CN115282330A - 一种脂肪间充质干细胞敷料的制备方法及其应用 - Google Patents
一种脂肪间充质干细胞敷料的制备方法及其应用 Download PDFInfo
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Abstract
本发明提出一种脂肪间充质干细胞敷料的制备方法及其应用,步骤为:将β‑甲壳素纳米纤维悬浮液加入细胞培养基中,β‑甲壳素纳米纤维悬浮液与细胞培养基的体积比为2‑4:1,使β‑甲壳素纳米纤维凝胶化,弃除凝胶外的多余液体,然后将胶原蛋白粉与β‑甲壳素纳米纤维凝胶按质量体积比为10%‑40%混合均匀,得到β‑甲壳素纳米纤维‑胶原水凝胶;在β‑甲壳素纳米纤维‑胶原水凝胶中,按照106‑107个/ml加入脂肪间充质干细胞,混合均匀后在脂肪间充质干细胞培养基中培养24‑48h,得到脂肪间充质干细胞敷料。本发明解决脂肪间充质干细胞创面救治应用过程中给药方式受限、细胞存活率低等问题。
Description
技术领域
本发明属于细胞药物制备的技术领域,尤其涉及一种脂肪间充质干细胞敷料的制备方法及其在促进糖尿病创面修复中的应用。
背景技术
皮肤是人体覆盖面积最大、具有重要屏障及保护作用的器官,皮肤也因此成为极易发生损伤的部位。在全球范围内,外伤、烧烫伤、溃疡或手术创伤等导致的皮肤缺损的病例日益增多,虽然大部分创口可以实现自然愈合,但一些大面积损伤和糖尿病创面等,依靠自身的力量却难以愈合,成为威胁患者生命的主要因素。伤口愈合是一个复杂的生物学过程,主要包括止血、炎症、增殖和重塑四个阶段;各阶段在时间上具有重叠性,通过不同类型细胞的活化和多种功能的调节有序进行。创面形成后首先进入止血阶段,该过程主要依赖于血小板进行止血。炎症期开始后,中性粒细胞和单核细胞向创面内富集以清除创面上的异物和受损细胞。此后,在多种细胞因子的调节下,单核细胞转化为巨噬细胞,吞噬死亡或感染的细胞,吸引其他细胞到创面部位释放细胞因子和生长因子,刺激细胞增殖和迁移,加速创面愈合。巨噬细胞已被证明对创面愈合至关重要,因为除了上述功能,它们在创面炎症阶段过渡到细胞增殖阶段同样起着关键作用。在增殖阶段,血液的成纤维细胞增殖和迁移至创面,能够促进创面肉芽新生和细胞外基质的形成。新形成的细胞外基质主要由胶原组成,除影响细胞增殖、迁移、分化和凋亡等活动,还调节巨噬细胞活化和多种细胞因子的释放,包括转化生长因子-β和血小板衍生生长因子等。此外,许多成纤维细胞被转化(分化)为肌成纤维细胞,以促进伤口愈合。伤口在基质形成后逐步发生重塑。此时,伤口组织进一步成熟,血管网恢复,上皮形成,修复皮肤初级结构,从而形成正常组织。当急性创面发生后,会触发上述创面愈合过程,但任何异常都可能导致慢性创面的形成。糖尿病代谢紊乱是慢性创面形成的常见因素之一。与急性创面相比,糖尿病创面无有序而及时的修复过程,或经1个月治疗仍不能完成创面愈合,也无愈合倾向,修复机制更为复杂糖尿病创面给患者和卫生保健系统带来显著负担。随着人口老龄化和糖尿病等慢性病发病率的上升,关于皮肤损伤机制及救治策略的研究也越来越受到重视。
随着再生医学的发展,干细胞移植在多种退行性疾病和损伤性疾病的治疗中显示出较为理想的疗效,其临床应用的需求日益增高。根据发育状态可将干细胞分为能够分化为几乎所有类型的胚胎干细胞和具有多向分化潜能的成体干细胞。其中,成体干细胞可来源于脂肪、脐带、软骨和牙龈等多种组织。获取的原代间充质干细胞在特定条件下可分化为脂肪、骨、软骨等多种类型细胞,在多种疾病的治疗中均被证实产生了较好的疗效。在多种成体干细胞中,脂肪间充质干细胞有因储量丰富、获取方便、生物学特性稳定、免疫原性低、生物安全性高及避免了伦理争议等优点,使其成为细胞治疗较为理想的种子细胞,在组织修复治疗方面具有广阔的应用前景,尤其是其可以分泌多种促进创面愈合的细胞因子的特性,为创面损伤的治疗中提供了新的可能。已有研究证实脂肪间充质干细胞注射后可向创伤区域聚集并分泌生长因子,促进创面血管和细胞外基质生成,改善血供,为创面局部创造良好的修复环境。脂肪干细胞为糖尿病创面、骨缺损等疾病的治疗提供了一种新的可能,但应用过程中尚存在给药方式受限、毛细血管阻塞、细胞存活率低、干细胞数量不足、二维培养局限明显、定向分化诱导效率低等问题。
发明内容
发明目的:本发明提出一种脂肪间充质干细胞敷料的制备方法及其应用,其目的在于,围绕糖尿病创面修复难题,解决脂肪间充质干细胞创面救治应用过程中给药方式受限、细胞存活率低等问题,本发明制备的脂肪间充质干细胞敷料,完善脂肪间充质干细胞应用方法,提高糖尿病创面救治能力。
技术方案:
一种脂肪间充质干细胞敷料的制备方法,步骤为:
步骤一、将β-甲壳素纳米纤维悬浮液加入细胞培养基中,β-甲壳素纳米纤维悬浮液与细胞培养基的体积比为2-4:1,使β-甲壳素纳米纤维凝胶化,弃除凝胶外的多余液体,然后将胶原蛋白粉与β-甲壳素纳米纤维凝胶按质量体积比为10%-40%混合均匀,得到β-甲壳素纳米纤维-胶原水凝胶;
步骤二、在β-甲壳素纳米纤维-胶原水凝胶中,按照106-107个/ml加入脂肪间充质干细胞,混合均匀后在脂肪间充质干细胞培养基中培养24-48h,得到脂肪间充质干细胞敷料。
优选的,所述β-甲壳素纳米纤维的制备方法为以鱿鱼软骨为原料,将鱿鱼软骨洗净干燥,分散在0.1mol/L盐酸溶液中脱矿,用清水洗涤至中性后,用4wt%氢氧化钠溶液在80℃下处理进行脱蛋白,然后用清水以18.2MΩ·cm的电阻率洗涤至中性,得到β-甲壳素,将纯化后的β-甲壳素加入到去离子水中,用乙酸酸化至pH 3,然后用超声波在冰水浴中19.5kHz和300W下处理,得到β-甲壳素纳米纤维。
优选的,步骤一中β-甲壳素纳米纤维悬浮液的浓度为0.15-0.35%wt%。
优选的,细胞培养基为DMED培养基、1640培养基或脂肪间充质干细胞专用培养基。
一种如权利要求1所述的制备方法制备的脂肪间充质干细胞敷料在制备促进糖尿病创面修复中的药物或生物活性物质的载体材料上的应用。
有益效果:
通过制备脂肪间充质干细胞水凝胶解决了脂肪间充质干细胞创面救治应用过程中给药方式受限、细胞存活率低等问题;针对糖尿病创面救治难,综合脂肪间充质干细胞和水凝胶湿性愈合的作用,为糖尿病创面修复带来新的可能策略,具体有益效果如下:
(1)本研究通过酸水解、碱提取成功制备出β-甲壳素纳米纤维悬浮液,该悬浮液能够与培养基反应形成果冻状凝胶。在此基础上,与胶原复合后制备出β-甲壳素纳米纤维-胶原水凝胶(图1)。
(2)脂肪间充质干细胞能够在β-甲壳素纳米纤维-胶原水凝胶内呈球状均匀生长,细胞可在凝胶中存活和增殖(图2)。与创面愈合相关生长因子TGFβ1和PDGFD的表达水平升高(图3)。
(3)脂肪间充质干细胞水凝胶能够更好地促进糖尿病小鼠皮肤创面愈合(图4)。
(4)脂肪间充质干细胞水凝胶能够促进糖尿病小鼠皮肤组织内VEGF和α-SMA蛋白表达水平(图5)。
综上所述,本研究制备了β-甲壳素纳米纤维胶原水凝胶,验证了其在糖尿病创面愈合中的作用,为寻找新的促创面愈合救治方法提供实验基础。
附图说明
图1为水凝胶状态;
图2为水凝胶中细胞增殖情况柱状图;
图3为创面愈合相关生长因子TGFβ1和PDGFD的表达水平;
图4为创面愈合情况,创面愈合率及小鼠血糖;
图5为血管生成相关蛋白VEGF和α-SMA表达量;
图6为创面组织HE染色。
具体实施方式
以下结合说明书附图更详细的说明本发明。本研究创造性的将β-甲壳素纳米纤维与胶原相结合,制备出适用于脂肪间充质干细胞培养和应用的水凝胶,将该脂肪间充质干细胞水凝胶涂抹于db/db糖尿病小鼠创面能够显著促进糖尿病创面愈合,使用方便且效果显著。
近年来,胶原蛋白凭借良好的生物相容性在美容祛皱、保护皮肤、硬组织修复、创面止血等医药卫生领域应用广泛,尤其是其改善皮肤的屏障功能,修复骨、肌腱损伤等作用成为国内外研究热点。甲壳素是一种生物活性良好的多功能材料,本研究将甲壳素与胶原复合制备脂肪间充质干细胞支架,制备出脂肪间充质干细胞敷料,为脂肪间充质干细胞局部应用和糖尿病创面修复提供新的方法。
实施例1
一种脂肪间充质干细胞敷料的制备方法,其步骤为:
步骤一、以来源广泛、可再生的鱿鱼软骨为原料,将鱿鱼软骨50g洗净干燥,分散在750mL 0.1mol/L盐酸溶液中脱矿20h。用清水洗涤至中性后,用750mL 4wt%氢氧化钠溶液在80℃下处理10h进行脱蛋白,然后用清水以18.2MΩ·cm的电阻率洗涤至中性,得到β-甲壳素。将纯化后的β-甲壳素以0.3wt%加入到的去离子水中,用乙酸酸化至pH 3,然后用超声波在冰水浴中19.5kHz和300W下处理6min,制备β-甲壳素纳米纤维悬浮液。以β-甲壳素纳米纤维悬浮液:细胞培养基2:1的比例(体积比)将β-甲壳素纳米纤维加入细胞培养基中,使β-甲壳素纳米纤维凝胶化,立刻弃除凝胶外的多余液体,然后将胶原蛋白粉与β-甲壳素纳米纤维凝胶按10%比例(10g/100ml)混合均匀。甲壳素纳米纤维除甲壳素自身优势外,亦可充分发挥纳米形态高长径比、高比表面积等特性而广泛应用于医药领域。鱿鱼软骨作为一种渔业废物现逐步被开发利用,具有来源广泛、可再生的特点。
细胞培养基为DMED培养基、1640培养基或脂肪间充质干细胞专用培养基等用于细胞培养的培养基均可。含酸碱指示剂、盐、营养、生长因子和激素等成分。
步骤二、在上述凝胶中,按照1×106个/ml加入脂肪间充质干细胞,混合均匀后在含10%胎牛血清、1%青霉素-链霉素和1%谷氨酰胺的脂肪间充质干细胞培养基中培养36h,得到脂肪间充质干细胞敷料。脂肪间充质干细胞培养基可购买于赛业生物科技有限公司,批号:MUBMD-03011-440。
实施例2
一种脂肪间充质干细胞敷料的制备方法,其步骤为:
步骤一、制备β-甲壳素纳米纤维悬浮液的方法同实施例1,其中β-甲壳素纳米纤维悬浮液的浓度为0.15wt%。以β-甲壳素纳米纤维悬浮液:细胞培养基3:1的比例(体积比)将β-甲壳素纳米纤维加入细胞培养基中,使β-甲壳素纳米纤维凝胶化,立刻弃除凝胶外的多余液体,然后将胶原蛋白粉与β-甲壳素纳米纤维凝胶按20%比例(10g/100ml)混合均匀。
步骤二、在上述凝胶中,按照1×107个/ml加入脂肪间充质干细胞,混合均匀后在含10%胎牛血清、1%青霉素-链霉素和1%谷氨酰胺的脂肪间充质干细胞培养基中培养48h,得到脂肪间充质干细胞敷料。
实施例3
一种脂肪间充质干细胞敷料的制备方法,其步骤为:
步骤一、制备β-甲壳素纳米纤维悬浮液的方法同实施例1,β-甲壳素纳米纤维悬浮液的浓度为0.35%wt%。以β-甲壳素纳米纤维悬浮液:细胞培养基4:1的比例(体积比)将β-甲壳素纳米纤维加入细胞培养基中,使β-甲壳素纳米纤维凝胶化,立刻弃除凝胶外的多余液体,然后将胶原蛋白粉与β-甲壳素纳米纤维凝胶按40%比例(10g/100ml)混合均匀。
步骤二、在上述凝胶中,按照1×106个/ml加入脂肪间充质干细胞,混合均匀后在含10%胎牛血清、1%青霉素-链霉素和1%谷氨酰胺的脂肪间充质干细胞培养基中培养36h,得到脂肪间充质干细胞敷料。
通过实施例1、2或者3制备出的β-甲壳素纳米纤维-胶原水凝胶状态见图1所示。脂肪间充质干细胞能够在β-甲壳素纳米纤维-胶原水凝胶内呈球状均匀生长,如图2所示,细胞可在凝胶中存活和增殖。
实施例4
按照ELISA说明书(酶科生物科技)操作,取细胞超声破碎后,10000转/分离心20min,收集上清;设空白孔、标准孔和待测样品孔;采用试剂盒自带封板膜将ELISA板封好置于37℃温控箱内30min;取出洗涤液按照说明书用蒸馏水进行30倍稀释,反复加满ELISA板进行洗涤;ELISA板进行5次洗涤后,空白孔不做处理,其余孔每孔加入50μl酶标试剂;再次将ELISA板封好并于37℃温控箱内平置30min,洗板5次,将板拍干后依次加入50μL显色剂A和50μL显色剂B,避光37℃反应10min;按照说明书将板内孔加50μL终止液,测定OD值并计算浓度。
实验结果如图3和图5所示,图3中A为生长因子TGFβ1的表达水平,B为生长因子PDGFD的表达水平,说明凝胶培养后,脂肪间充质干细胞中促创面愈合相关因子(TGFβ1和PDGFD)表达升高,有利于脂肪间充质干细胞更好的发挥生物学活性。图5中A为蛋白VEGF的表达水平,B为蛋白α-SMA的表达水平,说明脂肪间充质干细胞凝胶敷料干预后糖尿病创面组织血管生成相关蛋白(VEGF和α-SMA)表达增加,有利于血管生成,发挥促创面愈合作用。
实施例5
以db/db(12周)糖尿病小鼠为实验动物,于22±3℃、湿度45%~60%的环境中适应性饲养7天进行模型建立。小鼠麻醉后,进行脱毛备皮,通过模具在小鼠背部标记直径1cm圆形,剪取全层缺损皮肤组织进行模型建立。脂肪源间充质干细胞水凝胶300μL均匀涂抹于伤口表面,小鼠定期监测血糖,血糖值均大于15mmol/L。术后用Image J(美国国立卫生研究院)拍摄伤口并测量面积。伤口愈合率计算公式为:伤口愈合率(%)=(A0-At)/A0×100%。
实验结果如图4所示,图4中A为糖尿病小鼠创面愈合情况,使用脂肪间充质干细胞凝胶后,从第6天开始,从图中可以明显看出糖尿病小鼠创面愈合效果;图4中B为糖尿病小鼠创面愈合率,从图中可以明显看出,使用脂肪间充质干细胞凝胶干预后创面愈合率显著提高;图4中C为小鼠血糖情况,凝胶对于血糖水平无明显影响。图4结果表明,脂肪间充质干细胞凝胶能够促进糖尿病创面愈合,创面愈合率增加。
实施例6小鼠伤口愈合实验
取部分db/db糖尿病小鼠皮肤标本,10%甲醛固定,进行脱水处理。具体为:75%乙醇(4h)→80%乙醇(4h)→95%乙醇Ⅰ(2h)→95%乙醇Ⅱ(2h)→100%乙醇Ⅰ(40min)→100%乙醇Ⅱ(20min)→二甲苯与无水乙醇混合液(1h)→二甲苯(20min)。脱水后用石蜡包埋,再将蜡块切5μm厚切片,脱蜡:二甲苯Ⅰ(8min)→二甲苯Ⅱ(8min)→无水乙醇Ⅰ(5min)→无水乙醇Ⅱ(5min)→95%酒精(5min)。自来水洗5-8遍。苏木素-伊红染色:苏木素染液染色10min→自来水洗5-8遍,反复洗去多余苏木素染液至无色→1%盐酸酒精分化3s→自来水洗、返蓝5min→伊红染液染色3min→自来水洗5-8遍,反复洗去多余伊红染液至无色。乙醇梯度脱水、二甲苯透明:95%乙醇Ⅰ(30s)→95%乙醇Ⅱ(5min)→无水乙醇Ⅰ(5min)→无水乙醇Ⅱ(5min)→二甲苯Ⅰ(8min)→二甲苯Ⅱ(8min)。封片:待浸泡完毕的载玻片取出,滴中性树脂胶盖玻片封片。晾干后于光学显微镜下进行观察,选取每组样本组织的同一区域进行比较。模型组小鼠出现细胞碎片、坏死组织和表皮再生少。然而,在脂肪间充质干细胞水凝胶(200μl)创面局部覆盖干预后,大部分碎片和坏死组织被清除,更完整的表皮再生,皮肤组织恢复良好。
实验结果如图6所示,图6中A为血管生成相关蛋白VEGF表达水平,B为血管生成相关蛋白α-SMA表达水平,脂肪间充质干细胞凝胶干预后VEGF和α-SMA蛋白表达升高,说明脂肪间充质干细胞凝胶有效促进创面血管生成,加快创面愈合;HE染色结果表明,相比于模型对照,脂肪间充质干细胞水凝胶敷料干预后,糖尿病创面组织上皮化更加完整,毛囊、血管和其他皮肤附属物发育,脂肪逐步形成。
综上,本发明能够有利于脂肪间充质干细胞生长,更好的模拟细胞细胞体内生长环境使其呈球状。本发明的凝胶有利于促进脂肪间充质干细胞表达与创面愈合密切相关的TGF-β1、PDGFD的表达。通过小鼠伤口愈合实验,也证明了所述的脂肪间充质干细胞水凝胶具有在动物实验上显著的促进作用,具有较好的应用价值和前景。脂肪间充质干细胞水凝胶能够促进db/db糖尿病小鼠创面愈合相关蛋白VEGF和α-SMA表达水平。本发明提供了脂肪间充质干细胞在皮肤损伤修复中新的使用方法,提供了皮肤损伤修复新策略。
Claims (5)
1.一种脂肪间充质干细胞敷料的制备方法,其特征在于,步骤为:
步骤一、将β-甲壳素纳米纤维悬浮液加入细胞培养基中,β-甲壳素纳米纤维悬浮液与细胞培养基的体积比为2-4:1,使β-甲壳素纳米纤维凝胶化,弃除凝胶外的多余液体,然后将胶原蛋白粉与β-甲壳素纳米纤维凝胶按质量体积比为10%-40%混合均匀,得到β-甲壳素纳米纤维-胶原水凝胶;
步骤二、在β-甲壳素纳米纤维-胶原水凝胶中,按照106-107个/ml加入脂肪间充质干细胞,混合均匀后在脂肪间充质干细胞培养基中培养24-48h,得到脂肪间充质干细胞敷料。
2.根据权利要求1所述的一种脂肪间充质干细胞敷料的制备方法,其特征在于,所述β-甲壳素纳米纤维的制备方法为以鱿鱼软骨为原料,将鱿鱼软骨洗净干燥,分散在0.1mol/L盐酸溶液中脱矿,用清水洗涤至中性后,用4wt%氢氧化钠溶液在80℃下处理进行脱蛋白,然后用清水以18.2MΩ·cm的电阻率洗涤至中性,得到β-甲壳素,将纯化后的β-甲壳素加入到去离子水中,用乙酸酸化至pH 3,然后用超声波在冰水浴中19.5kHz和300W下处理,得到β-甲壳素纳米纤维。
3.根据权利要求1所述的一种脂肪间充质干细胞敷料的制备方法,其特征在于,步骤一中β-甲壳素纳米纤维悬浮液的浓度为0.15-0.35%wt%。
4.根据权利要求1所述的一种脂肪间充质干细胞敷料的制备方法,其特征在于,细胞培养基为DMED培养基、1640培养基或脂肪间充质干细胞专用培养基。
5.一种如权利要求1所述的制备方法制备的脂肪间充质干细胞敷料在制备促进糖尿病创面修复中的药物或生物活性物质的载体材料上的应用。
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