CN111195354A - Facial anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nano-liposome and preparation method thereof - Google Patents
Facial anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nano-liposome and preparation method thereof Download PDFInfo
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- CN111195354A CN111195354A CN202010047754.8A CN202010047754A CN111195354A CN 111195354 A CN111195354 A CN 111195354A CN 202010047754 A CN202010047754 A CN 202010047754A CN 111195354 A CN111195354 A CN 111195354A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 69
- 230000001153 anti-wrinkle effect Effects 0.000 title claims abstract description 25
- 239000000843 powder Substances 0.000 title claims abstract description 24
- 230000001815 facial effect Effects 0.000 title claims abstract description 19
- 239000002502 liposome Substances 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 26
- 239000008223 sterile water Substances 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims abstract description 13
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 13
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 13
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 13
- 235000019422 polyvinyl alcohol Nutrition 0.000 claims abstract description 13
- 229940083466 soybean lecithin Drugs 0.000 claims abstract description 13
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 11
- 229930195725 Mannitol Natural products 0.000 claims abstract description 11
- 239000000594 mannitol Substances 0.000 claims abstract description 11
- 235000010355 mannitol Nutrition 0.000 claims abstract description 11
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 3
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- 238000004108 freeze drying Methods 0.000 claims description 3
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- 238000000034 method Methods 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 11
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- 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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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K8/022—Powders; Compacted Powders
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- A61K8/8129—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal or ketal radical; Compositions of hydrolysed polymers or esters of unsaturated alcohols with saturated carboxylic acids; Compositions of derivatives of such polymers, e.g. polyvinylmethylether
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Abstract
The invention discloses a face anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nano-liposome, which comprises the following raw materials: according to the mass percentage, the SNAP25-Htt fusion gene nano-liposome is 0.1%, the mannitol is 5.5%, and the balance is sterile water, and the SNAP25-Htt fusion gene nano-liposome is composed of SNAP25-Htt fusion gene, sterile water, soybean lecithin, cholesterol and polyvinyl alcohol. The facial anti-wrinkle freeze-dried powder containing the SNAP25-Htt150Q fusion gene nanoliposome and the preparation method thereof can easily transfer the SNAP25-Htt150Q fusion gene to a synapse part to play a role, and finally the freeze-dried powder is prepared in a vacuum freeze-drying mode so as to keep the activity of the SNAP25-Htt150Q fusion gene for a long time and have good anti-wrinkle and wrinkle-removing effects.
Description
Technical Field
The invention relates to the technical field of medical cosmetology, in particular to facial anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nano-liposomes and a preparation method thereof.
Background
Skin wrinkles are the most prominent feature of female skin aging, and the earliest and most obvious signs of aging are the face. With the development of advanced technologies, the most effective way of removing facial wrinkles goes from traditional wrinkle removal to the current minimally invasive injection wrinkle removal, and the technology is more and more mature and advanced. However, both traditional incisions and minimally invasive surgical interventions pose a number of uncontrolled risks to the patient.
Facial skin aging is closely related to the overactivity of facial expression muscles, and when facial muscles contract, facial skin also moves along with the facial muscles to form dynamic wrinkles perpendicular to the muscle contraction direction. With age, facial muscle fibers add to the common expression lines, which become especially apparent after age 30. Reducing muscle contraction can prevent wrinkles from occurring or delay their appearance. However, no real and effective external wrinkle removing cosmetic exists in the market at present.
Muscle contraction is a physiological process that occurs in nerves and muscles at synaptic sites. Synapses are structures of mutual contact between neurons, where the impulse of one neuron is transmitted to another neuron or to another cell, and are functionally related and key parts of information transmission. The axonal terminals of neurons are branched multiple times, and finally the ends of each branch expand into a cup or sphere, called a synaptosome. The synaptic bodies can contact the cell bodies or dendrites of a plurality of neurons to form synapses. Synapses are composed of a presynaptic membrane, a synaptic cleft, and a postsynaptic membrane.
When nerve impulse is transmitted to the synaptosome through the axon, the permeability of the presynaptic membrane to calcium ions is increased, and the calcium ions in the synaptic cleft enter the synaptosome to promote the synaptophyte to be tightly fused with the presynaptic membrane and generate a fracture. Neurotransmitters in synaptic vesicles are released into the synaptic cleft and diffuse to the postsynaptic membrane to bind to protein receptors on the postsynaptic membrane, transmitting excitatory or inhibitory signals.
Disclosure of Invention
The invention aims to provide a face anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nanoliposomes and a preparation method thereof, which can easily transfer SNAP25-Htt150Q fusion genes to synapse parts to play a role, and finally adopts a vacuum freeze-drying mode to prepare the freeze-dried powder so as to keep the activity of the SNAP25-Htt150Q fusion genes for a long time and have good anti-wrinkle and wrinkle-removing effects.
In order to realize the purpose, the invention provides facial anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nano-liposomes, which comprises the following raw materials: according to the mass percentage, the SNAP25-Htt fusion gene nano-liposome is 0.1 percent, the mannitol is 5.5 percent, and the balance is sterile water.
The SNAP25-Htt fusion gene nanoliposome preferably comprises SNAP25-Htt fusion gene, sterile water, soybean lecithin, cholesterol and polyvinyl alcohol.
Preferably, the mass ratio of the soybean lecithin to the cholesterol is 8: 5, and the volume ratio of the polyvinyl alcohol to the sterile water is 3: 2.
A preparation method of facial anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nanoliposomes comprises the following steps:
s1, extracting an SNAP25-Htt fusion gene;
s2, wrapping the extracted SNAP25-Htt fusion gene by adopting a nanoliposome technology, specifically dissolving the obtained SNAP25-Htt fusion gene in sterile water, adding soybean lecithin and cholesterol, wherein the mass ratio of the soybean lecithin to the cholesterol is 8: 5, uniformly mixing by using polyvinyl alcohol as a solvent, and the volume ratio of the polyvinyl alcohol solvent to the sterile water is 3: 2 to prepare the SNAP25-Htt fusion gene nanoliposome;
s3, adding the prepared SNAP25-Htt fusion gene nanoliposome and mannitol into a sterile aqueous solution, and uniformly stirring, wherein the adding amount of the SNAP25-Htt fusion gene nanoliposome is 0.1%, and the adding amount of the mannitol is 5.5%;
s4, pre-freezing the solution in the S3 at-20 ℃ for 24 hours, and then putting the solution into a vacuum freeze dryer for freeze drying to obtain freeze-dried powder;
and S5, performing quality detection on the freeze-dried powder, and packaging after the detection is qualified.
Preferably, the extraction method of the SNAP25-Htt150Q fusion gene in the step S1 is as follows:
firstly, fusing a first exon of 150Q Htt gene with SNAP25 gene;
secondly, modifying a PRK5 vector, inserting a mouse prion promoter and Pmel and Spel enzyme cutting sites, and then constructing the SNAP25-Htt fusion gene on the modified PRK5 vector;
thirdly, transforming the plasmid vector into TOP10 competent cells, coating an ampicillin resistance plate for 24h, selecting 3-5 clones, shaking bacteria, extracting plasmids by using a plasmid miniprep kit, performing enzyme digestion identification on the plasmids extracted by the 3-5 clones by using Pmel and Spel, inoculating the bacterial clones which are subjected to the enzyme digestion identification correctly, expanding the bacterial clones to 250ml of bacterial liquid, and performing plasmid extraction by using a plasmid macroprep kit;
finally, the extracted plasmid was digested with Pmel and Spel, and the SNAP25-Htt fusion gene fragment was recovered by gel electrophoresis.
Preferably, the identification primers for enzyme digestion identification are divided into a forward primer and a reverse primer;
wherein the forward primer is 5'-ATGAAGGCCTTCGAGTCCCTCAAGTCCTTC-3', and the reverse primer is 5'-AAACTCACGGTCGGTGCAGCGGCTCCTCAG-3'.
Preferably, the particle size of the SNAP25-Htt fusion gene liposome prepared in the step S2 is 30 nm.
Therefore, the facial anti-wrinkle freeze-dried powder containing the SNAP25-Htt150Q fusion gene nanoliposome and the preparation method thereof can easily transfer the SNAP25-Htt150Q fusion gene to a synaptic site to play a role, and finally the freeze-dried powder is prepared in a vacuum freeze-drying mode so as to keep the activity of the SNAP25-Htt150Q fusion gene for a long time and have good anti-wrinkle and wrinkle-removing effects.
The technical solution of the present invention is further described in detail by the following examples.
Detailed Description
The embodiments of the present invention will be further explained below.
The invention provides a face anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nano-liposome, which comprises the following raw materials: according to the mass percentage, the SNAP25-Htt fusion gene nanoliposome is 0.1%, the mannitol is 5.5%, and the balance is sterile water, the SNAP25-Htt fusion gene nanoliposome is composed of SNAP25-Htt fusion gene, sterile water, soybean lecithin, cholesterol and polyvinyl alcohol, the mass ratio of the soybean lecithin to the cholesterol is 8: 5, and the volume ratio of the polyvinyl alcohol to the sterile water is 3: 2.
A preparation method of facial anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nanoliposomes comprises the following steps:
s1, extracting the SNAP25-Htt fusion gene, wherein the specific extraction method comprises the following steps:
firstly, fusing a first exon of 150Q Htt gene with SNAP25 gene;
secondly, modifying a PRK5 vector, inserting a mouse prion promoter and Pmel and Spel enzyme cutting sites, and then constructing the SNAP25-Htt fusion gene on the modified PRK5 vector;
thirdly, transforming a plasmid vector into TOP10 competent cells, coating an ampicillin resistant plate for 24h, selecting 3-5 clones, shaking bacteria, extracting plasmids by using a plasmid miniprep kit, carrying out enzyme digestion identification on the plasmids extracted by the 3-5 clones by using Pmel and Spel, wherein the identification primers for enzyme digestion identification are divided into a forward primer and a reverse primer, wherein the forward primer is 5'-ATGAAGGCCTTCGAGTCCCTCAAGTCCTTC-3', the reverse primer is 5'-AAACTCACGGTCGGTGCAGCGGCTCCTCAG-3', inoculating the bacterial clones which are subjected to enzyme digestion identification and are correct again, expanding the bacterial liquid to 250ml, and carrying out plasmid extraction by using a plasmid macroextraction kit;
finally, the extracted plasmid was digested with Pmel and Spel, and the SNAP25-Htt fusion gene fragment was recovered by gel electrophoresis.
S2, wrapping the extracted SNAP25-Htt fusion gene by adopting a nanoliposome technology, specifically dissolving the obtained SNAP25-Htt fusion gene in sterile water, adding soybean lecithin and cholesterol, wherein the mass ratio of the soybean lecithin to the cholesterol is 8: 5, uniformly mixing by using polyvinyl alcohol as a solvent, and the volume ratio of the polyvinyl alcohol solvent to the sterile water is 3: 2 to prepare the SNAP25-Htt fusion gene nanoliposome with the particle size of 30 nm.
S3, adding the prepared SNAP25-Htt fusion gene nanoliposome and mannitol into a sterile aqueous solution, and uniformly stirring, wherein the adding amount of the SNAP25-Htt fusion gene nanoliposome is 0.1%, and the adding amount of the mannitol is 5.5%.
S4, pre-freezing the solution in the S3 at the temperature of-20 ℃ for 24 hours, and then putting the solution into a vacuum freeze dryer for freeze drying to obtain freeze-dried powder.
And S5, performing quality detection on the freeze-dried powder, and packaging after the detection is qualified.
Example 1
Effect experiment: 200 healthy women of 35-45 years old were selected for a 30 day trial. 200 persons were randomly divided into 2 groups of 100 persons each. Set as control group and experimental group. The control group used lyophilized powder containing only mannitol, and the experimental group used lyophilized powder containing SNAP25-Htt150Q fusion gene. After 30 days, the anti-wrinkle effect around the mouth and the eye socket of the testers is tested by a visa measuring instrument. Wrinkles were measured by the following 3 criteria.
Shallow wrinkles: the elastic tissue is slightly denatured, i.e. subtle changes in the tissue are slightly deepened with skin lines.
Severe wrinkles: moderate degeneration of elastic tissue, marked papular-like degeneration of elastic tissue, yellow, translucent papules, and discoloration of the skin.
Severe wrinkles: elastic tissue is severely denatured, i.e. multi-papular and fused elastic tissue is denatured, and rhomboid skin is thickened, yellowish or pale.
| Group of | Is remarkable in that | Good effect | In general | Invalidation | Allergy (S) |
| Control group | 0 | 11 | 13 | 76 | 0 |
| Experimental group | 76 | 17 | 5 | 2 | 0 |
The statistical method is used for analysis, the experimental data are found to have obvious statistical difference, and the SNAP25-Htt150Q fusion gene nano liposome freeze-dried powder has good anti-wrinkle and wrinkle-removing effects.
Therefore, the facial anti-wrinkle freeze-dried powder containing the SNAP25-Htt150Q fusion gene nanoliposome and the preparation method thereof can easily transfer the SNAP25-Htt150Q fusion gene to a synaptic site to play a role, and finally the freeze-dried powder is prepared in a vacuum freeze-drying mode so as to keep the activity of the SNAP25-Htt150Q fusion gene for a long time and have good anti-wrinkle and wrinkle-removing effects.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.
Claims (7)
1. A facial anti-wrinkle freeze-dried powder containing SNAP25-Htt150Q fusion gene nanoliposomes is characterized by comprising the following raw materials: according to the mass percentage, the SNAP25-Htt fusion gene nano-liposome is 0.1 percent, the mannitol is 5.5 percent, and the balance is sterile water.
2. The facial anti-wrinkle lyophilized powder containing SNAP25-Htt150Q fusion gene nanoliposome of claim 1, which is characterized in that: the SNAP25-Htt fusion gene nano-liposome consists of SNAP25-Htt fusion gene, sterile water, soybean lecithin, cholesterol and polyvinyl alcohol.
3. The facial anti-wrinkle lyophilized powder containing SNAP25-Htt150Q fusion gene nanoliposome of claim 2, which is characterized in that: the mass ratio of the soybean lecithin to the cholesterol is 8: 5, and the volume ratio of the polyvinyl alcohol to the sterile water is 3: 2.
4. The preparation method of the facial anti-wrinkle lyophilized powder containing SNAP25-Htt150Q fusion gene nanoliposome as claimed in any one of claims 1 to 3, comprising the following steps:
s1, extracting an SNAP25-Htt fusion gene;
s2, wrapping the extracted SNAP25-Htt fusion gene by adopting a nanoliposome technology, specifically dissolving the obtained SNAP25-Htt fusion gene in sterile water, adding soybean lecithin and cholesterol, wherein the mass ratio of the soybean lecithin to the cholesterol is 8: 5, uniformly mixing by using polyvinyl alcohol as a solvent, and the volume ratio of the polyvinyl alcohol solvent to the sterile water is 3: 2 to prepare the SNAP25-Htt fusion gene nanoliposome;
s3, adding the prepared SNAP25-Htt fusion gene nanoliposome and mannitol into a sterile aqueous solution, and uniformly stirring, wherein the adding amount of the SNAP25-Htt fusion gene nanoliposome is 0.1%, and the adding amount of the mannitol is 5.5%;
s4, pre-freezing the solution in the S3 at-20 ℃ for 24 hours, and then putting the solution into a vacuum freeze dryer for freeze drying to obtain freeze-dried powder;
and S5, performing quality detection on the freeze-dried powder, and packaging after the detection is qualified.
5. The method for preparing the facial anti-wrinkle lyophilized powder containing SNAP25-Htt150Q fusion gene nanoliposome according to claim 4, wherein the method for extracting SNAP25-Htt150Q fusion gene in step S1 is as follows:
firstly, fusing a first exon of 150Q Htt gene with SNAP25 gene;
secondly, modifying a PRK5 vector, inserting a mouse prion promoter and Pmel and Spel enzyme cutting sites, and then constructing the SNAP25-Htt fusion gene on the modified PRK5 vector;
thirdly, transforming the plasmid vector into TOP10 competent cells, coating an ampicillin resistance plate for 24h, selecting 3-5 clones, shaking bacteria, extracting plasmids by using a plasmid miniprep kit, performing enzyme digestion identification on the plasmids extracted by the 3-5 clones by using Pmel and Spel, inoculating the bacterial clones which are subjected to the enzyme digestion identification correctly, expanding the bacterial clones to 250ml of bacterial liquid, and performing plasmid extraction by using a plasmid macroprep kit;
finally, the extracted plasmid was digested with Pmel and Spel, and the SNAP25-Htt fusion gene fragment was recovered by gel electrophoresis.
6. The preparation method of the facial anti-wrinkle lyophilized powder containing SNAP25-Htt150Q fusion gene nanoliposome according to claim 5, wherein the preparation method comprises the following steps:
the identification primers for enzyme digestion identification are divided into forward primers and reverse primers;
wherein the forward primer is 5'-ATGAAGGCCTTCGAGTCCCTCAAGTCCTTC-3', and the reverse primer is 5'-AAACTCACGGTCGGTGCAGCGGCTCCTCAG-3'.
7. The preparation method of the facial anti-wrinkle lyophilized powder containing SNAP25-Htt150Q fusion gene nanoliposome according to claim 4, wherein the preparation method comprises the following steps: the particle size of the SNAP25-Htt fusion gene liposome prepared in the step S2 is 30 nm.
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