CN111282018B - 一种用于创伤修复的脂肪干细胞组合物 - Google Patents
一种用于创伤修复的脂肪干细胞组合物 Download PDFInfo
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Abstract
一种用于创伤修复的脂肪干细胞组合物，其特征是所述组合物由作为活性成分的重量百分比1~2%的脂肪干细胞培养产物及至少一种可以外用的药用辅料组成，所述的脂肪干细胞培养产物的以如下方法制备：A）将脂肪间充质干细胞，接种于培养基中，B）将接种了脂肪间充质干细胞的培养皿于10~15%CO2，37℃的条件下培养C）培养完成后，去除培养基并收集细胞，清洗细胞，细胞匀浆，匀浆液离心取上清，将上清液置于透析袋中，以4℃PBS透析8~24h后，将透析袋内的溶液冻干，作为脂肪干细胞培养产物。
Description
技术领域
本发明涉及一种用于外用组合物。
背景技术
脂肪间充质干细胞( adipose-derived stemcells，ASCs) 系2001年首次报道从脂肪组织分离获得，因其与骨髓间充质干细胞( bone marrow-derived stem cells，BMSCs) 一样具有高度增殖和自我更新能力，可在脂肪组织中分离获得大量的ASCs，且能多向分化成为中胚层和非中胚层细胞，使其成为成体干细胞在再生医学应用研究的热点，被广泛应用于皮肤组织再生、软骨和骨组织再生、伤口愈合等众多研究领域。
在创伤修复的研究中，如何能抑制乃至消除瘢痕的产生，是现有临床医学的难题。中国专利CN107320768A公开了一种含有诱导多能干细胞提取物的用于创伤修复的辅料，其利用了多能诱生干细胞提取物能对成纤维细胞进行诱生转化能力，其主要效果为促进创伤修复且抑菌，但其对严重皮肤创伤及烫伤的效果较差，尤其是对严重创伤的修复过程中，对疤痕生产的抑制效果较差。
因此如何利用脂肪间充质干细胞（亦称脂肪干细胞）提供一种抑制乃至消除瘢痕的外用组合物成为现有技术中亟待解决的问题。
发明内容
为解决前述技术问题，我们提供的技术方案是：
一种用于创伤修复的脂肪干细胞组合物，所述组合物由作为活性成分的重量百分比1~2%的脂肪干细胞培养产物及至少一种可以外用的药用辅料组成，所述的脂肪干细胞培养产物以如下方法制备：
A）将脂肪间充质干细胞，以3~6×104/ml的密度接种于培养基中，所述培养基为含诱导培养试剂的基础培养基，所述基础培养基含有10%体积分数胎牛血清的低糖DMEM培养基，所述诱导培养试剂为地塞米松、甘草酸二铵、维生素D3和维甲酸，诱导培养试剂在培养基中的用量为：地塞米松0.2~0.3μmol/L，甘草酸二铵10~15μmol/L，维生素D36~10μmol/L，维甲酸1~3μmol/L；
B）将接种了脂肪间充质干细胞的培养皿置于15%~20%CO2、37℃的条件下培养,每3天更换一次培养基，共培养7~8天；
C）培养完成后，离心去除培养基并收集细胞，以PBS清洗细胞2~3次，在4℃条件下将细胞匀浆，将所得匀浆液冷冻至完全冻结，再置于42℃的温水彻底融化，然后离心取上清，将上清液置于透析袋中，以4℃的PBS透析8~24h后，将透析袋内的溶液冻干，作为脂肪干细胞培养产物。
所述的一种用于创伤修复的脂肪干细胞组合物，进一步的，所述组合物为液体敷料，所述可以外用的药用辅料包括壳聚糖、乙酸、羟丙基甲基纤维素、pH调节剂和余量的水。
所述的一种用于创伤修复的脂肪干细胞组合物，进一步的，所述壳聚糖用量占组合物重量百分比分数为1%~3%，优选为1.5%~2.5%；所述羟丙基甲基纤维素用量占组合物的重量百分比分数为0.5~1%；所述壳聚糖的脱乙酰度为85%~95%。
所述的一种用于创伤修复的脂肪干细胞组合物，进一步的所述pH调节剂为能将组合物pH调节为6.5~7.0的pH缓冲剂。
在研究中我们意外的发现，当采用特定浓度范围的地塞米松、甘草酸二铵、维生素D3和维甲酸对脂肪干细胞进行在特定条件下（15%~20%CO2）诱导培养时，可诱导脂肪干细胞产生能够有利于创伤修复的活性物质，以该培养产物作为活性成分，制成的壳聚糖凝胶，在对创伤动物模型的治疗中表现出了更好的创伤修复效果，且上述效果在以特定浓度范围的地塞米松、甘草酸二铵、维生素D3和维甲酸对脂肪干细胞进行诱导培养后较为明显，改变诱导培养试剂和培养条件将导致脂肪干细胞培养产物的创伤修复活性下降，此外，我们还意外的发现，在对兔耳瘢痕模型的治疗过程中，采用本发明提供的组合物，能够显著修复瘢痕，而改变细胞培养条件得到的脂肪干细胞培养产物其瘢痕修复效果也会明显下降。
具体实施方式
1、脂肪干细胞培养产物的制备采用以下方法：
A）将脂肪间充质干细胞，以5×104/ml的接种量接种于培养基中，所述培养基为含诱导培养试剂的基础培养基，所述基础培养基是含有10%体积分数胎牛血清的低糖DMEM培养基，所述诱导培养试剂为地塞米松、甘草酸二铵、维生素D3和维甲酸，诱导培养试剂在培养基中的用量为：地塞米松（A1）μmol/L，甘草酸二铵（A2）μmol/L，维生素D3（A3）μmol/L，维甲酸（A4）1~3μmol/L；
B）将接种了脂肪间充质干细胞的培养皿于B1%浓度的CO2、37℃的条件下培养,每3天更换一次培养基；共培养7天；
C）培养完成后，离心去除培养基并收集细胞，以PBS清洗细胞2~3次，在4℃条件下将细胞匀浆，所得匀浆液冷冻至完全冻结后，再置于42℃的温水彻底融化，然后离心取上清，将上清液置于透析袋中，以4℃的PBS透析12h，将透析袋内的溶液冻干，作为脂肪干细胞培养产物。
透析袋的截留分子量为8000~14000。
脂肪间充质干细胞为成人脂肪间充质干细胞，购自赛业（苏州）生物科技有限公司，产品货号（HUXMD-01001）。
脂肪干细胞培养产物的不同制备实施例参数见下表：
| 编号 | A<sub>1</sub> | A<sub>2</sub> | A<sub>3</sub> | A<sub>4</sub> | B<sub>1</sub> |
| 实施例1 | 0.2 | 10 | 6 | 1 | 10 |
| 实施例2 | 0.3 | 15 | 8 | 2 | 15 |
| 实施例3 | 0.2 | 15 | 10 | 3 | 10 |
| 实施例4 | 0.3 | 10 | 10 | 1 | 15 |
| 实施例5 | 0.2 | 10 | 8 | 2 | 10 |
| 实施例6 | 0.3 | 15 | 6 | 3 | 15 |
| 对比例1 | 0 | 15 | 8 | 2 | 15 |
| 对比例2 | 0.2 | 0 | 8 | 2 | 10 |
| 对比例3 | 0.3 | 15 | 0 | 2 | 15 |
| 对比例4 | 0.2 | 10 | 8 | 0 | 10 |
| 对比例5 | 0.3 | 15 | 8 | 2 | 5 |
| 对比例6 | 0.5 | 15 | 8 | 2 | 15 |
| 对比例7 | 0.1 | 10 | 8 | 2 | 10 |
| 对比例8 | 0.3 | 20 | 8 | 2 | 15 |
| 对比例9 | 0.2 | 5 | 8 | 2 | 10 |
| 对比例10 | 0.3 | 15 | 3 | 2 | 15 |
| 对比例11 | 0.2 | 10 | 15 | 2 | 10 |
| 对比例12 | 0.3 | 15 | 8 | 5 | 15 |
| 对比例13 | 0 | 0 | 0 | 0 | 5 |
2、药理实验——瘢痕修复效果对比
采用兔耳瘢痕模型，造模方法如下：
选取清洁级新西兰大耳白兔，体重介于2.5~3.5公斤，雌雄不限（不含孕兔），双侧兔耳发育良好，无畸形。单只分笼适应性饲养48小时，建模前一天去除双侧兔耳腹侧毛发，术前8小时禁食。用10%水合氯醛配制成的溶液进行腹腔注射麻醉。麻药显效后，固定于兔台上，并注意实验动物的保暖。常规消毒术区，每侧兔耳腹侧沿长轴避开大血管，制作直径约10mm的圆形创面6个，每个创面间隔至少在1cm以上，去除软骨膜，并止血。待实验动物麻醉苏醒后，于原环境下继续饲养。动态观察每只动物的生活及伤口情况，待术后28天创面完全上皮化，形成增生性瘢痕为造模成功。
选取造模成功的实验动物随机分组，每组2只，给药方法为将各实施例及对比例得到的脂肪干细胞培养产物溶于水制成2%重量百分比浓度的溶液，在实验动物瘢痕部位涂抹给药，每天三次，涂药后轻揉至药物吸收，于给药后28d取兔耳瘢痕标本并处死动物，标本切取自瘢痕组织中央与周边正常组织交界区。用10%中性福尔马林固定，石蜡包埋后作HE、VG染色及免疫组织化法检测，主要检测I型胶原、III型胶原表达情况，以及瘢痕增生指数（HI）。
瘢痕增生指数检测方法为：
将染色切片在低倍镜（40倍）下用显微测量标尺测量，并按公式HI=A/B计算瘢痕增生指数。其中A为瘢痕最突起点距离兔耳软骨表面的垂直厚度，而B为瘢痕周围的正常皮肤上界距离兔耳软骨表面的垂直厚度，每组均测得24个数值。
I型胶原、III型胶原表达情况检测方法为：
将已包埋好的蜡块固定于切片机上，做厚度5μm的连续切片并将切片裱于多聚赖氨酸防脱玻片上。将玻片置于60℃的烤箱内烤片2小时后取出，待其自然冷却，作为供视样品。免疫组化图像，将采集的图片以ImagePro-plus 6.0 图像分析系统进行分析，测定每个视野中I、III型胶原的平均光密度值AOD。
上述数据结果以均数±标准差的方式表示，以EXCEL对数据进行处理。
分组、给药与检测结果如下：
（means±s，n=24）
| 分组 | 给药 | I型胶原AOD | III型胶原AOD | HI |
| 实验组1 | 实施例1 | 0.860±0.069 | 1.343±0.093 | 1.544±0.205 |
| 实验组2 | 实施例2 | 0.858±0.074 | 1.341±0.130 | 1.530±0.234 |
| 实验组3 | 实施例3 | 0.835±0.070 | 1.402±0.137 | 1.416±0.170 |
| 实验组4 | 实施例4 | 0.864±0.079 | 1.333±0.089 | 1.525±0.203 |
| 实验组5 | 实施例5 | 0.826±0.063 | 1.382±0.127 | 1.415±0.206 |
| 实验组6 | 实施例6 | 0.831±0.056 | 1.401±0.139 | 1.439±0.169 |
| 实验组7 | 对比例1 | 1.148±0.101 | 1.272±0.091 | 2.155±0.351 |
| 实验组8 | 对比例2 | 1.115±0.110 | 1.274±0.096 | 2.119±0.260 |
| 实验组9 | 对比例3 | 1.107±0.107 | 1.265±0.123 | 2.087±0.283 |
| 实验组10 | 对比例4 | 1.101±0.109 | 1.275±0.086 | 2.121±0.306 |
| 实验组11 | 对比例5 | 0.974±0.077 | 1.302±0.123 | 1.858±0.289 |
| 实验组12 | 对比例6 | 1.024±0.082 | 1.280±0.086 | 1.956±0.260 |
| 实验组13 | 对比例7 | 1.069±0.106 | 1.288±0.099 | 1.932±0.306 |
| 实验组14 | 对比例8 | 1.020±0.088 | 1.300±0.107 | 1.942±0.311 |
| 实验组15 | 对比例9 | 1.014±0.079 | 1.280±0.099 | 1.932±0.232 |
| 实验组16 | 对比例10 | 1.029±0.078 | 1.297±0.095 | 1.960±0.227 |
| 实验组17 | 对比例11 | 1.024±0.068 | 1.293±0.108 | 1.962±0.252 |
| 实验组18 | 对比例12 | 1.017±0.087 | 1.298±0.104 | 1.952±0.282 |
| 实验组19 | 对比例13 | 1.275±0.092 | 1.202±0.102 | 2.398±0.315 |
| 阳性对照 | 生理盐水 | 1.432±0.130 | 1.165±0.114 | 2.714±0.395 |
根据现有技术已知，皮肤中的胶原类型以I型胶原和III型胶原为主，III型胶原属于重建型胶原，在瘢痕修复过程中，较多III型胶原的表达，可以促进瘢痕的修复，而上述实验数据表明，采用实施例1~6的实验组，其实验动物瘢痕中III型胶原的含量明显高于其他实验组，说明其瘢痕修复效果较好，且瘢痕增生指数也表明，采用实施例1~6的实验组瘢痕增生修复效果较好，而采用各对比例的实验组，其实验动物瘢痕愈合情况显著低于采用实施例1~6的实验组，说明改变诱导试剂的配比以及将改变培养条件（将CO2降低至5%）均会对得到的脂肪干细胞培养产物的瘢痕修复效果产生不良影响。
3、制剂实施例：
所述组合物制备成为液体敷料，以如下方法制备：
1）将脂肪干细胞培养产物溶于水，加入处方量的羟丙基甲基纤维素充分溶解后，得到A液；
2）将处方量壳聚糖溶解在乙酸溶液中，得到壳聚糖乙酸溶液，将A液缓慢加入壳聚糖乙酸溶液中，搅拌均匀，以0.1M NaOH溶液调节pH至6.5~6.8，补足余量的水，即得。
制剂实施例的配方见下表（单位：重量百分比%）
| 编号 | 脂肪干细胞培养产物/用量 | 壳聚糖用量/% | HPMC用量/% |
| 1 | 实施例1/1% | 1.5 | 0.5 |
| 2 | 实施例2/2% | 2.5 | 1 |
| 3 | 实施例3/1% | 2 | 0.5 |
| 4 | 实施例4/2% | 1.5 | 1 |
| 5 | 实施例5/2% | 2.5 | 0.5 |
| 6 | 实施例6/1% | 2 | 1 |
制剂实施例得到的组合物，采用壳聚糖和HPMC作为辅料，可以作为液体敷料，用于创伤修复。在研究中我们发现，与常见的壳聚糖液体敷料相比，加入了本发明提供的脂肪干细胞后的液体敷料，在用于兔耳瘢痕模型动物造模过程中的干预治疗（即手术后以每日两次涂敷制剂实施例得到的液体敷料）时，不但能够促进创伤的愈合，还能在愈合过程中显著抑制瘢痕的生成。
Claims (6)
1.一种用于创伤修复的脂肪干细胞组合物，其特征是所述组合物由作为活性成分的重量百分比1~2%的脂肪干细胞培养产物及至少一种可以外用的药用辅料组成，所述的脂肪干细胞培养产物以如下方法制备：
A）将脂肪间充质干细胞，以3~6×104/ml的接种量接种于培养基中，所述培养基为含诱导培养试剂的基础培养基，所述基础培养基含有10%体积分数胎牛血清的低糖DMEM培养基，所述诱导培养试剂为地塞米松、甘草酸二铵、维生素D3和维甲酸，诱导培养试剂在培养基中的用量为：地塞米松0.2~0.3μmol/L，甘草酸二铵10~15μmol/L，维生素D36~10μmol/L，维甲酸1~3μmol/L；
B）将接种了脂肪间充质干细胞的培养皿于10~15%CO2，37℃的条件下培养,每3天更换一次培养基；共培养7~8天；
C）培养完成后，离心去除培养基并收集细胞，以PBS清洗细胞2~3次，在4℃以下将细胞匀浆，所得匀浆液冻存至完全冻结后，再置于42℃以下的温水彻底融化，然后离心取上清，将上清液置于透析袋中，以4℃PBS透析8~24h后，将透析袋内的溶液冻干，作为脂肪干细胞培养产物。
2.如权利要求1所述的一种用于创伤修复的脂肪干细胞组合物，其特征是，所述组合物为液体敷料，所述可以外用的药用辅料包括壳聚糖、乙酸、羟丙基甲基纤维素、pH调节剂和余量的水。
3.如权利要求2所述的一种用于创伤修复的脂肪干细胞组合物，其特征是所述壳聚糖用量占组合物重量百分比分数为1%~3%；所述羟丙基甲基纤维素用量占组合物的重量百分比分数为0.5~1%。
4.如权利要求3所述的一种用于创伤修复的脂肪干细胞组合物，其特征是所述壳聚糖用量占组合物重量百分比分数为1.5%~2.5%。
5.如权利要求2~4任一所述的一种用于创伤修复的脂肪干细胞组合物，其特征是所述壳聚糖的脱乙酰度为85%~95%。
6.如权利要求2所述的一种用于创伤修复的脂肪干细胞组合物，其特征是所述pH调节剂为将组合物pH调节为6.5~7.0的pH调节剂。
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