgical method of eliminating wrinkles, which works well for eliminating skin expression wrinkles, but suffer from the disadvantage that the effect is temporary, lasting on average about 3-4 months, and the patient needs to receive injections periodically to maintain a smooth and youthful appearance of the skin, and wrinkles reappear if injections are not performed periodically. At the same time, repeated injections of botulinum toxin induce an immune response in the body, resulting in inefficiency.
Hundreds of millions of men, women and even children worldwide are afflicted with alopecia, the cause of which is numerous, such as inheritance, aging, labor, cancer treatment, burns and stress. Although hair loss does not cause serious health problems, it can bring emotional burden, trauma and even depression because it can permanently change the personal image.
At present, methods for treating alopecia are very limited, mainly comprise methods of drug treatment (minoxidil and finasteride), surgical treatment (hair implantation), physical therapy (traditional Chinese medicine acupuncture and low-frequency laser therapy) and the like, can not prevent alopecia for a long time, and can not realize hair follicle regeneration.
Disclosure of Invention
In view of the above problems, embodiments of the present application provide a skin reprogramming factor preparation, a biomaterial and use thereof, so as to solve the above technical problems.
In a first aspect, embodiments of the present application provide a skin reprogramming factor formulation comprising 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.
Alternatively, the skin reprogramming factor formulation comprises 3 molar parts of mRNA nucleic acid molecule encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding Lin28 transcription factor.
Optionally, the skin reprogramming factor preparation further comprises a first solvent, wherein the concentration of citrate in the first solvent is 8-12 mmol/L, and the concentration of sodium chloride in the first solvent is 120-140 mmol/L.
Optionally, the concentration of citrate in the first solvent is 10mmol/L, the concentration of sodium chloride in the first solvent is 130mmol/L, and the pH value of the first solvent is 7.5.
Alternatively, each of the mRNA nucleic acid molecules includes a 5' cap structure, a 5' utr sequence, an mRNA coding sequence for a transcription factor, a 3' utr sequence, and a polyadenylation sequence.
Optionally, some or all of the nucleotides in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are chemically modified to increase the stability of the mRNA nucleic acid molecule in vivo.
Alternatively, the chemical modification comprises replacing 100% of the cytosines in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor with 5-methylpseudouridine, and replacing 100% of the uracils in the mRNA nucleic acid molecule encoding Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor with N1-methylpseudouridine.
In a second aspect, embodiments of the present application provide a biomaterial that is a host skin cell comprising a skin reprogramming factor formulation as described above.
In a third aspect, embodiments of the present application provide an application of the skin reprogramming factor preparation or the biological material in preparing a skin cell reprogramming agent.
In a fourth aspect, the present application provides an application of the skin reprogramming factor preparation or the biological material in preparing skin anti-aging drugs or hair growth drugs.
The skin reprogramming factor preparation, the biological material and the application provided by the embodiment of the application comprise 2-4 mole parts of mRNA nucleic acid molecule encoding Oct4 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding Sox2 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding Klf4 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding Glis1 transcription factor and 1 mole part of mRNA nucleic acid molecule encoding Lin28 transcription factor; after the skin reprogramming factor preparation is introduced into skin, the skin aging is reversed in situ, so that the expression of the skin aging-related genes is regulated down, the skin wrinkle eliminating effect is better, and meanwhile, the hair follicle-related genes are regulated up, so that the hair follicle proliferation and hair growth can be promoted.
These and other aspects of the application will be more readily apparent from the following description of the embodiments.
Drawings
FIG. 1 is a graph showing the results of the fluorescence test of human in vitro skin in example 2 of the present application.
FIG. 2 is a graph showing the results of the in vitro skin injection experiment of human in example 3 of the present application.
FIG. 3 is a graph showing the results of skin injection experiments of skin defect models after skin transplantation of immunodeficient mice in example 4 of the present application.
Fig. 4 shows the results of skin injection experiments of different formulations of the skin reprogramming factor preparation in example 5 of the present application.
Detailed Description
Embodiments of the present application are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are exemplary only for explaining the present application and are not to be construed as limiting the present application.
In order to enable those skilled in the art to better understand the solution of the present application, the following description will make clear and complete descriptions of the technical solution of the present application in the embodiments of the present application with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the embodiments of the present application, it should be noted that, in this document, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
In describing embodiments of the present application, words such as "exemplary" or "such as" are used to mean illustrated, described, or described. Any embodiment or design described as "exemplary" or "such as" in an embodiment of the application is not necessarily to be construed as preferred or advantageous over another embodiment or design. The use of words such as "example" or "such as" is intended to present relative concepts in a clear manner.
In addition, the term "plurality" in the embodiments of the present application means two or more, and in view of this, the term "plurality" may be understood as "at least two" in the embodiments of the present application. "at least one" may be understood as one or more, for example as one, two or more. For example, including at least one means including one, two or more, and not limiting what is included, e.g., including at least one of A, B and C, then A, B, C, A and B, A and C, B and C, or A and B and C, may be included.
It should be noted that, in the embodiment of the present application, "and/or" describe the association relationship of the association object, which means that three relationships may exist, for example, a and/or B may be represented: a exists alone, A and B exist together, and B exists alone. The character "/", unless otherwise specified, generally indicates that the associated object is an "or" relationship.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
An embodiment of the present application provides a skin reprogramming factor formulation comprising 2 to 4 molar parts of mRNA nucleic acid molecule encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding Lin28 transcription factor.
In this example, by adjusting the molar ratio of mRNA nucleic acid molecules expressing different transcription factors, a specific skin reprogramming factor preparation is formed, and after the preparation is introduced into the skin, the skin aging is reversed in situ, so that the expression of the skin aging-related gene is down-regulated, and the skin wrinkle eliminating effect is better, and meanwhile, the hair follicle-related gene is up-regulated, so that the hair follicle proliferation and hair growth can be promoted.
As one embodiment, the skin reprogramming factor formulation of the present example comprises 3 molar parts of mRNA nucleic acid molecule encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding Lin28 transcription factor.
As an embodiment, the skin reprogramming factor preparation of the present example further comprises a first solvent, and the mRNA nucleic acid molecule encoding Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor described above are dissolved in the first solvent. The first solvent can be formed by citrate and normal saline, the concentration of the citrate in the first solvent is 8-12 mmol/L, and the concentration of the sodium chloride in the first solvent is 120-140 mmol/L.
In this embodiment, each mRNA nucleic acid molecule can be made less susceptible to degradation by the first solvent.
In some embodiments, the concentration of citrate in the first solvent is 10mmol/L, the concentration of sodium chloride in the first solvent is 130mmol/L, and the pH of the first solvent is 7.5.
As one embodiment, each of the mRNA nucleic acid molecules includes a 5' cap structure, a 5' utr sequence, an mRNA coding sequence for a corresponding transcription factor, a 3' utr sequence, and a polyadenylation sequence.
Specifically, mRNA nucleic acid molecules encoding Oct4 transcription factors include a 5' cap structure, a 5' utr sequence, an mRNA coding sequence for Oct4 transcription factors, a 3' utr sequence, and a polyadenylation sequence; mRNA nucleic acid molecules encoding Sox2 transcription factors include 5' cap structure, 5' UTR sequence, mRNA coding sequence for Sox2 transcription factor, 3' UTR sequence and polyadenylation sequence; mRNA nucleic acid molecules encoding the Klf4 transcription factor include 5' cap structure, 5' UTR sequence, mRNA coding sequence for the Klf4 transcription factor, 3' UTR sequence, and polyadenylation sequence; mRNA nucleic acid molecules encoding the Glis1 transcription factor include 5' cap structure, 5' UTR sequence, mRNA coding sequence for the Glis1 transcription factor, 3' UTR sequence and polyadenylation sequence; mRNA nucleic acid molecules encoding the Lin28 transcription factor include 5' cap structure, 5' UTR sequence, mRNA coding sequence for the Lin28 transcription factor, 3' UTR sequence, and polyadenylation sequence.
As one embodiment, a part or all of the nucleotides in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are chemically modified to improve the stability of the mRNA nucleic acid molecule in vivo.
In some embodiments, the chemical modification comprises replacing 100% of the cytosines in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor with 5-methylpseudouridine, and replacing 100% of the uracils in the mRNA nucleic acid molecule encoding Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor with N1-methylpseudouridine.
Example 1: preparation of skin reprogramming factor preparation
The skin reprogramming factor formulation of the present example can be prepared as follows:
s11, preparation of DNA templates of transcription factors
The Oct4 transcription factor DNA template comprises a T7 promoter shown as SEQ ID NO.10, a 5'UTR DNA sequence shown as SEQ ID NO.1, an Oct4 transcription factor DNA sequence shown as SEQ ID NO.3 and a 3' UTR DNA sequence shown as SEQ ID NO. 2;
the Sox2 transcription factor DNA template comprises a T7 promoter shown as SEQ ID NO.10, a 5'UTR DNA sequence shown as SEQ ID NO.1, a Sox2 transcription factor DNA sequence shown as SEQ ID NO.4 and a 3' UTR DNA sequence shown as SEQ ID NO. 2;
the Klf4 transcription factor DNA template comprises a T7 promoter shown as SEQ ID NO.10, a 5'UTR DNA sequence shown as SEQ ID NO.1, a Klf4 transcription factor DNA sequence shown as SEQ ID NO.5 and a 3' UTR DNA sequence shown as SEQ ID NO. 2;
the Glis1 transcription factor DNA template comprises a T7 promoter shown as SEQ ID NO.10, a 5'UTR DNA sequence shown as SEQ ID NO.1, a Glis1 transcription factor DNA sequence shown as SEQ ID NO.6 and a 3' UTR DNA sequence shown as SEQ ID NO. 2;
the Lin28 transcription factor DNA template comprises a T7 promoter shown as SEQ ID NO.10, a 5'UTR DNA sequence shown as SEQ ID NO.1, a Lin28 transcription factor DNA sequence shown as SEQ ID NO.7 and a 3' UTR DNA sequence shown as SEQ ID NO. 2;
DNA templates for transcription factors were synthesized based on the above sequence information and can be directly ordered by IDT company in U.S.A.
S12, preparation of DNA full templates of transcription factors
And adding poly- (a) tails on the Oct4 transcription factor DNA template, the Sox2 transcription factor DNA template, the Klf4 transcription factor DNA template, the Glis1 transcription factor DNA template and the Lin28 transcription factor DNA template by PCR to obtain an Oct4 transcription factor DNA full template, a Sox2 transcription factor DNA full template, a Klf4 transcription factor DNA full template, a Glis1 transcription factor DNA full template and a Lin28 transcription factor DNA full template respectively.
PCR premix (total volume 200. Mu.l, eight reactions 25. Mu.l each) was prepared according to Table 1
TABLE 1 composition of PCR premix
Component (A) | Dosage of | Final concentration |
Kapa PCR mix(2X) | 100μL | 1X |
Tailing primer-F1 μm | 6μL | 0.3μM |
Tailing primer-T120 [ mu ] m | 6μL | 0.3μM |
Water and its preparation method | 80μL | |
gBlock DNA 10μg/μl | 8μL | 40-400pg/μl |
Wherein, the tailing primer-F1 is shown as SEQ ID NO.11, and the tailing primer-T120 is shown as SEQ ID NO. 9.
PCR was performed using the conditions of table 2:
TABLE 2 tailing PCR conditions
Number of cycles | Denaturation (denaturation) | Annealing | Expansion of |
1 | 95℃,2–3min | ||
2-31 | 98℃,20s | 60℃,15s | 72℃,60s |
32 | 72℃,3min |
The quality of the PCR products was checked by gel electrophoresis.
Cut gel recovery PCR product (QIAquick PCR purification kit, qiagen, cat.no. 28106): the final concentration of the tail template was adjusted to 100 ng/. Mu.l as the whole template for each transcription factor DNA.
S13, in vitro transcription synthesis of mRNA nucleic acid molecules of transcription factors
13-1, mRNA cap structure and nucleotide mixture were assembled according to Table 3:
hat Structure N7mGpppGm RNA cap analog (New England Biolabs), methyl-5' -triphosphates (Me-CTP; trilink) 100% replaced CTP, N1-methyl pseudoudidine (Me-Pseudo-UTP; trilink), and 100% replaced UTP the other components were all from MEGAscript T7 kit (Ambion).
TABLE 3mRNA cap structures and nucleotide mixtures
13-2, an mRNA in vitro transcription system was assembled according to Table 4:
TABLE 3 in vitro mRNA transcription System
13-3, the reaction is placed in a PCR instrument and incubated for 3-6 h at 37 ℃.
13-4, 2. Mu.l of Turbo DNase (from MEGAscript T7 kit, ambion, cat. No. AM1334) was added to each sample.
13-5, gently mix and incubate at 37℃for 15min.
13-6, purifying the DNase-treated reaction using a MEGAclear kit (Ambion, cat.no. AM1908); the modified mRNA was eluted with a total of 100. Mu.l of elution buffer (50. Mu.l of elution buffer was eluted twice).
13-7, the purified mRNA nucleic acid molecules of the respective transcription factors were treated with phosphatase (Antarctic phosphatase (New England Biolabs, cat.no. M0289S).
13-8, to each sample (. About.100. Mu.l) 11. Mu.l of 10 Xphosphatase buffer was added followed by 2. Mu.l of phosphatase; the samples were gently mixed and incubated at 37℃for 0.5-1 h.
13-9, after elution, the concentration of modified mRNA was measured in a NanoDrop spectrophotometer. The expected total yield should be-50 ug (30-70 ug range; 100 ul elution volume of one 40 ul IVT reaction is 300-700 ng ul).
13-10, mRNA nucleic acid molecule encoding Oct4 transcription factor, mRNA nucleic acid molecule encoding Sox2 transcription factor, mRNA nucleic acid molecule encoding Klf4 transcription factor, mRNA nucleic acid molecule encoding Glis1 transcription factor, and mRNA nucleic acid molecule encoding Lin28 transcription factor at a molar ratio of 3:1:1:1:1:1, by addition of citric acid-normal saline (10 mmol/L citrate, 130mmol/L sodium chloride, nuclease-free distilled water, PH 7.5) to form a skin reprogramming factor formulation, the concentration was adjusted to 100ng/μl.
Example 2: verification of the intradermal injection of the mRNA preparation encoding firefly fluorescent protein
An mRNA nucleic acid molecule encoding a firefly fluorescent protein was prepared according to the method of example 1, the firefly fluorescent protein whole DNA template comprising a T7 promoter as shown in SEQ ID No.10, a 5'utr DNA sequence as shown in SEQ ID No.1, a firefly fluorescent protein DNA sequence as shown in SEQ ID No.8, a 3' utr DNA sequence as shown in SEQ ID No.2, and a poly- (a) tail; and then carrying out in vitro transcription on the whole DNA template of the firefly fluorescent protein to obtain an mRNA nucleic acid molecule for encoding the firefly fluorescent protein.
mRNA nucleic acid molecules encoding the firefly fluorescent protein were dissolved in citrate-normal saline (10 mmol/L citrate, 130mmol/L sodium chloride, nuclease free distilled water, pH 7.5) to form mRNA preparations encoding the firefly fluorescent protein.
The expression of the mRNA preparation encoding the firefly fluorescent protein is quantitatively detected by an IVIS imager by injecting the mRNA preparation encoding the firefly fluorescent protein into human isolated skin, and the specific steps are as follows: the volunteer operation incision redundant whole skin is transported to a laboratory, after washing with normal saline, adipose tissues are removed by using dissecting scissors, epidermis and dermis are reserved, the skin is cultured in a 10% fetal bovine serum DEME culture medium and a 5% CO2 cell incubator to form a human isolated skin culture model, an mRNA preparation encoding firefly fluorescent protein is injected intradermally, the injection part is the junction of the epidermis and the dermis, after 6 hours, a firefly fluorescent protein substrate is added, and the fluorescence intensity of the firefly fluorescent protein is detected in an IVIS in-vitro in-vivo imager.
Example 3: intradermal injection verification of skin reprogramming factor formulations
The skin reprogramming factor preparation prepared in example 1 was injected into human ex vivo skin (preparation of human ex vivo skin is described in example 2) to reverse skin aging in situ, and quantitative PCR assays were performed on aging index P16, skin epigenetic index H3K9me3, skin cell proliferation index K67, skin collagens I and VI, extracellular matrices MMP1 and MMP9, and oxygen radical SOD3 after collecting skin tissue with the empty mRNA preparation and the mRNA preparation encoding firefly fluorescent protein of example 2, respectively, as follows:
after collection, total mRNA was extracted by Trizol method and SuperScript was prepared according to In Vitrogen Co., ltd TM IV One-Step RT-PCR System Specification One-Step reverse transcription PCR was performed, and a One-Step reverse transcription PCR reaction System was prepared by SybrGreen method, inThe ABIQuantum studio 5 quantitative PCR instrument reacted to process data in double delta and output quantitative gene expression data.
Example 4: skin defect model establishment and skin reprogramming factor preparation skin injection experiment after skin transplantation of human in-vitro skin-immunodeficiency mice
As a comparison, a SCUB3mRNA preparation and a reporter mRNA preparation were used, wherein the SCUB3mRNA preparation and the reporter mRNA preparation were prepared in the same manner as in example 1.
The SCUB3-DNA template comprises a T7 promoter shown as SEQ ID NO.10, a 5'UTR DNA sequence shown as SEQ ID NO.1, a DNA sequence encoding SCUB3, a 3' UTR DNA sequence shown as SEQ ID NO.2 and a poly- (a) tail; and then carrying out in vitro transcription on the SCUB3-DNA template to obtain the mRNA nucleic acid molecule encoding the SCUB 3. The mRNA nucleic acid molecule encoding SCUB3 was dissolved in citrate-normal saline (10 mmol/L citrate, 130mmol/L sodium chloride, nuclease free distilled water, pH 7.5) to form a preparation of SCUB3 mRNA.
An mRNA nucleic acid molecule encoding a firefly fluorescent protein was prepared according to the method of example 1, the firefly fluorescent protein whole DNA template comprising a T7 promoter as shown in SEQ ID No.10, a 5'utr DNA sequence as shown in SEQ ID No.1, a firefly fluorescent protein DNA sequence as shown in SEQ ID No.8, a 3' utr DNA sequence as shown in SEQ ID No.2, and a poly- (a) tail; and then carrying out in vitro transcription on the whole DNA template of the firefly fluorescent protein to obtain an mRNA nucleic acid molecule for encoding the firefly fluorescent protein. mRNA nucleic acid molecules encoding the firefly fluorescent protein were dissolved in citrate-normal saline (10 mmol/L citrate, 130mmol/L sodium chloride, nuclease free distilled water, pH 7.5) to form mRNA preparations encoding the firefly fluorescent protein.
Skin injections were performed separately using the skin reprogramming factor formulation of example 1, the SCUB3mRNA formulation, the Luc mRNA formulation (the mRNA formulation encoding firefly fluorescent protein of example 2) to reverse skin aging in situ, promote skin repair in humans, and evaluate the effects, as follows:
first, human ex vivo skin-immunodeficiency mice skin following skin transplantation skin defect model establishment: NOD-Prkdcsccid/Smoc immunodeficient mice were purchased from the corresponding biological company and the health status and immunodeficient properties of the mice were determined prior to the start of the experiment.
Donor skin extraction: skin tissue surgically removed from the volunteer's body is taken as donor skin and strict aseptic procedures are followed during surgery to reduce the risk of infection.
Trimming donor skin: fat and other tissues were removed and the extracted skin tissue was trimmed to the appropriate 2X2 cm skin to accommodate the skin defect area of the mice. Treating the skin pieces may include washing, hair and adipose tissue removal steps to ensure the purity of the graft.
Preparation of mice: immunodeficient mice were anesthetized and local disinfection was done at the target skin defect area. Ensuring that the mice are in a painless and comfortable state.
Transplanting skin: the treated skin pieces were carefully placed on the skin defect areas of the mice. The skin pieces were fixed to the defect area using sutures or bio-glue, etc., and lesions of about 1 mm were formed on the isolated skin of the human body, covered with sterile dressing, observed periodically and sampled at the corresponding time points.
Second, HE staining:
firstly, obtaining a tissue slice fixed and embedded by 4% PFA, and finally obtaining the slice through dehydration and embedding; dewaxing and dehydrating: the slices of the wax block were immersed in a dewaxing agent Xylene to remove waxes.
Washing: the dewaxed slice is washed under running tap water to thoroughly remove the dewaxed agent residue.
Dyeing: the sections are sequentially immersed in a stain, first a heme stain (hemadyloxy) which stains the cell nucleus and the material surrounding the cell nucleus. The sections were then transferred to an acid wine red (Eosin) solution, which stained the cytosol and extracellular matrix.
Dehydrating: the stained sections were sequentially immersed in different concentrations of alcohol (e.g., 70% alcohol, 95% alcohol, and absolute alcohol) to dehydrate the sections.
And (3) cleaning agent treatment: the slices are immersed in a suitable cleaning agent, such as Xylene or other cleaning agent, to remove alcohol and make the slices transparent.
Sealing piece: the sections are placed in a suitable Medium, such as a clear coverslip (e.g., a moving Medium) between a glass slide and a cover slip, and then covered with a glass slide cover slip.
After the above steps are completed, the slice is ready. Using a microscope, the staining of the nuclei (blue or purple), the staining of the cytosol and extracellular matrix (pink), and details of skin tissue structure such as anagen hair follicles can be observed by HE-stained sections, quantification of the thickness of the epidermis and anagen hair follicles is performed with Image J software, and statistical and data output is performed with graphpad prism software.
Third, mass staining:
first, it is necessary to obtain tissue sections, dehydrated and embedded by 4% pfa fixation and embedding process, and finally obtain sections, dehydrated by wax removal: the slices of the wax block were immersed in an Xylene dewaxing agent to remove wax. Washing: the dewaxed slice is washed under running tap water to thoroughly remove the dewaxed agent residue.
Dyeing with an acidic dye: the sections are immersed in an acidic stain (e.g., acid magenta) for staining the cytoplasm and nucleus. The time of soaking in the stain was performed according to the standard procedure of the laboratory. Washing: the stained sections were washed under running tap water to remove excess stain.
Cobalt acid staining: the sections were immersed in a staining agent containing cobalt acid for staining the collagen fibers. The cobalt acid dye is a blue dye which can make the collagen fiber blue. Washing: the stained sections were washed under running tap water to remove excess stain. Acid washing: the slices are immersed in a dilute acidic detergent (e.g., acetic acid) for removal of the cobalt acid residue.
Blue dyeing: the sections were immersed in a stain containing hematoxylin and hematoxylin blue. Hematoxylin blue is a blue dye used to stain nuclei. Washing: the stained sections were washed under running tap water to remove excess stain. Dehydrating: the sections were sequentially immersed in different concentrations of alcohol (e.g., 70% alcohol, 95% alcohol, and absolute alcohol) to dehydrate the sections. And (3) cleaning agent treatment: the slices are immersed in a suitable cleaning agent, such as Xylene or other cleaning agent, to remove alcohol and make the slices transparent.
Sealing piece: the sections are placed in a suitable Medium, such as a clear coverslip (e.g., a moving Medium) between a glass slide and a cover slip, and then covered with a glass slide cover slip.
After the above steps are completed, the sections are ready for observation. Using a microscope, the staining of collagen fibers (blue) and the staining of nuclei (purple), as well as details of tissue structure, can be observed by Masson-stained sections, image J software quantification.
Immunohistochemistry: (a) obtaining a tissue slice: first, it is necessary to obtain a tissue section dehydrated and embedded by 4% pfa fixation and embedding process, and finally obtain a section. (b) wax block treatment: the slices of the wax block were immersed in a dewaxing agent to remove wax with Xylene. (c) antigen retrieval: heat treatment was used to restore antigen activity in the sections. (d) protein blocking: to reduce non-specific binding, it is desirable to block non-specific binding sites in tissue sections, using bovine serum albumin, which is coated on the sections. (e) incubation of antibodies: the target antibody anti-Oct 4 antibody (R & D system) solution was added dropwise to the sections, and then incubated at 4 degrees overnight to allow specific binding of the antibody to Oct4 protein in the sections. (f) washing: after incubation of the antibodies, multiple washes with buffer or PBS or the like are performed to remove unbound antibodies and other non-specific binders. (g) incubation of secondary antibody: the corresponding specific secondary antibodies (R & D systems) were incubated with the sections at 4℃overnight. (h) washing: after incubation of the secondary antibody, multiple washes with buffer or PBS or the like are performed to remove unbound secondary antibody and other non-specific binders. (i) visual staining: enzyme substrates were added to the sections using enzyme-labeled horseradish peroxidase, depending on the type of secondary antibody, resulting in a visible staining reaction. (j) washing and sealing: finally, a final washing step is carried out to remove excess dyeing substances and substrates. The sections are then placed in a suitable Medium, such as a clear coverslip (e.g., a mount Medium) between a glass slide and a cover slip, and then covered with a glass slide cover slip. (k) After this step, the sections were ready for observation, using a microscope, for determining the distribution and localization of Oct4, as well as other specific structures that bind specifically to the antibody.
Example 5: skin injection experiment of skin reprogramming factor preparations with different proportions
The present example is divided into the following four experimental groups:
experiment group 1 is reporter gene chemically modified mRNA: the formulation of example 2 was used;
experiment group 2 was OSKGL (3:1:1:1:1) chemically modified mRNA: the formulation of example 1 was used;
experiment group 3 was OSKGL (1:1:1:1:1) chemically modified mRNA: the molar ratio of the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor was 1:1:1:1, and the concentration was adjusted to 100 ng/. Mu.l by adding citric acid-physiological saline (10 mmol/L citrate, 130mmol/L sodium chloride, nuclease-free distilled water, pH 7.5) to form a skin reprogramming factor preparation.
Experiment group 4 was OSKGL (0.5:1:1:1:1) chemically modified mRNA: the molar ratio of the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor was 0.5:1:1:1 by adding citric acid-physiological saline (10 mmol/L citrate, 130mmol/L sodium chloride, nuclease-free distilled water, pH 7.5) to form a skin reprogramming factor preparation, and the concentration was adjusted to 100 ng/. Mu.l.
The method comprises the following specific steps of performing intradermal injection by adopting an experimental group 1, an experimental group 2, an experimental group 3 and an experimental group 4 respectively to reverse skin aging in situ, promote skin repair of a human body and evaluate the effect:
first, human ex vivo skin-immunodeficiency mice skin following skin transplantation skin defect model establishment: NOD-Prkdcsccid/Smoc immunodeficient mice were purchased from the corresponding biological company and the health status and immunodeficient properties of the mice were determined prior to the start of the experiment.
Donor skin extraction: skin tissue surgically removed from the volunteer's body is taken as donor skin and strict aseptic procedures are followed during surgery to reduce the risk of infection.
Trimming donor skin: fat and other tissues were removed and the extracted skin tissue was trimmed to the appropriate 2X2 cm skin to accommodate the skin defect area of the mice. Treating the skin pieces may include washing, hair and adipose tissue removal steps to ensure the purity of the graft.
Preparation of mice: immunodeficient mice were anesthetized and local disinfection was done at the target skin defect area. Ensuring that the mice are in a painless and comfortable state.
Transplanting skin: the treated skin pieces were carefully placed on the skin defect areas of the mice. The skin pieces were fixed to the defect area using sutures or bio-glue, etc., covered with sterile dressing, periodically observed and sampled at the corresponding time points.
Different mRNA formulation injections: four different treatment modes, namely experiment group 1, experiment group 2, experiment group 3 and experiment group 4, are injected into the transplanted human skin, sampling is carried out 10 days after injection, and quantitative PCR (polymerase chain reaction) of senescence index P16 gene, epigenetic index H3K9me3 gene, wrinkle index collagen VI gene, wrinkle metalloprotease gene and oxygen radical index SOD2 gene is carried out.
Example 2 experimental results:
referring to fig. 1, a portion a in fig. 1 is an in vitro culture schematic of the isolated skin, and a portion B in fig. 1 is an in vitro culture schematic of the isolated skin; part C of FIG. 1 shows that the firefly luciferin mRNA preparation can be efficiently expressed within 6 hours of intradermal injection.
Example 3 experimental results:
as shown in FIG. 2, the expression of each of the empty mRNA preparation (NC), the mRNA preparation encoding firefly fluorescent protein (Luc mRNA), the skin reprogramming factor preparation (OSKGL mRNA1 and OSKGL mRNA2, respectively, were the preparation of example 1) after 5 days of reprogramming of the human skin portion, and it was found that the aging index P16 gene expression of the skin tissue was down-regulated (A), the epigenetic younger index H3K9me3 gene expression was down-regulated (B), the cell proliferation index K67 expression was up-regulated (C), the collagen I and collagen VI were up-regulated (D), the extracellular matrix MMP1, the MMP2 gene expression was down-regulated (E), and the oxygen radical index SOD2 gene expression was down-regulated (F).
Example 4 experimental results:
as shown in fig. 3, a: on a human skin graft model, immunohistochemical detection suggests that the reprogramming factor OSKGL mRNA formulation of example 1 can be efficiently expressed in the whole layer of skin after 6 hours of injection, and the expression is localized to the skin part. B: on human skin graft models, HE and Masson fiber staining typically results suggest that injection of the reprogramming factor OSKGL mRNA formulation of example 1 can reprogram the full-thickness driving portion of the skin, increasing the thickness of the epidermis. C: quantitative determination by Image J software: the reprogramming factor OSKGL mRNA of example 1 increased the thickness of the epidermis and eliminated wrinkles compared to the empty mRNA preparations, firefly luciferin mRNA preparation and SCUBE3mRNA preparation. Quantitative determination by pathological section and Image J software: while firefly luciferin mRNA preparation and SCUBE3mRNA preparation significantly increased the number of anagen follicles to promote hair growth compared to the empty mRNA preparation, the reprogramming factor OSKGL mRNA preparation of example 1 more significantly increased the number of anagen follicles to promote hair growth compared to the SCUBE3mRNA preparation.
Example 5 experimental results:
as shown in fig. 4, the specific molar ratio of the reprogrammed OSKGL factor is 3:1:1:1:1 mRNA preparation versus reporter mRNA preparation, reprogramming OSKGL factor specific molar ratio is 1:1:1:1:1, reprogramming OSKGL factor specific molar ratio of 0.5: the mRNA preparation of 1:1:1:1 can obviously reduce the aging index P16 gene, obviously reduce the metalloproteinase gene of extracellular matrix, obviously reduce the oxygen radical index SOD2 gene, obviously improve the expression of the epigenetic young index H3K9me3 gene and obviously improve the expression of the skin collagen index collagen V I gene.
The skin reprogramming factor preparation prepared by the embodiment of the application can efficiently influence the expression mode of the whole skin gene, activate the related gene network of the skin stem cells, realize the function reset of the whole skin cells, reverse skin aging in situ, reconstruct the young state of the skin, recombine the functions of the whole skin cells, increase the collagen fibers of the skin, reduce the content of oxygen free radicals, rejuvenate the epigenetic markers and lower the related genes of the aging, and the overall effect can provide a safer and more effective method for eliminating wrinkles.
The skin reprogramming factor preparation prepared by the embodiment of the application can reverse skin aging in situ on skin, the hair follicle related genes are up-regulated, the number of hair follicles in anagen phase is obviously increased, and the effects of promoting hair growth and/or improving the curative effect of hair implantation surgery and/or accidentally improving the effect of chemically modifying mRNA of SCUBE3 protein (hair follicle growth promoting gene) to increase the number of hair follicles in anagen phase and promote hair growth can be improved.
While the application has been described with respect to the above embodiments, it should be noted that modifications can be made by those skilled in the art without departing from the inventive concept, and these are all within the scope of the application.
Claims (10)
1. A skin reprogramming factor formulation comprising 2 to 4 molar parts of mRNA nucleic acid molecule encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding Lin28 transcription factor.
2. The skin reprogramming factor formulation of claim 1, comprising 3 molar parts of mRNA nucleic acid molecule encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding Lin28 transcription factor.
3. The skin reprogramming factor formulation of claim 1, further comprising a first solvent, wherein the concentration of citrate in the first solvent is 8 to 12mmol/L and the concentration of sodium chloride in the first solvent is 120 to 140mmol/L.
4. A skin reprogramming factor formulation as claimed in claim 3, wherein the concentration of citrate in the first solvent is 10mmol/L, the concentration of sodium chloride in the first solvent is 130mmol/L, and the pH of the first solvent is 7.5.
5. The skin reprogramming factor formulation of claim 1, wherein each of the mRNA nucleic acid molecules comprises a 5' cap structure, a 5' utr sequence, an mRNA coding sequence for a corresponding transcription factor, a 3' utr sequence, and a polyadenylation sequence.
6. The skin reprogramming factor formulation of claim 1, wherein some or all of the nucleotides in the mRNA nucleic acid molecule encoding Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor are chemically modified to increase the stability of the mRNA nucleic acid molecule in vivo.
7. The skin reprogramming factor formulation of claim 6, wherein the chemical modification comprises replacing 100% of cytosine in the mRNA nucleic acid molecule encoding Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor with 5-methylpseudouridine, and replacing 100% of uracil in the mRNA nucleic acid molecule encoding Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor, and the mRNA nucleic acid molecule encoding Lin28 transcription factor with N1-methylpseudouridine.
8. A biomaterial, characterized in that it is a host skin cell comprising a skin reprogramming factor formulation as claimed in any one of claims 1 to 7.
9. Use of a skin reprogramming factor formulation of any of claims 1 to 7 or a biomaterial of claim 8 in the preparation of a skin cell reprogramming agent.
10. Use of a skin reprogramming factor formulation according to any of claims 1 to 7 or a biomaterial according to claim 8 in the manufacture of a skin anti-ageing medicament or a hair growth medicament.
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