CN105396124A - Medicine for treating ultraviolet-caused keratinocyte apoptosis - Google Patents
Medicine for treating ultraviolet-caused keratinocyte apoptosis Download PDFInfo
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- CN105396124A CN105396124A CN201510851693.XA CN201510851693A CN105396124A CN 105396124 A CN105396124 A CN 105396124A CN 201510851693 A CN201510851693 A CN 201510851693A CN 105396124 A CN105396124 A CN 105396124A
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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
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
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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
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
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Abstract
The invention discloses a medicine for treating ultraviolet caused keratinocyte apoptosis and belongs to the field of peptide-containing medicinal preparations. Active ingredients of the medicine comprise human recombinant epidermal growth factors and alkaline fibroblast growth factors. The final concentration of the recombinant epidermal growth factors is 100 micrograms per milliliter, and the final concentration of the alkaline fibroblast growth factors is 20 micrograms per milliliter. The problems that existing medicine is poor in epidermal cell apoptosis treatment effect and cannot meet clinical requirements are effectively solved. The medicine can achieve the purposes of removing apoptotic and senile keratinocytes in a biological treatment mode after keratinocyte apoptosis is caused by ultraviolet so as to increase the keratinocyte proliferation and differentiation rate, improve the keratinocyte proliferation and differentiation effect and restore skin damage, and has wide application prospects and high clinical significance.
Description
Technical field
The invention belongs to the pharmaceutical product field containing peptide, specifically a kind ofly treat the medicine that ultraviolet causes Keratinocyte apoptosis.
Background technology
Skin is divided into epidermis and corium.Epidermis is the shallow structure of skin, is made up of stratified squamous epithelium.Five layers can be divided into, i.e. basal layer, spinous layer, granular layer, clear layer and horny layer from basal layer to surface.Basal layer: the bottommost layer being positioned at epidermis, is connected with the corium of deep layer by means of basement membrane.Basal layer is the short columnar epithelial cell of one deck.Cell is less, marshalling, and core is normal containing melanin granule in oval kytoplasm.Melanocyte is had between short columnar epithelial cell.Melanocyte is slightly rounded, has dendritic processes, and karyon is less, can produce melanin granule.The number of melanin granule is relevant with the depth of skin color.Melanin granule can absorb ultraviolet, makes deep tissues from the infringement of ultraviolet radiation.The cell division of basal layer is more active, constantly produces new cell and also passes to shallow-layer, with supplementary aging, the horn cell that comes off.Therefore, also stratum germinativum is claimed.Horny layer is positioned at the most shallow-layer of epidermis, is made up of, is full of oxyphilous keratin in Cytoplasm which floor to the flat seedless horn cell of tens layers, and to acid, alkali, friction etc. are because have stronger resistance.Horn cell by epithelial cells propagation and differentiate.This process is subject to the meticulous adjustment of body, and along with a series of morphology and biochemical change.Cuticular superficial cell often comes off in small pieces, forms scurf.
Human body skin is exactly a dynamic process from fetal development, and ectoderm cell is by different propagation, and differentiation and death, develop into skin, make skin layering simultaneously, and area increases the whole body of covering, and epidermis appendages is also formed thereupon; Last skin is ripe again.Keratinocyte ripe is again one and upgrades and repair cell colony faster, and be divided into hair growth promoting, differentiation and keratinocyte tissue, this layer does not have activity, is the dead end product of epidermis cell differentiation.Ultraviolet (UV) causes apoptosis in epidermal cell to attract attention in recent years.Existing test proves that skin keratin forms the increasing and time lengthening that cell/epithelial cells (HaCaT) measure along with ultraviolet, the vigor decline of cell; Apoptosis rate and mortality rate raise.Apoptosis often causes skin circularity to decline.But the effective Therapeutic Method up to the present, reducing apoptosis of keratinocytes is also little.
Summary of the invention
The present invention is exactly the problem that the effect in order to solve existing Drug therapy apoptosis in epidermal cell can not meet clinical needs, and what propose a kind ofly treats the medicine that ultraviolet causes Keratinocyte apoptosis.
The present invention realizes according to following technical scheme.
Treat the medicine that ultraviolet causes Keratinocyte apoptosis, its active component comprises recombinant human epidermal growth factor (EGF) and basic fibroblast growth factor (b-FGF).
The final concentration of restructuring epithelical cell growth factor is 90-110 μ g/ml, and the final concentration of basic fibroblast growth factor is 10-30 μ g/ml.
Present invention obtains following beneficial effect.
The problem that the effect that the present invention effectively solves existing Drug therapy apoptosis in epidermal cell can not meet clinical needs.The present invention can reach after ultraviolet causes epithelial cells (HaCaT) apoptosis, biologic treatment mode is used to remove apoptosis, old and feeble keratinocyte, to improve Keratinocyte Proliferation, the speed of differentiation and effect, recover the object of skin injury, have broad application prospects and clinical meaning.
Accompanying drawing explanation
Fig. 1 is that inverted light microscope of the present invention observes 0Jm
2the metamorphosis figure of negative control group HaCaT cell injury after ultraviolet radiation;
Fig. 2 is that inverted light microscope of the present invention observes 0Jm
2the metamorphosis figure of EGF group HaCaT cell injury is added after ultraviolet radiation;
Fig. 3 is that inverted light microscope of the present invention observes 0Jm
2the metamorphosis figure of EGF and b-FGF group HaCaT cell injury is added after ultraviolet radiation;
Fig. 4 is that inverted light microscope of the present invention observes 300Jm
2the metamorphosis figure of negative control group HaCaT cell injury after ultraviolet radiation;
Fig. 5 is that inverted light microscope of the present invention observes 300Jm
2the metamorphosis figure of EGF group HaCaT cell injury is added after ultraviolet radiation;
Fig. 6 is that inverted light microscope of the present invention observes 300Jm
2the metamorphosis figure of EGF and b-FGF group HaCaT cell injury is added after ultraviolet radiation;
Fig. 7 is that inverted light microscope of the present invention observes 600Jm
2the metamorphosis figure of negative control group HaCaT cell injury after ultraviolet radiation;
Fig. 8 is that inverted light microscope of the present invention observes 600Jm
2the metamorphosis figure of EGF group HaCaT cell injury is added after ultraviolet radiation;
Fig. 9 is that inverted light microscope of the present invention observes 600Jm
2the metamorphosis figure of EGF and b-FGF group HaCaT cell injury is added after ultraviolet radiation;
Figure 10 is that inverted light microscope of the present invention observes 900Jm
2the metamorphosis figure of negative control group HaCaT cell injury after ultraviolet radiation;
Figure 11 is that inverted light microscope of the present invention observes 900Jm
2the metamorphosis figure of EGF group HaCaT cell injury is added after ultraviolet radiation;
Figure 12 is that inverted light microscope of the present invention observes 900Jm
2the metamorphosis figure of EGF and b-FGF group HaCaT cell injury is added after ultraviolet radiation;
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention will be further described.
1. reagent
RPMI1640 is the product of GENMED company, and its production code member is GMS12049.2A;
People AB blood plasma is purchased from BJ Red Cross Blood Center;
Injection restructuring recombinant human epidermal growth factor and basic fibroblast growth factor are purchased from American ScienCell company.Restructuring recombinant human epidermal growth factor article No.: 105-04; Recombination human basic fibroblast growth factor article No.: 104-02.
2. experimental technique
2.1 medicines of the present invention cause the impact of HaCaT cell proliferation on ultraviolet
(1) experiment grouping: negative control group (through ultraviolet radiation, not adding EGF and/or b-FGF); Ultraviolet radiation adds EGF (100 μ g/ml) group; Ultraviolet radiation adds EGF (100 μ g/ml) and b-FGF (20 μ g/ml) group;
(2) be inoculated in 6 well culture plates by HaCaT cell, inoculating cell number is 2 × 10
4individual/hole, culture medium is that RPMI1640 adds 10% people AB blood plasma.
(3) until cell grow to 70% ~ 80% to be paved with bottle at the bottom of time, with PBS liquid (about 3mL, lower with) replacement medium, be placed in 30cm place under ultraviolet B radiation lamp, ultraviolet presses 0Jm
2(irradiation), 300Jm
2, 600Jm
2, 900Jm
2irradiate HaCaT cell 72h.
(4) change culture medium after having irradiated and continue cultivation, add each group of medicine that should add according to experiment grouping, the metamorphosis damaged with inverted light microscope observation of cell after continuing to cultivate 72h.
Fig. 1-3 for ultraviolet be 0Jm
2time cell injury metamorphosis figure, wherein Fig. 1 is ultraviolet radiation HaCaT cell 72h, continues to cultivate inverted light microscope observed result after 72h; Fig. 2 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and continues to cultivate inverted light microscope observed result after 72h; Fig. 3 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and b-FGF20 μ g/ml and continues to cultivate inverted light microscope observed result after 72h.
Fig. 4-6 for ultraviolet be 300Jm
2time cell injury metamorphosis figure, wherein Fig. 4 is ultraviolet radiation HaCaT cell 72h, continues to cultivate inverted light microscope observed result after 72h; Fig. 5 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and continues to cultivate inverted light microscope observed result after 72h; Fig. 6 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and b-FGF20 μ g/ml and continues to cultivate inverted light microscope observed result after 72h.
Fig. 7-9 for ultraviolet be 600Jm
2time cell injury metamorphosis figure, wherein Fig. 7 is ultraviolet radiation HaCaT cell 72h, continues to cultivate inverted light microscope observed result after 72h; Fig. 8 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and continues to cultivate inverted light microscope observed result after 72h; Fig. 9 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and b-FGF20 μ g/ml and continues to cultivate inverted light microscope observed result after 72h.
Figure 10-12 for ultraviolet be 900Jm
2time cell injury metamorphosis figure, wherein Figure 10 is ultraviolet radiation HaCaT cell 72h, continues to cultivate inverted light microscope observed result after 72h; Figure 11 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and continues to cultivate inverted light microscope observed result after 72h; Figure 12 is ultraviolet radiation HaCaT cell 72h, adds EFG100 μ g/ml and b-FGF20 μ g/ml and continues to cultivate inverted light microscope observed result after 72h.
Result shows that medicine of the present invention facilitates the propagation of HaCaT cell after ultraviolet radiation.Cell counting testing result shows: compared with negative control group, adds medicine of the present invention after ultraviolet radiation, and the rate of increase of HaCaT cell is 43.6%.
2.2 medicines of the present invention cause the apoptotic impact of HaCaT to ultraviolet
(1) be inoculated in 6 well culture plates by HaCaT cell, inoculating cell number is 2 × 10
4individual/hole, culture medium is that RPMI1640 adds 10% people AB blood plasma.Until cell grow to 70% ~ 80% to be paved with bottle at the bottom of time, with Digestive system (0.025 pancreatin+0.02%EDTA) catapepsis at 37 DEG C, be diluted to single cell suspension, draw Digestive system in 5mL centrifuge tube, 4 DEG C, the centrifugal 10min of 2000r/min; Suck supernatant.
(2) individual cells suspension is made into, with every hole 1 × 10 by the RPMI1640 culture medium containing 10% people AB blood plasma
4individual cell is inoculated into 96 orifice plates, every pore volume 200ul.
(3) experiment grouping: blank group (non-irradiated with ultraviolet radiation does not add EGF and/or b-FGF); Negative control group (through ultraviolet radiation, not adding EGF and/or b-FGF); Ultraviolet radiation adds EGF (100 μ g/ml) group; Ultraviolet radiation adds b-FGF (20 μ g/ml) group; Ultraviolet radiation adds EGF (100 μ g/ml) and b-FGF (20 μ g/ml) group;
(4) until cell grow to 70% ~ 80% to be paved with bottle at the bottom of time, with PBS liquid (about 3mL, lower with) replacement medium, be placed in 30cm place under ultraviolet B radiation lamp, ultraviolet presses 0Jm
2(irradiation), 300Jm
2, 600Jm
2, 900Jm
2irradiate HaCaT cell 72h.
(5) after replaced medium, add each group of medicine that should add according to experiment grouping, after continuing to cultivate 72h, every hole adds MTT solution (5mg/ml PBS prepares, pH=7.4) 20ul, continues to hatch 4h.
(6) stop cultivating, careful suction abandons culture supernatant in hole, and every hole adds 150ulDMSO, and vibration 10min, makes crystal fully dissolve.
(7) on enzyme linked immunological monitor, measure each hole absorbance value, wavelength is 490nm, and record result take time as abscissa, and light absorption value is that vertical coordinate draws cell growth curve.
Result shows, medicine of the present invention significantly reduces ultraviolet and causes HaCaT apoptosis rate (see table 1).
Table 1 medicine of the present invention (process 72h) causes the apoptotic impact of HaCaT to ultraviolet
Claims (2)
1. treat the medicine that ultraviolet causes Keratinocyte apoptosis, it is characterized in that: its active component comprises recombinant human epidermal growth factor and basic fibroblast growth factor.
2. according to claim 1ly a kind ofly treat the medicine that ultraviolet causes Keratinocyte apoptosis, it is characterized in that: the final concentration of restructuring epithelical cell growth factor is 90-110 μ g/m1, and the final concentration of basic fibroblast growth factor is 10-30 μ g/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510851693.XA CN105396124A (en) | 2015-11-30 | 2015-11-30 | Medicine for treating ultraviolet-caused keratinocyte apoptosis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510851693.XA CN105396124A (en) | 2015-11-30 | 2015-11-30 | Medicine for treating ultraviolet-caused keratinocyte apoptosis |
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| Publication Number | Publication Date |
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| CN105396124A true CN105396124A (en) | 2016-03-16 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510851693.XA Pending CN105396124A (en) | 2015-11-30 | 2015-11-30 | Medicine for treating ultraviolet-caused keratinocyte apoptosis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966171A (en) * | 2019-04-28 | 2019-07-05 | 福建省海西细胞生物工程有限公司 | It is a kind of to inhibit to lead to the composition of Apoptosis because of ultraviolet light irradiation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002005828A1 (en) * | 2000-07-13 | 2002-01-24 | Gropep Limited | Compositions and methods for the treatment of skin damage |
| CN1813663A (en) * | 2005-12-02 | 2006-08-09 | 何秋莎 | Skin care product |
| CN104056258A (en) * | 2014-06-05 | 2014-09-24 | 武汉科隆生物医学有限公司 | Composition for promoting physiologically regulative regeneration of damaged tissue as well as preparation method and use thereof |
-
2015
- 2015-11-30 CN CN201510851693.XA patent/CN105396124A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002005828A1 (en) * | 2000-07-13 | 2002-01-24 | Gropep Limited | Compositions and methods for the treatment of skin damage |
| CN1813663A (en) * | 2005-12-02 | 2006-08-09 | 何秋莎 | Skin care product |
| CN104056258A (en) * | 2014-06-05 | 2014-09-24 | 武汉科隆生物医学有限公司 | Composition for promoting physiologically regulative regeneration of damaged tissue as well as preparation method and use thereof |
Non-Patent Citations (4)
| Title |
|---|
| MARK A. SEEGER等: "The Roles of Growth Factors in Keratinocyte Migration", 《ADVANCES IN WOUND CARE》 * |
| 侯锐等: "生长因子在组织工程中的应用", 《国外医学生物医学工程分册》 * |
| 杨宗城等: "《实用烧伤外科手册 第2版》", 30 September 2004, 中国医药科技出版社 * |
| 王宝玺等: "重组牛碱性成纤维细胞生长因子对体外培养人角质形成细胞促增殖作用", 《中国皮肤科杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966171A (en) * | 2019-04-28 | 2019-07-05 | 福建省海西细胞生物工程有限公司 | It is a kind of to inhibit to lead to the composition of Apoptosis because of ultraviolet light irradiation |
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Application publication date: 20160316 |