CN109833479A - It is a kind of to treat or prevent the drug and its application that Keratin 1 is degraded - Google Patents
It is a kind of to treat or prevent the drug and its application that Keratin 1 is degraded Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及治疗或预防角蛋白1降解的药物，具体是蛋白酶抑制剂作为治疗或预防角蛋白1降解的药物及其应用。The present invention relates to a drug for treating or preventing the degradation of keratin 1, in particular to a protease inhibitor as a drug for treating or preventing the degradation of keratin 1 and its application.
背景技术Background technique
阳光中的紫外线是人的皮肤受到紫外线辐射损伤的主要原因，紫外线辐射对于皮肤会产生多种病理作用，例如皮肤红肿，脱皮，炎症，溃烂，以及多种皮肤疾病等，并会产生大量活性氧化物，造成皮下细胞的DNA损伤，加速皮肤衰老。事实上，人体皮肤衰老90％的原因是由于紫外线照射，而紫外线诱导的最严重的疾病是皮肤癌，已有研究证明，90％以上的皮肤癌是由阳光中的紫外线照射引起的。皮肤癌患者占所有癌症患者中的40％，全世界每年有300多万新发皮肤癌病例，而皮肤癌基金会也指出，五分之一的美国人在一生中的某个阶段会得皮肤癌。随着人类工业化的进展，大气层中负责阻挡紫外线的臭氧层在不断变薄，有专家曾预测，臭氧层厚度减少1％，紫外线辐射强度增加2％。因此，对于皮肤紫外线损伤的检测对于人们提高皮肤的健康水平、防止皮肤癌等重大皮肤疾病的产生，都有着重大的社会经济意义和临床价值。Ultraviolet rays in sunlight are the main reason for human skin to be damaged by ultraviolet radiation. Ultraviolet radiation will have various pathological effects on the skin, such as skin redness, peeling, inflammation, ulceration, and various skin diseases, etc., and will produce a lot of active oxidation. It causes DNA damage in subcutaneous cells and accelerates skin aging. In fact, 90% of human skin aging is due to UV exposure, and the most serious UV-induced disease is skin cancer. It has been proven that more than 90% of skin cancers are caused by UV exposure in sunlight. Skin cancer accounts for 40% of all cancer patients, with more than 3 million new skin cancer cases worldwide each year, and the Skin Cancer Foundation states that one in five Americans will develop skin at some point in their lives cancer. With the progress of human industrialization, the ozone layer in the atmosphere responsible for blocking ultraviolet rays is continuously thinning. Some experts have predicted that the thickness of the ozone layer will decrease by 1%, and the intensity of ultraviolet radiation will increase by 2%. Therefore, the detection of skin UV damage has great social and economic significance and clinical value for people to improve skin health and prevent the occurrence of major skin diseases such as skin cancer.
而在我国，由于人口压力巨大，诊断和就医困难，紫外皮肤损伤尚未得到足够的重视，往往很多皮肤受创患者只有受到特别强的皮损后才会门诊就医，而使得近年来，我国乃至于全世界紫外创伤导致的皮肤病和皮肤癌患者数量逐渐增多。因此，有效的皮肤紫外损伤的检测方法和治疗手段，以及低成本的预防性药物或制剂，对于皮肤健康的监测、皮肤疾病特别是皮肤癌的诊断和治疗，意义重大。In my country, due to the huge population pressure and the difficulty of diagnosis and medical treatment, UV skin damage has not been paid enough attention. Often many patients with skin trauma will only seek medical treatment in outpatient clinics after suffering from particularly strong skin lesions. The number of skin diseases and skin cancers caused by UV trauma is increasing worldwide. Therefore, effective detection methods and treatment methods for skin UV damage, as well as low-cost preventive drugs or preparations, are of great significance for monitoring skin health, and diagnosing and treating skin diseases, especially skin cancer.
发明内容SUMMARY OF THE INVENTION
本发明提供了靶向角蛋白1的物质在制备治疗或预防紫外光诱导的角蛋白1降解的药物、保健品或试剂中的用途，其中，所述药物、保健品或试剂能够抑制紫外光诱导的角蛋白1降解，或维持细胞或组织中角蛋白1的含量。The present invention provides the use of a substance targeting keratin 1 in the preparation of a medicine, health product or reagent for treating or preventing UV-induced degradation of keratin 1, wherein the medicine, health product or reagent can inhibit UV-induced degradation of keratin 1 Degradation of keratin 1, or maintenance of keratin 1 content in cells or tissues.
所述角蛋白1还包括缺失部分氨基酸序列的角蛋白1片段。The keratin 1 also includes a keratin 1 fragment with a partial amino acid sequence deleted.
在一个实施例中，其中所述靶向角蛋白1的物质选自除了半胱氨酸蛋白酶抑制剂以外的蛋白酶抑制剂、角蛋白1的抗体、抑制角蛋白1降解的小核苷酸、激活角蛋白1的小核苷酸中的至少一种。In one embodiment, wherein the substance targeting keratin 1 is selected from the group consisting of protease inhibitors other than cysteine protease inhibitors, antibodies to keratin 1, small nucleotides that inhibit keratin 1 degradation, activation At least one of the small nucleotides of keratin 1.
在一个实施例中，所述药物、保健品或试剂是一种组合物，其包括药学上可接受的药用辅料。In one embodiment, the medicine, health product or reagent is a composition comprising pharmaceutically acceptable pharmaceutical excipients.
在一个具体实施例中，所述药用辅料包括药学上可接受的载体、稀释剂或赋形剂In a specific embodiment, the pharmaceutical excipients include pharmaceutically acceptable carriers, diluents or excipients
在一个具体实施例中，所述组合物的剂型可以是固体制剂、液体制剂或半固体制剂，优选片剂、胶囊剂、颗粒剂、滴丸剂、混悬剂、糖浆剂、各种肠溶制剂、注射剂、膜制剂、乳膏剂或喷雾剂，包括缓释片剂、缓释胶囊、缓释注射剂。In a specific embodiment, the dosage form of the composition can be a solid preparation, a liquid preparation or a semi-solid preparation, preferably tablets, capsules, granules, dropping pills, suspensions, syrups, various enteric preparations , injections, film formulations, creams or sprays, including sustained-release tablets, sustained-release capsules, and sustained-release injections.
本发明还提供了蛋白酶抑制剂在制备用于诊断和检测紫外线诱导的角蛋白1降解的试剂或试剂盒中的用途。The present invention also provides the use of a protease inhibitor in the preparation of a reagent or kit for diagnosing and detecting UV-induced keratin 1 degradation.
在本发明中，所述紫外光选自UVA、UVB和UVC的一种以上，优选UVB。In the present invention, the ultraviolet light is selected from one or more of UVA, UVB and UVC, preferably UVB.
在本发明中，所述紫外光为包括UVB的紫外光，可以是UVB与UVA的组合、UVB和UVC的组合、UVB与UVA以及UVC的组合。In the present invention, the ultraviolet light is ultraviolet light including UVB, which may be a combination of UVB and UVA, a combination of UVB and UVC, or a combination of UVB and UVA and UVC.
在本发明中，所述角蛋白1降解的过程为角蛋白1经过紫外光照射后降解成缺失氨基酸的角蛋白1片段。In the present invention, the process of degrading keratin 1 is that keratin 1 is degraded into keratin 1 fragments lacking amino acids after being irradiated with ultraviolet light.
其中，在本发明中，所述角蛋白1降解物包括基因水平或蛋白质水平角蛋白1中的一种或多种。其中，角蛋白1降解物优选是缺失部分氨基酸序列的角蛋白1片段，或上述片段蛋白和全长角蛋白1的混合，检测上述蛋白不限于蛋白质水平，还可以是基因水平，包括磷酸化、乙酰化等修饰水平。Wherein, in the present invention, the keratin 1 degradants include one or more of keratin 1 at the gene level or protein level. Wherein, the keratin 1 degraded product is preferably a keratin 1 fragment that lacks a partial amino acid sequence, or a mixture of the above-mentioned fragment protein and full-length keratin 1. The detection of the above-mentioned protein is not limited to the protein level, but also the gene level, including phosphorylation, Modification levels such as acetylation.
在一个实施例中，所述角蛋白1降解物还包括蛋白结构变化，其中，所述蛋白结构包括角蛋白1降解物与其他蛋白例如角蛋白10所形成的二聚体结构，其中角蛋白1降解物优选是其缺失部分氨基酸序列的角蛋白1片段，例如缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个氨基酸或20个以上的角蛋白1片段。In one embodiment, the keratin 1 degradant further includes changes in protein structure, wherein the protein structure includes a dimer structure formed by the keratin 1 degradant and other proteins such as keratin 10, wherein keratin 1 The degradant is preferably a fragment of keratin 1 whose partial amino acid sequence is deleted, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20 amino acids or more than 20 keratin 1 fragments.
在本发明中，缺失部分氨基酸序列的角蛋白1片段也称作角蛋白1降解物，其选自缺失1至644之间任意数个氨基酸的角蛋白1片段，该缺失可以是角蛋白1的N端的缺失，也可以是角蛋白1的C端的缺失，还可以是角蛋白1全序列中任意氨基酸的组合的缺失。例如缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、49/、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644个氨基酸的角蛋白1片段中的一个或多个。In the present invention, a keratin 1 fragment with a partial amino acid sequence deletion is also referred to as a keratin 1 degrader, which is selected from a keratin 1 fragment with any number of amino acids between 1 and 644 deleted, and the deletion may be of keratin 1 The deletion of the N-terminus may also be the deletion of the C-terminus of keratin 1, or the deletion of any combination of amino acids in the entire sequence of keratin 1. For example, missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 49/, 498, 499, 500, 501 , 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526 , 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, One or more of the 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644 amino acid keratin 1 fragments.
在一个实施例中，所述蛋白酶抑制剂在制备治疗或预防紫外光诱导的角蛋白1降解的药物、保健品或试剂中的用途或在制备用于诊断和检测紫外线诱导的角蛋白1降解的试剂或试剂盒中的用途即可以是体外，也可以是体内的。In one embodiment, the use of the protease inhibitor in the preparation of a drug, health product or reagent for treating or preventing UV-induced degradation of keratin 1 or in the preparation of a drug for diagnosis and detection of UV-induced degradation of keratin 1 The use in the reagent or kit can be either in vitro or in vivo.
在本发明中，所述蛋白酶抑制剂选自丝氨酸蛋白酶抑制剂、苏氨酸蛋白酶抑制剂、天冬氨酸蛋白酶抑制剂、谷氨酸蛋白酶抑制剂所组成的组中的至少一种。In the present invention, the protease inhibitor is at least one selected from the group consisting of serine protease inhibitor, threonine protease inhibitor, aspartic protease inhibitor, and glutamate protease inhibitor.
在一个实施例中，所述检测角蛋白1或其角蛋白1降解物含量、结构的方法包括免疫印迹、免疫组化、免疫荧光、荧光光谱、拉曼光谱、光学检测、或物理性质检测的一种或多种。In one embodiment, the method for detecting the content and structure of keratin 1 or its keratin 1 degradation product comprises one of immunoblotting, immunohistochemistry, immunofluorescence, fluorescence spectroscopy, Raman spectroscopy, optical detection, or physical property detection. one or more.
在一个实施例中，增加角蛋白1的含量，可以减少紫外光诱导的皮肤损伤。In one embodiment, increasing the content of keratin 1 can reduce UV-induced skin damage.
本发明的另一方面，还提供了一种模型，该模型用于紫外线诱导皮肤损伤的抑制或激动药物的筛选，其特征在于，使用本发明所述的酶抑制剂构建该筛选模型。In another aspect of the present invention, a model is also provided, which is used for the screening of UV-induced skin damage inhibitory or agonistic drugs, characterized in that the screening model is constructed by using the enzyme inhibitor of the present invention.
本发明进一步提供了所述模型在制备用于治疗紫外线诱导皮肤损伤的药物中的应用。The present invention further provides the application of the model in the preparation of a medicament for treating UV-induced skin damage.
附图说明Description of drawings
图1示出UVC诱导皮肤角蛋白1含量下降且下降的程度与UVC的剂量呈正相关的免疫印迹法代表图。Figure 1 shows a representative graph of immunoblotting for UVC-induced reduction in skin keratin 1 content and the extent of the reduction is positively correlated with the dose of UVC.
图2示出UVC诱导皮肤角蛋白1含量下降且下降的程度与UVC的剂量呈正相关的免疫印迹法量化图。Figure 2 shows a graph of immunoblotting quantification of UVC-induced reduction in skin keratin 1 content and the extent of the reduction was positively correlated with the dose of UVC.
图3示出UVB诱导皮肤角蛋白1含量下降且下降的程度与UVB处理的时间呈正相关的免疫印迹法代表图。Figure 3 shows a representative graph of immunoblotting for UVB-induced reduction in skin keratin 1 content and the extent of the reduction is positively correlated with duration of UVB treatment.
图4示出UVB诱导皮肤角蛋白1含量下降且下降的程度与UVB处理的时间呈正相关的免疫印迹法量化图。Figure 4 shows a graph of immunoblotting quantification of UVB-induced reduction in skin keratin 1 content and the extent of the reduction is positively correlated with duration of UVB treatment.
具体实施方式Detailed ways
下面将通过具体描述，对本发明作进一步的说明。The present invention will be further illustrated by the specific description below.
除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
如本文所用，“角蛋白1”、“keratin 1”、“KRT1”可互换，其是包括能够使用角蛋白1抗体识别检测的角蛋白，其可以是单一物质，也可以是混合物；在本发明中，角蛋白1包括其缺失部分氨基酸序列的角蛋白1片段，所述缺失部分氨基酸序列的角蛋白1片段为角蛋白1的N端或C端缺失1至644个氨基酸中任一种的角蛋白1片段。As used herein, "keratin 1", "keratin 1", "KRT1" are interchangeable and include keratin proteins that can be recognized and detected using keratin 1 antibodies, which may be a single substance or a mixture; In the present invention, keratin 1 includes a keratin 1 fragment whose partial amino acid sequence is deleted, and the keratin 1 fragment whose partial amino acid sequence is deleted is any one of 1 to 644 amino acids deleted from the N-terminus or C-terminus of keratin 1. Keratin 1 fragment.
如本文所用，“缺失部分氨基酸序列的角蛋白1片段”、“角蛋白1降解物”可互换，其是指角蛋白1的N端或C端缺失1至644个氨基酸中任一种的角蛋白1片段。As used herein, "keratin 1 fragment with partial amino acid sequence deletion" and "keratin 1 degrader" are interchangeable, and refer to a keratin 1 with any one of 1 to 644 amino acids deleted from the N-terminus or C-terminus of keratin 1. Keratin 1 fragment.
如本文所用，“蛋白酶抑制剂、“酶抑制剂”可互换，其是指除胱氨酸之外的蛋白酶抑制剂。As used herein, "protease inhibitor, "enzyme inhibitor" are interchangeable and refer to protease inhibitors other than cystine.
实施例1紫外照射下角蛋白1的含量变化Example 1 Content change of keratin 1 under ultraviolet irradiation
根据本发明，使用雄性C57小鼠，UVC、照射的小鼠重量在15-25g范围之间，UVB/UVA照射的小鼠重量在18-25g范围之间。紫外线辐射完成后，小鼠在动物房中进行饲养，条件为22-24℃，12小时的明/暗循环，并可自由进食取水。According to the present invention, using male C57 mice, the weight of UVC, irradiated mice is in the range of 15-25 g, and the weight of UVB/UVA irradiated mice is in the range of 18-25 g. After completion of UV irradiation, mice were housed in an animal room at 22-24°C with a 12-hour light/dark cycle and had free access to food and water.
24小时内，使用激光共聚焦显微镜对紫外线照射的皮肤进行无创成像。其后一天或五天后牺牲小鼠，取损伤皮肤组织，进行H&E(苏木精&伊红染色)检测。小鼠的皮肤组织用激光共聚焦显微镜成像，其中激光共聚焦显微镜的激发波长为440-600nm。利用双光子显微镜760nm-800nm激发，测定荧光物质光谱。Non-invasive imaging of UV-irradiated skin using confocal laser microscopy within 24 hours. The mice were sacrificed one day or five days later, and the damaged skin tissue was collected for H&E (hematoxylin & eosin staining) detection. The skin tissue of the mice was imaged with a confocal laser with an excitation wavelength of 440-600 nm. Using a two-photon microscope to excite at 760nm-800nm, measure the spectrum of the fluorescent substance.
皮肤组织的储存：皮肤取出后，取出适量组织浸泡于4％多聚甲醛，用于制作石蜡切片，剩余组织用铝箔纸包裹好，使用液氮冷冻，之后转移到-80℃冰箱长期保存。Storage of skin tissue: After removing the skin, take out an appropriate amount of tissue and soak it in 4% paraformaldehyde to make paraffin sections. Wrap the remaining tissue in aluminum foil, freeze it in liquid nitrogen, and then transfer it to a -80°C refrigerator for long-term storage.
皮肤组织石蜡切片：将皮肤组织浸泡4％多聚甲醛溶液24h，然后依照石蜡切片的制作方法依次浸自来水、蒸馏水、梯度酒精、二甲苯、石蜡，制作成石蜡切片。Paraffin section of skin tissue: soak the skin tissue in 4% paraformaldehyde solution for 24 hours, then immerse it in tap water, distilled water, graded alcohol, xylene, and paraffin in sequence according to the method for making paraffin sections to make paraffin sections.
皮肤石蜡切片的H&E染色：将石蜡切片浸二甲苯脱蜡，然后依次浸于梯度酒精、蒸馏水，苏木素染色10分钟，自来水流水冲洗30min，蒸馏水浸泡30s，95％乙醇10s，伊红复染30s，70％酒精洗涤2次，依次浸梯度酒精、二甲苯，中性树脂封片。H&E staining of paraffin sections of skin: immerse the paraffin sections in xylene for dewaxing, then immerse in gradient alcohol, distilled water, hematoxylin staining for 10 minutes, rinse with running water for 30 minutes, soak in distilled water for 30 seconds, 95% ethanol for 10 seconds, and counterstain with eosin for 30 seconds. Washed twice with 70% alcohol, then immersed in gradient alcohol, xylene, and sealed with neutral resin.
对染色结果拍照、量化：对上述两种染色进行拍照。并针对表皮角质化厚度，表皮厚度等指标进行量化。Photograph and quantify the staining results: Take photographs of the above two types of staining. And quantified for epidermal keratinization thickness, epidermal thickness and other indicators.
免疫蛋白印迹实验(western blot)：Western blot:
1.蛋白样品制备1. Protein Sample Preparation
用水合氯醛过量麻醉动物后，取耳朵组织，放入称重的EP管中，加入裂解液，置于冰上，研磨，加入裂解液，离心取上清分装后存于-80度冰箱。After anesthetizing the animal with chloral hydrate overdose, take the ear tissue, put it into a weighed EP tube, add the lysate, put it on ice, grind it, add the lysate, centrifuge to get the supernatant and store it in a -80°C freezer .
2.蛋白浓度测定2. Protein Concentration Determination
按照BCA试剂盒的说明书，配制标准浓度蛋白。Prepare standard concentration protein according to the instructions of BCA kit.
3.SDS聚丙烯酰胺凝胶电泳3. SDS polyacrylamide gel electrophoresis
将配制好的凝胶固定在电泳装置中，分别将蛋白样品加入。电泳、转膜，牛奶室温封闭、TBST洗涤后加入一抗过夜。TBST洗后加入HRP标记的二抗。最后用显色液对硝酸纤维膜进行显色，在成像系统中曝光拍照。The prepared gel was fixed in the electrophoresis device, and the protein samples were added separately. Electrophoresis, membrane transfer, blocking with milk at room temperature, washing with TBST, and adding primary antibody overnight. After washing with TBST, HRP-conjugated secondary antibody was added. Finally, the nitrocellulose membrane was developed with a chromogenic solution, and then exposed and photographed in an imaging system.
免疫组织荧光染色：Immunohistofluorescence staining:
1.样品准备1. Sample Preparation
动物样片使用二甲苯脱蜡和梯度酒精复水。抗原修复后的细胞样品使用4％PFA固定、PBS洗涤。Animal swatches were dewaxed with xylene and rehydrated with graded alcohol. Cell samples after antigen retrieval were fixed with 4% PFA and washed with PBS.
2.染色：10％BSA常温封闭。用一抗孵育，4℃过夜。阴性对照用PBS液。经过PBS洗涤后加入二抗并避光孵育。PBS洗涤后，DAPI染色并封片。2. Staining: 10% BSA sealed at room temperature. Incubate with primary antibody overnight at 4°C. Negative control with PBS solution. After washing with PBS, secondary antibodies were added and incubated in the dark. After washing with PBS, DAPI stained and mounted.
本发明的利用激发光激发表皮角蛋白以及角蛋白相关蛋白的结构及含量的方式包括使用普通的连续光输出进行激发的方式、使用电调制进行调制激发的方式或者使用脉冲激光进行激发的方式中的至少一种。The methods of using excitation light to excite the structures and contents of epidermal keratin and keratin-related proteins include the excitation method using ordinary continuous light output, the modulation excitation method using electrical modulation, or the excitation method using pulsed laser light. at least one of.
在本发明的检测方法中，优选的是，在被实验对象的皮肤受到紫外光照射后的72小时之内进行检测；更优选的是，在48小时之内进行检测，最优选的，在24小时之内进行检测。In the detection method of the present invention, preferably, the detection is performed within 72 hours after the subject's skin is irradiated with ultraviolet light; more preferably, the detection is performed within 48 hours, and most preferably, within 24 hours Check within hours.
本发明的活体无创检测紫外线诱导皮肤损伤的方法，根据表皮角蛋白以及角蛋白相关蛋白的结构及含量与皮肤损伤程度呈正比的规律，预测被实验对象的皮肤损伤程度。The method for non-invasive detection of UV-induced skin damage in vivo of the present invention predicts the skin damage degree of the subject according to the law that the structure and content of epidermal keratin and keratin-related proteins are proportional to the degree of skin damage.
选取了角蛋白的两个亚型，角蛋白1以及角蛋白10进行免疫印迹实验，对经过紫外光照射的皮肤中的蛋白进行检测。发现，皮肤接受紫外光照射后，1小时内角蛋白1的含量会降低，但是角蛋白10的含量没有显著性差异。所以进一步提示紫外光(例如UVC)诱导的皮肤自荧光上升和角蛋白1有关(图1和图2)。另外，发现UBV照射后，角蛋白1的含量也会降低(图3和图4)。紫外造成的角蛋白1的降解并不是影响了蛋白的转录翻译水平，因为在UVC照射1小时或UVB照射2小时时即检测到角蛋白1含量的显著性下降。紫外造成的角蛋白1的降解的机理为紫外光会激活本发明中发现的具有抑制角蛋白1降解的蛋白酶的表达，继而导致角蛋白1的降解，而造成皮肤损伤。Two subtypes of keratin, keratin 1 and keratin 10, were selected for immunoblotting experiments to detect the proteins in skin irradiated with UV light. It was found that the content of keratin 1 decreased within 1 hour after the skin was exposed to UV light, but the content of keratin 10 was not significantly different. Therefore, it is further suggested that the increase of skin autofluorescence induced by ultraviolet light (such as UVC) is related to keratin 1 (Figure 1 and Figure 2). In addition, it was found that the content of keratin 1 was also decreased after UBV irradiation (Figures 3 and 4). The degradation of keratin 1 by UV did not affect the level of transcription and translation of the protein, as a significant decrease in keratin 1 content was detected after 1 h of UVC irradiation or 2 h of UVB irradiation. The mechanism of UV-induced degradation of keratin 1 is that UV light activates the expression of proteases that inhibit the degradation of keratin 1 found in the present invention, thereby causing the degradation of keratin 1 and causing skin damage.
实施例2蛋白酶抑制剂的筛选Example 2 Screening of protease inhibitors
在所有的蛋白酶抑制剂中，由于角蛋白1与其相互作用蛋白之间结构复杂，发明人在经过大量的筛选后，最终发现四种蛋白酶抑制剂具有防止紫外皮肤损伤的能力，其明显好于其他的蛋白酶抑制剂。其中丝氨酸蛋白酶抑制剂：4-(2-氨乙基)苯磺酰氟盐酸盐(AEBSF)，丁基硼酸这两个药物可以抑制角蛋白1的降解，并显著防止紫外光诱导的皮肤损伤。Among all protease inhibitors, due to the complex structure between keratin 1 and its interacting proteins, after extensive screening, the inventors finally found that four protease inhibitors have the ability to prevent UV skin damage, which is significantly better than others of protease inhibitors. Among them, serine protease inhibitors: 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), butylboronic acid, these two drugs can inhibit the degradation of keratin 1 and significantly prevent UV-induced skin damage .
苏氨酸蛋白酶抑制剂：发现可以通过抑制苏氨酸蛋白酶的活力，从而抑制角蛋白1的降解，防止紫外光诱导的皮肤损伤。Threonine protease inhibitor: It is found that it can inhibit the degradation of keratin 1 by inhibiting the activity of threonine protease and prevent UV-induced skin damage.
天冬氨酸蛋白酶抑制剂：(1S，2R)-(-)-顺式-1-氨基-2-茚满醇发现，这个药物可以通过抑制天冬氨酸蛋白酶的活力，从而抑制角蛋白1的降解，防止紫外光诱导的皮肤损伤。Aspartic protease inhibitor: (1S,2R)-(-)-cis-1-amino-2-indanol It was found that this drug can inhibit keratin 1 by inhibiting the activity of aspartic protease degradation, preventing UV-induced skin damage.
谷氨酸蛋白酶发现可以通过抑制谷氨酸蛋白酶的活力，从而抑制角蛋白1的降解，防止紫外光诱导的皮肤损伤。Glutamate protease was found to prevent UV-induced skin damage by inhibiting the activity of glutamate protease, thereby inhibiting the degradation of keratin 1.
本领域的技术人员应当明了，尽管为了举例说明的目的，本文描述了本发明的具体实施方式，但可以对其进行各种修改而不偏离本发明的精神和范围。因此，本发明的具体实施方式和实施例不应当视为限制本发明的范围。本发明仅受所附权利要求的限制。本申请中引用的所有文献均完整地并入本文作为参考。It will be apparent to those skilled in the art that although specific embodiments of the invention are described herein for illustrative purposes, various modifications can be made therein without departing from the spirit and scope of the invention. Therefore, the specific descriptions and examples of the present invention should not be construed as limiting the scope of the present invention. The invention is limited only by the appended claims. All documents cited in this application are incorporated by reference in their entirety.
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| US4906457A (en) * | 1988-09-06 | 1990-03-06 | Washington State University Research Foundation, Inc. | Compositions and methods for reducing the risk of sunlight and ultraviolet induced skin cancer |
| US20070104746A1 (en) * | 2005-07-29 | 2007-05-10 | Seishiro Fujii | Methods and compositions for reducing skin damage |
| US20070243520A1 (en) * | 2006-04-18 | 2007-10-18 | Kao Corporation | Method for screening an agent for preventing or ameliorating wrinkles |
| CN104007056A (en) * | 2013-02-22 | 2014-08-27 | 株式会社芳珂 | Ultraviolet ray damage resistance evaluating method |
| CN108355134A (en) * | 2017-01-26 | 2018-08-03 | 上海交通大学 | Cystine protease inhibitors are preparing the application in treating or preventing UV light-induced skin injury drug |
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|---|---|---|---|---|
| US4906457A (en) * | 1988-09-06 | 1990-03-06 | Washington State University Research Foundation, Inc. | Compositions and methods for reducing the risk of sunlight and ultraviolet induced skin cancer |
| US20070104746A1 (en) * | 2005-07-29 | 2007-05-10 | Seishiro Fujii | Methods and compositions for reducing skin damage |
| US20070243520A1 (en) * | 2006-04-18 | 2007-10-18 | Kao Corporation | Method for screening an agent for preventing or ameliorating wrinkles |
| CN104007056A (en) * | 2013-02-22 | 2014-08-27 | 株式会社芳珂 | Ultraviolet ray damage resistance evaluating method |
| CN108355134A (en) * | 2017-01-26 | 2018-08-03 | 上海交通大学 | Cystine protease inhibitors are preparing the application in treating or preventing UV light-induced skin injury drug |
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