CN113209271A - 一种可以促进成纤维细胞增殖的组合物及其制备方法 - Google Patents
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Abstract
本发明公开了一种可以促进成纤维细胞增殖的组合物及其制备方法，该组合物主要由序列为Gly‑Asn‑Pro‑Asn‑Asn‑Cly‑Ile、Gly‑Pro‑Hyp‑Gly‑Ser‑Pro、Gly‑Leu‑Hyp‑Gly‑Hyp‑Ala、Gly‑His‑Lys‑Val‑Lys的肽与锁链素、异锁链素组成。制备方法包括将鳕鱼皮与牛心脉管按照3:1的比例混合，加入10‑30倍体积的氢氧化钠(1％)溶液脱脂，然后粉碎，加入5‑20倍体积的去离子水，100‑300U/L木瓜蛋白酶，100‑300U/L胰蛋白酶、100‑300U/L碱性蛋白酶，保持40℃‑50℃搅拌2‑4小时，10000r/min离心后取上清液，先利用截留分子量1000Da的超滤膜过滤，取透过液利用截留分子量为200Da的滤膜过滤，取截留液加入D101大孔树脂吸附，50％乙醇洗脱，后经浓缩、干燥，得到上述组合物。该组合物对成纤维细胞的增殖具有明显的促进作用，可用于食品、化妆品与药品领域。
Description
技术领域
本发明涉及生物技术领域，具体是一种可以促进成纤维细胞增殖的组合物及其制备方法。
背景技术
成纤维细胞是疏松结缔组织的主要细胞成分，由胚胎时期的间充质细胞(mesenchymal cell)分化而来。成纤维细胞是构成皮肤真皮层的主要细胞，是真皮层产生胶原及其他细胞间质的主要细胞，其正常的分化、增殖维系着皮肤正常的结构和生理功能。在间质的更新和创伤修复过程中，具有十分重要的作用。所以在衰老发生的时候，成纤维细胞的数量和质量也会受到严重的影响，导致它在皮肤中的含量较少，造成皮肤的衰老，所以能够使成纤维细胞在母体的环境中自然生存并增殖，从而使皮肤真皮层的厚度和密度增加的物质都具有潜在的舒展皱纹，填平凹陷，消除疤痕，恢复皮肤弹性和光泽的作用。
发明内容
本发明要解决的技术问题就是克服以上的技术缺陷，提供一种可以促进成纤维细胞增殖的组合物及其制备方法。
为了解决上述问题，本发明的技术方案为：一种可以促进成纤维细胞增殖的组合物，主要由序列为Gly-Asn-Pro-Asn-Asn-Cly-Ile、Gly-Pro-Hyp-Gly-Ser-Pro、Gly-Leu-Hyp-Gly-Hyp-Ala、Gly-His-Lys-Val-Lys的肽与锁链素、异锁链素组成。
作为改进，包括以下步骤：将鳕鱼皮与牛心脉管按照3:1的比例混合，加入10-30倍体积的氢氧化钠溶液脱脂，然后粉碎，加入5-20倍体积的去离子水，100-300U/L木瓜蛋白酶，100-300U/L胰蛋白酶、100-300U/L碱性蛋白酶，保持40℃-50℃搅拌2-4小时，10000r/min离心后取上清液，先利用截留分子量1000Da的超滤膜过滤，取透过液利用截留分子量为200Da的滤膜过滤，取截留液加入D101大孔树脂吸附，50％乙醇洗脱，后经浓缩、干燥得到所述组合物。
作为改进，所述Gly-Asn-Pro-Asn-Asn-Cly-Ile的含量为1％-4％，Gly-Pro-Hyp-Gly-Ser-Pro的含量为1-5％，Gly-Leu-Hyp-Gly-Hyp-Ala的含量为1％-6％，Gly-His-Lys-Val-Lys的含量为2-6％，锁链素的含量为0.1-2％，异锁链素的含量为0.1-2％。
作为改进，所述组合物具有促进成纤维细胞增殖的作用。
作为改进，所述组合物可用于食品、化妆品和药品领域中。
作为改进，所述氢氧化钠溶液的浓度为1％。
本发明与现有的技术相比的优点在于：本发明公开的组合物对成纤维细胞的增殖明显具有促进作用，可用于食品、化妆品与药品领域。
具体实施方式
以下通过具体实施例进一步描述本发明，但本发明不仅仅限于以下实施例。在本发明的范围内或者在不脱离本发明的内容、精神和范围内，对本发明进行的变更、组合或替换，对于本领域的技术人员来说是显而易见的，且包含在本发明的范围之内。
实施例一
取新鲜鳕鱼皮150克，新鲜牛心脉管50g，用清水清洗去除尘土等杂质；然后加入2000ml氢氧化钠(w/w，1％)溶液，轻微搅拌4小时，过滤后将固体粉碎成浆，加入1000ml去离子水，100U木瓜蛋白酶，100U胰蛋白酶、100U碱性蛋白酶，40℃搅拌2小时，然后离心(10000r/min，10min)分离，取上清液，先利用截留分子量1000Da的超滤膜过滤，取透过液利用截留分子量为200的纳滤膜过滤，取截留液中加入100g D101大孔树脂，轻微搅拌2小时后过滤，将大孔树脂装入柱子中，用500ml含量为50％(v/v)的乙醇-水溶液洗脱，洗脱液减压浓缩至50ml，冷冻干燥后得到1.0g白色固体，经检测白色固体主要由列为Gly-Asn-Pro-Asn-Asn-Cly-Ile、Gly-Pro-Hyp-Gly-Ser-Pro、Gly-Leu-Hyp-Gly-Hyp-Ala与Gly-His-Lys-Val-Lys的肽与锁链素、异锁链素组成，含量分别为3.1％，3.0％，4.2％，4.3％，1.2％，0.8％。
实施例二
取新鲜鳕鱼皮150克，新鲜牛心脉管50g，用清水清洗去除尘土等杂质；然后加入3000ml氢氧化钠(w/w，1％)溶液，轻微搅拌4小时，过滤后将固体粉碎成浆，加入2000ml去离子水，300U木瓜蛋白酶，300U胰蛋白酶、300U碱性蛋白酶，40℃搅拌2小时，然后离心(10000r/min，10min)分离，取上清液，利用截留分子量1000Da的超滤膜过滤，取透过液利用截留分子量为200的纳滤膜过滤，取截留液中加入100g D101大孔树脂，轻微搅拌2小时后过滤，将大孔树脂装入柱子中，用500ml含量为50％(v/v)的乙醇-水溶液洗脱，洗脱液减压浓缩至50ml，冷冻干燥后得到1.2g白色固体，经检测白色固体主要由列为Gly-Asn-Pro-Asn-Asn-Cly-Ile、Gly-Pro-Hyp-Gly-Ser-Pro、Gly-Leu-Hyp-Gly-Hyp-Ala与Gly-His-Lys-Val-Lys的肽与锁链素、异锁链素组成，含量分别为2.8％，3.2％，4.5％，4.1％，1.1％，1.5％。
实施例三
取新鲜鳕鱼皮150克，新鲜牛心脉管50g，用清水清洗去除尘土等杂质；然后加入1500ml氢氧化钠(w/w，1％)溶液，轻微搅拌4小时，过滤后将固体粉碎成浆，加入1500ml去离子水，200U木瓜蛋白酶，200U胰蛋白酶、200U碱性蛋白酶，40℃搅拌2小时，然后离心(10000r/min，10min)分离，取上清液，利用截留分子量1000Da的超滤膜过滤，取透过液利用截留分子量为200的纳滤膜过滤，取截留液中加入100g D101大孔树脂，轻微搅拌2小时后过滤，将大孔树脂装入柱子中，用500ml含量为50％(v/v)的乙醇-水溶液洗脱，洗脱液减压浓缩至50ml，冷冻干燥后得到1.1g白色固体，经检测白色固体主要由列为Gly-Asn-Pro-Asn-Asn-Cly-Ile、Gly-Pro-Hyp-Gly-Ser-Pro、Gly-Leu-Hyp-Gly-Hyp-Ala与Gly-His-Lys-Val-Lys的肽与锁链素、异锁链素组成，含量分别为2.9％，3.4％，4.1％，4.4％，1.4％，1.1％。
实施例四
人包皮成纤维细胞(HFF)培养基由15％FBS的DMEM基础培养基、1％100U/mL青霉素和100μg/mL链霉素混合而配制。将HFF细胞37℃、5％CO2的培养箱中培养，换液按照每隔两天到三天的频率。当细胞培养至细胞密度达到80％以上，再用在胰蛋白酶消化培养瓶中的细胞进行传代培养，为后续实验做准备。采用第5代至第15代的细胞进行实验。
使用UV光度计测量和校准UVB剂量。用配制好的PBS充分洗涤接种在96孔板的细胞，每孔细胞数为5*104。然后进行紫外线照射(5J/cm2)。完成紫外线照射后，再次用PBS洗涤。充分洗涤细胞后，细胞置于培养基中培养24小时，温度为37℃，CO2为25％。
将成纤维细胞(第6-8代)以5×104个细胞/cm2接种在含有含10％(v/v)FBS的DMEM的24孔板中，并在37℃，湿润的5％CO2气氛中培养24小时。随后，细胞将在含有样品的培养基中再培养24小时。然后将细胞置于薄层DPBS中并暴露于UV，使用紫外灯在离细胞30cm的距离处进行均匀照射之后将细胞暴露于UV后，分别用用含有不同浓度实施例1得到的组合物样品的培养基替换PBS，并在37℃下的二氧化碳培养箱中孵育24小时。然后，将正常对照组在相同条件下在没有UV的培养基中孵育，并在分析前将样品孵育，并且将没有样品处理的模型对照组以相同剂量暴露于UV。本实验细胞活力采用MTT方法进行检测，检测吸光度为570nm。计算方法如下：
细胞存活率＝{1-(样品OD-空白OD)/样品空白OD}×100
其中样品的OD和样品空白的OD代表在样品的存在和不存在下的吸光值，空白的OD是在没有照射UV的吸光值。
以上所述仅为本发明专利的较佳实施例而已，并不用以限制本发明专利，凡在本发明专利的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明专利的保护范围之内。
Claims (6)
1.一种可以促进成纤维细胞增殖的组合物，其特征在于：主要由序列为Gly-Asn-Pro-Asn-Asn-Cly-Ile、Gly-Pro-Hyp-Gly-Ser-Pro、Gly-Leu-Hyp-Gly-Hyp-Ala、Gly-His-Lys-Val-Lys的肽与锁链素、异锁链素组成。
2.一种可以促进成纤维细胞增殖的组合物的制备方法，其特征在于，包括以下步骤：将鳕鱼皮与牛心脉管按照3:1的比例混合，加入10-30倍体积的氢氧化钠溶液脱脂，然后粉碎，加入5-20倍体积的去离子水，100-300U/L木瓜蛋白酶，100-300U/L胰蛋白酶、100-300U/L碱性蛋白酶，保持40℃-50℃搅拌2-4小时，10000r/min离心后取上清液，先利用截留分子量1000Da的超滤膜过滤，取透过液利用截留分子量为200Da的滤膜过滤，取截留液加入D101大孔树脂吸附，50％乙醇洗脱，后经浓缩、干燥得到所述组合物。
3.根据权利要求1所述的一种可以促进成纤维细胞增殖的组合物，其特征在于：所述Gly-Asn-Pro-Asn-Asn-Cly-Ile的含量为1％-4％，Gly-Pro-Hyp-Gly-Ser-Pro的含量为1-5％，Gly-Leu-Hyp-Gly-Hyp-Ala的含量为1％-6％，Gly-His-Lys-Val-Lys的含量为2-6％，锁链素的含量为0.1-2％，异锁链素的含量为0.1-2％。
4.根据权利要求1-3任意一项所述的一种可以促进成纤维细胞增殖的组合物，其特征在于：所述组合物具有促进成纤维细胞增殖的作用。
5.根据权利要求1-4任意一项所述的一种可以促进成纤维细胞增殖的组合物，其特征在于：所述组合物可用于食品、化妆品和药品领域中。
6.根据权利要求2所述的一种可以促进成纤维细胞增殖的组合物的制备方法，其特征在于：所述氢氧化钠溶液的浓度为1％。
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