WO2021134913A1 - 一种人隐花色素蛋白I(hCRY1)在制备抗紫外辐射的制剂中的应用 - Google Patents

一种人隐花色素蛋白I(hCRY1)在制备抗紫外辐射的制剂中的应用 Download PDF

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WO2021134913A1
WO2021134913A1 PCT/CN2020/078516 CN2020078516W WO2021134913A1 WO 2021134913 A1 WO2021134913 A1 WO 2021134913A1 CN 2020078516 W CN2020078516 W CN 2020078516W WO 2021134913 A1 WO2021134913 A1 WO 2021134913A1
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hcry1
radiation
preparation
ultraviolet
group
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黄文林
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广州达博生物制品有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations

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  • the invention belongs to the field of biopharmaceutical technology, and specifically relates to the application of human cryptochrome protein I (hCRY1) in the preparation of anti-ultraviolet radiation preparations.
  • sunscreen products on the market are mainly divided into physical sunscreens and chemical sunscreens.
  • Physical sunscreens usually reflect or scatter ultraviolet and visible light. Commonly used substances are talc, zinc oxide, titanium dioxide, magnesium oxide, kaolin and so on.
  • Chemical sunscreens usually absorb ultraviolet rays, and they have different effects depending on the wavelength of the ultraviolet rays.
  • the sunscreens that absorb long-wave ultraviolet (wavelength range 320-400nm) include benzophenone, 4-tert-butyl-4-methoxymethane and 4-isopropyl diphenylmethane; absorb medium-wave ultraviolet (wavelength range) (290-320nm) sunscreens include p-aminobenzoic acid and its derivatives, cinnamate and salicylate.
  • physical sunscreens are some opaque substances that cannot selectively absorb ultraviolet rays. Chemical sunscreens have the characteristics of toxic side effects and non-broad-spectrum absorption. Therefore, it is urgent to study a safe, non-toxic and good UV absorption effect. .
  • Ultraviolet radiation causes skin aging, which is mainly manifested as skin pigmentation, dryness, loss of skin elasticity, thickening of texture and deepening of wrinkles, dull or grayish-yellow skin.
  • NF-kB nuclear factor kappa-B
  • the activation of NF-kB transcription factors promotes the expression of pro-inflammatory cytokines and growth factors, such as interleukin-1 (IL1), tumor necrosis factor (TNF) and epidermal growth factor (EGF).
  • IL1 interleukin-1
  • TNF tumor necrosis factor
  • EGF epidermal growth factor
  • the mitogen-activated protein kinase (MAPK) pathway is affected and upregulated.
  • MMPs matrix metalloproteinase family
  • AP-1 transcription factor activator protein 1
  • MMPs matrix metalloproteinase family
  • UV radiation can up-regulate MMPs, thereby accelerating the degradation of skin collagen and ultimately leading to aging.
  • CN103834677B discloses a recombinant expression method of human cryptochrome protein I (hCRY1) and its application in the preparation of a radiotherapy protective agent.
  • the examples prove that the H2AXFOCI produced by the cells added with hCRY1 is significantly reduced when they are damaged by radiation, and the reduction is not caused by hCRY1 causing damage to the cells. Therefore, it can be proved that hCRY1 has a protective effect on cells from UV damage. It shows that the prepared recombinant hCRY1 can be used as a radiotherapy protective agent, such as a radiotherapy protective agent, especially a skin radiotherapy protective agent.
  • the present invention provides an application of human cryptochrome protein I (hCRY1) in the preparation of anti-ultraviolet radiation preparations to determine whether hCRY1 has an ultraviolet protection function. Respective cell experiments and animal experiments have shown that hCRY1 has a significant protective effect on ultraviolet radiation, and is effective as a skin care.
  • hCRY1 human cryptochrome protein I
  • a human cryptochrome protein I (hCRY1) emulsion The components of the emulsion include the following raw materials: water, glycerin, polyethylene glycol-100 stearate, salicylate, sodium polyacetate, silicon dioxide, ethyl acetate Glycerin, glyceryl stearate, methyl paraben, xanthan gum, fragrance, butylated hydroxytoluene and sodium ethylenediaminetetraacetate.
  • the concentration range of the human cryptochrome protein I (hCRY1) in the formulation is 1.6-50 ⁇ g/mL, and further, the concentration range of the human cryptochrome protein I (hCRY1) in the formulation is 6.25 25 ⁇ g/mL, further preferably, the concentration of the human cryptochrome protein I (hCRY1) in the preparation is 12.5 ⁇ g/mL or 25 ⁇ g/mL.
  • human cryptochrome protein I hCRY1
  • hCRY1 human cryptochrome protein I
  • the human cryptochrome protein I (hCRY1) is used as an ultraviolet sunscreen, and further preferably, the human cryptochrome protein I (hCRY1) is used as a broad-spectrum ultraviolet sunscreen.
  • human cryptochrome protein I (hCRY1) is used in the preparation of sunscreen cosmetics or additives.
  • human cryptochrome protein I (hCRY1) is used in the preparation of face creams, body lotions, isolation creams, cushions, and sunscreen sprays.
  • human cryptochrome protein I (hCRY1) is used in the preparation of drugs for breeding plants against ultraviolet radiation.
  • hCRY1 as a potential new biological ultraviolet sunscreen in this application, has the characteristics of safety, light transmission, water solubility, and broad spectrum absorption of 100-400nm ultraviolet rays.
  • This application mainly uses tissue morphology staining and immunohistochemical staining, and the mRNA expression level of related genes to reveal changes in skin photoaging and gene expression differences in observed animal models.
  • Figure 1 The radiation resistance test results of different concentrations of hCRY1 to 30mJ/cm 2 dose of UVC 257nm radiation treatment.
  • Figure 2 Detection results of hCRY1 after UVB 320nm radiation treatment.
  • a 12.5 ⁇ g/mL hCRY1 protection group staining results B 25 ⁇ g/mL hCRY1 protection group staining results, C 50 ⁇ g/mL hCRY1 protection group staining results, D radiation control group, E is no radiation control group.
  • Figure 3 The results of detection of gene regulation in HaCaT cells after UVB 320nm radiation treatment, the results of differential expression of HSP70 gene in A HaCaT cells, the differential expression of MMP-1 in B HaCaT cells, and the differential expression of TP53 in C HaCaT cells.
  • Figure 4 The results of the detection of pathological changes in rat skin tissue after UVB 320nm radiation treatment.
  • A is stained in the control group without radiation
  • B is stained in the non-protective radiation group
  • C is stained in the 12.5 ⁇ g/mL hCRY1 protection group
  • D is stained in the commercial SPF30 radiation protection group .
  • FIG. 5 After UVB 320nm radiation treatment, the detection result of matrix metalloproteinase MMP-1 antibody immunohistochemical staining of rat skin. Among them, A was immunostained for the control group without radiation, B was immunostained for the control group without radiation, C was immunostained for the 12.5 ⁇ g/mL hCRY1 protection group, and D was immunostained for the commercial SPF30 radiation protection group.
  • Fig. 6 HE staining of rat skin after UVB 320nm radiation treatment, A non-radiation control group staining, B non-radiation protection group staining, C 12.5 ⁇ g/mL hCRY1 emulsion protection group staining, and D commercial SPF30 radiation protection group staining .
  • Figure 7 After UVB 320nm radiation treatment, the matrix metalloproteinase MMP-1 antibody immunohistochemical staining of rat skin, where A is immunostaining without radiation control group, B is immunostaining without radiation protection group, C 12.5 ⁇ g/mL hCRY1 emulsion Protection group immunostaining, D commercial SPF30 protection radiation group immunostaining.
  • Figure 8 Comparison of cell density under UVB 320nm radiation, where A hCRY1, B PBS negative control group, and C non-radiation group.
  • UVA ultraviolet lamp Guanya Optoelectronics, wave peak 360nm
  • UVB ultraviolet lamp Guanya Optoelectronics, wave peak 320nm
  • UVC ultraviolet lamp Beijing Stone, wave peak 257nm
  • UV illuminance meter Tiwan Xianchi
  • enzyme label Instrument Bio-Rad
  • fluorescence quantitative PCR instrument Stratagene MX 3000P
  • hCRY1 recombinant human cryptochrome protein solution Dubbio, E.
  • UV irradiation box use polystyrene foam box as the outer shell of the irradiation box, fix the ultraviolet lamp and lamp holder in the foam box, and paste and cover the shell with tin foil.
  • UVC 257nm radiation treatment Dilute the cells in the logarithmic growth phase and dilute to 1 ⁇ 10 5 cells/mL, inoculate 100 ⁇ L per well into a 96-well plate, 37°C, 5% CO 2 , and incubate for 24 hours .
  • Radiation experiment group settings Dilute the hCRY1 protein solution with PBS, set 0, 1.6, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/mL total 8 concentration gradients, use 50 ⁇ g/ml BSA, PBS solution as the radiation control group, HaCaT without UVA radiation was used as a non-radiation control group.
  • the radiation treatment operation is as follows: Take the HaCaT cells out of the incubator, remove the culture medium, add the above-prepared experimental group to the cells, do 6 repetitions for each group, after replacement, incubate at 37°C for 5 minutes, with a radiation intensity of 50 ⁇ W /cm 2 UVC ultraviolet lamp irradiates 600s, the total radiation dose is 30mJ/cm 2 . After irradiation, the liquid in the 96-well plate was aspirated and replaced with 100 ⁇ L of 1% FBS DMEM medium per well. After culturing for 24 hours, add 10 ⁇ L of CCK-8 reagent to each well and incubate for 60min, set the wavelength OD 450nm to measure the reading.
  • UVB 320nm radiation treatment Dilute the cells in the logarithmic growth phase and dilute to 1.5 ⁇ 10 5 cells/mL, inoculate 1 mL per well into a 24-well plate, 37°C, 5% CO 2 , and incubate for 24 hours .
  • Radiation experiment group setting Dilute the hCRY1 protein solution with DMEM medium, set 3 concentration gradients of 12.5, 25, and 50 ⁇ g/mL, use 10% FBS DMEM as the radiation control group, and use HaCaT without UVA radiation as the non-radiation control group. Radiation control group.
  • the radiation treatment operation is as follows: Take the HaCaT cells out of the incubator, replace the medium, do 5 replicates for each group, incubate at 37°C, 5% CO 2 for 30 minutes, radiate UVB 320nm with a radiation intensity of 250 ⁇ W/cm 2 for 80 seconds, and radiation dose It is 20mJ/cm 2 . After irradiation, the liquid in the 24-well plate was aspirated and replaced with 1 mL of 10% FBS DMEM medium per well. After culturing for 24 hours, rinse with PBS once, fix with 100% methanol for 10 minutes, stain with 1% crystal violet for 10 minutes, and soak in PBS for 5 minutes 3 times.
  • Radiation experiment group setting Dilute the hCRY1 protein solution with DMEM medium, set 2 concentration gradients of 12.5 and 25 ⁇ g/mL, use 10% FBS DMEM as the radiation control group (0 ⁇ g/mL hCRY1), use no UVA radiation HaCaT as a non-radiation control group.
  • the operation of the radiation treatment is as follows: Take the HaCaT cells out of the incubator, replace the medium, do 3 replicates for each group, discard the medium after 24 hours of culture, and extract total RNA. Use the HSP70, TP53, and MMP-1 genes in the table below. The primers were detected by fluorescence quantitative PCR to compare the expression levels of genes involved in the repair mechanism of HaCaT cells after radiation treatment by hCRY1.
  • the operation of the radiation treatment is as follows: the skin of the air-lifted cultured rat is irradiated with UVB 320nm with a radiation intensity of 1500 ⁇ W/cm 2 for 60 minutes, the medium is changed after the radiation treatment, and the culture is continued for 3 days, and then rinsed twice with PBS for 30 minutes. Polyoxymethylene is fixed for 24 hours. Prepare paraffin specimens and slice them for HE staining and matrix metalloproteinase MMP-1 antibody immunohistochemical staining.
  • OD 450nm measured by CCK-8 The results of OD 450nm measured by CCK-8 are shown in Figure 1:
  • the OD 450nm of 6.25 ⁇ g/mL and 12.5 ⁇ g/mL hCRY1 solutions are 0.857 ⁇ 0.103 and 0.836 ⁇ 0.095, respectively, and the OD 450nm of the PBS control group is 0.652 ⁇ 0.066.
  • Comparison with the control group, hCRY1 protein protects against UVC 257nm caused by proliferation (P ⁇ 0.05, n 6 ).
  • the cells in the 12.5 ⁇ g/mL hCRY1 protein treatment group have obvious resistance to UVC compared to the PBS and 50 ⁇ g/mL BSA control groups.
  • Hacat cells with a cell density of 80% confluence were incubated with different concentrations of hCRY1 protein for 30 minutes, and then treated with 20mJ/cm 2 radiation dose and 250 ⁇ W/cm 2 radiation intensity of UVB radiation. Cells cultured for 24 hours were stained with crystal violet The result is shown in Figure 2.
  • HaCaT cells maintained normal proliferation with the cells in the non-radiation control group after UVB 320nm radiation treatment.
  • the cells in the radiation control group maintained their normal proliferation state.
  • the cell morphology is shrunken, the cytoplasm has vesicular bodies, and the cells are arranged loosely.
  • the cells in the wells of the radiation control group were sparse, while the staining ratios of A, B, C, and E in Figure 2 were close to 90%.
  • Hacat cells When the density of Hacat cells reached 80% confluence, they were incubated with different concentrations of hCRY1 protein for 30 minutes and then treated with 20mJ/cm 2 radiation dose and 250 ⁇ W/cm 2 UVB radiation for 80 seconds. After 24 hours of culture, total RNA was extracted and RT- The qPCR method detects the expression differences of HSP70, TP53, and MMP-1, as shown in Figure 3.
  • HSP70 and TP53 are important genes that mediate repair after DNA damage, and MMP-1 is an important gene that mediates collagen degradation.
  • Figure 3 indicates that UVB radiation caused a significant down-regulation of HSP70 protein in the unprotected group, and an up-regulation in the hCRY1 protected group.
  • the HE staining of rat skin is shown in Figure 4.
  • a non-radiation control group, C 12.5 ⁇ g/mL hCRY1 protection group and D commercial SPF30 radiation protection group observed under the microscope, the dermal fibrous tissue is dense, orderly arranged, and evenly distributed.
  • the dermal fibers had cell swelling and degeneration, and the dermal tissues were arranged disorderly and distributed unevenly or sparsely.
  • the matrix metalloproteinase MMP-1 antibody immunohistochemical staining of rat skin is shown in Figure 5.
  • the expression of MMP-1 in group C was not significant; the immunostained hair follicles in group B without radiation showed outward infiltration, and the score of MMP-1 expression was +++.
  • Source of raw materials Experimental animals: SD rats (purchased from Guangzhou Youdi Bio), 30, MMP-1 antibody (purchased from Bioss).
  • QPCR reagents purchased from YEASEN: TRIZOL, reverse transcriptase, SYBR MIX, MMP-1, MMP-3, GAPDH real-time quantitative PCR primers.
  • Specimen preparation and immunohistochemistry chloral hydrate, paraformaldehyde, paraffin, dewaxing agent, mounting tablets, slides, absolute ethanol, HE staining kit, immunohistochemistry secondary antibody kit, immunohistochemistry tool kit .
  • UVA UV lamp 15W (Guanya Optoelectronics, wave peak 360nm), UVB UV lamp 15W (Guanya Optoelectronics, wave peak 320nm), dimmer, ultraviolet light meter (Taiwan Xianchi), rat fixator, electric razor, QPCR instrument (Purchased from STRATAGENE).
  • a human cryptochrome protein I (hCRY1) emulsion which includes the following raw materials: water, glycerin, polyethylene glycol-100 stearate, salicylate, sodium polyacetate, silicon dioxide, ethyl Hexylglycerol, glyceryl stearate, methyl paraben, xanthan gum, fragrance, butylated hydroxytoluene and sodium ethylenediaminetetraacetate.
  • the concentration of human cryptochrome protein I (hCRY1) in the preparation is 12.5 ⁇ g/mL.
  • UVB 320nm group non-protective radiation group
  • UVB 320nm hCRY1 smear group hCRY1 emulsion protection group
  • RNA extraction sample Take the mung bean-sized skin tissue and grind it in a liquid nitrogen mortar, and use trizol as the RNA extraction sample. Extract different groups of RNA and reverse transcribe cDNA. The expression differences of MMP-1, MMP-3 and MMP-9 were detected by qPCR instrument.
  • Hacat cells with a cell density of 80% confluence were incubated with 12.5 ⁇ g/mL hCRY1 emulsion for 30 minutes, and then treated with 20mJ/cm 2 radiation dose and 250 ⁇ W/cm 2 radiation intensity of UVB radiation. Cells cultured for 24 hours were used Crystal violet staining result.

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Abstract

一种人隐花色素蛋白I（hCRY1）在制备抗紫外辐射的制剂中的应用。细胞实验和动物实验中，通过组织形态染色和免疫组化染色，关联基因mRNA表达水平揭示观察动物模型上的皮肤光衰老变化和基因表达差异，确定hCRY1有紫外防护功能。hCRY1作为潜在的生物紫外遮光剂，相比物理遮光剂不透光、化学性遮光剂有毒副作用且非常广谱吸收的特点，hCRY1具有安全、透光、水溶性、光谱吸收100-400nm紫外线的特点。

Description

一种人隐花色素蛋白I(hCRY1)在制备抗紫外辐射的制剂中的应用 技术领域
本发明属于生物制药工艺领域，具体涉及一种人隐花色素蛋白I(hCRY1)在制备抗紫外辐射的制剂中的应用。
背景技术
目前，市面上的防晒产品主要分为物理性遮光剂和化学性遮光剂。物理性遮光剂通常是反射或散射紫外线及可见光，常用的物质有滑石粉、氧化锌、二氧化钛、氧化镁、白陶土等。化学性遮光剂通常是吸收紫外线，具体根据其吸收紫外线波长的不同而表现不同的作用。比如吸收长波紫外线(波长范围320-400nm)的遮光剂有二苯甲酮、4-特丁基-4-甲氧基甲烷和4-异丙基二苯甲烷等；吸收中波紫外线(波长范围290-320nm)的遮光剂有对氨基苯甲酸及其衍生物、肉桂酸酯和水杨酸酯等。但是物理性遮光剂是一些不透光的物质，不能选择性的吸收紫外线，化学性遮光剂有毒副作用且非广谱吸收的特点，所以亟需研究一种安全无毒吸收紫外线效果好的遮光剂。
紫外线照射造成皮肤老化，老化主要表现为皮肤色素沉着、干燥、皮肤弹性丧失、纹理变粗皱纹加深、皮肤无光泽或呈灰黄色。皮肤老化有诸多作用机制，其中最主要的机制是紫外线辐射触发活性氧的形成，进而激活NF-kB(nuclear factor kappa-B)的信号通路。NF-kB转录因子的激活促进了促炎细胞因子和生长因子的表达，如白细胞介素-1(IL1)、肿瘤坏死因子(TNF)和表皮生长因子(EGF)的表达。丝裂原活化蛋白激酶(MAPK)通路进而受到影响上调表达。MAPK通道的主要靶点是转录因子激活蛋白1(AP-1)。通过激活AP-1上调了基质金属蛋白酶家族(MMPs)的表达，从而导致胶原降解和前胶原抑制。由于这种结构的破坏，最终会导致表皮/上皮细胞损伤特征性皮肤皱纹或色斑出现。MMPs是引起皮肤老化的主要因素，紫外线照射可使MMPs上调，从而加速皮肤胶原的降解，最终导致老化。
CN103834677B公开了一种人隐花色素蛋白I(hCRY1)的重组表达方法及其在制备放疗保护剂中的应用。通过实施例证明了添加有hCRY1的细胞在受到射线损伤时所产生H2AXFOCI明显减少，并且该减少并非由hCRY1对细胞造成了损伤所引起的，因此可以证实hCRY1对细胞有紫外损伤防护作用。说明制备的重组hCRY1可用作放疗保护剂，如制备成放疗保护剂类药物，特别是皮肤放疗保护剂。
为了研究出安全、环保、无毒的防晒遮光剂，研发人员进行了大量的研究，但是，目前的遮光剂存在有毒副作用和非广谱吸收等特点，遮光效果不佳和不透光等问题，因此，亟需研发一种制备工艺简单、安全、透光、水溶性和广谱吸收性能的制备工艺。
发明内容
针对上述背景技术指出的不足，本发明提供了一种人隐花色素蛋白I(hCRY1)在制备抗紫外辐射的制剂中的应用，进而确定hCRY1是否有紫外防护功能。分别进行细胞实验和动物实验都显示出hCRY1对紫外线辐射具有显著的保护作用，作为皮肤护理效果明显。
为了实现上述目的，本发明采用以下技术方案：
一种人隐花色素蛋白I在制备抗紫外辐射的制剂中的应用。
一种人隐花色素蛋白I(hCRY1)乳液剂，乳液剂成分包括以下原料：水、甘油、聚乙二醇-100硬脂酸酯、水杨酸盐、聚乙酸钠、二氧化硅、乙基己基甘油、硬脂酸甘油酯、尼泊金甲酯、黄原胶、香料、丁基羟基甲苯和乙二胺四乙酸钠。
进一步地，所述人隐花色素蛋白I(hCRY1)在制剂中的浓度范围为1.6-50μg/mL，进一步地，所述人隐花色素蛋白I(hCRY1)在制剂中的浓度范围为6.25-25μg/mL，进一步优选地，所述人隐花色素蛋白I(hCRY1)在制剂中的浓度为12.5μg/mL或25μg/mL。
进一步地，所述人隐花色素蛋白I(hCRY1)在制备体外抗紫外辐射的化妆品或医药配置品中的应用。
进一步地，所述人隐花色素蛋白I(hCRY1)在制备体外抗紫外辐射的水剂、膏剂、霜剂中的应用。
进一步地，所述人隐花色素蛋白I(hCRY1)用作紫外遮光剂，进一步优选地，所述人隐花色素蛋白I(hCRY1)用作广谱紫外线遮光剂。
进一步地，所述人隐花色素蛋白I(hCRY1)应用于制备防晒化妆品或其添加剂中。
进一步地，所述人隐花色素蛋白I(hCRY1)应用于制备面霜、身体乳、隔离霜、气垫、防晒喷雾中。
进一步地，所述人隐花色素蛋白I(hCRY1)应用于制备植物抗紫外辐射育种的药物。
进一步地，所述人隐花色素蛋白I(hCRY1)在制备吸收波长为100-400nm的抗紫外辐射的制剂中的应用。
与现有技术相比，本发明的有益效果为：
hCRY1在本申请中作为潜在的新型的生物紫外遮光剂具有安全、透光、水溶性、广谱吸 收100-400nm紫外线的特点。本申请主要通过组织形态染色和免疫组化染色，关联基因mRNA表达水平揭示观察动物模型上的皮肤光衰老变化和基因表达差异。
附图说明
图1不同浓度的hCRY1对30mJ/cm 2剂量的UVC 257nm辐射处理的抗辐射试验结果。
图2 hCRY1对UVB 320nm辐射处理后的检测结果。A 12.5μg/mL hCRY1防护组染色结果，B 25μg/mL hCRY1防护组染色结果，C 50μg/mL hCRY1防护组染色结果，D辐射对照组，E为无辐射对照组。
图3 UVB 320nm辐射处理后在HaCaT细胞基因调控上的检测结果，A HaCaT细胞HSP70基因表达差异结果，B HaCaT细胞MMP-1表达差异结果，C HaCaT细胞TP53表达差异结果。
图4 UVB 320nm辐射处理后大鼠皮肤组织病理形态学变化检测结果，其中A无辐射对照组染色，B无防护辐射组染色，C 12.5μg/mL hCRY1防护组染色，D商用SPF30防护辐射组染色。
图5在UVB 320nm辐射处理后，大鼠皮肤的基质金属蛋白酶MMP-1抗体免疫组化染色检测结果。其中A无辐射对照组免疫染色，B无防护辐射组免疫染色，C 12.5μg/mL hCRY1防护组免疫染色，D商用SPF30防护辐射组免疫染色。
图6在UVB 320nm辐射处理后，大鼠皮肤的HE染色，其中A无辐射对照组染色，B无防护辐射组染色，C 12.5μg/mL hCRY1乳液剂防护组染色，D商用SPF30防护辐射组染色。
图7在UVB 320nm辐射处理后，大鼠皮肤的基质金属蛋白酶MMP-1抗体免疫组化染色，其中A无辐射对照组免疫染色，B无防护辐射组免疫染色，C 12.5μg/mL hCRY1乳液剂防护组免疫染色，D商用SPF30防护辐射组免疫染色。
图8 UVB 320nm辐射的细胞密度比较，其中A hCRY1，B PBS阴性对照组，C无辐射组。
具体实施方式
为了更好地理解本发明，下面结合具体实施例对本发明作进一步的描述，其中实施例中使用的术语是为了描述特定的具体实施方案，不构成对本发明保护范围的限制。
实施例1 细胞实验
原料来源：UVA紫外灯(冠雅光电，波峰360nm)，UVB紫外灯(冠雅光电，波峰320nm)，UVC紫外灯(北京四通，波峰257nm)，紫外照度计(台湾先驰)，酶标仪(Bio-Rad)，荧光定量PCR仪(Stratagene MX 3000P)，hCRY1重组人隐花色素蛋白溶液(达博生物，大肠杆菌表达发酵纯化)，CCK-8试剂盒(上海翊圣生物)，6-8周龄SD大鼠(广州优迪生物)， Anti-MMP-1抗体(Bioss)，SPF30防晒霜(购自玫凯娜)，SYBR Green MIX(上海翊圣生物)。
制备方法：
(1)细胞培养：将HaCaT细胞用含10％FBS的DMEM培养基复苏培养，连续传代，取第3-10代细胞进行实验。
(2)UV辐照箱制备：用聚苯乙烯泡沫盒做辐照箱外壳，将紫外灯和灯架固定在泡沫盒内，用锡纸对外壳进行粘贴覆盖。
(3)UVC 257nm辐射处理：取对数生长期的细胞经消化后稀释至1×10 5cells/mL，以每孔100μL接种至96孔板中，37℃，5％CO 2，培养24小时。
辐射实验组设置：将hCRY1蛋白溶液用PBS稀释，设置0、1.6、3.125、6.25、12.5、25、50、100μg/mL共8个浓度梯度，用50μg/ml BSA、PBS溶液做辐射对照组，用不经UVA辐射的HaCaT作为无辐射对照组。
辐射处理操作如下：将HaCaT细胞取出培养箱，移取培养基，用以上配制的实验组加入细胞中，每个组做6个重复，置换好后37℃静置孵育5分钟，用辐射强度50μW/cm 2的UVC紫外灯辐照600s，辐射总剂量为30mJ/cm 2。辐射后将96孔板内液体吸出更换成100μL每孔的1％FBS的DMEM培养基。培养24小时后用每孔加入10μL CCK-8试剂孵育60min，设定波长OD 450nm测定读数。
(4)UVB 320nm辐射处理：取对数生长期的细胞经消化后稀释至1.5×10 5cells/mL，以1mL每孔接种至24孔板中，37℃，5％CO 2，培养24小时。
辐射实验组设置：将hCRY1蛋白溶液用DMEM培养基配制稀释，设置12.5、25、50μg/mL共3个浓度梯度，用10％FBS的DMEM做辐射对照组，用不经UVA辐射的HaCaT作为无辐射对照组。
辐射处理操作如下：将HaCaT细胞取出培养箱，置换培养基，每个组做5个重复，37℃，5％CO 2孵育30分钟，辐射强度250μW/cm 2的UVB 320nm辐射80秒，辐射剂量为20mJ/cm 2。辐射后将24孔板内液体吸出更换成1mL每孔的10％FBS的DMEM培养基。培养24小时后用PBS漂洗1遍，用100％甲醇固定10分钟，用1％结晶紫染色10分钟，用PBS浸泡5分钟3次。
(5)UVB 320nm辐射处理后在HaCaT细胞基因调控上的影响：取对数生长期的细胞经消化后稀释至1.5×10 5cells/mL，以1mL每孔接种至24孔板中，37℃，5％CO 2，培养24小时。
辐射实验组设置：将hCRY1蛋白溶液用DMEM培养基配制稀释，设置12.5、25μg/mL共2个浓度梯度，用10％FBS的DMEM做辐射对照组(0μg/mL hCRY1)，用不经UVA辐射的HaCaT作为无辐射对照组。
辐射处理操作如下：将HaCaT细胞取出培养箱，置换培养基，每个组做3个重复，培养24小时后弃去培养基，提取总RNA，用下表中HSP70，TP53，MMP-1基因的引物进行荧光定量PCR检测，比较hCRY1对辐射处理后HaCaT细胞修复机制所涉及基因的表达水平。
Figure PCTCN2020078516-appb-000001
(6)UVB 320nm辐射处理气提大鼠皮肤：用10％水合氯醛0.5mL注射大鼠，剃去背部毛发，饲养一天后背部轻微机械损伤恢复，脱臼处死。用0.1％次氯酸进行背部消毒。用手术刀和手术剪背部取材，分成约1.5×1.5cm的皮肤。用含1％青链霉素的PBS漂洗3分钟3次。用镊子接至transwell上室培养，下室为5％FBS的DMEM培养基，下室预先接种5x10 5个3T3细胞作为饲养层。
辐射实验组设置：PBS+UVB(无防护辐射组)、12.5μg/mL hCRY1蛋白+UVB(12.5μg/mL hCRY1防护组)、SPF30防晒霜+UVB(商用SPF30防护辐射组)；以无辐射对照组作为空白对照，每组做3个重复。
辐射处理操作如下：将气提培养大鼠皮肤用辐射强度为1500μW/cm 2的UVB 320nm辐射60分钟，辐射处理后换液，继续培养3天后用PBS震荡漂洗30分钟两次，用4％多聚甲醛固定24小时。制作石蜡标本并切片进行HE染色和基质金属蛋白酶MMP-1抗体免疫组化染色。
(7)统计学方法：数据的统计分析采用Microsoft Excel软件。数据用x±SD表示。图片制作软件采用GraphPad Prism 8软件。多组间比较采用单因素方差分析，组间两两比较采用q检验，P＜0.05则差异有统计学意义。
测试结果：
(1)hCRY1蛋白对UVC 257nm辐射处理的HaCaT细胞增殖影响
通过CCK-8测定OD 450nm结果显示，见图1：6.25μg/mL和12.5μg/mL hCRY1溶液的OD 450nm分别0.857±0.103，0.836±0.095，PBS对照组的OD 450nm为0.652±0.066。与对照组对比，hCRY1蛋白能防护UVC 257nm带来的增殖抑制(P＜0.05，n＝6)。
其中12.5μg/mL hCRY1蛋白处理组的细胞相对PBS、50μg/mL BSA的对照组具有明显抵抗 UVC的能力。
(2)hCRY1对UVB 320nm辐射处理后的凋亡影响
细胞密度为80％汇合度的Hacat细胞经不同浓度hCRY1蛋白保护性孵育30分钟后，再经20mJ/cm 2辐射剂量、250μW/cm 2辐射强度的UVB辐射处理，培养24h的细胞用结晶紫染色结果，如图2所示。
HaCaT细胞经不同浓度hCRY1蛋白孵育处理后在UVB 320nm辐射处理后与无辐射对照组的细胞维持正常增殖状态，形态饱满，呈现梭状紧密排列，辐射对照组大部分细胞凋亡飘浮于板孔中间，细胞形态皱缩，胞浆有泡状小体，细胞之间排列疏松。辐射对照组板孔中细胞稀少，而图2中的A、B、C和E染色比例接近90％。
结果显示不同浓度的hCRY1蛋白对UVB有很强的防护作用。
(3)UVB 320nm辐射处理后在HaCaT细胞基因调控上的影响
Hacat细胞密度达80％汇合度时经不同浓度hCRY1蛋白孵育30分钟再经20mJ/cm 2辐射剂量、250μW/cm 2辐射强度的UVB辐射处理80秒，培养24小时后提取总RNA，采用RT-qPCR方法检测HSP70，TP53，MMP-1的表达差异，如图3所示。
HSP70、TP53是DNA损伤后介导修复的重要基因，MMP-1是介导胶原降解的重要基因。图3提示了UVB辐射导致无防护组HSP70蛋白显著下调，hCRY1保护组有所上调。
(4)UVB 320nm辐射处理后大鼠皮肤组织病理形态学变化
在UVB 320nm辐射处理后，大鼠皮肤的HE染色，如图4所示。A无辐射对照组、C 12.5μg/mL hCRY1防护组和D商用SPF30防护辐射组显微镜下观察，真皮纤维组织致密、排列有序，分布均匀。B无防护辐射组的真皮纤维发生细胞肿胀变性，真皮组织排列紊乱，分布不均匀或稀疏。
在UVB 320nm辐射处理后，大鼠皮肤的基质金属蛋白酶MMP-1抗体免疫组化染色，如图5所示。A无辐射对照组免疫染色、C 12.5μg/mL hCRY1防护组免疫染色和D商用SPF30防护辐射组防护组免疫染色在不同组的MMP-1表达状态。C组的MMP-1表达不显著；B无防护辐射组免疫染色毛囊呈向外浸润，MMP-1表达分值+++。结果提示了hCRY1能够减少由于UVB辐射引起的MMP-1基质金属蛋白酶的过度表达。
实施例2 动物实验
原料来源：实验动物：SD大鼠(购自广州优迪生物)，30只，MMP-1抗体(购自Bioss)。
QPCR试剂(购自YEASEN)：TRIZOL、反转录酶、SYBR MIX、MMP-1、MMP-3、GAPDH实时定量PCR引物。
标本制备及免疫组化：水合氯醛、多聚甲醛、石蜡、脱蜡剂、封片剂、玻片、无水乙醇、HE染色试剂盒、免疫组化二抗试剂盒、免疫组化工具盒。
UVA紫外灯15W(冠雅光电，波峰360nm)、UVB紫外灯15W(冠雅光电，波峰320nm)、调光器、紫外照度计(台湾先驰)、大鼠固定器、电推剃刀、QPCR仪(购自STRATAGENE)。
一种人隐花色素蛋白I(hCRY1)乳液剂，乳液剂包括以下原料：水、甘油、聚乙二醇-100硬脂酸酯、水杨酸盐、聚乙酸钠、二氧化硅、乙基己基甘油、硬脂酸甘油酯、尼泊金甲酯、黄原胶、香料、丁基羟基甲苯和乙二胺四乙酸钠。
其中，人隐花色素蛋白I(hCRY1)在制剂中的浓度为12.5μg/mL。
制备方法：
(1)动物处理：将大鼠置于大鼠固定器中，用记号笔标记背部照射范围，用电推剪剔毛，剔除干净使皮肤暴露，隔天重剃。
(2)制备紫外辐射箱体：用紫外照度计测量不同高度位置的辐射强度，计算出最合适的高度。
(3)预实验：确定大鼠紫外辐射损伤的合适剂量(50mJ，100mJ，200mJ)。通过不同剂量对同一只大鼠的皮肤进行辐射并做肉眼观察比较形态差异。必要时做组织切片并做HE染色。预实验大鼠数量为2只。
(4)UVB 320nm组(无防护辐射组)、UVB 320nm hCRY1涂抹组(hCRY1乳液剂防护组)：1.5W/m 2的辐射强度照射合适剂量。每组大鼠5只。
(5)对正常对照组(无辐射对照组)、UVB 320nm组、UVB 320nm hCRY1涂抹组、市售防晒霜组(商用SPF30防护辐射组)进行拍照，肉眼观察比较。
(6)实验取材：在大鼠腹腔内注射水合氯醛(350mg/kg)，大鼠无反抗后立即剥离背部皮肤，将取出的皮肤组织迅速放入4％多聚甲醛溶液中固定，然后脱水、透明、浸没蜡然后进行石蜡包埋做组织切片，HE染色做组织形态学观察；用MMP-1抗体做免疫组化。
(7)取绿豆大小皮肤组织放入液氮研钵中研磨，用trizol做RNA提取样品。提取不同组的RNA，反转录cDNA。用qPCR仪检测MMP-1、MMP-3、MMP-9的表达差异。
测试实验：
(1)大鼠皮肤的HE染色
在UVB 320nm辐射处理后，见图6，在显微镜下观察A无辐射对照组、C12.5μg/mL hCRY1乳液剂和D商用SPF30防护辐射组，发现真皮纤维组织致密、排列有序，分布均匀，而B无防护辐射组的真皮纤维发生细胞肿胀变性，真皮组织排列紊乱，分布不均匀或稀疏。实验结果 显示hCRY1乳液剂具有抗紫外辐射的功能。
(2)大鼠皮肤的基质金属蛋白酶MMP-1表达状态
在UVB 320nm辐射处理后，见图7，A无辐射对照免疫染色组、C 12.5μg/mL hCRY1乳液剂免疫染色和D商用SPF30防护辐射免疫染色组在不同组的MMP-1表达状态与B无防护辐射免疫染色组对比，实验结果显示hCRY1乳液剂C组的MMP-1表达不显著；而B无防护辐射组毛囊呈向外浸润，MMP-1表达分值+++。实验结果显示了hCRY1乳液剂能够减少由于UVB辐射引起的MMP-1基质金属蛋白酶的过度表达。
(3)细胞密度比较
细胞密度为80％汇合度的Hacat细胞经12.5μg/mL hCRY1乳液剂保护性孵育30分钟后，再经20mJ/cm 2辐射剂量、250μW/cm 2辐射强度的UVB辐射处理，培养24h的细胞用结晶紫染色结果。
结果如图8所示，12.5μg/mL hCRY1乳液剂保护的细胞密度与无辐射的细胞密度几乎没有差异，而B无防护辐射组细胞密度非常低，与A组和C组呈现出极其显著的差异，显示出hCRY1乳液剂对UVB有很强的防护作用。

Claims (10)

  1. 一种人隐花色素蛋白I在制备抗紫外辐射的制剂中的应用。
  2. 根据权利要求1所述的应用，其特征在于：人隐花色素蛋白I在制备体外抗紫外辐射的化妆品或医药配置品中的应用。
  3. 根据权利要求2所述的应用，其特征在于：人隐花色素蛋白I在制备体外抗紫外辐射的水剂、膏剂、霜剂中的应用。
  4. 根据权利要求1所述的应用，其特征在于：所述人隐花色素蛋白I在制剂中的浓度范围为1.6-50μg/mL。
  5. 根据权利要求4所述的应用，其特征在于：所述人隐花色素蛋白I在制剂中的浓度范围为6.25-25μg/mL。
  6. 根据权利要求1所述的应用，其特征在于：所述人隐花色素蛋白I用作紫外遮光剂。
  7. 根据权利要求6所述的应用，其特征在于：所述人隐花色素蛋白I用作广谱紫外线遮光剂。
  8. 根据权利要求1所述的应用，其特征在于：所述人隐花色素蛋白I应用于制备防晒化妆品或其添加剂中。
  9. 根据权利要求1所述的应用，其特征在于：所述人隐花色素蛋白I应用于制备植物抗紫外辐射育种的药物。
  10. 根据权利要求1所述的应用，其特征在于：所述人隐花色素蛋白I在制备吸收波长为100-400nm的抗紫外辐射的制剂中的应用。
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