CN116942701A - 用于大伤口止血愈合的干细胞外泌体与prp组合物制剂 - Google Patents
用于大伤口止血愈合的干细胞外泌体与prp组合物制剂 Download PDFInfo
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- CN116942701A CN116942701A CN202310853407.8A CN202310853407A CN116942701A CN 116942701 A CN116942701 A CN 116942701A CN 202310853407 A CN202310853407 A CN 202310853407A CN 116942701 A CN116942701 A CN 116942701A
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Abstract
本发明提供了一种用于大伤口止血愈合的干细胞外泌体与prp组合物制剂,组合物制剂包括A剂为粉状制剂,包括干细胞外泌体富集冻干粉、prp富集冻干粉、壳聚糖季铵盐以及硅铝酸盐;B剂为胶状制剂,包括a‑氰基丙烯酸正辛酯、聚丙烯酸酯、聚丙烯醇以及异丙醇。本发明在使用时不需要对伤口进行完全止血处理,反而在流血状态下会更加促进血小板的凝固,由此使本发明的应用场景可不限制于医院清洁等级较高的术后清创缝合处理,也可作为家庭常用应对一般破损的外伤制剂。本发明不仅对伤口具有粘接作用,还具有止血、促愈合效果,且流血可促进制剂发挥作用,因此可针对较长、较深的大伤口,而这种伤口是家庭存储创可贴所不能胜任的。
Description
技术领域
本发明涉及生物手段促进干细胞外泌体收集技术领域,具体为应用富集干细胞外泌体与prp组合物制剂。
背景技术
干细胞外泌体在应用方面表现出众多的优势,包括皮肤修复及抗衰老、无菌性炎症、组织损伤修复、免疫调节。干细胞所分泌的外泌体,富含一百多种胞外基质活性蛋白、mRNA信使核糖核酸和多种生长因子。活性蛋白和生长因子能直接穿透皮肤间隙,快速达到基底层,有效促进皮肤成纤维细胞中Ⅰ型胶原蛋白、成纤维蛋白,促进皮肤有效快速再生;此外,还能够促进皮肤成纤维细胞(HDFs)弹性蛋白生成,保持皮肤饱满和弹性;有效降低皮肤成纤维细胞(HDFs)中胶原蛋白酶(MMP-1)的生成从而防止皮肤间质中I、II、III型胶原蛋白被降解;富含活性物质能够促进皮肤毛细血管的修复与重建再生。
富血小板血浆(Platelet-rich plasma,PRP)是高度浓缩的血小板,其中富含多种生长因子,它可以促进细胞增生和分化。PRP在伤口愈合、关节炎软骨组织修复和皮肤损伤修复等方面表现出显著的效果。但是将干细胞外泌体与富血小板血浆联合应用于皮肤创面的应用较少。
目前在皮肤创面整形过程中,逐渐使用医用胶水来取代伤口缝线手术。使用医用胶水相比缝线可减轻伤者的痛苦,不需麻醉,愈后效果好,不留缝线疤痕。但是医用胶水的应用有一定的限制,如使用前需保持伤口干净,无明显流血以防止流血影响胶粘效果,且医用胶水仅具备粘合作用,缺乏对伤口的促愈合作用,因此对现有医用胶水的研制还具有较广阔的前景,尤其是目前领域内较少有结合干细胞外泌体与富血小板血浆的相关研究。
发明内容
本申请的目的在于克服现有技术的不足之处,提供一种用于大伤口止血愈合的干细胞外泌体与prp组合物制剂。
一种用于大伤口止血愈合的干细胞外泌体与prp组合物制剂,组合物制剂包括搭配使用的A剂和B剂;
其中,A剂为粉状制剂,包括干细胞外泌体富集冻干粉、prp富集冻干粉、壳聚糖季铵盐以及硅铝酸盐;
B剂为胶状制剂,包括a-氰基丙烯酸正辛酯、聚丙烯酸酯、聚丙烯醇以及异丙醇;
所述A剂的制备方法包括以下步骤:
S1、扩增培养人间充质干细胞,培养至第三代时,每隔48h进行一次传代,对第三次传代后的悬浮细胞进行电击刺激,电压为500V,电击3次,每次电击间隔5s;收集第三代至第六代的电击后干细胞加入添加有20%小牛血清和电转缓冲液的MEM培养基继续培养24h获得富外泌体培养液;对富外泌体培养液通过差速离心法收集干细胞外泌体沉淀,向干细胞外泌体沉淀中加入PBS溶液以及2%海藻糖溶液,混匀经0.22μm除菌膜后在-80℃条件下急速冷冻,后经蒸发干燥制得干细胞外泌体富集冻干粉;
其中传代培养所用培养基为添加了100U/ml青霉素以及100μg/ml链霉素的MEM培养基;
S2、将获得的全血中加入抗凝剂,经二次离心获得浓缩血小板血浆以及贫血小板血浆;调控浓缩血小板血浆的计数大于1,000,000/μl时,加入2%溶液体积的海藻糖溶液混匀经0.22μm除菌膜后在-80℃条件下急速冷冻8h以上,得到血小板预冻品;向每1.2ml贫血小板血浆中加入10%的氯化钙50μl制成与浓缩血小板血浆体积相同的血小板激活剂;将血小板激活剂同样经0.22μm除菌膜后在-80℃条件下急速冷冻8h以上,得到血小板激活剂预冻品;将血小板预冻品以及血小板激活剂预冻品一同蒸发干燥后混匀得到prp富集冻干粉;
S3、将干细胞外泌体富集冻干粉以及prp富集冻干粉按照重量比1:1混合在一起,并且向其中加入0.2重量比的粉状吸水剂,粉状吸水剂包括等重量的壳聚糖季铵盐和硅铝酸盐,将上述所有粉剂混匀并真空包装,包装规格为5g~20g,作为一次性用量;
S4、以异丙醇为溶剂,向其中添加聚丙烯酸酯以及聚丙烯醇,其中聚丙烯酸酯、聚丙烯醇以及异丙醇的重量比为1:0.1:1制成胶基体;向胶基体中加入a-氰基丙烯酸正辛酯,胶基体与a-氰基丙烯酸正辛酯的体积比为1:1;将制得的B剂真空包装,包装规格为10g~50g,作为一次性用量;一次性用量的B剂重量至少是A剂重量的2倍。
进一步地,富外泌体培养液的离心条件为:
300×g离心10min,弃掉沉淀;
2000×g离心10min,弃掉沉淀;
10000×g离心10min,弃掉沉淀;
1000000×g离心85min,收集沉淀。
进一步地,将全血经200×g离心10min,保留上层血浆;再将上层血浆经1509×g离心10min,分离出2/3上层血浆制作激活剂,保留沉淀和剩余1/3上层血浆并将沉淀在剩余1/3上层血浆中吹匀获得浓缩血小板血浆;将多组制备的浓缩血小板血浆混合在一起至血小板计数大于1,000,000/μl。
进一步地,蒸发干燥的条件为:
升温至-50~-48℃,真空度0.01~0.02Torr并维持100~120min;
以1.0℃/min的速度升温至-15~-13℃,真空度0.10~0.15Torr并维持100~120min;
以1.5℃/min的速度升温至18~20℃,真空度0.15~0.2Torr并维持120~150min。
进一步地,使用时首先将A剂的真空包装打开并将粉剂沿着伤口走势均匀播撒;然后将B剂的真空包装打开并将胶剂涂抹在伤口上,涂抹过程中使胶水不仅要覆盖在有A剂的伤口表面上,还要接触到伤口旁边的完整皮肤。
干细胞外泌体富集冻干粉可向伤口中释放胶原蛋白、成纤维蛋白等多种促进毛细血管修复和再生的促进分子,对伤口起到抗菌消炎的作用,可促进伤口的愈合。另外在干细胞培养阶段对干细胞进行电击刺激可增加干细胞膜的通透性,增加培养基营养物质浓度,可加快细胞内外营养物质的输送,促进外泌体囊泡的形成。
血流会冲掉血小板的聚集,减慢血小板凝聚时间,因此常规的止血操作均是以降低伤口流血速度,等待血液凝固为基本原理。prp富集冻干粉中血小板的浓度高,经钙离子激活后可与伤口周围血液中血小板共同作用参与凝血,且prp富集冻干粉中的钙离子激活剂增加了伤口处钙离子的浓度,对伤口周围血液中的血小板同样起到激活作用。
壳聚糖季铵盐具有良好的水溶性,可将伤口流血中的水分迅速吸收并形成具有抗菌效果的膜,对伤口起到保护抗菌作用。
硅铝酸盐作为一种分子筛,化学通式为(M'2M)O·Al2O3·xSiO2·y H2O,M′、M分别为一价、二价阳离子如K+、Na+和Ca2+、Ba2+等。可将除水分子之外的大分子物质如血细胞、血小板等截留下来,使血液接触到硅铝酸盐后只有水分子能通过,而血小板被截留堆积在伤口创面,从而有利于血栓的形成。并且配合prp富集冻干粉中的激活剂,激活截留的血小板活力,促进血小板的凝聚。
A剂为粉剂,一方面脱水处理可保证A剂中的活性成分不会失活,也不会提前反应消耗。另一方面粉剂颗粒之间具有孔隙使之本身具有良好的吸水性能,吸水后可成团聚集在伤口处,从而迅速阻止血液外流,防止血流将血小板冲走。由此A剂在伤口处起到快速止血、血小板快速凝聚的效果。
B剂中a-氰基丙烯酸正辛酯为无色透明液体,其遇水或人体组织液在数秒钟内固化成膜,对组织产生较大的胶接强度。具有无毒、不致突变及畸形、无菌、无致敏等特性。可保护伤口与外界隔离,并且可防止伤口撕裂。有助于伤口的愈后愈合。聚丙烯酸酯具有粘合性和包覆性,能在伤口处形成保护层,从而促进伤口愈合。聚丙烯醇具有粘合性和包覆性,对胶体整体起到胶质化作用。异丙醇作为聚丙烯酸酯和聚丙烯醇的良性溶剂,有助于两种成分混合均匀,使胶体易于涂抹和定型。由此B剂的作用是封闭以及粘合伤口,保护伤口在愈合期间抗菌消炎不发生感染,并且保护伤口不因运动或外力二次撕裂,配合A剂作用可加快伤口的愈合。
本发明的有益效果:本发明在使用时不需要对伤口进行完全止血处理,反而在流血状态下会更加促进血小板的凝固,由此使本发明的应用场景可不限制于医院清洁等级较高的术后清创缝合处理,也可作为家庭常用应对一般破损的外伤制剂。本发明不仅对伤口具有粘接作用,还具有止血、促愈合效果,且流血可促进制剂发挥作用,因此可针对较长、较深的大伤口,而这种伤口是家庭存储创可贴所不能胜任的。
附图说明
图1为富集处理后与未经富集处理的外泌体透射电镜照片。
图2为Western blot印记法复水后干细胞外泌体囊泡上的标志物CD63和TSG101电泳图谱。
图3为prp富集冻干粉的OD560吸光值变化曲线图。
图4为制剂的粘合强度随时间变化直方图。
具体实施方式
下面将结合本发明实施例中的附图,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。
实施例一、富外泌体培养液的制备方法及步骤
通过购买获得人间充质干细胞。用添加了100U/ml青霉素以及100μg/ml链霉素的MEM培养基对人间充质干细胞进行培养。2~3d全量换液1次,直至贴壁细胞生长至汇聚度达到80%,弃掉培养液,用5ml PBS洗涤一遍,加入2ml 0.25%胰酶EDTA钠溶液,置于37℃培养箱消化5min,加入3ml MEM培养基中和胰酶活性,获得原代人间充质干细胞。
人间充质干细胞的传代培养:原代人间充质干细胞培养至贴壁细胞生长密度为4.0×103个/cm2时进行分瓶。每瓶加入含有100U/ml青霉素以及100μg/ml链霉素的MEM培养基,贴壁细胞生长至汇聚度达到80%时进行传代。
传代培养至第三代时,培养48h后进行传代,至第六代。对第三代至第六代的悬浮细胞进行电击。取悬浮细胞400×g离心5-7min收集细胞,用PBS重悬至密度为1~5×106cell/ml。在电压为500V的条件下对重悬细胞液进行电击刺激,电击3次,每次电击间隔5s。向电击后的细胞液中加入添加了20%小牛血清、100U/ml青霉素以及100μg/ml链霉素的MEM培养基,并向MEM培养基中加入BIO-RAD伯乐电转缓冲液。继续培养24h获得富外泌体培养液。用低温透射电镜观察富外泌体培养液中外泌体的形态及密度。结果如图1A所示,透射电镜显示富外泌体培养液中外泌体直径为100nm左右,形状呈圆形或椭圆形,密度相比未经富集处理(图1B)的密度更大,说明对人间充质干细胞经过电击刺激后,干细胞的细胞膜通透性增加,配合培养基中增大了浓度的营养物,可加快干细胞内外营养物质的输送,促进外泌体囊泡的形成,最终使外泌体的分泌量增加。
表1为人间充质干细胞培养过程中使用的材料
材料名称 | 生产公司 |
人间充质干细胞 | 青岛奥克生物开发有限公司 |
青霉素 | 上海跃腾生物技术有限公司 |
链霉素 | 上海跃腾生物技术有限公司 |
MEM培养基 | 赛默飞世尔科技中国 |
PBS | 赛默飞世尔科技中国 |
0.25%胰酶EDTA钠溶液 | 北京百奥莱博科技有限公司 |
小牛血清 | 赛默飞世尔科技中国 |
BIO-RAD伯乐电转缓冲液 | 安诺伦(北京)生物科技 |
实施例二、干细胞外泌体富集冻干粉的制备方法
将富外泌体培养液进行差速离心,离心条件为:
300×g离心10min,弃掉沉淀;
2000×g离心10min,弃掉沉淀;
10000×g离心10min,弃掉沉淀;
1000000×g离心85min,收集沉淀,获得富集干细胞外泌体。
向干细胞外泌体沉淀中加入PBS溶液重悬以及2%溶液体积的海藻糖溶液,混匀经0.22μm除菌膜后在-80℃条件下急速冷冻8h以上,得到预冻品。
将预冻品放入冻干机中,干燥条件为:
升温至-50~-48℃,真空度0.01~0.02Torr并维持100~120min;
以1.0℃/min的速度升温至-15~-13℃,真空度0.10~0.15Torr并维持100~120min;
以1.5℃/min的速度升温至18~20℃,真空度0.15~0.2Torr并维持120~150min。从冻干机中取出样品即制得干细胞外泌体富集冻干粉。
向干细胞外泌体富集冻干粉中添加生理盐水使干细胞外泌体富集冻干粉复水完全融化,并通过Western印记分析复水后干细胞外泌体(fMSC)的活性,通过外泌体囊泡上的标志物CD63和TSG101作为活性标志,以未经冻干的人间充质干细胞(hMSC)作为对照。结果如图2所示,复水后干细胞外泌体表面标志物CD63和TSG101均呈阳性表达,说明冻干过程对干细胞外泌体活性的负面影响较小。
表2为干细胞外泌体富集冻干粉制备过程用到的材料
材料名称 | 生产公司 |
海藻糖 | 上海麦克林生化科技股份 |
PBS | 赛默飞世尔科技中国 |
CD63ELISA检测试剂盒 | 上海江莱生物科技有限公司 |
TSG101ELISA检测试剂盒 | 上海江莱生物科技有限公司 |
实施例三、prp富集冻干粉的制备方法
向每100ml的全血中加入2.5%枸橼酸钠10ml。
将200ml的全血经200×g离心10min,保留上层血浆;再将上层血浆经1509×g离心10min,分离出2/3上层血浆制作激活剂,保留沉淀和剩余1/3上层血浆并将沉淀在剩余1/3上层血浆中吹匀获得浓缩血小板血浆。
重复上述步骤制备多瓶浓缩血小板血浆,并将多瓶浓缩血小板血浆混合在一起,通过血小板计数控制浓缩血小板血浆中血小板的计数大于1,000,000/μl时用于后续冻干。过程中通过自动血液分析仪进行血小板计数。若血小板浓度不足,将浓缩血小板血浆再次经1509×g离心10min,弃去适当上层血浆后,再将血小板沉淀和剩余血浆混匀,减少溶剂体积以增加血小板浓度。
向浓度合适的浓缩血小板血浆中加入2%体积的海藻糖溶液,混匀经0.22μm除菌膜后在-80℃条件下急速冷冻8h以上,得到血小板预冻品。
制备血小板激活剂向分离出的离心后上层血浆中加入10%的氯化钙50μl每1.2ml血浆。血小板激活剂的溶液体积与血小板预冻品的浓缩血小板血浆的体积相同。将血小板激活剂同样经0.22μm除菌膜后在-80℃条件下急速冷冻8h以上,得到血小板激活剂预冻品。
将血小板预冻品以及血小板激活剂预冻品一同放入冻干机中,干燥条件为:
升温至-50~-48℃,真空度0.01~0.02Torr并维持100~120min;
以1.0℃/min的速度升温至-15~-13℃,真空度0.10~0.15Torr并维持100~120min;
以1.5℃/min的速度升温至18~20℃,真空度0.15~0.2Torr并维持120~150min。从冻干机中取出样品并混合即制得prp富集冻干粉。
prp富集冻干粉的聚集活性验证:
取100mg的prp富集冻干粉并加入3ml生理盐水混匀制成复水组富集血小板血浆(f-prp),通过移液枪将3ml复水组富集血小板血浆转移到96孔板中,每孔300μl,共5个孔。
取3ml贫血小板血浆(ppp),通过移液枪将贫血小板血浆转移到96孔板中,每孔300μl,共5个孔。
取3ml清水(blank),通过移液枪将清水转移到96孔板中,每孔300μl,共5个孔。
将上述96孔板放入酶标仪中测量560nm下的光吸收值,并且在0min、5min、10min时刻下分别测量一次。取5个孔的平均值绘制变化曲线如图3所示。随着时间的延长复水组富集血小板血浆(f-prp组)发生聚集,说明经冻干处理后的血小板活性仍然存在,并且可在钙离子的激活下发生聚集,使用在伤口上时可起到血液促凝作用。
表3prp富集冻干粉的制备过程用到的材料
材料名称 | 生产公司 |
2.5%枸橼酸钠 | 上海麦克林生化科技股份 |
海藻糖 | 上海麦克林生化科技股份 |
10%氯化钙 | 潍坊达康化工有限公司 |
实施例四、A剂和B剂的制作方法及应用
A剂的制作方法:将干细胞外泌体富集冻干粉以及prp富集冻干粉按照重量比1:1混合在一起,并且向其中加入0.2重量比的粉状吸水剂,粉状吸水剂包括等重量的壳聚糖季铵盐和硅铝酸盐,将上述所有粉剂混匀并真空包装,包装规格为5g~20g,作为一次性用量。
B剂的制作方法:以异丙醇为溶剂,向其中添加聚丙烯酸酯以及聚丙烯醇,其中聚丙烯酸酯、聚丙烯醇以及异丙醇的重量比为1:0.1:1。并通过搅拌使聚丙烯酸酯与聚丙烯醇充分溶解在异丙醇中,制成胶基体。向胶基体中加入a-氰基丙烯酸正辛酯,胶基体与a-氰基丙烯酸正辛酯的体积比为1:1。并通过搅拌的方式使a-氰基丙烯酸正辛酯与胶基体混合均匀。将制得的胶体B剂真空包装,包装规格为10g~50g,作为一次性用量。一次性用量的B剂重量至少是A剂重量的2倍。
使用时,可对伤口进行大概的去血处理,不必保持伤口无血整洁。首先将A剂的真空包装打开并将粉剂沿着伤口走势均匀播撒。然后将B剂的真空包装打开并将胶剂涂抹在伤口上,涂抹过程中使胶水不仅要覆盖在有A剂的伤口表面上,还要接触到伤口旁边的完整皮肤,胶剂吸收血液中的水分迅速固化,在伤口及附近皮肤上形成坚硬的胶壳,不仅对伤口起到隔离效果,还可以固定伤口两侧皮肤有效防止伤口裂开。
实施例五、组合物的粘合性能测试
以无毛猪皮为生物基材,测试组合物的粘结性。从市场购买新鲜猪皮,经去毛、去油、去污处理。在一块洁净猪皮表面绘制2.5cm×2.5cm的测试区域。在测试区域的猪皮表面先涂上一层新鲜猪血液,后按照实施例四中的方法先后在测试区域内涂抹A剂和B剂。后迅速将另一块洁净猪皮的表面覆盖在测试区域内,使用100g的砝码压在测试区域上使两块猪皮充分接触。待组合物固化后,通过拉伸搭接剪切测试法考察组合物的粘结性。通过拉伸测试测定最大粘附力F,拉伸速率为5mm/min,通过以下公式计算粘合强度。
结果如图4所示,组合物的粘合强度随着粘合时间的增加而增加。粘合强度最大可达190kPa,与已知的氰基丙烯酸酯粘合强度相当(160~200kPa)。由于a-氰基丙烯酸正辛酯分子中有两个强烈的吸电子基团-CN(氰基)和酯基连接在同一个碳原子上,很容易在水或弱碱物质催化下迅速发生阴离子聚合反应。而大伤口的流血量大,A剂呈粉状,对血液具有迅速吸收的作用。且A剂中的干细胞外泌体富集冻干粉以及prp富集冻干粉在短时间内向伤口内释放大量的胶原蛋白、成纤维蛋白以及促凝因子使血液加快凝固,迅速在伤口处形成血栓结构,可防止血液将A剂或B剂冲走。同时A剂中的硅铝酸盐在遇到新鲜血液后形成分子筛,将除水以外的大分子结构如血细胞、血小板等截留,截留的血小板帮助伤口血栓的形成,而血液中的水分被硅铝酸盐吸附并与后来B剂中的a-氰基丙烯酸正辛酯接触,a-氰基丙烯酸正辛酯遇水迅速发生阴离子聚合反应,使B剂迅速固化,可将伤口与外部环境隔离,并依靠较强的粘合强度保护伤口稳定。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
Claims (5)
1.一种用于大伤口止血愈合的干细胞外泌体与prp组合物制剂,其特征在于,所述组合物制剂包括搭配使用的A剂和B剂;
其中,A剂为粉状制剂,包括干细胞外泌体富集冻干粉、prp富集冻干粉、壳聚糖季铵盐以及硅铝酸盐;
B剂为胶状制剂,包括a-氰基丙烯酸正辛酯、聚丙烯酸酯、聚丙烯醇以及异丙醇;
所述A剂的制备方法包括以下步骤:
S1、扩增培养人间充质干细胞,培养至第三代时,每隔48h进行一次传代,对第三次传代后的悬浮细胞进行电击刺激,电压为500V,电击3次,每次电击间隔5s;收集第三代至第六代的电击后干细胞加入添加有20%小牛血清和电转缓冲液的MEM培养基继续培养24h获得富外泌体培养液;对富外泌体培养液通过差速离心法收集干细胞外泌体沉淀,向干细胞外泌体沉淀中加入PBS溶液以及2%海藻糖溶液,混匀经0.22μm除菌膜后在-80℃条件下急速冷冻,后经蒸发干燥制得干细胞外泌体富集冻干粉;
其中传代培养所用培养基为添加了100U/ml青霉素以及100μg/ml链霉素的MEM培养基;
S2、将获得的全血中加入抗凝剂,经二次离心获得浓缩血小板血浆以及贫血小板血浆;调控浓缩血小板血浆的计数大于1,000,000/μl时,加入2%溶液体积的海藻糖溶液混匀经0.22μm除菌膜后在-80℃条件下急速冷冻8h以上,得到血小板预冻品;向每1.2ml贫血小板血浆中加入10%的氯化钙50μl制成与浓缩血小板血浆体积相同的血小板激活剂;将血小板激活剂同样经0.22μm除菌膜后在-80℃条件下急速冷冻8h以上,得到血小板激活剂预冻品;将血小板预冻品以及血小板激活剂预冻品一同蒸发干燥后混匀得到prp富集冻干粉;
S3、将干细胞外泌体富集冻干粉以及prp富集冻干粉按照重量比1:1混合在一起,并且向其中加入0.2重量比的粉状吸水剂,粉状吸水剂包括等重量的壳聚糖季铵盐和硅铝酸盐,将上述所有粉剂混匀并真空包装,包装规格为5g~20g,作为一次性用量;
S4、以异丙醇为溶剂,向其中添加聚丙烯酸酯以及聚丙烯醇,其中聚丙烯酸酯、聚丙烯醇以及异丙醇的重量比为1:0.1:1制成胶基体;向胶基体中加入a-氰基丙烯酸正辛酯,胶基体与a-氰基丙烯酸正辛酯的体积比为1:1;将制得的B剂真空包装,包装规格为10g~50g,作为一次性用量;一次性用量的B剂重量至少是A剂重量的2倍。
2.如权利要求1所述的用于大伤口止血愈合的干细胞外泌体与prp组合物制剂,其特征在于,富外泌体培养液的离心条件为:
300×g离心10min,弃掉沉淀;
2000×g离心10min,弃掉沉淀;
10000×g离心10min,弃掉沉淀;
1000000×g离心85min,收集沉淀。
3.如权利要求1所述的用于大伤口止血愈合的干细胞外泌体与prp组合物制剂,其特征在于,将全血经200×g离心10min,保留上层血浆;再将上层血浆经1509×g离心10min,分离出2/3上层血浆制作激活剂,保留沉淀和剩余1/3上层血浆并将沉淀在剩余1/3上层血浆中吹匀获得浓缩血小板血浆;将多组制备的浓缩血小板血浆混合在一起至血小板计数大于1,000,000/μl。
4.如权利要求1所述的用于大伤口止血愈合的干细胞外泌体与prp组合物制剂,其特征在于,蒸发干燥的条件为:
升温至-50~-48℃,真空度0.01~0.02Torr并维持100~120min;
以1.0℃/min的速度升温至-15~-13℃,真空度0.10~0.15Torr并维持100~120min;
以1.5℃/min的速度升温至18~20℃,真空度0.15~0.2Torr并维持120~150min。
5.如权利要求1所述的用于大伤口止血愈合的干细胞外泌体与prp组合物制剂,其特征在于,使用时首先将A剂的真空包装打开并将粉剂沿着伤口走势均匀播撒;然后将B剂的真空包装打开并将胶剂涂抹在伤口上,涂抹过程中使胶水不仅要覆盖在有A剂的伤口表面上,还要接触到伤口旁边的完整皮肤。
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