CN112716976B - 含脐带间充质干细胞的纳米复合水凝胶及其制备方法 - Google Patents
含脐带间充质干细胞的纳米复合水凝胶及其制备方法 Download PDFInfo
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Abstract
本发明属于干细胞技术领域,特别涉及含脐带间充质干细胞的纳米复合水凝胶及其制备方法。本发明将脐带间充质干细胞和纳米技术进行结合,并且对脐带间充质干细胞培养液中的细胞因子进行有效的利用,经过纳米技术处理的透明质酸纳米凝胶能够有效地促进营养成分透过皮层,增强皮肤对营养物质的吸收。本发明制备方法工艺简单,反应条件温和,易于操作,成本较低。
Description
技术领域
本发明属于干细胞技术领域,特别涉及含脐带间充质干细胞的纳米复合水凝胶及其制备方法。
背景技术
间充质干细胞(MSC)是一类具有多向分化潜能的成体干细胞,可以从骨髓、外周血、脂肪、脐带等多种组织中获得,间充质干细胞不仅可以分泌大量的生物活性因子,而且细胞内也含有丰富的生物活性物质,可以有效调控机体信号传导、活化人体干细胞,进而生理性修复或替代机体损伤、衰老细胞等,目前间充质干细胞在细胞移植、基因治疗、美容护肤等方面越来越受到人们的关注。研究发现干细胞生长因子如成纤维细胞生长因子(FGF),角质细胞生长因子(KGF),表皮生长因子(EGF),血管内皮细胞生长因子(VEGF)等能诱导皮肤细胞包括成纤维细胞与角质细胞的增殖及皮肤附属器官的再生,具有良好的美容修复能力。但作为蛋白大分子,这些细胞因子透皮性能很差,常规涂抹无法达到有效透皮剂量,难以取得理想的皮损修复效果,且细胞因子体外稳定性差、保存条件苛刻,常温易失活,因此其临床应用受到了极大的限制。
发明内容
针对上述现有技术的不足,本发明提供了含脐带间充质干细胞的纳米复合水凝胶的制备方法,目的是为了解决现有技术中,干细胞的营养因子皮性能很差,常规涂抹无法达到有效透皮剂量,难以取得理想的皮损修复效果,且细胞因子体外稳定性差、保存条件苛刻,常温易失活,因此其临床应用受到了极大的限制的技术问题。
本发明提供的含脐带间充质干细胞的纳米复合水凝胶的制备方法,具体技术方案如下:
脐带间充质干细胞的纳米复合水凝胶的制备方法,包括如下步骤:
S1,脐带间充质干细胞的原代分离和培养;
S2,将脐带间充质干细胞培养混合液中的细胞与上清液进行分离,获取培养液和脐带间充质干细胞;
S3,将步骤S2中的培养液与透明质酸钠进行混合,采用高压均质法制备透明质酸钠纳米水凝胶;
S4,将纳米水凝胶与步骤S2中分离的脐带间充质干细胞进行孵育,加入胰酶消化,离心,用PBS重悬,获得含脐带间充质干细胞的纳米复合水凝胶。
在某些实施方式中,步骤S1中,脐带间充质干细胞的原代分离和培养的具体步骤如下:
S11,获取去掉血管组织的脐带组织,将脐带组织剪碎,加入含FBS和双抗的培养基中培养,得到原代人脐带间充质干细胞;
S12,当细胞融合度为80%-90%时,胰蛋白酶消化细胞传代培养,取传代细胞,继续培养;
S13,当细胞融合度80%-90%时,弃含FBS的培养上清液,PBS洗涤细胞,加入无血清培养基继续培养。
在某些实施方式中,步骤S2中,将第5-10代的脐带间充质干细胞培养混合液中的细胞与上清液进行分离,所述上清液经过浓缩、过滤获得培养液。
在某些实施方式中,步骤S3包括如下步骤:
S31,将透明质酸钠和聚乙二醇-400溶解于步骤S2中的培养液中,形成水相;
S32,将步骤S31中的水相加入有机相中,经过高压均质处理得到初乳;
S33,步骤S32中的初乳经过减压蒸发除去有机溶剂后加入水性介质,再经过高压均质处理,获得纳米水凝胶。
在某些实施方式中,步骤S31中,所述透明质酸钠的浓度为0.2-0.4%,所述聚乙二醇-400的浓度为0.1%-10%;
步骤S32中,所述水相与所述有机相的体积比为1:1-1:8,所述有机相为二氯甲烷、丙酮、环己烷中的一种或多种;
步骤S33中,所述高压均质处理的工艺参数如下:压力为800-1200bar,循环3-10次。
在某些实施方式中,步骤S4中,所述孵育时间为3-5h,所述纳米水凝胶的浓度为0.05-0.2mM。
本发明还提供了根据上述的方法制备的含脐带间充质干细胞的纳米复合水凝胶。
此外,本发明还提供了含脐带间充质干细胞的纳米复合水凝胶的应用,上述方案中的含脐带间充质干细胞的纳米复合水凝胶,将所述含脐带间充质干细胞的纳米复合水凝胶附在微针上,用于肌肤的皮下注射。
本发明具有以下有益效果:本发明将脐带间充质干细胞和纳米技术进行结合,并且对脐带间充质干细胞培养液中的细胞因子进行有效的利用,经过纳米技术处理的透明质酸纳米凝胶能够有效地促进营养成分透过皮层,增强皮肤对营养物质的吸收。本发明制备方法工艺简单,反应条件温和,易于操作,成本较低。本发明提供的含脐带间充质干细胞的纳米复合水凝胶有预防和修复皮肤损伤,延缓衰老等功效,注入皮肤真皮层可以促进皮肤细胞新陈代谢,修复和替换老化细胞,具有良好的应用前景。
附图说明
图1是本发明提供的含脐带间充质干细胞的纳米复合水凝胶的制备方法的流程图。
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白,以下结合具体实施例,并参照附图1,对本发明进一步详细说明。
实施例1
本实施例提供的含脐带间充质干细胞的纳米复合水凝胶的制备方法,具体的方案如下:
一、脐带间充质干细胞的原代分离和培养
收集健康产妇的脐带到无菌管中,在超净工作台内,将新鲜人脐带剪成5cm的小段,洗净脐带表面残余的血液;剥离脐动脉和脐静脉后,将脐带置于含双抗的DEME培养基中,剪成3mm左右的组织块;将剪碎的组织转移到50mL离心管中,300g离心5min;小心弃去上层培养基,加入与组织等体积的混合消化酶,于37℃摇床中,振荡消化1.5h。混合消化酶含Ⅱ型胶原酶2g/L、中性蛋白酶0.8g/L、透明质酸酶0.03g/L;将消化后的组织液于300g离心5min,小心弃去上清,PBS洗1次,弃上清,沉淀用DEME完全培养基重悬,接种于数个75cm2培养瓶中,37℃、5%CO2培养箱中培养,每隔3天换液,细胞融合度为85%时,取传代细胞,继续培养。当细胞融合度85%时,弃含FBS的培养上清液,PBS洗涤细胞3次,以3000个/cm2密度接种到T75培养瓶中,加入无血清培养基继续培养。
二、获取培养液和脐带间充质干细胞
取P6代脐带间充质干细胞在血清的培养基中培养,3天后将脐带间充质干细胞与上清液通过离心的方法进行分离,上清液经过0.22μm的微孔过滤,用30kd超滤膜进行浓缩,获得培养液。
三、制备透明质酸钠纳米水凝胶
S31,将透明质酸钠和聚乙二醇-400溶解于培养液中,形成水相,其中透明质酸钠的浓度为0.3%,聚乙二醇-400的浓度为5%;
S32,将步骤S31中的水相加入有机相(二氯甲烷)中,经过高压均质处理得到初乳,其中水相与所述有机相的体积比为1:4;
S33,步骤S32中的初乳经过减压蒸发除去有机溶剂后加入水性介质,再经过高压均质处理(压力为1000bar,循环5次),获得纳米水凝胶。
四、获得含脐带间充质干细胞的纳米复合水凝胶
将浓度为0.05mM纳米水凝胶与脐带间充质干细胞(10000个/ml)进行孵育4h,加入胰酶消化2min,1200r/min离心5min,用PBS重悬,获得含脐带间充质干细胞的纳米复合水凝胶。
本实施例还提供利用上述方法制备的含脐带间充质干细胞的纳米复合水凝胶。
实施例2
透明质酸钠纳米乳
实施例3
本实施例提供的含脐带间充质干细胞的纳米复合水凝胶的制备方法,具体的方案如下:
一、脐带间充质干细胞的原代分离和培养
收集健康产妇的脐带到无菌管中,在超净工作台内,将新鲜人脐带剪成5cm的小段,洗净脐带表面残余的血液;剥离脐动脉和脐静脉后,将脐带置于含双抗的DEME培养基中,剪成3mm左右的组织块;将剪碎的组织转移到50mL离心管中,300g离心5min;小心弃去上层培养基,加入与组织等体积的混合消化酶,于37℃摇床中,振荡消化1.5h。混合消化酶含Ⅱ型胶原酶2g/L、中性蛋白酶0.8g/L、透明质酸酶0.03g/L;将消化后的组织液于300g离心5min,小心弃去上清,PBS洗1次,弃上清,沉淀用DEME完全培养基重悬,接种于数个75cm2培养瓶中,37℃、5%CO2培养箱中培养,每隔3天换液,细胞融合度为90%时,取传代细胞,继续培养。当细胞融合度90%时,弃含FBS的培养上清液,PBS洗涤细胞3次,以3000个/cm2密度接种到T75培养瓶中,加入无血清培养基继续培养。
二、获取培养液和脐带间充质干细胞
取P6代脐带间充质干细胞在血清的培养基中培养,3天后将脐带间充质干细胞与上清液通过离心的方法进行分离,上清液经过0.22μm的微孔过滤,用30kd超滤膜进行浓缩,获得培养液。
三、制备透明质酸钠纳米水凝胶
S31,将透明质酸钠和聚乙二醇-400溶解于步骤S2中的培养液中,形成水相,其中透明质酸钠的浓度为0.4%,聚乙二醇-400的浓度为10%;
S32,将步骤S31中的水相加入有机相(二氯甲烷、丙酮、环己烷)中,经过高压均质处理得到初乳,其中水相与所述有机相的体积比为1:8;
S33,步骤S32中的初乳经过减压蒸发除去有机溶剂后加入水性介质,再经过高压均质处理(压力为1200bar,循环3次),获得纳米水凝胶。
四、获得含脐带间充质干细胞的纳米复合水凝胶
将浓度为 0.2mM纳米水凝胶与脐带间充质干细胞(10000个/ml)进行孵育3h,加入胰酶消化2min,1200r/min离心5min,用PBS重悬,获得含脐带间充质干细胞的纳米复合水凝胶。
本实施例还提供利用上述方法制备的含脐带间充质干细胞的纳米复合水凝胶。
实施例4
选取体重0.5公斤左右豚鼠42只,雌雄各半。然后将豚鼠分为6组,每组7只。除去第6组的豚鼠,其余经脱毛、局麻后,用波长1064nm激光、光斑直径3mm、能量密度12J/cm2、光皮距离7cm、时间30秒照射豚鼠背部,造成深达真皮的损伤。第1组为模型对照组:只照激光不给药;第2组为实施例1组:微针注射实施例1的含脐带间充质干细胞的纳米复合水凝胶0.5uL/cm2,每天给药一次;第3组为实施例2组:微针注射实施例2的含脐带间充质干细胞的纳米复合水凝胶0.5uL/cm2,每天给药一次;第4组为实施例3组:微针注射实施例3的含脐带间充质干细胞的纳米复合水凝胶0.5uL/cm2,每天给药一次;第5组为对比例组:微针注射实施例1脐带间充质干细胞与浓缩后的培养液中5uL/cm 2,每天给药一次;第6组为空白对照组:不照激光不给药。每天肉眼观察上述6组豚鼠的创面脱痂时间和愈合时间。
表1 各组豚鼠创面脱痂时间
表2各组豚鼠愈合时间
从表1和表2中可知,本发明提供的含脐带间充质干细胞的纳米复合水凝胶有效促进受损皮肤的修复与愈合,具有意料不到的效果。
上述仅本发明较佳可行实施例,并非是对本发明的限制,本发明也并不限于上述举例,本技术领域的技术人员,在本发明的实质范围内,所作出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (3)
1.含脐带间充质干细胞的纳米复合水凝胶的制备方法,其特征在于,包括如下步骤:
S1,脐带间充质干细胞的原代分离和培养;
S2,将脐带间充质干细胞培养混合液中的细胞与上清液进行分离,获取培养液和脐带间充质干细胞;
S3,将步骤S2中的培养液与透明质酸钠进行混合,采用高压均质法制备透明质酸钠纳米水凝胶;
S31,将透明质酸钠和聚乙二醇-400溶解于步骤S2中的培养液中,形成水相;所述透明质酸钠的浓度为0.2-0.4%,所述聚乙二醇-400的浓度为0.1%-10%;
S32,将步骤S31中的水相加入有机相中,经过高压均质处理得到初乳;所述水相与所述有机相的体积比为1:1-1:8,所述有机相为二氯甲烷、丙酮、环己烷中的一种或多种;
S33,步骤S32中的初乳经过减压蒸发除去有机溶剂后加入水性介质,再经过高压均质处理,获得纳米水凝胶;所述高压均质处理的工艺参数如下:压力为800-1200bar,循环3-10次;
S4,将纳米水凝胶与步骤S2中分离的脐带间充质干细胞进行孵育,所述孵育时间为3-5h,所述纳米水凝胶的浓度为0.05-0.2mM,加入胰酶消化,离心,用PBS重悬,获得含脐带间充质干细胞的纳米复合水凝胶。
2.根据权利要求1所述的含脐带间充质干细胞的纳米复合水凝胶的制备方法,其特征在于,步骤S1中,脐带间充质干细胞的原代分离和培养的具体步骤如下:
S11,获取去掉血管组织的脐带组织,将脐带组织剪碎,加入含FBS和双抗的培养基中培养,得到原代人脐带间充质干细胞;
S12,当细胞融合度为80%-90%时,胰蛋白酶消化细胞传代培养,取传代细胞,继续培养;
S13,当细胞融合度80%-90%时,弃含FBS的培养上清液,PBS洗涤细胞,加入无血清培养基继续培养。
3.根据权利要求2所述的含脐带间充质干细胞的纳米复合水凝胶的制备方法,其特征在于,步骤S2中,将第5-10代的脐带间充质干细胞培养混合液中的细胞与上清液进行分离,所述上清液经过浓缩、过滤获得培养液。
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Title |
---|
马自超等著.《姜黄素 功能制备及应用研究》.北京:中国轻工业出版社,2020,132-134. * |
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