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Vaccine for preventing and treating scars and preparation method thereof

A vaccine and scar technology, applied in the field of vaccines, can solve the problems of lack of pathological site specificity, complicated purification process, and many times of medication, so as to prevent and treat scar formation, reduce toxic side effects, and reduce dosage

Inactive Publication Date: 2012-12-26
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Renovo's use of recombinant hIL-10 as a candidate drug for the treatment of scar hyperplasia and improvement of scar appearance has entered phase III clinical research, but there are disadvantages such as lack of specificity to pathological sites, frequent administration, long time and complicated purification process.

Method used

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  • Vaccine for preventing and treating scars and preparation method thereof
  • Vaccine for preventing and treating scars and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Obtaining hIL-10-CBD recombinant gene

[0027] (1) Obtaining hIL-10cDNA

[0028] 1. Extraction of total RNA

[0029] Mononuclear cells were separated from human peripheral blood by lymphocyte separation medium, washed twice with PBS, resuspended in 10% RPMI 1640 culture medium, added ConA with a final concentration of 10g / l, placed in a 5% CO2 incubator, and incubated at 37°C for 48h. Collect cells. According to the method of RNA extraction kit, total RNA was extracted and stored at -80°C for later use.

[0030] 2. Extraction of total RNA and amplification of hIL-10cDNA

[0031] Use the RNA extraction and inversion kit from Takara Company to extract total RNA according to the instructions, and perform RT-PCR reaction: total RNA 500ng, 5× reverse transcription Buffer 2μl, oligo dT 0.5μl, 6mer 0.5μl, AMV reverse transcriptase 0.5μl, DEPC water was added to 10 μl. Incubate at 37°C for 30 minutes, then incubate at 85°C for 3 minutes to obtain cDNA.

[0032]...

Embodiment 2

[0043] Embodiment 2: Preparation and quality standard control of nucleic acid vaccine

[0044] (1) Preparation of vaccine

[0045] 1. Vaccine Amplification

[0046] The above-mentioned pcDNA-hIL10-CBD / DH5α bacteria were inoculated in LB medium containing 100 μg / ml ampicillin, cultured with shaking at 37°C for 7-8 hours, and inoculated into the same medium with 2% inoculation amount the next day, and continued at 37 ℃ overnight culture.

[0047] 2. vaccine preparation

[0048] Operate in accordance with the instructions of the "Gold Medal Excess Endotoxin-Free Plasmid Extraction Kit" produced by Beijing Kangwei Biotechnology Co., Ltd. Preparation and extraction of endotoxin-free plasmids—that is, nucleic acid vaccines, the specific steps are as follows:

[0049] ① Take 150ml of overnight cultured bacterial solution, add it to a centrifuge tube, centrifuge at 10,000rpm for 2-3min to collect bacteria, and discard all supernatant as much as possible.

[0050] ② Add 12ml of ...

Embodiment 3

[0062] Embodiment 3: the establishment of Balb / C mouse wound healing model

[0063] (1) Establishment of wound healing model in Balb / C mice

[0064] 1. Pretreatment of Balb / C mice and injection of the first vaccine

[0065] Seven-week-old, 18 Balb / C male mice were divided into 3 groups to remove the soft hair on the back by electric clippers and powerful depilatory cream. Draw a 0.4cm×1cm rectangular area with India ink in the middle of the back of the mouse. After 24 hours, both sides of the region were subcutaneously injected: ① The blank control group was injected with 50 μl of normal saline; ② The negative control group was injected with 50 μg / 50 μl (empty vector pcDNA3. +) Empty vector; ③ The experimental group was injected with 50 μg / 50 μl (the nucleic acid vaccine pcDNA3.1-hIL10-CBD prepared by diluting with physiological saline) nucleic acid vaccine.

[0066] 2. Preparation of mouse wounds and injection of vaccine again

[0067] After 24 hours, the 0.4 cm × 1 cm ar...

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Abstract

The invention relates to a vaccine for preventing and treating scars and a preparation method thereof. The vaccine is formed by fusing an encoded human interlenkin 10 (hIL-10) gene, a polypeptide CBD gene specifically combined with collagen, and a eukaryotic expression vector pcDNA3.1(+) on the gene level. As proved by Balb / c mouse wound healing model testing, the vaccine can be used for continuously inducing synthetic secretion of hIL-10-CBD by a mouse in a certain period of time. A secreted hIL-10-CBD protein can be specifically combined with collagen generated by cells in a wound healing process, the vaccine quantity, toxic and side effects and treatment cost can be reduced, and the pharmacological action can be brought into play specifically and effectively at scar positions, so that fibrosis caused by excessive deposition of extracellular matrixes is prevented, and the curative effects of preventing and treating the formation of scars and improving scar appearances are achieved. The vaccine is formed by fusing human interlenkin 10 (hIL-10) with a CBD polypeptide which is specifically combined with collagen in a gene engineering measure on the gene level, and fusing with the eukaryotic expression vector pcDNA3.1(+) on the gene level.

Description

technical field [0001] The invention relates to a vaccine, in particular to a vaccine for preventing and treating scars and a preparation method thereof, belonging to the field of medicine. Background technique [0002] Interleukin 10 (IL-10) is an important immunoregulatory cytokine produced by a variety of cells. It has a wide range of biological activities, including immunosuppressive, anti-inflammatory and immune regulatory properties. The main biological function is to limit and terminate the inflammatory response. Regulates the differentiation and proliferation of a variety of immune cells. In vitro and in vivo and animal experiments have shown that IL-10 plays an important role in inflammation, malignant transformation and autoimmune diseases. It is clinically used in the treatment of acute and chronic inflammatory diseases, autoimmune diseases such as inflammatory bowel disease, rheumatoid arthritis, Crohn's disease, multiple sclerosis, psoriasis, etc. [0003] IL-...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P17/02
Inventor 胡大海石继红朱雄翔韩军涛胡晓龙方小兵白晓智蔡维霞汤朝武
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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