CN115531410B - miR-632在制备治疗皮肤创伤修复药物中的应用 - Google Patents
miR-632在制备治疗皮肤创伤修复药物中的应用 Download PDFInfo
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Abstract
本发明公开了miR‑632在制备治疗皮肤创伤修复药物中的应用，所述药物上调miR‑632的表达。本发明首次发现了miR‑632在治疗皮肤创伤修复中的用途，miR‑632及其上调剂及衍生物等可用于制备或筛选促皮肤创伤修复的有效药物，为治疗皮肤创伤修复的药物提供了新的靶点，为正常皮肤创面愈合和慢性创面的发病机制的研究提供了新的思路和方向。
Description
技术领域
本发明属于生物技术领域，具体涉及miR-632在制备治疗皮肤创伤修复药物中的应用。
背景技术
皮肤是人体最大的器官，不仅是机体首要的天然保护性屏障，而且在众多生理过程中发挥着至关重要的作用。皮肤一旦损伤后机体即刻启动级联且受精密调控的创伤修复进程，以尽快恢复皮肤的屏障功能和维持内环境稳定。然而创伤皮肤再生过程易受到自身疾病和外界环境的影响进而导致慢性不愈合伤口或伤口过度愈合。慢性不愈合伤口和伤口过度愈合不仅损害皮肤的正常生理功能，而且临床治疗花费高，受影响的患者人数也在逐年增加，为患者带来了巨大的心理压力和经济负担，也给全球的医疗保健系统带来沉重的负担。因此，皮肤受创后的快速高效再生修复对于机体正常生理功能重建、内环境稳态恢复以及减轻患者、社会的心理和经济负担具有重要作用。
近年来干细胞治疗，组织工程技术以及角质形成细胞移植为创伤修复提供了新型治疗策略，由于价格昂贵，治疗周期长，难以得到广泛推广。药物治疗在皮肤创伤治疗中仍发挥着关键的作用，常见的临床创伤修复治疗药物主要包括:小分子化合物，生长因子（rb-FGF, EGF），动植物来源药物，低粘性敷料等。这些药物虽然具有不错的效果，但也存在缺陷如，难以合成，合成成本较高，储存条件苛刻，药物活性成分不稳定以及容易导致瘢痕形成等，难以满足临床对皮肤创伤治疗的药物需求。因此新型高效的促皮肤组织再生先导分子的发掘显得尤为重要且必要。
miRNAs 是一类 18~25 nt 的单链内源性非编码 RNA 分子，通过影响靶基因的的翻译或稳定进而调控相关目的基因的表达。miRNAs 参与细胞增殖、分化、衰老、凋亡及各种疾病的生理及病理过程，且有研究表明，miRNAs 在正常皮肤创面愈合和慢性创面的发病机制中都发挥着重要作用，有望成为新型的治疗靶点以及核酸类药物。但相关研究仍处于起步阶段，相关药物研发以及作用机制知之甚少。
发明内容
本发明的目的在于提供miR-632在制备治疗皮肤创伤修复药物中的应用。
本发明的目的是这样实现的，miR-632分应用为在制备治疗皮肤创伤修复药物中的应用，所述药物上调miR-632的表达。
本发明的有益效果为：本发明首次发现了miR-632在治疗皮肤创伤修复中的用途，miR-632及其上调剂及衍生物等可用于制备或筛选促皮肤创伤修复的有效药物，为治疗皮肤创伤修复的 药物提供了新的靶点，为正常皮肤创面愈合和慢性创面的发病机制的研究提供了新的思路和方向。
附图说明
图1为本发明miR-632上调表达后对角质形成细胞的促增殖，迁移以及细胞划痕修复的影响；其中，A为miR-632 mimics与miR-632 mimics negative control转染角质形成细胞24 h后miR-632的相对表达量；B为miR-632 mimics与miR-632 mimics negativecontrol转染角质形成细胞后，角质形成细胞划痕修复在0 h 和24 h的划痕情况图；C为miR-632 mimics与miR-632 mimics negative control转染角质形成细胞后，24 h细胞划痕修复率；D为miR-632 mimics与miR-632 mimics negative control转染角质形成细胞后，培养24 h时角质形成细胞细胞活力图；E为miR-632 mimics与miR-632 mimicsnegative control转染角质形成细胞后，角质形成细胞在24 h时细胞迁移情况图；F为角质形成细胞在转染miR-632 mimics与miR-632 mimics negative control 24 h后，角质形成细胞的迁移数；G为miR-632 mimics与miR-632 mimics negative control转染角质形成细胞后，连续培养两周后细胞菌落形成图；H为miR-632 mimics与miR-632 mimics negativecontrol转染角质形成细胞后，培养两周后的细胞菌落数；
图2为本发明上调miR-632表达对SD大鼠全皮层创伤的修复作用图；其中，A为SD大鼠的造模示意图，给药方式，时间以及取材处理图；B为在SD大鼠创伤区域皮下注射miR-632agomir与miR-632 agomir negative control后伤口第0天和第9天伤口代表图；C为miR-632 agomir与miR-632 agomir negative control处理SD大鼠全皮层伤口后，第9天伤口修复情况图；D为miR-632 agomir与miR-632 agomir negative control处理SD大鼠全皮层伤口9天后，伤口区域组织HE染色结果；E为miR-632 agomir与miR-632 agomir negativecontrol处理SD大鼠全皮层伤口9天后，伤口区域再生表皮厚度图；F为miR-632 agomir与miR-632 agomir negative control处理SD大鼠全皮层伤口9天后，伤口区域再生肉芽组织厚度图；G为miR-632 agomir与miR-632 agomir negative control处理SD大鼠全皮层伤口9天后，伤口区域组织Ki67免疫组化染色结果；H为miR-632 agomir与miR-632 agomirnegative control处理SD大鼠全皮层伤口9天后，伤口区域Ki67阳性表达强度图。
具体实施方式
下面结合附图和实施例对本发明作进一步的详细说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或改进，均落入本发明的保护范围。
本发明提供了miR-632在制备治疗皮肤创伤修复药物中的应用，所述药物上调miR-632的表达。
所述药物的活性成分为：
（1）miR-632；
（2）miR-632 mimics；
（3）miR-632 agomir；
（4）前体miRNA，所述的前体miRNA能在宿主内加工成miR-632；
（5）多核苷酸，所述的多核苷酸能被宿主转录成步骤4中所述的前体miRNA，并加工形成miR-632，或能在宿主内加工成miR-632；
（6）表达构建物，所述的表达构建物含有步骤1中所述的miR-632、或步骤4中所述的前体miRNA、或步骤5中所述的多核苷酸。
所述皮肤创伤为慢性皮肤创伤或急性皮肤创伤。
实施例1 上调miR-632表达对角质形成细胞细胞迁移、增殖和划痕修复的影响
一、体外细胞划痕实验
实验方法：角质形成细胞在含10%胎牛血清和1% 抗生素(100 U/mL 青霉素，100U/mL链霉素)的DMEM/F12培养基中培养，温度为37℃，湿度为5% CO2。将HaCaT接种于24孔板（2×105细胞/孔），当细胞密度达到60%时，根据锐博转染试剂说明书进行转染，上调miR-632表达（riboFect™ CP转染试剂，miR-632 mimics，miR-632 mimics NC, 锐博，中国）。当角质形成细胞形成汇合的单层，使用200 μL无菌吸头划伤细胞单层模拟伤口。PBS洗尽细胞碎屑后，分别在转染组和空白组中加入500 μL无血清培养基置于37℃，5% CO2环境下继续培养24 h。
结果：从图1 B和图1C可知，上调miR-632的表达可以显著促进角质形成细胞划痕修复。
二、MTS实验
实验方法：将角质形成细胞接种于96孔板中(5×103 /孔)，待细胞贴壁后连续培养4 h后，根据博转染试剂说明书进行转染，上调miR-632表达组（miR-632 mimic组）和空白对照组（mimic NC组），连续培养24 h后，使用MTS试剂检测细胞增殖变化。
结果：从图1D可知，上调miR-632的表达可以显著促进角质形成细胞增殖。
三、Trans-well实验
实验方法：以滤膜孔径为8 μm的 trans-well 板作为上室插入下室24孔板中，再将角质形成细胞以细胞密度 1×104 /孔接种到上室培养4 h后，根据博转染试剂说明书进行转染，上调miR-632表达组（miR-632 mimic组）和空白对照组（mimic NC组），然后在24孔板下室中加入含10% FBS 的培养基。37 °C培养24 h，PBS清洗后使用4%多聚甲醛固定细胞并使用1mg/mL结晶紫染色20 min。PBS清洗上室并去除非迁移细胞后在倒置显微镜下拍摄。拍照记录完成后使用33%乙酸完全洗脱结晶紫，并用酶标仪在570 nm处检测吸光度值（OD值），计算细胞迁移率。
结果：从图1E和图1F可知，上调miR-632的表达可以显著促进角质形成细胞迁移。
综上可知，上调miR-632表达能够显著促进角质形成细胞的划痕修复、增殖和迁移，表明miR-632具有良好的促创伤修复活性。
实施例2 上调miR-632表达对体内皮肤创伤修复的影响
实验方法：小鼠全皮层创伤实验
1、用1%戊巴比妥钠（60mg/kg）麻醉SD大鼠，去除背部皮毛后，在背部两侧使用活检穿刺器创建两个直径为10 mm的全层伤口。将小鼠随机分为两组，分别为miR-632 agomirNC组和miR-632 agomir组。根据锐博动物转染试剂说明书操作（micrON™ miRNA agomir,锐博, 中国)，每隔4天在皮肤创伤区域注射一次miR-632转染试剂（miR-632 agomir）和空白对照(miR-632 agomir NC)，术后分别在第0天和第9天拍照记录。于术后9天取大鼠背部创口组织进行病理组织学分析。
2、为了评估大鼠创口组织在不同组别处理后的再生情况，进行hematoxylin andeosin (HE)染色分析。将皮肤组织切成厚度为5 μm的组织片，依次脱蜡、梯度水化然后再进行HE染色处理。使用光学显微镜在相同的放大倍数记录创伤组织再生情况，用软件Image J计算不同病理组织皮肤的表皮层、新生组织及上皮化形成的变化，并将数据在prism软件中进行统计分析。
结果：由图2B和图2C可知，miR-632 agomir 上调miR-632表达能够显著促进小鼠背部全皮层创口修复，且治疗9天后伤口修复率达到94%，显著优于agomir NC组。图2D-F则显示上调miR-632表达明显促进了大鼠表皮再生和肉芽组织再生。此外，图2G和图2H表明上调miR-632表达能显著促进大鼠表皮细胞增殖与细胞水平结果相一致。
综上可知，通过上调miR-632的表达，可以达到促进创伤修复的作用，说明miR-632可以作为治疗皮肤创伤修复的药物靶点。
Claims (1)
1.miR-632在制备治疗皮肤创伤修复药物中的应用，其特征在于，所述药物能够促进角质形成细胞的细胞迁移、增殖和划痕修复。
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Non-Patent Citations (3)
| Title |
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| miR-632 promotes gastric cancer progression by accelerating angiogenesis in a TFF1-dependent manner;Ying Shi等;《BMC Cancer》;第19卷(第14期);第1-9页 * |
| miR-632 Promotes Laryngeal Carcinoma Cell Proliferation, Migration, and Invasion Through Negative Regulation of GSK3β;Zhong-xin Zhou等;《Oncology Research》;第28卷;第21-31页，尤其是第21页摘要，第25页右栏倒数第1段，第26页左栏第1段 * |
| 玉糊浆促进大鼠背部创面愈合的实验研究;徐浩等;《中国中西医结合杂志》;第41卷(第2期);第224-228页，尤其是第224页摘要 * |
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