CN117599245A - 一种改性壳聚糖基水凝胶包覆干细胞组织工程材料及其制备方法和应用 - Google Patents
一种改性壳聚糖基水凝胶包覆干细胞组织工程材料及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种改性壳聚糖基水凝胶包覆干细胞组织工程材料及其制备方法和应用,属于生物医药技术领域。将壳聚糖双胍盐酸盐溶液、壳聚糖溶液、β‑甘油磷酸钠溶液和鱼皮胶原蛋白溶液混合,调整所得混合溶液的pH值至7.0~7.3,得到改性壳聚糖基水凝胶前液;向所述改性壳聚糖基水凝胶前液中加入脐带间充质干细胞使所述脐带间充质干细胞处于悬浮状态,进行成胶,得到改性壳聚糖基水凝胶包覆干细胞组织工程材料。动物实验结果表明改性壳聚糖基水凝胶包覆干细胞组织工程材料在糖尿病小鼠模型中具有较好的促进胰岛β细胞增殖和降低胰岛炎症潜力。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种改性壳聚糖基水凝胶包覆干细胞组织工程材料及其制备方法和应用。
背景技术
二型糖尿病(type2diabetesmilluts,T2DM)是一种常见的慢性代谢性疾病,其主要特征为胰岛素抵抗、胰岛素相对缺乏所造成的高血糖。长期的胰岛素抵抗和炎症可导致β细胞应激和代偿,持续的高血糖和高血脂会导致β细胞糖脂毒性和β细胞衰竭,进而导致广泛的健康问题,累及血管、肾、眼、足等多个器官慢性损伤,心血管疾病、肾脏疾病和视力下降等并发症不可避免地发生在体内。
T2DM目前的主要治疗方法为合理饮食、适度运动及口服降糖药物,常用的降糖药物主要通过增强外周组织的胰岛素敏感性(如噻唑烷二酮类)、抑制肝脏糖异生(如双胍类)、以葡萄糖依赖方式增加胰岛素分泌(如胰高血糖素样),仅针对减轻症状,即通过增强剩余β细胞的胰岛素分泌和改善T2DM中的胰岛素敏感性,以及通过严格控制的外源性胰岛素疗法使血糖正常化。这并不能起到改善胰岛β细胞损伤、降低慢性炎症来达到保护胰岛的目的。
为了保护胰岛,目前临床上选择直接输注干细胞,虽然来源广泛、成本低廉、对2型糖尿病有一定治疗作用,但是单纯的干细胞移植目前还存在未分化干细胞易形成肿瘤、无法特定停留在目标组织以及存活率低的问题。因此传统的干细胞输注难以满足日常和临床需求。
发明内容
有鉴于此,本发明的目的在于提供一种改性壳聚糖基水凝胶包覆干细胞组织工程材料的制备方法及其应用。本发明提供的方法制得的改性壳聚糖基水凝胶包覆干细胞组织工程材料能够免受患者免疫系统的影响,干细胞存活率高,可以定点停留生长,避免未分化干细胞形成肿瘤的风险发生。
为了解决上述技术问题,本发明提供以下技术方案:
本发明提供了一种改性壳聚糖基水凝胶包覆干细胞组织工程材料的制备方法,包括以下步骤:
将壳聚糖双胍盐酸盐溶液、壳聚糖溶液、β-甘油磷酸钠溶液和鱼皮胶原蛋白溶液混合,调整所得混合溶液的pH值至7.0~7.3,得到改性壳聚糖基水凝胶前液;
向所述改性壳聚糖基水凝胶前液中加入脐带间充质干细胞使所述脐带间充质干细胞处于悬浮状态,进行成胶,得到改性壳聚糖基水凝胶包覆干细胞组织工程材料;
所述壳聚糖、壳聚糖双胍盐酸盐和鱼皮胶原蛋白的质量比为1~8:6~15:0.2~1;
所述β-甘油磷酸钠和壳聚糖双胍盐酸盐的质量比为1.1~1.8:0.08~0.13;所述脐带间充质干细胞的数量与改性壳聚糖基水凝胶前液的体积比为5×105~1.5×106个:1mL。
优选地,所述壳聚糖双胍盐酸盐溶液的质量体积浓度为1.5~2%,所述壳聚糖双胍盐酸盐溶液由壳聚糖双胍盐酸盐溶于水得到;
所述壳聚糖溶液的质量体积浓度为1.5~2%,所述壳聚糖溶液由壳聚糖溶于醋酸得到;
所述β-甘油磷酸钠溶液的质量体积浓度为48~50%,所述β-甘油磷酸钠溶液由β-甘油磷酸钠溶于水得到;
所述鱼皮胶原蛋白溶液的质量体积浓度为0.5~1%,所述鱼皮胶原蛋白溶液由鱼皮胶原蛋白溶于水得到。
优选地,所述壳聚糖双胍盐酸盐的制备方法包括以下步骤:
将壳聚糖溶于盐酸中,进行质子化反应,得到壳聚糖盐酸盐溶液;
将所述壳聚糖盐酸盐溶液与胍基化试剂混合,进行加成反应,得到壳聚糖双胍盐酸盐。
优选地,所述壳聚糖与盐酸的固液比为2~6g:70~120mL;所述胍基化试剂与壳聚糖盐酸盐溶液的固液比为:1~5g:120~170mL。
优选地,所述胍基化试剂为异硫脲、异脲、异硫氰酸酯和双氰胺中的一种或几种。
优选地,制备所述壳聚糖双胍盐酸盐所用壳聚糖为95%脱乙酰度的壳聚糖;
所述盐酸的浓度为0.2~0.3mol/L。
优选地,所述加成反应的温度为80~110℃,时间为0.5~2h;
所述质子化反应的温度为40~60℃,时间为0.5~2h。
优选地,所述成胶的温度为35~45℃,保温时间为3~10min。
本发明还提供了上述技术方案所述的制备方法制备得到的改性壳聚糖基水凝胶包覆干细胞组织工程材料,所述改性壳聚糖基水凝胶包覆干细胞组织工程材料为多孔网状结构;所述改性壳聚糖基水凝胶包覆干细胞组织工程材料中脐带间充质干细胞的浓度为5×105~1.5×106个/mL。
本发明还提供了上述技术方案所述的改性壳聚糖基水凝胶包覆干细胞组织工程材料在制备减轻胰岛炎症、促进胰岛β细胞增殖药物中的应用。
本发明提供了一种改性壳聚糖基水凝胶包覆干细胞组织工程材料的制备方法,包括以下步骤:将壳聚糖双胍盐酸盐溶液、壳聚糖溶液、β-甘油磷酸钠溶液和鱼皮胶原蛋白溶液混合,调整所得混合溶液的pH值至7.0~7.3,得到改性壳聚糖基水凝胶前液;向所述改性壳聚糖基水凝胶前液中加入脐带间充质干细胞使所述脐带间充质干细胞处于悬浮状态,进行成胶,得到改性壳聚糖基水凝胶包覆干细胞组织工程材料;所述壳聚糖、壳聚糖双胍盐酸盐和鱼皮胶原蛋白的质量比为1~8:6~15:0.2~1;所述β-甘油磷酸钠和壳聚糖双胍盐酸盐的质量比为1.1~1.8:0.08~0.13;所述脐带间充质干细胞的数量与改性壳聚糖基水凝胶前液的体积比为5×105~1.5×106个:1mL。本发明使用壳聚糖双胍盐酸盐可降低胰岛素抵抗作用,利用壳聚糖中的胺基与鱼胶原蛋白中的羧基、与β-甘油磷酸钠磷酸根之间产生静电相互作用力,将胶原蛋白分子穿插在成胶网络中,发挥其生物学功能,利用壳聚糖双胍盐酸盐(为壳聚糖改性后得到),增加胺基数量,进一步增强静电相互作用力,并提高了水凝胶材料的亲水性,利于细胞生长;壳聚糖的羟基、胺基使壳聚糖分子间以及与水分子形成氢键,氢键的强度在壳聚糖改性后增强,同时鱼胶原蛋白羧基端、β-甘油磷酸钠的甘油部分与水分子之间也会形成氢键;壳聚糖分子链上疏水基团(-CH2)之间会发生疏水相互作用,在静电相互作用、氢键作用、疏水相互作用三种物理作用力交联,得到的水凝胶内部均呈多孔网状结构且孔径大小不均一,内部结构更加致密,且具有流变性,实现细胞正常的营养吸收和信息交流,利于细胞的黏附以及细胞间的相互作用,使其具有良好的生物相容性;同时得到的改性壳聚糖基水凝胶包覆干细胞组织工程材料在体外是可流动的溶胶状态,在37℃的条件下就会固化形成凝胶,通过定点注射,让溶胶在体内特定部位形成凝胶,实现定点停留,利用改性壳聚糖基水凝胶包裹干细胞,避免干细胞过度生长,进而避免了致癌性。
溶胀率测定结果表明改性壳聚糖基水凝胶保水性能良好;体外降解测定结果表明改性壳聚糖基水凝胶生物降解性能良好;流变学特征表明其储能模量>损耗模量,呈固态弹性特征;细胞黏附实验和活死细胞染色实验结果表明改性壳聚糖基水凝胶对小鼠脐带间充质干细胞具有良好的促进黏附和正常存活分裂效果;动物实验结果表明改性壳聚糖基水凝胶包覆干细胞组织工程材料在糖尿病小鼠模型中具有较好的促进胰岛β细胞增殖和降低胰岛炎症潜力。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中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性的前提下,还可以根据这些附图获得其他的附图。
图1为壳聚糖双胍盐酸盐X射线衍射、傅里叶变换红外光谱图;
图2为改性壳聚糖基水凝胶外观图;
图3为改性壳聚糖基水凝胶的扫描电镜、孔隙率、溶胀性、体外降解率图;
图4为改性壳聚糖基水凝胶的流变学性能储能模量(G’)和损耗模量(G”)图;
图5为改性壳聚糖基水凝胶的皮下注射皮肤组织病理切片H&E染色图;
图6为改性壳聚糖基水凝胶的细胞粘附和活死细胞染色实验以及改性壳聚糖基水凝胶的细胞骨架染色图;
图7为改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗三个月后小鼠的血糖变化和糖耐量测试,以及小鼠肝脏、胰岛和肾脏的H&E、油红O和马松染色图;
图8为改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗三个月后小鼠的胰岛素、胰高血糖素、Ki67、PDX1的免疫荧光染色图及蛋白杂交图;
图9为改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗三个月后小鼠胰岛iNOS、CD163的免疫荧光染色图及蛋白杂交图。
具体实施方式
本发明提供了一种改性壳聚糖基水凝胶包覆干细胞组织工程材料的制备方法,包括以下步骤:
将壳聚糖双胍盐酸盐溶液、壳聚糖溶液、β-甘油磷酸钠溶液和鱼皮胶原蛋白溶液混合,调整所得混合溶液的pH值至7.0~7.3,得到改性壳聚糖基水凝胶前液;
向所述改性壳聚糖基水凝胶前液中加入脐带间充质干细胞使所述脐带间充质干细胞处于悬浮状态,进行成胶,得到改性壳聚糖基水凝胶包覆干细胞组织工程材料;
所述壳聚糖、壳聚糖双胍盐酸盐和鱼皮胶原蛋白的质量比为1~8:6~15:0.2~1;
所述β-甘油磷酸钠和壳聚糖双胍盐酸盐的质量比为7.0~7.3;所述脐带间充质干细胞的数量与改性壳聚糖基水凝胶前液的体积比为5×105~1.5×106个:1mL。
在本发明中,未经特殊说明,所用原料均为本领域熟知的市售商品。
本发明将壳聚糖双胍盐酸盐溶液、壳聚糖溶液、β-甘油磷酸钠溶液和鱼皮胶原蛋白溶液混合,调整所得混合溶液的pH值至7.0~7.3,得到改性壳聚糖基水凝胶前液。
在本发明中,所述壳聚糖双胍盐酸盐溶液的质量体积浓度优选为1.5~2%,更优选为1.8~2%,进一步优选为2%;所述壳聚糖双胍盐酸盐溶液由壳聚糖双胍盐酸盐溶于水得到。
在本发明中,所述壳聚糖双胍盐酸盐的制备方法优选包括以下步骤:
将壳聚糖溶于盐酸中,进行质子化反应,得到壳聚糖盐酸盐溶液;
将所述壳聚糖盐酸盐溶液与胍基化试剂混合,进行加成反应,得到壳聚糖双胍盐酸盐。
本发明将壳聚糖溶于盐酸中,进行质子化反应,得到壳聚糖盐酸盐溶液。
在本发明中,所述壳聚糖优选为95%脱乙酰度的壳聚糖。
在本发明中,所述盐酸的浓度优选为0.2~0.3mol/L,在本发明中,所述壳聚糖与盐酸的固液比优选为2~6g:70~120mL,更优选为3~5g:90~110mL。所述质子化反应的温度优选为40~60℃,更优选为45~55℃,进一步优选为50℃,时间优选为0.5~2h,更优选为1~1.5h,进一步优选为1h。在本发明中,所述质子化反应优选在搅拌条件下进行。本发明在所述质子化反应过程中,壳聚糖中的氨基在盐酸的作用下质子化,形成壳聚糖盐酸盐。
得到壳聚糖盐酸盐溶液后,本发明将所述壳聚糖盐酸盐溶液与胍基化试剂混合,进行加成反应,得到壳聚糖双胍盐酸盐。
在本发明中,所述胍基化试剂优选为异硫脲、异脲、异硫氰酸酯和双氰胺中的一种或几种,更优选为双氰胺。在本发明中,所述胍基化试剂与壳聚糖盐酸盐溶液的固液比优选为1~5g:120~170mL,更优选为2~4g:130~160mL。
在本发明中,所述加成反应的温度优选为80~110℃,更优选为90~100℃,时间优选为0.5~2h,更优选为1~1.5h。在本发明中,所述加成反应优选在pH值为2~3的条件下进行。本发明优选向壳聚糖盐酸盐溶液与胍基化试剂的混合体系中加入盐酸调节pH值至2~3。在本发明中,所述盐酸的浓度优选为0.1~0.4M。本发明将加成反应的pH值控制在上述范围,能够增加壳聚糖的溶解度,使壳聚糖与双氰胺充分反应,增加胍的取代度。
以双氰胺为例,所述壳聚糖经质子化反应和加成反应形成壳聚糖双胍盐酸盐的具体反应方程式如下:
在本发明中,所述加成反应结束后优选还包括将反应后料液依次进行冷却、减压抽滤、向所得滤液中加入无水乙醇析出沉淀、固液分离,对所得固体进行洗涤、干燥和研磨,得到壳聚糖双胍盐酸盐。本发明对所述固液分离的方式没有特殊要求,采用本领域熟知的固液分离方式即可,具体如抽滤。在本发明中,所述洗涤采用的洗涤剂优选为无水乙醇。本发明对所述减压抽滤、干燥和研磨没有特殊要求,使用本领域熟知的方式均可。
在本发明中,所述壳聚糖溶液的质量体积浓度优选为1.5~2%,更优选为1.8~2%,进一步优选为2%,所述壳聚糖溶液优选由壳聚糖溶于醋酸得到。壳聚糖不溶于水,将壳聚糖溶于醋酸溶液中一方面有利于将最后成胶的凝胶pH值控制在7.2~7.3,接近人体pH范围。另一方面醋酸可将壳聚糖完全溶解,使溶液清澈。
在本发明中,所述β-甘油磷酸钠溶液的质量体积浓度优选为48~50%,更优选为50%,所述β-甘油磷酸钠溶液优选由β-甘油磷酸钠溶于水得到。β-甘油磷酸钠在凝胶中作为物理交联剂,提供了羧基和羟基,与壳聚糖的胺基和羟基一起形成氢键,使凝胶前体液在37℃形成凝胶。
在本发明中,所述鱼皮胶原蛋白溶液的质量体积浓度优选为0.5~1%,更优选为0.5%,所述鱼皮胶原蛋白溶液优选由鱼皮胶原蛋白溶于水得到。鱼皮胶原蛋白作为短链穿插在壳聚糖形成的凝胶网络中,一方面提高凝胶的机械强度,使凝胶的机械强度更加接近适合体内注射需要的强度;另一方面利用鱼皮胶原蛋白的亲水性使凝胶具备更好的生物相容性且更适合包裹的干细胞的生长。
在本发明中,所述壳聚糖、壳聚糖双胍盐酸盐和鱼皮胶原蛋白的质量比为1~8:6~15:0.2~1,优选为5~7:12~15:0.3~0.5。所述β-甘油磷酸钠和壳聚糖双胍盐酸盐的质量比优选为1.1~1.8:0.08~0.13,更优选为1.2~1.7:0.09~0.11。
在本发明中,所述调整所得混合溶液的pH值优选至7.0~7.3,更优选7.1~7.2。本发明优选使用碳酸氢钠调节所述混合溶液的pH值。
在本发明中,所述将壳聚糖双胍盐酸盐溶液、壳聚糖溶液、β-甘油磷酸钠溶液和鱼皮胶原蛋白溶液混合优选包括:将壳聚糖双胍盐酸盐和壳聚糖混合后,依次加入β-甘油磷酸钠和鱼皮胶原蛋白。
胶原蛋白优选在加完壳聚糖-壳聚糖双胍盐酸盐、β-甘油磷酸钠后加入,使壳聚糖中的胺基与鱼胶原蛋白中的羧基、与β-甘油磷酸钠磷酸根之间产生静电相互作用力。将胶原蛋白分子穿插在成胶网络中,发挥其生物学功能。在本发明中,壳聚糖双胍盐酸盐(为壳聚糖改性后得到),增加胺基数量,进一步增强静电相互作用力;壳聚糖的羟基、胺基使壳聚糖分子间以及与水分子形成氢键,氢键的强度在壳聚糖改性后增强,同时鱼胶原蛋白羧基端、β-甘油磷酸钠的甘油部分与水分子之间也会形成氢键;壳聚糖分子链上疏水基团(-CH2)之间会发生疏水相互作用。
得到改性壳聚糖基水凝胶前液后,本发明向所述改性壳聚糖基水凝胶前液中加入脐带间充质干细胞使所述脐带间充质干细胞处于悬浮状态,进行成胶,得到改性壳聚糖基水凝胶包覆干细胞组织工程材料。
在本发明中,所述脐带间充质干细胞通过购买市售产品或自行制备获得均可,当自行制备得到时,本发明对所述脐带间充质干细胞的制备方法没有特殊要求,采用本领域熟知的制备方法即可。在本发明的实施例中,所述脐带间充质干细胞的制备方法优选为:将怀孕母鼠麻醉后解剖,取出小鼠并剪下脐带,取出其中的华通氏胶,无菌处理后加入胶原酶,酶解后再次无菌处理后先润洗培养瓶底部,将细胞接种于细胞培养瓶中,利用鼠脐带间充质干细胞专用培养基培养并纯化细胞,待细胞融合到80%,经过鉴定、传代、培养后得干细胞。
在本发明中,所述脐带间充质干细胞优选以细胞培养液的形式使用,培养液组分优选为:胎牛血清、高糖培养基和双抗,体积比优选为10~15%:80~85%:0.8~1%。
在本发明中,所述细胞培养液中脐带间充质干细胞的浓度优选为1.0×106个/mL。在本发明中,所述脐带间充质干细胞的数量与改性壳聚糖基水凝胶前液的体积比为5×105~1.5×106个:1mL,更优选为1.0×106个:1mL。本发明对所述脐带间充质干细胞处于悬浮状态采用的手段没有特殊要求,使用本领域熟知将细胞均匀分布在溶液中的方式均可。在本发明中,向所述改性壳聚糖基水凝胶前液中加入脐带间充质干细胞后,优选使用移液枪轻轻吹打几次,使得细胞均匀分布在于改性壳聚糖基水凝胶前液中,细胞分布均匀也能够有利于细胞的生长。
在本发明中,所述成胶的温度优选为35~45℃,更优选为37~42℃,保温时间优选为3~10min,更优选为5~7min。
本发明还提供了上述方案所制备的改性壳聚糖基水凝胶包覆干细胞组织工程材料,所述改性壳聚糖基水凝胶包覆干细胞组织工程材料为多孔网状结构;所述改性壳聚糖基水凝胶包覆干细胞组织工程材料中脐带间充质干细胞的浓度为5×105~1.5×106个/mL。
本发明还提供了上述改性壳聚糖基水凝胶包覆干细胞组织工程材料在制备减轻胰岛炎症、促进胰岛β细胞增殖药物中的应用。
本发明对所述应用的具体方式没有特殊的限定,采用本领域技术人员熟知的方式即可。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合具体实例对本发明的技术方案进行清楚、完整地描述,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。凡是依据本发明的技术实质和一般原理,在没有做出创造性劳动前提下对本发明实施方式作的任何修改、等同替换、改进等均应在本发明的保护范围之内。
实施例1
制备脐带间充质干细胞
将怀孕母鼠麻醉后解剖,取出小鼠并剪下脐带,取出其中的华通氏胶,无菌处理后加入胶原酶,酶解后再次无菌处理后先润洗培养瓶底部,将细胞接种于细胞培养瓶中,培养液的组分为15%的胎牛血清,84%的高糖培养基,1%的双抗,利用鼠脐带间充质干细胞专用培养液培养并纯化细胞,待细胞融合到80%,经过鉴定、传代、培养后得干细胞。其中,脐带间充质干细胞在培养液中的浓度为1×106个/mL。
实施例2
改性壳聚糖基水凝胶的制备
向100mL浓度为0.2mol/L的盐酸中加入4.0g壳聚糖,50℃水浴1h搅拌至完全溶解,加入3g双氰胺,在90℃温度条件下搅拌1h至完全反应,同时滴加2mL 0.1M盐酸调节pH值2~3,冷却后进行减压抽滤,抽滤后的滤饼再用无水乙醇洗涤2~3遍,进行真空干燥后研磨,得到壳聚糖双胍盐酸盐粉末。
称取2g壳聚糖双胍盐酸盐粉末溶解于100mL去离子水中,制得壳聚糖双胍盐酸盐溶液。
称取2g壳聚糖溶解于100mL醋酸溶液中,制得壳聚糖溶液。
称取0.125g鱼皮胶原蛋白溶解于25mL去离子水中,制得鱼皮胶原蛋白溶液。
称取17.714gβ-甘油磷酸钠,用去离子水定容至25mL,制得β-甘油磷酸钠溶液。
称取2g碳酸氢钠溶解于100mL去离子水中,制得碳酸氢钠溶液。
将6mL 2%(m/v)壳聚糖双胍盐酸盐溶液与3mL 2%(m/v)壳聚糖溶混合均匀,依次加入3mL 50%(m/v)β-甘油磷酸钠溶液、1mL 0.5%(m/v)鱼皮胶原蛋白溶液和2mL 2%(m/v)碳酸氢钠溶液,制得改性壳聚糖基水凝胶前液,加热到37℃条件下反应5min成胶,得到改性壳聚糖基水凝胶,记为Gel-3。
对比例1
10mL 2%(m/v)的壳聚糖,4mL 50%(m/v)的β-甘油磷酸钠、1mL 0.5%(m/v)的鱼皮胶原蛋白和8mL 1%(m/v)碳酸氢钠溶液,加热到37℃条件下反应5min成胶,记为Gel-1。
对比例2
6mL 2%(m/v)的壳聚糖双胍盐酸盐,3mL 2%(m/v)的壳聚糖、3mL50%(m/v)的甘油磷酸钠和2mL 2%(m/v)碳酸氢钠溶液,制得改性壳聚糖基水凝胶前液,加热到37℃条件下反应5min成胶,记为Gel-2。
实施例3
改性壳聚糖基水凝胶包覆干细胞组织工程材料的制备
取3mL 1×106个/mL培养液中的脐带间充质干细胞进行离心,得到细胞沉淀块,将细胞沉淀块加到3mL改性壳聚糖基水凝胶前液中,加入脐带间充质干细胞后用移液枪抽吸几次轻轻混匀,当看到细胞均匀的悬浮在改性壳聚糖基水凝胶前液中,加热到37℃条件下反应5min成胶,得到改性壳聚糖基水凝胶包覆干细胞组织工程材料。
理化性能测定指标评定
扫描电镜:为观察到改性壳聚糖基水凝胶内部完整结构,先将水凝胶样品冷冻干燥48h后得到干燥凝胶样品。用液氮脆断干燥凝胶样品,真空喷金20min于其表面以提高导电率。用扫描电镜进行观察,电压10kV。
孔隙率测试:孔隙率通过溶剂替代法测定,将水凝胶冻干到恒重(Wd),并将干燥后的样品浸泡在无水乙醇中过夜,次日取出样品用滤纸拭去表面水分并立即称重(Ww)。材料的孔隙率(ε%)通过以下公式计算:
其中,V:测试样品体积;
ρ:无水乙醇溶液密度。
溶胀性能测试:利用称重法测定溶胀性。取冻干的水凝胶样品适量,准确称量其质量(Wd),室温下浸泡在磷酸盐(PBS)缓冲液中,每隔2h将样品拿出,滤纸吸干其表面水分,准确称取其质量(Ws)。溶胀率(SWw)按以下公式计算:
其中,Ws:溶胀后凝胶的质量;
Wd:冻干凝胶的初始质量。
体外降解性能测试:制备改性壳聚糖基水凝胶样品并冻干称重,将冻干后的水凝胶置于2%溶菌酶溶液中(以pH为7.2的PBS缓冲液溶解溶菌酶)。于37℃,100rpm的条件下进行降解,在1、3、5、7、11、15天取出水凝胶,冻干后计算质量损失。
流变学性能测试:运用高级旋转流变仪(MCR92 Anton Paar奥地利)运用振荡模式,仪器的工作频率为1Hz,应变振幅0.01%,测定从15℃到45℃水凝胶样品的的储能模量(G’)、耗能模量(G”)随温度的变化关系。
以动态频率扫描法(0.01%应变)测量改性壳聚糖基水凝胶的储能模量(G’)和损耗模量(G”)。参数:温度37℃;扫描范围0.1~10Hz。
图3为改性壳聚糖基水凝胶的扫描电镜、孔隙率、溶胀性、体外降解率图。如图3所示,水凝胶均呈现疏松多孔网络样结构,孔隙率达到85%,网孔之间的相互贯通性较好。水凝胶疏松多孔结构不仅能够贮存细胞、有利于水分子的进出,还可以保证细胞之间的营养交换,有利于细胞代谢和存活。有利于进一步的3D培养细胞。
通过溶胀测试得出,改性壳聚糖基水凝胶在10h后的溶胀率为169%,在3h后基本达到溶胀平衡。溶胀率更高的原因是因为壳聚糖双胍盐酸盐的加入使凝胶的交联更紧密,使材料具备了更高的溶胀率。较高的溶胀率使凝胶具备较高的保水性,有利于提高水凝胶的生物相容性。
体外降解实验采用溶菌酶溶液进行,通过计算改性壳聚糖基水凝胶降解率分析水凝胶的降解行为,水凝胶三天后的降解率达到81%,在第5、7、11、15天分别为85%、88%、90%、92%。表明制备的水凝胶拥有良好的可生物降解性能,在体内多种酶的作用下,可很好代谢出体内。
图4为改性壳聚糖基水凝胶的流变学性能储能模量(G’)和损耗模量(G”)图。结果如图4所示,改性壳聚糖基水凝胶的G’远高于G”,因此改性壳聚糖基水凝胶呈现出弹性固态的特质。改性壳聚糖基水凝胶当温度超过一定温度时,G’和G”的曲线相交,这表明水凝胶经历了溶胶-凝胶转变。随着温度的进一步升高,G’远大于G”,这表明水凝胶在形成凝胶状态时表现出固体性质。对比例1、2中Gel-1和Gel-2的相转化温度分别在42℃、37℃,而改性壳聚糖基水凝胶Gel-3转化温度在34℃左右。结果表明,Gel-3比对比例1~2得到的水凝胶更适合在生理条件下的应用。
实施例4
改性壳聚糖基水凝胶皮下注射实验
将C57BL/6J小鼠作为体内安全性评价的模型,将配制改性壳聚糖基水凝胶的原料用0.22μm滤膜除菌,把改性壳聚糖基水凝胶前液注射到小鼠皮下特定位置,在小鼠体温温度下,特定位置成胶。在第1、7、14、21天处死小鼠,收集注射周围皮肤组织,通过H&E染色观察皮下注射区域周围皮肤组织炎症浸润情况。
图5为改性壳聚糖基水凝胶的皮下注射皮肤组织病理切片H&E染色图。从图5可以看出,改性壳聚糖基水凝胶前液植入小鼠皮下1、7、14、21天,在材料周围均未见炎性细胞浸润,皮肤组织排列整齐,形态正常,说明改性壳聚糖基水凝胶生物相容性良好,可以用于体内移植实验。
实施例5
改性壳聚糖基水凝胶的细胞粘附和活死细胞染色实验及细胞骨架染色实验
改性壳聚糖基水凝胶表面的细胞黏附:将脐带间充质干细胞浓度调整为1×106个/mL。将配制改性壳聚糖基水凝胶的原料过0.22μm滤膜除菌。在六孔板中加入3mL改性壳聚糖基水凝胶前液,将改性壳聚糖基水凝胶前液放入37℃培养箱成胶,得到改性壳聚糖基水凝胶;成胶后每孔加入1mL培养液中的脐带间充质干细胞和4mL培养液,培养两天后吸出3mL原培养液加入3mL新培养液。细胞在改性壳聚糖基水凝胶表面培养三天后吸出培养基,再加入0.5mL4%多聚甲醛作组织固定液,固定30min,然后进行乙醇梯度脱水(10%,30%,50%,70%,85%,90%,100%)。冻干后置于扫描电镜下观察。
活死细胞染色实验:将配制改性壳聚糖基水凝胶前液的原料用0.22μm滤膜除菌,取6mL培养液中的脐带间充质干细胞离心后得到的胞沉淀块,与6mL改性壳聚糖基水凝胶前液混匀后置于24孔板中,每孔500μL,然后将24孔板放入37℃培养箱成胶,成胶后每孔加入2mL培养液,在培养的第2、4、8天进行活死细胞染色。
细胞骨架染色实验:将配制改性壳聚糖基水凝胶前液的原料用0.22μm滤膜除菌,每个共聚焦皿加入500μL水凝胶前体液,37℃培养箱成胶,得到改性壳聚糖基水凝胶;成胶后将1mL培养液中的脐带间充质干细胞接种于水凝胶表面,加入2mL培养基,培养24h后补充1mL培养液继续培养,在培养48h后对细胞核和细胞骨架进行染色,在共聚焦显微镜下观察。
图6为改性壳聚糖基水凝胶的细胞粘附和活死细胞染色实验以及改性壳聚糖基水凝胶的细胞骨架染色图。实验结果如图6所示,接种到改性壳聚糖基水凝胶表面的脐带间充质干细胞,培养3天后可见脐带间充质干细胞黏附表面,且伸展良好,细胞舒展地平铺在凝胶表面。胞体伸出突起且出现分枝和伪足,提示复合水凝胶具有良好的脐带间充质干细胞黏附能力,说明该改性壳聚糖基水凝胶能为细胞提供良好的生长环境。
细胞活死染色结果显示,绿色荧光所负染的细胞为活细胞,在改性壳聚糖基水凝胶包覆中的脐带间充质细胞形态正常,细胞在各组水凝胶中都呈现相对均匀分布的状态,形态为椭圆形,在不同接种浓度下,在培养的2、4、8天都有大量细胞存活,与细胞增殖实验的结果一致,该结果进一步说明了改性壳聚糖基水凝胶的生物相容性良好。
改性壳聚糖基水凝胶和接种到改性壳聚糖基水凝胶表面的脐带间充质干细胞,共培养48h后利用鬼笔环肽对细胞染色后共聚焦显微镜观察,镜下可见脐带间充质干细胞形成丝状伪足,明显伸展呈现出椭圆形或多边形形态。同时可观察到细胞中有两个细胞核,说明水凝胶不仅可以让细胞黏附且不影响细胞的分裂。
空腹血糖测定:将建模成功的糖尿病小鼠随机分为3组,每组10只。开始正式给药之前,首先将各小鼠进行称重并测量血糖,然后对应编号。随机分为糖尿病对照组,不进行干细胞治疗,只注射PBS缓冲液(即2型糖尿病组);脐带间充质干细胞治疗组(即脐带间充质干细胞+PBS组)、改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗组(即脐带间充质干细胞+水凝胶组)。注射量为每只小鼠50μL。并选取健康小鼠作为正常对照组,注射PBS缓冲液作为参照。各组小鼠分别在实验前及给药后第0天及每七天禁食不禁水后,同一时间段监测小鼠尾静脉空腹血糖水平直到实验结束。
腹腔注射糖耐量实验:治疗8周后各组小鼠完成空腹血糖值测定后,经自由饮水但禁食12h后,给予小鼠腹腔注射糖耐量试验。尾静脉采血测量0min时的空腹血糖值,然后每组小鼠给予注射相同剂量(2g/kg)的葡萄糖溶液,测定30min、60min和120min时间点的血糖值。
组织切片染色:为了探索改性壳聚糖基水凝胶包覆干细胞组织工程材料对2型糖尿病模型小鼠的肝脏和胰脏在形态学方面的作用和影响,本发明对小鼠肝脏、胰脏和肾脏组织进行了H&E染色。
免疫荧光染色:为了探索改性壳聚糖基水凝胶包覆干细胞组织工程材料对2型糖尿病模型小鼠的胰岛的作用和影响,本发明对小鼠胰岛进行了免疫荧光染色。
蛋白杂交:为了探索改性壳聚糖基水凝胶包覆干细胞组织工程材料对2型糖尿病模型小鼠的胰岛胰岛素、Ki67、PDX1、iNOS、CD163表达量的影响,本发明对小鼠胰岛进行了蛋白杂交染色。
图7为改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗三个月后小鼠的血糖变化和糖耐量测试,以及小鼠肝脏、胰岛和肾脏的H&E、油红O和马松染色图。如图7所示,健康组小鼠的血糖一直处于比较稳定的状态,2型糖尿病组、脐带间充质干细胞+PBS组、脐带间充质干细胞+水凝胶组小鼠在给药前与给药期间其空腹血糖始终保持高血糖状态,说明糖尿病病情逐渐加。脐带间充质干细胞+PBS组、脐带间充质干细胞+水凝胶组空腹血糖水平比2型糖尿病+PBS组低,说明干细胞治疗对糖尿病血糖有一定程度的疗效及控制作用。
注射葡萄糖后各个实验组小鼠的血糖值在30min时均出现最高值。健康组小鼠的腹腔注射糖耐量正常,即在60min时基本恢复正常值,90min后能恢复正常。对比而言,脐带间充质干细胞+PBS组、2型糖尿病组、脐带间充质干细胞+水凝胶组小鼠血糖在30min出现最高值后一直持续保持在较高水平,该特征为糖耐量降低的具体表现。脐带间充质干细胞+水凝胶组小鼠血糖值在30min达到最高水平后,随着时间的推移而逐渐降低,到120min时基本可恢复至接受糖负荷前的水平。
如图7所示,治疗13周结束时,四组的肝脏和胰腺的H&E染色结果。正常对照组的肝细胞排列整齐,而2型糖尿病组、脐带间充质干细胞+PBS组和脐带间充质干细胞+水凝胶组的小鼠均具有不同程度的肝细胞小泡性脂变。在2型糖尿病组中,肝细胞出现以细胞核为中心的泡状脂肪变性,细胞质中充满小的脂滴。与2型糖尿病组肝组织变性相比,脐带间充质干细胞+PBS组和脐带间充质干细胞+水凝胶组的肝组织变性有一定程度的改善。从胰腺组织的分析来看,正常对照组胰腺中的胰岛结构保持了完整性,具有许多β细胞的胰岛清晰可见。2型糖尿病组胰岛β细胞肥大,有空泡变性。脐带间充质干细胞+PBS组和脐带间充质干细胞+水凝胶组,胰岛形态退化得到改善,胰岛β细胞形态更清晰,细胞大小更均匀。
如图7所示,由于脂肪量的不同,四个实验组显示出明显的不同颜色。在治疗13周结束时,正常对照组小鼠肝细胞排列有序,细胞边界清晰,无脂肪肝细胞内可见沉积物。相反2型糖尿病组的脂肪呈暗红色,这表明2型糖尿病小鼠已经有严重的脂肪肝损伤。脐带间充质干细胞+PBS组也观察到脂肪沉积,但程度与2型糖尿病组不同。与2型糖尿病组和脐带间充质干细胞+PBS组的病理现象相比,脐带间充质干细胞+水凝胶组的肝细胞形态和大小更均匀,脂肪沉积更少。
如图7所示,治疗3个月后,正常组小鼠肾小球细胞结构完整清晰,可见正常近曲小管。细胞的边界清晰,排列整齐致密,形状和大小正常规则。2型糖尿病组肾小球细胞数量减少,肾小球和肾小管之间的边界不清楚,肾小球周围的染色变浅,表明肾小管坏死。近曲小管管腔增大。与2型糖尿病组相比,脐带间充质干细胞+PBS和脐带间充质干细胞+水凝胶组肾小球细胞数量大量恢复,肾小管坏死大大减少,肾小管形态恢复正常。
如图7所示,治疗3个月后,马松染色结果显示,正常组小鼠肾脏中胶原纤维的分布非常小,2型糖尿病组胶原纤维的分配多于其他三组,治疗后,与2型糖尿病组相比,脐带间充质干细胞+PBS和脐带间充质干细胞+水凝胶组中肾胶原纤维的沉积显著减少。结果表明,脐带间充质干细胞+PBS、脐带间充质干细胞+水凝胶治疗在一定程度上减轻了肾损伤。
图8为改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗三个月后小鼠的胰岛素、胰高血糖素、Ki67、PDX1的免疫荧光染色图及蛋白杂交图。从图8可以看出,正常对照组胰岛结构完整,呈粗糙的圆形。在胰岛周围,有一圈分泌胰高血糖素的α细胞。在2型糖尿病组中,每片β胰岛的数量和大小明显减少,胰岛的形态和结构完整性也受到破坏。脐带间充质干细胞+PBS和脐带间充质干细胞+水凝胶组均恢复了胰岛形态和结构完整性。与2型糖尿病组相比,脐带间充质干细胞+PBS和脐带间充质干细胞+水凝胶组的胰岛β细胞数量显著增加,表明脐带间充质干细胞治疗对胰岛β细胞具有显著的修复作用。
如图8所示,另一种免疫荧光染色是胰岛核抗原Ki67,反映胰岛β细胞的增殖和胰岛素分泌。正常对照组的胰岛中有一定数量的Ki67阳性细胞和大量的胰岛素分泌,而2型糖尿病组的Ki67阴性细胞和胰岛素分泌很少。与2型糖尿病组相比,脐带间充质干细胞+PBS和脐带间充质干细胞+水凝胶组胰岛中Ki67阳性细胞的数量和胰岛素分泌明显增加。特别是脐带间充质干细胞+水凝胶组胰岛中Ki67阳性细胞显著增加,提示脐带间充质干细胞+水凝胶治疗可提高胰岛β细胞对2型糖尿病的增殖能力。
如图8所示,在正常对照组中,大多数胰岛的β细胞复制水平较低(2-3PDX1/胰岛)。在2型糖尿病组中,PDX1阳性细胞很少(0.88PDX1/胰岛)。脐带间充质干细胞+PBS组的PDX1阳性细胞数量增加(5.8个PDX1/胰岛),与脐带间充质干细胞+PBS组相比,脐带间充质干细胞+水凝胶组的PDX1阳性细胞数量进一步增加(11.7个PDX1+胰岛),这表明脐带间充质干细胞+水凝胶处理可显著提高β细胞的增殖和分化能力。蛋白杂交表现出相同的趋势,进一步证明脐带间充质干细胞+水凝胶处理可显著提高β细胞的增殖和分化能力。
如图8所示的蛋白质印迹分析显示了类似的结果。2型糖尿病组的胰岛素表达水平低于其他三组。脐带间充质干细胞+PBS组和脐带间充质干细胞+水凝胶组的胰岛素表达水平显著高于正常对照组和2型糖尿病组,证明脐带间充质干细胞治疗可以保护β细胞。2型糖尿病组、脐带间充质干细胞+PBS组和脐带间充质干细胞+水凝胶组的Ki67表达水平显著高于正常对照组。脐带间充质干细胞+水凝胶组Ki67的表达水平明显高于2型糖尿病组和脐带间充质干细胞+PBS组,表明脐带间充质干细胞+水凝胶可以恢复β细胞的增殖能力。
如图8所示β细胞转录因子PDX1的蛋白质印迹分析结果表明,脐带间充质干细胞+水凝胶可提高β细胞PDX1的表达水平,保护小鼠胰岛功能,与上述免疫荧光染色结果一致。
图9为改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗三个月后小鼠胰岛iNOS,CD163的免疫荧光染色图及蛋白杂交图。如图9所示,每个胰岛iNOS阳性细胞的免疫荧光检测统计显示,正常对照组iNOS阳性率为4.1%,2型糖尿病组为13.8%,脐带间充质干细胞+PBS组为13.2%,脐带间充质干细胞+水凝胶组为10%。除正常对照组组外,脐带间充质干细胞+水凝胶组iNOS阳性细胞率在三个实验组中最低。表明使用改性壳聚糖基水凝胶包覆干细胞组织工程材料治疗,可以在一定程度上减轻2型糖尿病小鼠的炎症反应。
对每个胰岛中CD163阳性细胞的免疫荧光检测统计显示,正常对照组CD163阳性率为6.35%,2型糖尿病组为3.4%,脐带间充质干细胞+PBS组为9.5%,脐带间充质干细胞+水凝胶组为9.4%。脐带间充质干细胞+PBS和脐带间充质干细胞+水凝胶组CD163细胞的阳性率无显著差异,但两组CD163阳性率均显著高于2型糖尿病组,提示脐带间充质干细胞治疗可提高2型糖尿病小鼠抗炎细胞因子的表达,减轻2型糖尿病小鼠的炎症反应。
如图9所示,与2型糖尿病组和正常对照组相比,脐带间充质干细胞+PBS组和脐带间充质干细胞+水凝胶组的胰岛iNOS的表达水平显著降低。结果表明,脐带间充质干细胞治疗可降低2型糖尿病小鼠M1型巨噬细胞的表达和胰岛的炎症反应。CD163蛋白质印迹结果显示,脐带间充质干细胞+PBS组和脐带间充质干细胞+水凝胶组的CD163表达水平明显高于2型糖尿病组。表明治疗可增加2型糖尿病小鼠M2型巨噬细胞的表达,减轻小鼠胰岛炎症,这与上述免疫荧光染色的结果一致。
图1为壳聚糖双胍盐酸盐X射线衍射、傅里叶变换红外光谱图。壳聚糖双胍盐酸盐的结构测定步骤如下:将壳聚糖双胍盐酸盐磨成粉末,备用,采用X-射线单晶衍射仪,将壳聚糖和壳聚糖双胍盐酸盐的粉末进行测试,扫描范围5~80°。通过傅里叶红外光谱利用溴化钾压片将壳聚糖和壳聚糖双胍盐酸盐的粉末进行测试。结果如图1所示。壳聚糖双胍盐酸盐在2θ为12°和24°时衍射峰的强度都明显减弱,说明了壳聚糖的结晶度要明显高于壳聚糖双胍盐酸盐,而壳聚糖双胍盐酸盐中胍基取代所形成的化学键破坏了分子内的氢键。傅里叶红外光谱结果表明壳聚糖双胍盐酸盐在1643cm-1处出现了强吸收峰,为胍基C=N·HCl的C=N伸缩振动峰,在1643cm-1附近的C=N伸缩振动吸收峰明显强于壳聚糖,说明产物仲胺类基团增多,壳聚糖发生胍基化反应。此外3387cm-1的宽峰是-OH的伸缩振动吸收峰与N-H的伸缩振动吸收峰重叠而成的多重吸收峰。这个位置的宽峰还说明,这些羟基和氨基存在着强弱不同的分子内和分子间氢键,峰宽的差异,又反映了氢键的强弱。而壳聚糖双胍盐酸盐中明显地见到这个峰变尖锐了,即壳聚糖分子内部氢键遭破坏。由此可以得出,壳聚糖中的-NH2发生了取代反应,壳聚糖双胍盐酸盐中含有胍基C=N·HCl基团。同样表明了壳聚糖氨基上发生了胍化反应。
改性壳聚糖基水凝胶包覆干细胞组织工程材料在用于糖尿病小鼠治疗过程中,内脏组织切片、胰岛免疫荧光染色、蛋白杂交结果均显示其对于促进胰岛β细胞修复具有显著效果。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种改性壳聚糖基水凝胶包覆干细胞组织工程材料的制备方法,其特征在于,包括以下步骤:
将壳聚糖双胍盐酸盐溶液、壳聚糖溶液、β-甘油磷酸钠溶液和鱼皮胶原蛋白溶液混合,调整所得混合溶液的pH值至7.0~7.3,得到改性壳聚糖基水凝胶前液;
向所述改性壳聚糖基水凝胶前液中加入脐带间充质干细胞使所述脐带间充质干细胞处于悬浮状态,进行成胶,得到改性壳聚糖基水凝胶包覆干细胞组织工程材料;
所述壳聚糖、壳聚糖双胍盐酸盐和鱼皮胶原蛋白的质量比为1~8:6~15:0.2~1;
所述β-甘油磷酸钠和壳聚糖双胍盐酸盐的质量比为1.1~1.8:0.08~0.13;所述脐带间充质干细胞的数量与改性壳聚糖基水凝胶前液的体积比为5×105~1.5×106个:1mL。
2.根据权利要求1所述的制备方法,其特征在于,所述壳聚糖双胍盐酸盐溶液的质量体积浓度为1.5~2%,所述壳聚糖双胍盐酸盐溶液由壳聚糖双胍盐酸盐溶于水得到;
所述壳聚糖溶液的质量体积浓度为1.5~2%,所述壳聚糖溶液由壳聚糖溶于醋酸得到;
所述β-甘油磷酸钠溶液的质量体积浓度为48~50%,所述β-甘油磷酸钠溶液由β-甘油磷酸钠溶于水得到;
所述鱼皮胶原蛋白溶液的质量体积浓度为0.5~1%,所述鱼皮胶原蛋白溶液由鱼皮胶原蛋白溶于水得到。
3.根据权利要求1所述的制备方法,其特征在于,所述壳聚糖双胍盐酸盐的制备方法包括以下步骤:
将壳聚糖溶于盐酸中,进行质子化反应,得到壳聚糖盐酸盐溶液;
将所述壳聚糖盐酸盐溶液与胍基化试剂混合,进行加成反应,得到壳聚糖双胍盐酸盐。
4.根据权利要求3所述的制备方法,其特征在于,所述壳聚糖与盐酸的固液比为2~6g:70~120mL;所述胍基化试剂与壳聚糖盐酸盐溶液的固液比为:1~5g:120~170mL。
5.根据权利要求3所述的制备方法,其特征在于,所述胍基化试剂为异硫脲、异脲、异硫氰酸酯和双氰胺中的一种或几种。
6.根据权利要求3所述的制备方法,其特征在于,制备所述壳聚糖双胍盐酸盐所用壳聚糖为95%脱乙酰度的壳聚糖;
所述盐酸的浓度为0.2~0.3mol/L。
7.根据权利要求3所述的制备方法,其特征在于,所述加成反应的温度为80~110℃,时间为0.5~2h;
所述质子化反应的温度为40~60℃,时间为0.5~2h。
8.根据权利要求1所述的制备方法,其特征在于,所述成胶的温度为35~45℃,保温时间为3~10min。
9.权利要求1~8所述的制备方法制备得到的改性壳聚糖基水凝胶包覆干细胞组织工程材料,其特征在于,所述改性壳聚糖基水凝胶包覆干细胞组织工程材料为多孔网状结构;所述改性壳聚糖基水凝胶包覆干细胞组织工程材料中脐带间充质干细胞的浓度为5×105~1.5×106个/mL。
10.权利要求9所述改性壳聚糖基水凝胶包覆干细胞组织工程材料在制备减轻胰岛炎症、促进胰岛β细胞增殖药物中的应用。
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