CN110404119A - 羊膜组织工程去免疫原皮肤支架的制备方法 - Google Patents
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Abstract
本发明公开一种羊膜组织工程去免疫原皮肤支架的制备方法,对羊膜进行漂洗消毒,然后浸泡于商品化羊膜细胞培养基中,于37℃的CO2培养箱内培养,72h后将培养的羊膜从培养基中取出,培养后的培养液经离心及0.22um滤膜过滤后分装,于‑80℃冰箱冻存;取出的羊膜用乙醇进行连续梯度脱水3d,无菌风干,按临床需求截成不同规格羊膜贴片,无菌真空包装后备用。本发明所采用的方法能够实现简便、快速、高效、大规模制备医用人工皮肤的目的,且使用的材料为人或猪羊膜,来源广泛,多为生产后的废弃物,成本较低且不存在伦理问题。本发明目前已分别在大鼠及猪(大动物模型)上进行烧伤后皮肤再生验证,效果显著且未见不良免疫反应。
Description
技术领域
本发明涉及一种羊膜组织工程去免疫原皮肤支架的制备方法。
背景技术
产期废弃的羊膜组织是重要的再生医学种子细胞来源之一,其来源广泛、易于获取且无伦理道德争议,是近年的研究热点。羊膜作为一种免疫豁免生物移植物,具有抗炎、抗粘连,减轻疼痛,促进上皮化等生物学特性,早在1910年开始就被应用于烧伤患者及其他外科手术,用于促进表皮化、减轻疼痛和预防感染。近年来,随着对羊膜的进一步认知及羊膜保存制备技术的发展,羊膜再次成为研究及应用的热点,被广泛应用于角膜重建、整形、烧伤救治等领域。通过美国NIH临床数据库检索到利用人羊膜进行创伤修复的临床试验有30余例,其中包含了糖尿病足溃疡、静脉溃疡、皮肤缺损、皮肤烧伤、角膜灼伤等多种难治愈创伤。由此可以看出人羊膜与羊膜细胞外基质作为创伤修复组织工程材料有良好的应用前景。
人羊膜为同种异体天然可降解材料,具有良好的细胞及组织相容性,无血管基质中含有I、Ⅳ型胶原蛋白、层粘连蛋白、纤维粘连蛋白及硫酸肝素蛋白多糖等成分有利于创周细胞的黏附、移行及上皮化,防止细胞凋亡,且无免疫原性。其内富含多种内源性生物活性因子,如碱性成纤维细胞生长因子(bFGF)、血小板生长因子(PDGF)、血管内皮生长因子(VEGF)、血管生成因子、转化生长因子β(TGF-β)、组织金属蛋白酶抑制因子1(TIMP-1)、组织金属蛋白酶抑制因子2(TIMP-2)、肝细胞生长因子(HGF)等,可以促进上皮形成、增生,使伤口快速愈合;通过清除炎症细胞降低炎症反应;抑制纤维组织过度增生,减少疤痕。既可以用作为伤口创面的覆盖物,也可以植入创口,做成引导皮肤再生的组织工程皮肤。
目前羊膜作为皮肤组织修复材料,常用的技术为新鲜羊膜或深低温冷冻羊膜,但仍存在异体细胞引入免疫原性以及保存不便无法随用随取等缺陷。后续一系列脱细胞羊膜皮肤支架应运而生(如CN105688287A,CN105797212A,CN106668955A,CN107019816A,CN107254431A,101330935A,CN107233623A,CN1757717A,CN102327640A等),但这些技术多采用胰酶消化等方法达到去除羊膜细胞的作用,其操作较为繁琐,且难以克服胰酶消化后羊膜呈胶冻状,内含细胞蛋白质等杂质,引起过敏反应,以及覆盖缺损皮肤无法包扎伤口等缺陷。
发明内容
针对上述现有技术存在的问题,本发明提供一种羊膜组织工程去免疫原皮肤支架的制备方法,能够简便、快速、大规模的制备出便于存储及运输的生物类去免疫原性的人工皮肤支架材料。
为了实现上述目的,本发明采用的一种羊膜组织工程去免疫原皮肤支架的制备方法,包括以下步骤:
1)取无病毒和细菌感染的胎盘,在无菌条件下从胎盘内层剥下羊膜,放置到无菌生理盐水中,反复冲洗除去羊膜表面的血迹,并用镊子自羊膜与绒毛膜间的潜在空隙钝性剥离羊膜,用无菌PBS冲洗,上述取材均在无菌条件下完成;
2)将剥离后的羊膜用生理盐水清洗,随后置于含抗生素的生理盐水中浸泡消毒;
3)将消毒后的羊膜置于商品化羊膜细胞培养基中,于37℃的CO2培养箱中培养制备羊膜贴片复性营养液,3天后过滤,分装后置于-80℃保存备用;
4)取出羊膜,经生理盐水清洗后用不同浓度乙醇-生理盐水梯度脱水3天,最后于75%乙醇中浸泡1-24h;
5)将脱水后的羊膜置于无菌超净工作台内风干;
6)按需求裁剪成不同规格羊膜贴片,进行无菌真空包装,阴凉干燥,室温保存备用。
作为改进,所述步骤1)中的胎盘来源于人体或猪。
作为改进,所述步骤2)中采用的抗生素是庆大霉素,加入量是3u/ml,浸泡消毒时间是30min。
作为改进,所述步骤3)中采用0.22um滤膜过滤。
作为改进,所述步骤4)中的乙醇-生理盐水梯度脱水依次是95%乙醇、75%乙醇、50%乙醇。
与现有技术相比,本发明借助羊膜在商品化羊膜细胞培养基(HYCLONE-DME/F-12)中浸泡培养72h,将羊膜内羊膜干细胞及其他细胞所产生的促增殖细胞因子充分释放到干细胞专用培养基中,同时借助乙醇系列(95%乙醇、75%乙醇、50%乙醇)梯度脱水,最终使羊膜完全浸泡于75%乙醇之中,制成方便保存运输的羊膜贴片,同时使羊膜中的细胞及免疫原、蛋白充分变性,且不会对羊膜本身造成机械损伤,还可以有效提高羊膜的韧性;使用前将独立保存的羊膜贴片浸泡于羊膜贴片营养液中(复性2h),在能够有效、快速软化贴片的同时,使多种原始的细胞因子贴附于复性的羊膜贴片(人工皮肤)上,直接敷于患处有助于伤口的快速愈合。
本发明所采用的方法能够实现简便、快速、高效、大规模制备医用人工皮肤的目的,且使用的材料为人或猪羊膜,来源广泛,多为生产后的废弃物,成本较低且不存在伦理问题。本发明目前已分别在大鼠及猪(大动物模型)上进行烧伤后皮肤再生验证,效果显著且未见不良免疫反应。
附图说明
图1为本发明中羊膜的延展效果。
图2为本发明中大鼠的治疗过程;图中,A:大鼠去表皮(面积为4*4.5cm),露出肌肉层;B:贴上人工皮肤支架;C:植入支架后3天,局部水肿;D:植入支架后7天,水肿消失,羊膜贴片与组织完全相容;E:植入支架后15天,创口缩小,局部干燥;F:植入支架后18天,形成皮肤覆盖物;G:植入支架后28天,伤口愈合,表皮已长毛发;H:植入支架后35天,创面毛发正常,浓密光亮;I:为H中大鼠创伤局部剃毛放大,创面疤痕平坦。
图3为本发明中猪皮肤的治疗过程;图中,A:雄性小耳猪,深III度烫伤,烫伤面积11*12cm;B:烫伤3天后开始手术,伤口深1.5cm至肌膜层,植入皮肤支架;C:植入支架后10天,伤口面积6*8cm,伤口无感染,干燥,红润,组织相容,无排斥反应;D:植入支架后20天,伤口面积1*2cm,伤口周围已长毛发;E:植入支架后35天,伤口完全愈合,毛发浓密有光泽。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明了,下面对本发明进行进一步详细说明。但是应该理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限制本发明的范围。
除非另有定义,本文所使用的所有的技术术语和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同,本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
实施例1
一种羊膜组织工程去免疫原皮肤支架的制备方法,包括以下步骤:
1)选取离体胎盘,经血清检测排除病毒和细菌感染,如排除HIV、梅毒、HBV和HCV等感染,在无菌条件下从胎盘的内层剥下羊膜,放置到无菌生理盐水中,反复冲洗除去羊膜表面的血迹,并用镊子自羊膜与绒毛膜间的潜在空隙钝性剥离羊膜,用无菌生理盐水清洗、冲洗,前述取材均在无菌条件下完成;
2)将羊膜用生理盐水清洗2~3遍,随后置于含抗生素(庆大霉素)的生理盐水中浸泡30min,进行消毒处理;
3)将消毒后的羊膜,放置于商品化羊膜细胞培养基HYCLONE-DME/F-12中,在37℃的CO2培养箱内培养(释放生长因子),制备羊膜贴片复性营养液,3天后用0.22um滤膜过滤(去除胶原蛋白,免疫原),分装后置于-80℃保存备用;
其中,采用的商品化羊膜细胞培养基HYCLONE-DME/F-12是在基础细胞培养基(pH6.8±0.3,渗透压299±5%)中添加了体积百分比为2~5%的血浆,基础细胞培养基的具体成分如下表1所示。
表1基础细胞培养基的成分
4)取出羊膜,经生理盐水清洗后依次用95%乙醇-生理盐水、75%乙醇-生理盐水、50%乙醇-生理盐水梯度脱水3天(各浓度分别脱水1天,目的是去除蛋白,灭活免疫原),最终浸泡于75%乙醇中1-24h,该过程在不破坏羊膜基质结构的前提下,有效提高羊膜的韧性,同时还能够有效灭活异体细胞,去除大量异源蛋白及未知免疫原,大大降低了移植过程中发生不良免疫反应的可能性;
5)将乙醇脱水后的羊膜置于无菌超净工作台内风干;
6)按临床需求裁剪成不同规格羊膜贴片,进行无菌真空包装,阴凉干燥处室温保存备用。
使用前,提前取出羊膜贴片,浸泡于分装保存的羊膜贴片复性营养液中,2-24h内方可用于皮肤缺损病人。
本发明中羊膜为单层上皮细胞互相连接构成的薄膜。单层上皮细胞具有分泌羊水的作用。随着胚胎的生长发育,羊膜与绒毛密切紧贴,形成胎膜。胎膜随着羊膜腔逐渐扩大而呈半透明的薄膜,无血管,富有韧性(胎膜也就是羊膜腔的囊壁)。羊膜是胎盘的最内层,其光滑,无血管、神经及淋巴,具有一定的弹性,厚约0.1~0.2mm,在电镜下,其分为五层:上皮层、基底膜、致密层、纤维母细胞层和海绵层,羊膜基底膜和基质层含有大量不同的胶元,主要为i、iii、iv、v、vii型胶原和纤维粘连蛋白、层粘连蛋白等成份,正是这些成份使羊膜可以充当“移植的基底膜”而发挥一种新的健康合适的基质作用来促进上皮化。羊膜移植于缺损的皮表,提供一个含基底和基质成份的胶质支架,使增殖的上皮细胞可在其上扩展及移行;羊膜产生的gb4、gb9、gb11等单克隆抗体,有助上皮细胞的增殖、移行和分化,上皮缺损的修复;并且,羊膜有助于干细胞的增殖并提供一个适合其生长的微环境,羊膜作为一种新型的生物材料,取得了神奇的效果,而且它来源广泛,价格低廉,取材和保存简便,手术操作简单而且几乎不发生免疫排斥反应,十分适宜在基层医院开展。
本发明中羊膜的相关性能测试:
延展性:原始平铺3.5cm,拉开延伸后8.5cm,厚约0.1~0.2mm;延展效果如图1。
应用实施例1
采用本发明的制备方法,先制备羊膜贴片,然后选择8周龄,450g雄性SD大鼠,去表皮露出肌肉层,再植入该羊膜贴片后,最终治愈。
具体如图2所示,A:大鼠去表皮(面积为4*4.5cm),露出肌肉层;B:贴上人工皮肤支架;C:植入支架后3天,局部水肿;D:植入支架后7天,水肿消失,羊膜贴片与组织完全相容;E:植入支架后15天,创口缩小,局部干燥;F:植入支架后18天,形成皮肤覆盖物;G:植入支架后28天,伤口愈合,表皮已长毛发;H:植入支架后35天,创面毛发正常,浓密光亮;I:为H中大鼠创伤局部剃毛放大,创面疤痕平坦。
应用实施例2
采用本发明的制备方法,先制备羊膜贴片,然后选择皮肤烧伤模型,动物编号152707,114天龄,体重11.6kg的雄性滇南小耳猪,在烧伤处植入该羊膜贴片后,最终治愈。
具体如图3所示,A:雄性小耳猪,深III度烫伤,烫伤面积11*12cm;B:烫伤3天后开始手术,伤口深1.5cm至肌膜层,植入皮肤支架;C:植入支架后10天,伤口面积6*8cm,伤口无感染,干燥,红润,组织相容,无排斥反应;D:植入支架后20天,伤口面积1*2cm,伤口周围已长毛发;E:植入支架后35天,伤口完全愈合,毛发浓密有光泽。
采用本发明的制备方法,原料来源丰富,价格低廉,有效减少皮肤供需矛盾;操作便捷,便于储存、运输、商品化制备及临床应用;能够最大限度保存羊膜基质完整性,具有延展性和可塑性(羊膜相关性能测试);去除异体胶原蛋白、相关免疫原,相容性好、无排异反应;有效保存了羊膜活性因子,更有助于伤口愈合,疤痕平整,且能够促进毛囊再生;一次性植入,永久性修复,可减轻病人反复植皮带来的痛苦。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种羊膜组织工程去免疫原皮肤支架的制备方法,其特征在于,包括以下步骤:
1)取无病毒和细菌感染的胎盘,在无菌条件下从胎盘内层剥下羊膜,放置到无菌生理盐水中,反复冲洗除去羊膜表面的血迹,并用镊子自羊膜与绒毛膜间的潜在空隙钝性剥离羊膜,用无菌PBS冲洗,上述取材均在无菌条件下完成;
2)将剥离后的羊膜用生理盐水清洗,随后置于含抗生素的生理盐水中浸泡消毒;
3)将消毒后的羊膜置于商品化羊膜细胞培养基中,于37℃的CO2培养箱中培养制备羊膜贴片复性营养液,3天后过滤,分装后置于-80℃保存备用;
4)取出羊膜,经生理盐水清洗后用不同浓度乙醇-生理盐水梯度脱水3天,最后于75%乙醇中浸泡1-24h;
5)将脱水后的羊膜置于无菌超净工作台内风干;
6)按需求裁剪成不同规格羊膜贴片,进行无菌真空包装,阴凉干燥,室温保存备用。
2.根据权利要求1所述的一种羊膜组织工程去免疫原皮肤支架的制备方法,其特征在于,所述步骤1)中的胎盘来源于人体或猪。
3.根据权利要求1所述的一种羊膜组织工程去免疫原皮肤支架的制备方法,其特征在于,所述步骤2)中采用的抗生素是庆大霉素,加入量是3u/ml,浸泡消毒时间是30min。
4.根据权利要求1所述的一种羊膜组织工程去免疫原皮肤支架的制备方法,其特征在于,所述步骤3)中采用0.22um滤膜过滤。
5.根据权利要求1所述的一种羊膜组织工程去免疫原皮肤支架的制备方法,其特征在于,所述步骤4)中的乙醇-生理盐水梯度脱水依次是95%乙醇、75%乙醇、50%乙醇。
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