CN113616675A - 含间充质干细胞的组合物及其在治疗退行性关节炎的应用 - Google Patents
含间充质干细胞的组合物及其在治疗退行性关节炎的应用 Download PDFInfo
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Abstract
本申请涉及间充质干细胞生物制剂的技术领域，具体公开了一种含间充质干细胞的组合物及其在治疗退行性关节炎的应用。所述组合物中包括：间充质干细胞2×106个/ml‑5×106个/ml；所述组合物以重量分数计，还包括以下组分：透明质酸或其药用盐3％‑5％、人血红蛋白4％‑6％、E‑钙粘附蛋白0.5％‑0.9％、胰岛素生长因子3％‑5％、生理盐水余量。本申请的组合物可用于骨关节炎、退行性关节炎的治疗，其具有治疗效果好，治疗后复发率低的优点。
Description
技术领域
本申请涉及间充质干细胞生物制剂的技术领域，更具体地说，它涉及一种含间充质干细胞的组合物及其在治疗退行性关节炎的应用。
背景技术
退行性骨关节炎称骨关节炎、退行性关节炎、老年性关节炎、肥大性关节炎，是一种退行性病变，系由于增龄、肥胖、劳损、关节先天性异常、关节畸形等诸多因素引起的关节软骨退化损伤、关节边缘和软骨下骨反应性增生。根据调查研究显示，在我国，骨关节患者达到了一亿以上，而骨关节炎中最为常见的便是膝关节炎，根据相关数据显示，膝关节炎致残率全球第二，且发病率会随着年龄的增长而变高，可见，膝关节炎已经是伴随和困扰现代人的一个常见病症。
骨关节炎的特征在于，在数年间软骨发生缓慢的降解。在正常软骨中，在机制的合成和降解之间存在微妙的平衡，但是在骨关节炎中，软骨的降解超过了合成。软骨合成与降解之间的平衡收到年龄的影响，并由滑膜和软骨细胞产生的几种因子，如细胞因子、生长因子、聚集蛋白聚糖酶和基质金属蛋白酶等调控。除了水之外，胞外基质还含有蛋白聚糖，其由连接到透明质酸或其药用盐所形成骨架上的糖胺聚糖形成，并包封于胶原框架或纤丝基质中。关节软骨中一种重要蛋白聚糖是聚集蛋白聚糖，其与透明质酸或其药用盐结合并有助于赋予软骨以可压缩性和弹性。聚集蛋白聚糖被聚集蛋白聚糖酶所切割，这导致其降解以后随后的软骨被侵蚀，软骨基质中聚集蛋白聚糖的损失是骨关节炎中观察到的最初病理生理变化之一。
透明质酸或其药用盐为作为润滑剂和减震器的滑液提供粘弹性，在滑液中，透明质酸或其药用盐覆盖在关节软骨表面并与胶原纤丝和硫酸蛋白聚糖共用软骨中的深部空间，它保护软骨并阻止蛋白聚糖从软骨基质中流失到滑液中，维持正常的软骨基质。在患有骨关节炎的患者膝关节滑液中，透明质酸或其药用盐、糖胺聚糖和硫酸角质素的浓度比正常膝关节滑液中的要低。
间充质干细胞是一种源于基质的异质细胞群，可以从人体的大多数组织获取，大量实验研究显示，间充质干细胞具有表皮细胞分化潜能，可促进受创皮肤的愈合。骨髓源性间充质干细胞由于存在病毒污染的隐患，且随着供者年龄增大，其细胞数量和扩增、分化能力出现明显下降趋势，不适合批量制备。而来源于人脐带、胎盘或羊膜的间充质干细胞，能够避开胚胎干细胞来源缺乏、异体排斥、道德伦理等诸多限制，成为了骨髓源间充质干细胞很好的替代物。间充质干细胞具有自我更新、组织修复、免疫调节能力，可以向中胚层谱系分化，例如分化成脂肪细胞、骨细胞和软骨细胞等，此外还能够向及其他胚层谱系细胞分化，例如向表皮细胞、血管内皮细胞分化，而且间充质干细胞在体外易于扩增，扩增后分化能力、增殖能力保持稳定，适合大规模制备。
现有将间充质干细胞、透明质酸或其药用盐用于治疗骨关节炎的文献报道，然而当间充质干细胞和透明质酸或其药用盐结合使用时，产生的效果却并不好。
发明内容
为了得到治疗骨关节炎的一种含有间充质干细胞和透明质酸或其药用盐的组合物，本申请提供一种含间充质干细胞的组合物及其在治疗退行性关节炎的应用。
第一方面，本申请提供一种含间充质干细胞的组合物，采用如下的技术方案：
一种含间充质干细胞的组合物，所述组合物中包括：间充质干细胞2×106个/ml-5×106个/ml；所述组合物以重量分数计，还包括以下组分：透明质酸或其药用盐3％-5％、人血红蛋白4％-6％、E-钙粘附蛋白0.5％-0.9％、胰岛素生长因子3％-5％、生理盐水余量。
通过采用上述技术方案，间充质干细胞的密度过大会由于组合物营养不足，导致干细胞存活率大幅度降低，而间充质干细胞的密度过小时，会导致组合物在应用过程中，所需要的剂量过大；透明质酸或其药用盐又称为玻尿酸、玻璃酸，是一种高分子的聚合物，透明质酸或其药用盐是构成人体细胞间质、眼玻璃体、关节滑液等结缔组织的主要成分，在体内发挥保水、维持细胞内外空间、调节渗透压、润滑、促进细胞修复的重要生理功能，在组合物中添加透明质酸或其药用盐可以为间充质干细胞提供合适的基质环境，维持间充质干细胞的存活率，且利于间充质干细胞在治疗骨关节炎的过程中，分化为软骨组织；人血红蛋白是人体红细胞内运输氧的特殊蛋白质，由珠蛋白和血红素组成，其珠蛋白部分是由两对不同的珠蛋白链组成的四聚体，在组合物中加入血红蛋白，能够提高组合物进入人体组织液后的氧负载能力，提高间充质干细胞的存活率；E-钙粘附蛋白是一种同亲型结合、钙依赖的细胞粘着糖蛋白，对细胞识别、迁移和组织分化有重要作用；胰岛素生长因子是一类多功能细胞增殖调控因子，在细胞的分化、增殖和生长发育中有重要的促进作用，且胰岛素生长因子有促进骨骼生长的作用；本申请组合物呈水混悬液，生理盐水与人体细胞的渗透压一致，以生理盐水作为溶剂，不会对间充质干细胞产生损伤。
优选的，所述间充质干细胞为脐带来源间充质干细胞。
本申请中脐带来源间充质干细胞的获取方法包括以下步骤：
步骤1、将新鲜干净的人脐带组织，用PBS缓冲液清洗干净后，用镊子玻璃脐带表面的羊膜和血管，用剪刀将脐带组织胶质剪成1-3mm3的组织块，将组织块平铺在培养器皿中；
步骤2、在培养器皿中加入包含10％FBS，100U/ml青霉素，100U/ml链霉素的α-MEM培养基中，将培养器皿置于二氧化碳浓度5％，37℃的培养箱中培养；
步骤3、当培养器皿中贴壁细胞融合率达到70％-80％时，使用消化酶使贴壁细胞脱离培养器皿底部；离心去除上层清液，加入PBS缓冲液重悬后，接种在Xeno-Free人间充质干细胞培养基中传代并扩增培养；培养至细胞融合率达到80％-90％后，即得脐带来源间充质干细胞。
优选的，所述间充质干细胞为脂肪来源间充质干细胞。
本申请中脂肪来源间充质干细胞的获取方法包括以下步骤：
步骤1、抽取人体来源新鲜脂肪，将脂肪转移至分液容器中，加入PBS缓冲液后剧烈摇晃，静止分层后去除下层液体，重复两次后，将上层油层转移至离心管中；
步骤2、在离心管内加入PBS缓冲液后，用1500rpm离心5min后，弃去下层液体，在上层脂肪油层内加入等体积的Ⅰ型胶原酶后，转移至恒温震荡机中，在37℃的温度下恒温消化至脂肪完全消失，再转移至离心机中用1500rpm离心5min，去除上清液；
步骤3、加入PBS缓冲液重悬后，加入间充质干细胞培养基后，间充质干细胞培养基内包括EGF10ng/ml，将其放置在37℃，二氧化碳浓度5％，湿度95％的培养箱中培养至细胞贴壁融合率80％后，进行细胞传代扩增，得到脂肪来源间充质干细胞。
优选的，所述间充质干细胞包含20-30％的成纤维细胞。
优选的，所述间充质干细胞为P3-P10代间充质干细胞。
第二方面，本申请提供一种含间充质干细胞的组合物在治疗退行性关节炎的应用。
综上所述，本申请具有以下有益效果：本申请中的组合物作为水混悬液，通过注射方式注入人体关节处时，能够对骨关节炎起到抑制作用，促进软骨生长，对退行性关节炎有一定的治疗作用。
具体实施方式
以下结合实施例对本申请作进一步详细说明。
制备例
制备例1
本制备例1制备脐带来源间充质干细胞，包括以下步骤：
步骤1、将新鲜干净的人脐带组织，用PBS缓冲液清洗干净后，用镊子玻璃脐带表面的羊膜和血管，用剪刀将脐带组织胶质剪成1-3mm3的组织块，将组织块平铺在培养器皿中；
步骤2、在培养器皿中加入包含10％FBS，100U/ml青霉素，100U/ml链霉素的α-MEM培养基中，将培养器皿置于二氧化碳浓度5％，37℃的培养箱中培养；
步骤3、当培养器皿中贴壁细胞融合率达到70％-80％时，使用消化酶使贴壁细胞脱离培养器皿底部；离心去除上层清液，加入PBS缓冲液重悬后，接种在Xeno-Free人间充质干细胞培养基中传代并扩增培养；培养至细胞融合率达到80％-90％后，即得脐带来源间充质干细胞。
制备例2
本制备例2制备脂肪来源间充质干细胞，包括以下步骤：
步骤1、抽取人体来源新鲜脂肪，将脂肪转移至分液容器中，加入PBS缓冲液后剧烈摇晃，静止分层后去除下层液体，重复两次后，将上层油层转移至离心管中；
步骤2、在离心管内加入PBS缓冲液后，用1500rpm离心5min后，弃去下层液体，在上层脂肪油层内加入等体积的Ⅰ型胶原酶后，转移至恒温震荡机中，在37℃的温度下恒温消化至脂肪完全消失，再转移至离心机中用1500rpm离心5min，去除上清液；
步骤3、加入PBS缓冲液重悬后，加入间充质干细胞培养基后，间充质干细胞培养基内包括EGF10ng/ml，将其放置在37℃，二氧化碳浓度5％，湿度95％的培养箱中培养至细胞贴壁融合率80％后，进行细胞传代扩增，得到脂肪来源间充质干细胞。
实施例
实施例1
本实施例1中含间充质干细胞的组合物，包括间充质干细胞2×106个/ml，且组合物以重量分数计，还包括以下组分，透明质酸或其药用盐3％、人血红蛋白4％、E-钙粘附蛋白0.5％、胰岛素生长因子3％、生理盐水余量。其中，间充质干细胞为制备例1中制备的脐带来源间充质干细胞，且间充质干细胞中包含20-30％的成纤维细胞，且为P3-P10代间充质干细胞。
实施例2
本实施例2中含间充质干细胞的组合物，包括间充质干细胞5×106个/ml，且组合物以重量分数计，还包括以下组分，透明质酸或其药用盐5％、人血红蛋白6％、E-钙粘附蛋白0.9％、胰岛素生长因子5％、生理盐水余量。其中，间充质干细胞为制备例1中制备的脐带来源间充质干细胞，且间充质干细胞中包含20-30％的成纤维细胞，且为P3-P10代间充质干细胞。
实施例3
本实施例3中含间充质干细胞的组合物，包括间充质干细胞4×106个/ml，且组合物以重量分数计，还包括以下组分，透明质酸或其药用盐4％、人血红蛋白5％、E-钙粘附蛋白0.7％、胰岛素生长因子4％、生理盐水余量。其中，间充质干细胞为制备例2中制备的脂肪来源间充质干细胞，且间充质干细胞中包含20-30％的成纤维细胞，且为P3-P10代间充质干细胞。
性能检测试验
试验方法
将实施例1-3中制备的组合物用于临床疗效对比实验。
招募患有骨关节炎的志愿者90人，所有志愿者经临床诊断标准诊断为同等程度或相似程度的疾病患者，将90位志愿者随机均分成3组，分别编号为实验1-3组，分别采用本申请中实施例1-3中的组合物进行治疗，每两个月注射一次，跟踪六个月后，统计无效、有效及有效后复发，其判断标准参照临床标准，统计结果如表1所示。
表1骨关节炎志愿者六个月后的治疗情况
| 组别 | 无效/(人数，％) | 有效不复发/(人数，％) | 有效但复发/(人数，％) |
| 实验1组 | 0，0％ | 23，76.7％ | 7，23.3％ |
| 实验2组 | 0，0％ | 23，76.7％ | 7，23.3％ |
| 实验3组 | 0，0％ | 22，73.3％ | 8，26.7％ |
由表1可知，本申请制备的组合物，能够用于骨关节炎的治疗，且有效不复发率达到了70％以上。
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。
Claims (6)
1.一种含间充质干细胞的组合物，其特征在于，所述组合物中包括：间充质干细胞2×106个/ml-5×106个/ml；所述组合物以重量分数计，还包括以下组分：透明质酸或其药用盐3％-5％、人血红蛋白4％-6％、E-钙粘附蛋白0.5％-0.9％、胰岛素生长因子3％-5％、生理盐水余量。
2.根据权利要求1所述的一种含间充质干细胞的组合物，其特征在于，所述间充质干细胞为脐带来源间充质干细胞。
3.根据权利要求1所述的一种含间充质干细胞的组合物，其特征在于，所述间充质干细胞为脂肪来源间充质干细胞。
4.根据权利要求1-3任一所述的一种含间充质干细胞的组合物，其特征在于：所述间充质干细胞包含20-30％的成纤维细胞。
5.根据权利要求4所述的一种含间充质干细胞的组合物，其特征在于：所述间充质干细胞为P3-P10代间充质干细胞。
6.一种如权利要求1-5任一所述组合物在治疗退行性关节炎的应用。
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