CN112516324A - 一种可降低干细胞悬浮液注射剪切力损伤的添加剂及干细胞悬浮液 - Google Patents
一种可降低干细胞悬浮液注射剪切力损伤的添加剂及干细胞悬浮液 Download PDFInfo
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Abstract
本发明公开了一种可降低干细胞悬浮液注射剪切力损伤的添加剂，其特征在于由以下重量份数的原料组成：透明质酸0.1～5份、II型胶原蛋白水解物1～5份、胆固醇0.01～0.1份、谷胱甘肽0.1～2份和神经酰胺0.1～1份。一种干细胞悬浮液，添加有所述的添加剂。本发明添加了所述添加剂的干细胞悬浮液，不管是通过何种规格的针管，其细胞活率和细胞完整率相对于对照组均得到大幅提升，从而说明添加剂能够保护细胞，降低针头剪切力的损害，进一步保证了细胞的活性，维持较高的细胞活性，能够在到达人体后更好的发挥作用，提升治疗效果，进而说明其在治疗疾病过程中具有重要作用。
Description
技术领域
本发明属于干细胞技术领域，具体的说，涉及一种可降低干细胞悬浮液注射剪切力损伤的添加剂及干细胞悬浮液。
背景技术
干细胞疗法是将祖细胞引入组织或器官，作为对各种疾病和损伤进行治疗的一种手段，利用生物材料作为载体将细胞植入局部组织中以达到再生目的。在过去的四十年中，由于其独特的生物相容性，化学和物理上多样化的水凝胶已经成为再生生物学的标准材料。透明质酸(HA)水凝胶是一种合成的生物材料，可以保护封装的细胞免受炎症和周围巨噬细胞的侵害，并为细胞附着，存活，迁移，生长和增殖提供生物相容性环境。
因为使用目的的不同，我们一般会选择不同的注射器针头。短而粗的针头，可以降低针头中细胞悬液的剪切力对细胞造成的伤害，提高细胞存活率，但是由于进入的深度较浅，干细胞也会向其他器官迁徙，导致靶向器官的干细胞浓度不足。长而细的针头能较好的使干细胞从进入点到达特定的部位，但是针头中细胞悬液的剪切力对细胞造成的伤害较大，也会影响靶向器官的干细胞浓度。
发明内容
为解决以上技术问题，本发明的目的在于提供一种可降低干细胞悬浮液注射剪切力损伤的添加剂及干细胞悬浮液。
本发明目的是这样实现的：
一、一种可降低干细胞悬浮液注射剪切力损伤的添加剂，其关键在于由以下重量份数的原料组成：透明质酸0.1～5份、II型胶原蛋白水解物1～5份、胆固醇0.01～0.1份、谷胱甘肽0.1～2份和神经酰胺0.1～1份。
优选地，上述一种可降低干细胞悬浮液注射剪切力损伤的添加剂，由以下重量份数的原料组成：所述透明质酸1份、II型胶原蛋白水解物2份、胆固醇0.05份、谷胱甘肽1份和神经酰胺0.5份。
优选地，上述透明质酸的分子量为40万
二、一种可降低干细胞悬浮液注射剪切力损伤的添加剂用于制备治疗关节炎、运动性软骨损伤、糖尿病足疮褥疮药物的应用。
三、一种可降低注射剪切力损伤的干细胞悬浮液，其关键在于：添加有上述的添加剂。
优选地，上述添加剂的浓度为1.31～13.1％(质量分数)。
进一步地，上述添加剂的浓度为4.55％，其中所述透明质酸：II型胶原蛋白水解物：胆固醇：谷胱甘肽1份：神经酰胺＝1:2:0.05:1:0.5。
优选地，剂型为注射剂。
四、实验：
(1)细胞和三维细胞培养
离体的骨髓抽吸物通过Ficoll-Paque梯度法分离出BM-MSC。将MSC在补充有10％胎牛血清(FBS)和1x Glutamax的AlphaMEM培养基中培养。将细胞培养物保持在37℃的5％CO 2潮湿培养箱中每两天更换一次气氛和培养基；细胞在相收缩光学显微镜下监测生长。细胞阳性表达常见的MSC特异性细胞表面标记CD105，CD73和CD90，而CD34，CD45，CD19和CD14阴性。一旦细胞达到亚汇合，就用TrypLE收获细胞，并放入新的烧瓶中。为了进行冷冻保存，将BM-MSC重悬于含有PlasmaLyte、10％血清白蛋白和2.5％DMSO的冷冷冻培养基中，等分入冷冻小瓶中，然后通过逐步降低温度至-196℃的最终冰点的步骤冷冻。将冷冻保存的细胞保存在液氮中。在所有实验中均使用了第6代细胞。
(2)细胞活力测定
a、将等量、细胞密度1*107的细胞分别混合所述添加剂、生理盐水作为实验组和对照组，而无任何添加的作为空白组。
b、实验组和对照组分别通过针管一(内径0.7mm*长度32mm)、针管二(1.2*38)、针管三(1.6*38)后，测定其细胞的存活率及完整率；空白组不通过针管直接测定其存活率及细胞完整率。
c、具体测定原理及方法：
通过
活力/细胞毒性试剂盒(Invitrogen，Carlsbad，CA)分析HyStem-C中的细胞存活率，该试剂盒基于可渗透细胞的活细胞染色和不可渗透细胞的活细胞染色。死亡和垂死的细胞，其特征是细胞膜受损。活细胞的特征是存在普遍存在的细胞内酯酶活性，这是通过将几乎无荧光的可透过细胞的钙黄绿素AM酶促转化为钙黄绿素而确定的，钙黄绿素在活细胞中表现为强烈，均匀的绿色荧光(例如495nm)/515nm)。红色成分(乙锭同二聚体-1，EthD-1)是可透过细胞的，因此仅会进入细胞膜受损的细胞。在垂死和死亡的细胞中，与DNA结合后会产生主要的不清楚的红色荧光(ex/em 570nm/602nm)，这是细胞死亡和细胞毒性的有力指标。用这种测定技术，背景荧光水平固有地较低，因为染料在与细胞相互作用之前实际上是无荧光的。培养48小时后，将含有细胞和凝胶的插入片段在1×PBS，pH 7.4中洗涤3次，然后与1×PBS，pH 7.4中的染色溶液(2μM钙黄绿素AM和4μM EthD-1)一起孵育30分钟在室温下。孵育后，将样品在1x PBS中再次洗涤3次，然后在1x PBS中覆盖。用装有Olympus DP71 CCD(Olympus America Inc.，San Jose，CA)的尼康E600荧光显微镜(Nikon Instruments Inc.，Melville，NY)对细胞进行成像，并用普通的绿色和红色成像滤镜对FITC进行染色，放大倍数为10倍。结果见图2、图3、图4、图5、图6、图7；通过图2～7总结得出表1及图1。
表1 1*107cell通过不同规格针管后，细胞活率、细胞完整情况
结果说明：不管是通过何种规格的针管，在添加有本申请添加剂后的干细胞悬浮液，其细胞活率和细胞完整率相对于对照组均得到大幅提升，从而说明添加剂能够保护细胞，降低针头剪切力的损害。
有益效果：
1、经研究发现适当浓度的透明质酸与Ⅱ型胶原蛋白水解物，具有良好的组织相容性，还具有较高的水渗透性，具备一定的强度，类似于生物体的软组织，可以自然的对包裹其中的细胞进行固定，降低针头剪切力的损伤。而且植入体内以后，容易充满整个具有不规则形状的缺损部位，避免细胞流动影响而治疗效果。
2、胆固醇是细胞膜和亚细胞膜的组成部分，可调节细胞的新陈代谢，对细胞起到保护作用。谷胱甘肽有谷氨酸、半胱氨酸和甘氨酸组成，在维持细胞生物功能方面起重要作用，能够激活多种酶，促进糖、脂肪及蛋白质代谢，从而影响细胞的代谢过程。神经酰胺具有抗炎症促进炎症细胞、坏死细胞凋亡的作用，可以增加体内TNF的量，进而引发多种细胞凋亡，从而起到抗炎作用。
3、透明质酸、Ⅱ型胶原蛋白水解物、胆固醇、谷胱甘肽与神经酰胺结合使用，几乎无免疫原性，且具有较好的组织相容性，可以保护细胞免受剪切力的损害，半固体凝胶注射剂可使细胞固定在特定部位，避免细胞的流动造成的磨损，加之该几种试剂对细胞的保护作用，进一步保证了细胞的活性，维持较高的细胞活性，能够在到达人体后更好的发挥作用，提升治疗效果，进而说明其在治疗疾病过程中具有重要作用。
附图说明
图1为实验组、对照组及空白组1*107cell通过不同规格针管后，细胞活率、细胞完整情况的柱状图；
图2为空白组的采样图；
图3为空白组的荧光显微镜成像图；
图4为针管一实验组的采样图；
图5为针管一实验组的荧光显微镜成像图；
图6为针管一对照组的采样图；
图7为针管一对照组的荧光显微镜成像图；
图8为针管二实验组的采样图；
图9为针管二实验组的荧光显微镜成像图；
图10为针管二对照组的采样图；
图11为针管二对照组的荧光显微镜成像图；
图12为针管三实验组的采样图；
图13为针管三实验组的荧光显微镜成像图；
图14为针管三对照组的采样图；
图15为针管三对照组的荧光显微镜成像图；
具体实施方式
下面结合实施例和附图对本发明作进一步说明。
实施例1
一种干细胞悬浮液添加有由以下重量分数的原料组成的添加剂：分子量为40万的透明质酸5％、II型胶原蛋白水解物1％、胆固醇0.1％、谷胱甘肽0.1％和神经酰胺1％。
实施例2
一种干细胞悬浮液添加有由以下重量分数的原料组成的添加剂：分子量为40万的透明质酸0.1％、II型胶原蛋白水解物5％、胆固醇0.01％、谷胱甘肽2％和神经酰胺0.1％。
实施例3
一种干细胞悬浮液添加有由以下重量分数的原料组成的添加剂：分子量为40万的透明质酸3％、II型胶原蛋白水解物4％、胆固醇0.06％、谷胱甘肽1.2％和神经酰胺0.7％。
实施例4
一种干细胞悬浮液添加有由以下重量分数的原料组成的添加剂：分子量为40万的透明质酸1％、II型胶原蛋白水解物2％、胆固醇0.05％、谷胱甘肽1％和神经酰胺0.5％。
实施例5
一种干细胞悬浮液添加有由以下重量分数的原料组成的添加剂：分子量为40万的透明质酸0.1％、II型胶原蛋白水解物1％、胆固醇0.01％、谷胱甘肽0.1％和神经酰胺0.1％。
实施例6
一种干细胞悬浮液添加有由以下重量分数的原料组成的添加剂：分子量为40万的透明质酸5％、II型胶原蛋白水解物5％、胆固醇0.1％、谷胱甘肽2％和神经酰胺1％。
最后需要说明的是，上述描述仅仅为本发明的优选实施例，本领域的普通技术人员在本发明的启示下，在不违背本发明宗旨及权利要求的前提下，可以做出多种类似的表示，这样的变换均落入本发明的保护范围之内。
Claims (8)
1.一种可降低干细胞悬浮液注射剪切力损伤的添加剂，其特征在于由以下重量份数的原料组成：透明质酸0.1～5份、II型胶原蛋白水解物1～5份、胆固醇0.01～0.1份、谷胱甘肽0.1～2份和神经酰胺0.1～1份。
2.根据权利要求1所述的一种可降低干细胞悬浮液注射剪切力损伤的添加剂，其特征在于由以下重量份数的原料组成：所述透明质酸1份、II型胶原蛋白水解物2份、胆固醇0.05份、谷胱甘肽1份和神经酰胺0.5份。
3.根据权利要求1或2所述的一种可降低干细胞悬浮液注射剪切力损伤的添加剂，其特征在于：所述透明质酸的分子量为40万。
4.一种可降低干细胞悬浮液注射剪切力损伤的添加剂在用于制备治疗关节炎、运动性软骨损伤、糖尿病足疮褥疮药物的应用。
5.一种干细胞悬浮液，其特征在于：添加有任一项权利要求1～3所述的添加剂。
6.根据权利要求5所述的一种干细胞悬浮液，其特征在于：所述添加剂的浓度为1.31～13.1％(质量分数)。
7.根据权利要求6所述的一种干细胞悬浮液，其特征在于：所述添加剂的浓度为4.55％，其中所述透明质酸：II型胶原蛋白水解物：胆固醇：谷胱甘肽1份：神经酰胺＝1:2:0.05:1:0.5。
8.根据权利要求5或6或7所述的一种干细胞悬浮液，其特征在于：剂型为注射剂。
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Application publication date: 20210319 |
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