CN113491713A - 一种脂肪间充质干细胞培养上清凝胶及其制备方法 - Google Patents
一种脂肪间充质干细胞培养上清凝胶及其制备方法 Download PDFInfo
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- CN113491713A CN113491713A CN202110704651.9A CN202110704651A CN113491713A CN 113491713 A CN113491713 A CN 113491713A CN 202110704651 A CN202110704651 A CN 202110704651A CN 113491713 A CN113491713 A CN 113491713A
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- Molecular Biology (AREA)
Abstract
本发明属于医用生物材料技术领域,公开了一种脂肪间充质干细胞培养上清凝胶及其制备方法,所述干细胞培养上清凝胶按照质量份数计,由干细胞培养上清30~50份、甲基纤维素20~30份、壳聚糖15~30份、生物活性玻璃体10~15份、透明质酸钠8~12份、植物精油6~10份、医用凡士林3~5份、凝胶固化剂2~4份、卡波姆0.1~0.5份以及灭菌蒸馏水余量组成。本发明提供的干细胞培养上清凝胶,具备良好的生物学性能,将间充质干细胞、无机活性玻璃和植物精油相结合,能够促进皮肤创面愈合,为创面提供良好的湿润密闭愈合环境,适用于各病种的各种创面及难愈合创面,无异物感,且操作方便。
Description
技术领域
本发明属于医用生物材料技术领域,尤其涉及一种脂肪间充质干细胞培养上清凝胶及其制备方法。
背景技术
目前,创面愈合是一个精细复杂、严格有序的动态过程,包括炎症期、增生期和重塑期,其中任何一个过程出现问题,都会导致创面愈合障碍。目前,修复创面的方法主要是手术,即将开放创口通过手术缝合转变为闭合伤口,从而加速创面愈合。其中,在创面愈合中的关键作用,以调节性功能细胞的方式,间接参与组织损伤的修复重建,主要涉及干细胞激活后以旁分泌形式分泌大量生物活性分子,促进其他修复细胞的功能。
生物活性玻璃研发应用最早源于美国,其主要目的在于研发一种对皮肤及骨组织均具有修复作用的药物。生物活性玻璃是由Hench教授于1971年首次提出并研制出,它是目前唯一一种“同时对软组织与骨组织具有修复和键合作用的”生物活性材料。该材料与其它生物材料相比,有更好的相容性和安全性。临床上,生物活性玻璃主要应用于骨填充、口腔方面,促进胶原、生长因子的分泌,促进上皮化。但现有技术中将间充质干细胞以及生物活性玻璃共同应用于组织创面修复的技术方案尚未见报道。因此,需要一种新的干细胞培养上清凝胶及其制备方法。
通过上述分析,现有技术存在的问题及缺陷为:现有技术中将间充质干细胞以及生物活性玻璃共同应用于组织创面修复的技术方案尚未见报道。
发明内容
针对现有技术存在的问题,本发明提供了一种脂肪间充质干细胞培养上清凝胶及其制备方法。
本发明是这样实现的,一种脂肪间充质干细胞培养上清凝胶的制备方法,所述脂肪间充质干细胞培养上清凝胶的制备方法,包括以下步骤:
步骤一,进行干细胞培养上清的制备:于不含血清的干细胞培养基中培养间充质干细胞;用乙酸乙酯溶液萃取纯化得到的无细胞上清液,得到乙酸乙酯提取液,再经过离心、过滤,冻干,即得干细胞培养上清,备用;
步骤二,进行生物活性玻璃体的制备:将硅凝胶、硝酸钠、磷酸三乙酯、四水硝酸钙和模板剂依次加入至盐酸水溶液中,置于50~60℃环境中,搅拌,得溶胶;将所述溶胶置于55~66℃环境中陈化,得湿凝胶;将所述湿凝胶置于100~120℃环境中干燥,得干凝胶;将所述干凝胶置于450~550℃环境中焙烧2~4h,得到半成品;将所述半成品经气流粉碎,即得纳米级的生物活性玻璃体,备用;
步骤三,进行植物精油的制备:依次称取薰衣草、迷迭香、花梨木和肉豆蔻在内的植物原料;将所述植物原料和所述添加剂置于萃取釜中,于35~60℃和5~30MPa的条件下,注入所述超临界二氧化碳溶剂萃取2~5h得到萃取液;将所述萃取液进行分离提纯处理,萃取液先进入第一级分离器中进行降温处理,再进入第二级分离器中去除二氧化碳,制得所述植物精油,并进行去味,备用;
步骤四,进行凝胶固化剂的制备:依次称取硅基骨架材料、填充剂、引发剂、pH调节剂和乙醇;将硅基骨架材料、填充剂、引发剂、pH调节剂和乙醇在反应釜中进行混溶,设置温度为250~350℃,压力为2~4Mpa,反应4~6h;反应结束后,加水稀释、过滤,滤液静置24~36h后,加入相应量表面活性剂,搅拌、过滤,即得凝胶固化剂,备用;
步骤五,按照质量份数依次称取干细胞培养上清、甲基纤维素、壳聚糖、生物活性玻璃体、透明质酸钠、植物精油、医用凡士林、凝胶固化剂、卡波姆以及灭菌蒸馏水;
步骤六,将制备得到的干细胞培养上清用灭菌蒸馏水溶解,依次加入甲基纤维素、壳聚糖、生物活性玻璃体、透明质酸钠、植物精油、医用凡士林、凝胶固化剂和卡波姆,搅拌混合均匀,即得干细胞培养上清凝胶。
进一步,步骤二中,所述硅凝胶、硝酸钠、磷酸三乙酯、四水硝酸钙和模板剂的质量比为:50~60份:15~20份:2~3份:15~25份。
进一步,步骤二中,所述模板剂为聚乙二醇400、吐温60和/或聚氧乙烯-聚氧丙烯共聚物中的任意一种。
进一步,步骤三中,所述添加剂为聚氧乙烯醚类化合物RO(CH2CH2O)nH,聚合度n的取值为5~10。
进一步,步骤四中,所述硅基骨架材料为甲醇硅烷三醇,所述填充剂为羟基磷灰石、水合铝盐、氯化钾、氯化钠、三氯化铝、氯化钙、水性钛白粉中的任意一种或多种,所述引发剂为过硫酸钾,所述pH调节剂为氨水、碳酸钠、碳酸氢钠、氢氧化钠、氢氧化钙、氢氧化钾中的任意一种,所述表面活性剂为吐温60。
本发明另一目的在于提供一种利用所述脂肪间充质干细胞培养上清凝胶的制备方法制备的脂肪间充质干细胞培养上清凝胶,所述脂肪间充质干细胞培养上清凝胶按照质量份数计,由干细胞培养上清30~50份、甲基纤维素20~30份、壳聚糖15~30份、生物活性玻璃体10~15份、透明质酸钠8~12份、植物精油6~10份、医用凡士林3~5份、凝胶固化剂2~4份、卡波姆0.1~0.5份以及灭菌蒸馏水余量组成。
进一步,所述植物精油由茶树精油、薰衣草精油、迷迭香精油、肉豆蔻精油、花梨木精油、肉桂精油、快乐鼠尾草精油、丁香精油、天竺葵精油、薄荷精油以及柠檬精油中的任意两种或多种复配而成。
进一步,所述灭菌蒸馏水包括重蒸水和/或三蒸水。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:本发明提供的干细胞培养上清凝胶,将间充质干细胞、无机活性玻璃和植物精油相结合,能够促进皮肤创面愈合,能够提高骨细胞诱导分化效果,成脂细胞诱导分化效果、成软骨细胞诱导分化效果,适用于各病种的各种创面及难愈合创面,无异物感,且操作方便。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例中所需要使用的附图做简单的介绍,显而易见地,下面所描述的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的脂肪间充质干细胞培养上清凝胶的制备方法流程图。
图2是本发明实施例提供的生物活性玻璃体的制备方法流程图。
图3是本发明实施例提供的植物精油的制备方法流程图。
图4是本发明实施例提供的凝胶固化剂的制备方法流程图。
图5是本发明实施例提供的骨细胞诱导分化效果示意图。
图6是本发明实施例提供的成脂细胞诱导分化效果示意图。
图7是本发明实施例提供的成软骨细胞诱导分化效果示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种脂肪间充质干细胞培养上清凝胶及其制备方法,下面结合附图对本发明作详细的描述。
本发明实施例提供的脂肪间充质干细胞培养上清凝胶按照质量份数计,由干细胞培养上清30~50份、甲基纤维素20~30份、壳聚糖15~30份、生物活性玻璃体10~15份、透明质酸钠8~12份、植物精油6~10份、医用凡士林3~5份、凝胶固化剂2~4份、卡波姆0.1~0.5份以及灭菌蒸馏水余量组成。
本发明实施例提供的植物精油由茶树精油、薰衣草精油、迷迭香精油、肉豆蔻精油、花梨木精油、肉桂精油、快乐鼠尾草精油、丁香精油、天竺葵精油、薄荷精油以及柠檬精油中的任意两种或多种复配而成。
本发明实施例提供的灭菌蒸馏水包括重蒸水和/或三蒸水。
如图1所示,本发明实施例提供的脂肪间充质干细胞培养上清凝胶的制备方法包括以下步骤:
S101,进行干细胞培养上清的制备;
S102,进行生物活性玻璃体的制备;
S103,进行植物精油的制备;
S104,进行凝胶固化剂的制备;
S105,按照质量份数依次称取干细胞培养上清、甲基纤维素、壳聚糖、生物活性玻璃体、透明质酸钠、植物精油、医用凡士林、凝胶固化剂、卡波姆以及灭菌蒸馏水;
S106,将制备得到的干细胞培养上清用灭菌蒸馏水溶解,依次加入甲基纤维素、壳聚糖、生物活性玻璃体、透明质酸钠、植物精油、医用凡士林、凝胶固化剂和卡波姆,搅拌混合均匀,即得干细胞培养上清凝胶。
本发明实施例提供的不含血清的干细胞培养基按照质量分数计,由1%的胰蛋白胨、0.5%的酵母提取物以及1%的NaCl组成,调节pH值为7~7.5。
如图2所示,本发明实施例提供的步骤S102中,所述生物活性玻璃体的制备,包括:
S301,将硅凝胶、硝酸钠、磷酸三乙酯、四水硝酸钙和模板剂依次加入至盐酸水溶液中,置于50~60℃环境中,搅拌,得溶胶;
S302,将所述溶胶置于55~66℃环境中陈化,得湿凝胶;
S303,将所述湿凝胶置于100~120℃环境中干燥,得干凝胶;
S304,将所述干凝胶置于450~550℃环境中焙烧2~4h,得到半成品;将所述半成品经气流粉碎,即得纳米级的生物活性玻璃体。
本发明实施例提供的硅凝胶、硝酸钠、磷酸三乙酯、四水硝酸钙和模板剂的质量比为:50~60份:15~20份:2~3份:15~25份。
本发明实施例提供的模板剂为聚乙二醇400、吐温60和/或聚氧乙烯-聚氧丙烯共聚物中的任意一种。
如图3所示,本发明实施例提供的步骤S103中,所述植物精油的制备,包括:
S401,依次称取薰衣草、迷迭香、花梨木和肉豆蔻在内的植物原料;
S402,将所述植物原料和所述添加剂置于萃取釜中,于35~60℃和5~30MPa的条件下,注入所述超临界二氧化碳溶剂萃取2~5h得到萃取液;
S403,将所述萃取液进行分离提纯处理,萃取液先进入第一级分离器中进行降温处理,再进入第二级分离器中去除二氧化碳,制得所述植物精油,并进出去味处理。
本发明实施例提供的添加剂为聚氧乙烯醚类化合物RO(CH2CH2O)nH,聚合度n的取值为5~10。
如图4所示,本发明实施例提供的步骤S104中,所述凝胶固化剂的制备,包括:
S501,依次称取硅基骨架材料、填充剂、引发剂、pH调节剂和乙醇;
S502,将硅基骨架材料、填充剂、引发剂、pH调节剂和乙醇在反应釜中进行混溶,设置温度为250~350℃,压力为2~4Mpa,反应4~6h;
S503,反应结束后,加水稀释、过滤,滤液静置24~36h后,加入相应量表面活性剂,搅拌、过滤,即得凝胶固化剂。
本发明实施例提供的硅基骨架材料为甲醇硅烷三醇,所述填充剂为羟基磷灰石、水合铝盐、氯化钾、氯化钠、三氯化铝、氯化钙、水性钛白粉中的任意一种或多种,所述引发剂为过硫酸钾,所述pH调节剂为氨水、碳酸钠、碳酸氢钠、氢氧化钠、氢氧化钙、氢氧化钾中的任意一种,所述表面活性剂为吐温60。
下面结合具体实验对本发明的技术方案作进一步的描述。
骨细胞诱导分化
间充质干细胞经传统成骨诱导培养基诱导培养,应具备分化为成骨细胞的功能。并随着代数的增加,成骨能力逐渐减弱。
成脂细胞诱导分化
间充质干细胞经传统成脂诱导培养基诱导培养,应具备分化为成脂细胞的功能。并随着代数的增加,成脂能力逐渐减弱。
成软骨细胞诱导分化
间充质干细胞经传统成软骨诱导培养基诱导培养,应具备分化为成软骨细胞的功能。并随着代数的增加,成软骨能力逐渐减弱。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
Claims (8)
1.一种脂肪间充质干细胞培养上清凝胶的制备方法,其特征在于,所述脂肪间充质干细胞培养上清凝胶的制备方法,包括以下步骤:
步骤一,进行干细胞培养上清的制备:于不含血清的干细胞培养基中培养间充质干细胞;用乙酸乙酯溶液萃取纯化得到的无细胞上清液,得到乙酸乙酯提取液,再经过离心、过滤,冻干,即得干细胞培养上清,备用;
步骤二,进行生物活性玻璃体的制备:将硅凝胶、硝酸钠、磷酸三乙酯、四水硝酸钙和模板剂依次加入至盐酸水溶液中,置于50~60℃环境中,搅拌,得溶胶;将所述溶胶置于55~66℃环境中陈化,得湿凝胶;将所述湿凝胶置于100~120℃环境中干燥,得干凝胶;将所述干凝胶置于450~550℃环境中焙烧2~4h,得到半成品;将所述半成品经气流粉碎,即得纳米级的生物活性玻璃体,备用;
步骤三,进行植物精油的制备:依次称取薰衣草、迷迭香、花梨木和肉豆蔻在内的植物原料;将所述植物原料和所述添加剂置于萃取釜中,于35~60℃和5~30MPa的条件下,注入所述超临界二氧化碳溶剂萃取2~5h得到萃取液;将所述萃取液进行分离提纯处理,萃取液先进入第一级分离器中进行降温处理,再进入第二级分离器中去除二氧化碳,制得所述植物精油,并进行去味,备用;
步骤四,进行凝胶固化剂的制备:依次称取硅基骨架材料、填充剂、引发剂、pH调节剂和乙醇;将硅基骨架材料、填充剂、引发剂、pH调节剂和乙醇在反应釜中进行混溶,设置温度为250~350℃,压力为2~4Mpa,反应4~6h;反应结束后,加水稀释、过滤,滤液静置24~36h后,加入相应量表面活性剂,搅拌、过滤,即得凝胶固化剂,备用;
步骤五,按照质量份数依次称取干细胞培养上清、甲基纤维素、壳聚糖、生物活性玻璃体、透明质酸钠、植物精油、医用凡士林、凝胶固化剂、卡波姆以及灭菌蒸馏水;
步骤六,将制备得到的干细胞培养上清用灭菌蒸馏水溶解,依次加入甲基纤维素、壳聚糖、生物活性玻璃体、透明质酸钠、植物精油、医用凡士林、凝胶固化剂和卡波姆,搅拌混合均匀,即得干细胞培养上清凝胶。
2.如权利要求1所述的脂肪间充质干细胞培养上清凝胶的制备方法,其特征在于,步骤二中,所述硅凝胶、硝酸钠、磷酸三乙酯、四水硝酸钙和模板剂的质量比为:50~60份:15~20份:2~3份:15~25份。
3.如权利要求1所述的脂肪间充质干细胞培养上清凝胶的制备方法,其特征在于,步骤二中,所述模板剂为聚乙二醇400、吐温60和/或聚氧乙烯-聚氧丙烯共聚物中的任意一种。
4.如权利要求1所述的脂肪间充质干细胞培养上清凝胶的制备方法,其特征在于,步骤三中,所述添加剂为聚氧乙烯醚类化合物RO(CH2CH2O)nH,聚合度n的取值为5~10。
5.如权利要求1所述的脂肪间充质干细胞培养上清凝胶的制备方法,其特征在于,步骤四中,所述硅基骨架材料为甲醇硅烷三醇,所述填充剂为羟基磷灰石、水合铝盐、氯化钾、氯化钠、三氯化铝、氯化钙、水性钛白粉中的任意一种或多种,所述引发剂为过硫酸钾,所述pH调节剂为氨水、碳酸钠、碳酸氢钠、氢氧化钠、氢氧化钙、氢氧化钾中的任意一种,所述表面活性剂为吐温60。
6.一种利用如权利要求1~5任意一项所述脂肪间充质干细胞培养上清凝胶的制备方法制备的脂肪间充质干细胞培养上清凝胶,其特征在于,所述脂肪间充质干细胞培养上清凝胶按照质量份数计,由干细胞培养上清30~50份、甲基纤维素20~30份、壳聚糖15~30份、生物活性玻璃体10~15份、透明质酸钠8~12份、植物精油6~10份、医用凡士林3~5份、凝胶固化剂2~4份、卡波姆0.1~0.5份以及灭菌蒸馏水余量组成。
7.如权利要求6所述脂肪间充质干细胞培养上清凝胶,其特征在于,所述植物精油由茶树精油、薰衣草精油、迷迭香精油、肉豆蔻精油、花梨木精油、肉桂精油、快乐鼠尾草精油、丁香精油、天竺葵精油、薄荷精油以及柠檬精油中的任意两种或多种复配而成。
8.如权利要求7所述脂肪间充质干细胞培养上清凝胶,其特征在于,所述灭菌蒸馏水包括重蒸水和/或三蒸水。
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