CN115137751A - 一种富血小板血浆的处理方法及其在抑制妊娠纹中的应用 - Google Patents
一种富血小板血浆的处理方法及其在抑制妊娠纹中的应用 Download PDFInfo
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Abstract
本发明公开了一种富血小板血浆的处理方法及其在抑制妊娠纹中的应用,其特征在于将外周血采用离心机进行分离得到富血小板血浆,所得到富血小板血浆经过激活剂激活,所述激活剂中含有CaCl2和凝血酶受体激动剂肽6。本发明将富血小板血浆应用于治疗妊娠纹具有很高的成功率;不仅能够帮助大量女性减轻妊娠纹方面的苦恼,还能够获得经济效益。
Description
技术领域
本发明属于医药生物技术领域,具体涉及一种富血小板血浆的处理方法及其在抑制妊娠纹中的应用。
背景技术
妊娠纹(Striaegravidarum,SG)是女性孕期增大的子宫及激素变化导致局部皮肤弹性纤维蛋白分解、断裂而出现的红色、紫色或白色条纹样病损。初产妇妊娠纹患病率高达50%以上,且一旦形成将终生不退,严重破坏腹部美观。妊娠纹早期治疗以外用药物为主,近年来出现了一些先进治疗模式,如二氧化碳剥脱性点阵激光治疗、射频技术和微针治疗等,但相关报道较少,效果也各不相同,缺乏系统性研究成果,因此目前尚不存在可有效治疗妊娠纹且副作用小的共识模式,也尚无临床治疗指南供参考。由此可见,系统评价妊娠纹治疗方案的临床疗效,并进一步研究治疗方案的作用机制,对于指导妊娠纹治疗意义重大。
治疗妊娠纹的核心目标是促进皮肤组织生长、恢复皮肤弹性、避免感染和消除疤痕,这些过程与伤口愈合十分相似,而富血小板血浆(platelet-richplasma,PRP)则是非常匹配的治疗方法。PRP是一种自体全血经离心后得到的血小板浓缩物,主要包含高浓度血小板、白细胞和纤维蛋白,其中血小板被激活后,能够释放多种生长因子如血小板源性生长因子(PDGF)、转化生长因子(TGF-β)、胰岛素样生长因子(IGF)、表皮生长因子(EGF)和血管内皮细胞生长因子(VEGF)以及一些促凝因子等,这些因子能够加速损伤组织的修复过程,因此被广泛应用于骨科、心脏外科、妇科、整形外科等很多不同医学领域。在皮肤科领域,PRP已被发现具有促进组织再生,伤口愈合,疤痕修复,皮肤更新等功能,并且由于PRP具有自源性,能够避免外源性生长因子引起的免疫排斥反应或疾病传染风险,因此越来越受到关注。
PRP的功能与治疗妊娠纹的核心目标很一致。在促进皮肤组织生长方面,PRP中TGF-β可提高皮肤角化细胞的增殖能力和迁移速度,EGF能够促进表皮细胞和成纤维细胞的分裂增殖作用,IGF、PDGF和VEGF可促进内皮细胞增殖和向创伤部位迁移,并诱导新生血管形成,从而为其他细胞生长和毛细血管网的建立提供营养;在恢复皮肤弹性方面,PRP中TGF-β、EGF、VEGF等因子均可促进Ⅰ、Ⅲ型胶原蛋白和透明质酸的合成,TGF-β还可抑制细胞外基质降解,激活纤维蛋白构成皮肤支架,由此提高皮肤弹性,缓解皮肤失水和松弛;在避免感染方面,PRP中含有大量的白细胞和单核细胞,可清除局部病原体和局部坏死组织,能够显著增强局部抗感染能力;以上作用均可推动疤痕消除。由此可见,PRP非常适合治疗妊娠纹,很可能具有显著疗效,但目前将PRP应用于妊娠纹临床治疗的报道极少,因此需要开展更加系统和深入的研究。
在中国专利申请CN109069360A,其公开了一种用于透皮施用血液产品的组分的皮肤护理制剂,所述制剂包含血液产品、透皮载体;和脂质体基质,其中所述血液产品和透皮载体的至少一部分包含在所述脂质体基质的脂质体内。其虽然泛泛提及了富血小板血浆的透皮施用有可能能够改善妊娠纹,但其并未提供任何实验数据证明这一点,特别是其并未针对妊娠纹对富血小板血浆的提取方法进行研究。
针对PRP的外用制剂,中国专利申请CN112915264A公开了用于明胶-海藻酸钠-PRP复合材料的制作方法,在10%明胶、4%海藻酸钠的混合预凝胶中,其他条件、交联剂、打印工艺不变,在交联前混入不同浓度(初步预实验发现5%浓度效果最佳)的富血小板血浆,混合均匀,该混合体系作为皮肤替代物3D打印的墨水材料。从而对原有的墨水做了改性,提高了材料的细胞相容性、组织修复能力,更有利于临床转化应用。
PRP中的血小板浓度和血小板激活后释放的活性物质是其发挥功效的首要影响因素,特殊人群和服用药物等情况会影响血小板功能,其浓缩后的生物学效用差别极大,这可能导致PRP的治疗效果不明显。因此为了更稳定地应用PRP治疗妊娠纹,PRP中关键药效成分的含量与治疗效果的关系需要受到关注。
发明内容
基于上述原因,本发明提出一种富血小板血浆的处理方法及其在抑制妊娠纹中的应用。具体而言,为了实现本发明的目的,本发明拟采用如下的技术方案:
本发明一方面涉及一种富血小板血浆的处理方法,其特征在于将外周血采用离心机进行分离得到富血小板血浆,所得到富血小板血浆经过激活剂激活,所述激活剂中含有CaCl2和凝血酶受体激动剂肽6。
在本发明的一个优选实施方式中,所述激活剂中CaCl2的浓度为4~12wt%;优选为4~6wt%。
在本发明的一个优选实施方式中,所述激活剂中凝血酶受体激动剂肽6的浓度0.5~1.5wt%;优选为0.8~1.2wt%。
在本发明的一个优选实施方式中,所述激活剂和富血小板血浆的体积比为8~10:1。
在本发明的一个优选实施方式中,所述富血小板血浆的血小板浓度为1.1~1.5×1012/L。
在本发明的一个优选实施方式中,所述激活包括如下步骤:将激活剂与富血小板血浆混合,室温静止8-12min,离心,提取萃取液。
在本发明的一个优选实施方式中,所述萃取液中PDGF、TGF-β、VEGF的浓度分别为370-400ng/ml、1800-1950ng/ml、1120-1200pg/ml。
本发明另一方面涉及上述处理方法所获得的经激活的富血小板血浆。
本发明还涉及上述经激活的富血小板血浆在抑制皮肤瘢痕中的应用,优选的,所述皮肤瘢痕是指妊娠纹。
在本发明的一个优选实施方式中,所述经激活的富血小板血浆是自源性的。
有益效果
本发明将PRP应用于治疗妊娠纹可有效利用PRP的核心优势,如:①PRP具有自源性,可避免外源性生长因子进入人体后引起的免疫排斥反应或疾病传染风险;②PRP制备简单、快捷、方法成熟,易于操作和推广;③PRP中含多种高浓度生长因子协同作用,可弥补单一因子的不足;④PRP的制备只需采集患者的外周血,对患者损伤小;⑤PRP具有激活免疫反应、促进血管生成、推动组织再生等多种与治疗妊娠纹非常匹配的功能,因此既具有创新性,又具有很高的成功率;不仅能够帮助大量女性减轻妊娠纹方面的苦恼,还能够获得经济效益。
具体实施方式
为了进一步理解本发明,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
如无特殊说明,本发明实施例中所涉及的试剂均为市售产品,均可以通过商业渠道购买获得。
实施例1:
收集30名妊娠纹患者的外周血,采用离心机进行离心,第1次离心1600r/min,10min;第2次离心5000r/min,5min。离心后提取PRP,第1次离心后收集全部血浆层、白膜层及2~3mm范围的红细胞层移至另一采血管;第2次离心后抽取3/4血浆层,即贫血小板血浆(platelet-poolplasma,PPP),余下的1/4即PRP。离心获得的PRP经检验血小板浓度为1.2×1012/L,符合实验要求。提取PRP后,将PRP分为4组:第1组未激活PRP;第2组只加10%CaC1激活;第3组加5wt%CaCl2和1wt%凝血酶受体激动剂肽6激活;第4组加10wt%CaCl2和1wt%凝血酶受体激动剂肽6激活。3种激活剂均按9:1(v/v)与PRP混合,室温静止10min,离心12000r/min,5min,提取萃取液。ELISA方法比较不同激活方法获得的PRP的PDGF、TGF-β、VEGF的浓度差异。其中,均采用ELISA方法进行检测,检测重复3次,检测结果如表1所示。
表1:PRP激活后PDGF、TGF-β、TGF-β的浓度
实验结果表明,激活组相比于未激活组,PRP的PDGF、TGF-β、VEGF的浓度都有所提高,特别是5wt%CaCl2+凝血酶受体激动剂肽6相比于其它组,PRP的PDGF、TGF-β、VEGF的浓度提高更为明显,相比于其它组具有显著性差异(P<0.05)。
实验例2:药效学实验
(1)入组标准
2021年10月1日至2022年1月31日在东莞市妇幼保健院皮肤科就诊的妊娠纹患者40例;
①18-50岁的女性妊娠纹受试者;
②腹部有妊娠纹(红纹或白纹均可)。
(2)剔除标准
①凝血功能障碍;
②具有瘢痕体质患者;
③半年内曾对治疗部位有治疗者;
④2个月内曾使用过维A酸类药物及其衍生物;
⑤孕妇、哺乳期妇女;
⑥对治疗期望值过高者;
⑦对局部涂抹麻药过敏者;
⑧有严重肝、肾功能损害等系统疾病的患者。
(3)PRP治疗
就医者取仰卧位,注射区域外涂复方利多卡因乳膏8~10g,用塑料保鲜膜封包1h进行表面麻醉后,采用套装中配套的注射器配27G针头抽取PRP(5wt%CaCl2+凝血酶受体激动剂肽6激活),进行腹部皮下注射,每点注射量0.1mL,每点针距约1cm,与皱纹平行刺入真皮浅层,边退针边注射,使PRP尽量填充皮肤凹陷处,注射过程中按压止血。嘱患者注射后当日腹部避免沾水及按摩揉捏,1周内禁饮酒及禁食辛辣刺激食物,避免蒸桑拿等可能诱发腹部毛细血管扩张的行为。每个月一次,共三次。
(4)CO2剥脱性点阵激光治疗
治疗前患者清洁腹部,外用复方利多卡因乳膏涂抹治疗区,封包40~60min,治疗前清洗局麻乳膏。治疗时光斑不重叠,治疗结束后立即用冰袋冷敷10~15min,采用10600nmCO2点阵激光治疗。治疗频率:每次治疗间隔2个月,共治疗3次,末次治疗结束后随访3个月。
(5)临床评价
①总有效率评价:由2名医师和患者共同评价妊娠纹的外观表现和数码照片,分为显效(妊娠纹明显改善,患者满意)、有效(妊娠纹改善,患者较满意)和无效3类(妊娠纹无明显变化,患者不满意)。总有效率=(显效+有效)例数/总例数×100%。
②皮肤弹性评价:采用皮肤弹性测量仪测量患者腹部妊娠纹区域皮肤总弹性指数,指数越接近1,表示皮肤弹性越好。
临床评价结果如表2所示。
实验结果表明,相比于激光治疗组,PRP治疗组的总有效率更高,对于皮肤弹性指数的改善也更明显,两组间具有显著性差异(P<0.05)。
以上描述了本发明优选实施方式,然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。
Claims (10)
1.一种富血小板血浆的处理方法,其特征在于将外周血采用离心机进行分离得到富血小板血浆,所得到富血小板血浆经过激活剂激活,所述激活剂中含有CaCl2和凝血酶受体激动剂肽6,将激活剂与富血小板血浆混合,室温静止8-12min,离心,提取萃取液,所述萃取液中PDGF、TGF-β、VEGF的浓度分别为370-400ng/ml、1800-1950ng/ml、1120-1200pg/ml。
2.根据权利要求1所述的处理方法,所述激活剂中CaCl2的浓度为4~12wt%。
3.根据权利要求1所述的处理方法,所述激活剂中CaCl2的浓度为4~6wt%。
4.根据权利要求1所述的处理方法,所述激活剂中凝血酶受体激动剂肽6的浓度0.5~1.5wt%。
5.根据权利要求1所述的处理方法,所述激活剂和富血小板血浆的体积比为8~10:1。
6.根据权利要求1所述的处理方法,所述富血小板血浆的血小板浓度为1.1~1.5×1012/L。
7.权利要求1-6任意一项所述处理方法所获得的经激活的富血小板血浆。
8.权利要求7所述的经激活的富血小板血浆在抑制皮肤瘢痕中的应用。
9.根据权利要求8所述的应用,所述皮肤瘢痕是指妊娠纹。
10.根据权利要求8或9所述的应用,所述经激活的富血小板血浆是自源性的。
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