CN111961645A - 一种高效扩增人表皮成纤维细胞的培养基 - Google Patents
一种高效扩增人表皮成纤维细胞的培养基 Download PDFInfo
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Abstract
本发明提供一种高效扩增人表皮成纤维细胞的培养基，所述细胞培养基中含有miRNA，本发明提供了一种高效扩增人表皮成纤维细胞的培养基，所述培养基包含新型载体，所述新型载体包栽miRNA，可以促进干细胞体外增殖，因而可以用于人表皮成纤维细胞的增殖。
Description
技术领域
本发明涉干细胞培养领域，特别地涉及一种高效扩增人表皮成纤维细胞的培养基。
背景技术
人皮肤成纤维细胞是组成皮肤真皮的主要结构成分，其可合成和分泌胶原纤维、弹性纤维、网状纤维、透明质酸等细胞外基质，对于保持皮肤强度和弹性、修复损伤及美容美体具有重要作用。是维持皮肤年轻态的决定性因素，也是维持皮肤结构稳定的重要组成部分。而由此形成的皮肤胶原蛋白修复体系一旦形成，它的生命旺盛周期为10-15年。人随着年龄的增长，成纤维细胞数量越来越少，活性越来越低，相应的其产生的胶原蛋白、弹性纤维、透明质酸这些物质就会减少，不足以支撑皮肤结构，导致真皮层变薄，皮肤开始松弛，失去弹性，出现皱纹。
成纤维细胞摄取所需的氨基酸，如脯氨酸和赖氨酸等，在粗面内质网的核蛋白体上合成前α多肽链，多肽链输送到高尔基复合体后，组成前胶原分子。前胶原分子由分泌囊泡带到细胞表面，然后通过胞吐作用释放到细胞外。在前胶原肽酶催化下，将每一前α多肽链的尾段除去，成为原胶原分子。许多原胶原分子成行平行排列，结合成具有周期性横纹的胶原原纤维。由胶原原纤维互相结合形成胶原纤维。
成纤维细胞在一般创伤修复中的表现各种创伤均会造成不同程度的细胞变性、坏死和组织缺损，必须通过细胞增生和细胞间基质的形成来进行组织修复。在此修复过程中，成纤维细胞起着十分重要的作用。以伤口愈合过程为例，成纤维细胞通过有丝分裂大量增殖，并从4～5天或6天开始合成和分泌大量的胶原纤维和基质成分，与新生毛细血管等共同形成肉芽组织，填补伤口组织缺损，为表皮细胞的覆盖创造条件。在伤口愈合中，成纤维细胞主要来源于真皮乳头层的局部成纤维细胞和未分化的间充质细胞，以及血管周围的成纤维细胞和周细胞。
发明内容
为了解决上述技术问题，本发明提供一种高效扩增人表皮成纤维细胞的培养基。
本发明是以如下技术方案实现的：
一种高效扩增人表皮成纤维细胞的培养基，所述细胞培养基中含有miRNA。
进一步地，所述细胞培养基中还含有细胞生长因子。
进一步地，所述细胞培养基中还含有特制载体。
进一步地，所述miRNA选自hsa-miR-214、miR-UL112-3p、hsa-miR-168a、hsa-miR-156a、miR-1260b。
优选地，所述细胞生长因子为转化生长因子FGF9。
进一步地，所述特制载体的制备方法包括：用多聚甲醛和二硫化物还原剂二硫苏糖醇封闭巯基以促进细胞产生细胞外囊泡。
优选地，所述miRNA包含于特制载体中。
进一步地，所述细胞培养基含有转铁蛋白、转化生长因子FGF9、地塞米松、烟酸肌醇酯、装载有miRNA的载体、DMEM培养基和胎牛血清。
进一步地，所述细胞培养基含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-214的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
优选地，所述细胞培养基含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有has-miR-UL112-3p的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
优选地，所述细胞培养基含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-168a的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
优选地，所述细胞培养基含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-156a的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
优选地，所述细胞培养基含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有miR-1260b的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
本发明的有益效果：本发明提供了一种高效扩增人表皮成纤维细胞的培养基，所述培养基包含新型载体，所述新型载体包栽miRNA，可以促进干细胞体外增殖，因而可以用于人表皮成纤维细胞的增殖。
具体实施方式
为了有助于更清楚的理解本发明的内容，现结合具体的实施例详细介绍如下。如未明确指出，以下实施例中涉及的实验操作方法为本领域常规的分子生物学操作方法，涉及的试剂均为分析纯级试剂，涉及的试剂或仪器均可从正规渠道商购获得。除非特别指出，以下各实施例中涉及的各种实验方法和操作，包括细胞培养，RNA的提取，RCR扩增，荧光定量PCR，细胞染色等等可参考分子克隆，所需技术为本领域技术人员所知悉的。
实施例1：
在细胞培养过程中，细胞会释放天然产生的空载体，并带有大量杂质，例如蛋白质、细胞碎片、短肽、脂质、氨基酸等，为提高载体的制备效率，本发明提供一种高效制备载体的方法。用多聚甲醛和二硫化物还原剂二硫苏糖醇封闭巯基以促进细胞产生细胞外囊泡，具体地，用18mM多聚甲醛(PFA)和1mM DTT处理EL4细胞，共孵育45分钟，将上述共孵育产物离心(12000,1min)并收集空载体，去上清后，用添加了PFA和DTT的磷酸盐缓冲盐水清洗，获得富含载体的上清液。通过巯基阻断作用产生的新型载体的尺寸范围为25-40nm，平均直径约为30nm，将所得新型载体与miRNA片段共孵育1h，miRNA信息及序列详见表1，分别获得装载有hsa-miR-214、miR-UL112-3p、hsa-miR-168a、hsa-miR-156a、miR-1260b的载体。
表1miR序列信息
| miR名称 | miR序列信息 |
| has-miR-214 | ugacggacagacacggacggaca |
| has-miR-UL112-3p | aagugacggugagauccaggcu |
| hsa-miR-168a | ucgcuuggugcaggucgggaa |
| hsa-miR-156a | ugacagaagagagugagcac |
| miR-1260b | accaccgucuccacccua |
实施例2：待测试实验组配方
测试实验组编码为S1-S6,Y1-Y6
S1组含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-214的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
S2组含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有has-miR-UL112-3p的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
S3组含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-168a的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
S4组含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-156a的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
S5组含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有miR-1260b的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
S6组含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、DMEM培养基和体积分数为8.5％的胎牛血清。
Y1组含终浓度为3.5mg/L转铁蛋白、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-214的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
Y2组含终浓度为3.5mg/L转铁蛋白、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有miR-UL112-3p的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
Y3组含终浓度为3.5mg/L转铁蛋白、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-MiR-168a的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
Y4组含终浓度为3.5mg/L转铁蛋白、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-156a的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
Y5组含终浓度为3.5mg/L转铁蛋白、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有miR-1260b的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
Y6组含终浓度为3.5mg/L转铁蛋白、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、DMEM培养基和体积分数为8.5％的胎牛血清。
实施例3：测定培养基对干细胞体外增殖活力的影响
取复苏传代后融合率约87-92％的人表皮成纤维细胞T25细胞，离心后弃上清液，加入体积比0.25％的胰酶，37℃消化10分钟，之后加入等体积L-DMEM培养基终止消化，离心后弃上清液，用完全培养基重悬沉积物，制成终浓度为1×105/mL的细胞混悬液，之后接种于96孔板，每孔100μL，在37℃、5％CO2恒温培养箱中培养，12h后将诱导培养基分别更换为S1-S6,Y1-Y6培养基(每个编码对应8个孔位)，对照组更换为新的完全培养基，在37℃、5％CO2恒温培养箱中继续培养3天后，每孔加20μL 5g/L的MTT溶液，孵育4h弃上清，每孔加入150μL二甲基亚砜，混均后于酶标仪490nm波长处检测吸光度值，以对照组OD值计为增殖率100％，按照实验组与对照组的比值*100％计算得到实验组的细胞增殖率，结果如表2所示。
表2细胞增值率
| 编码 | 细胞增值率 |
| 对照组 | 100％ |
| S1 | 121.53％ |
| S2 | 146.64％ |
| S3 | 123.31％ |
| S4 | 167.45％ |
| S5 | 142.34％ |
| S6 | 111.34％ |
| Y1 | 114.76％ |
| Y2 | 148.60％ |
| Y3 | 129.52％ |
| Y4 | 155.32％ |
| Y5 | 130.91％ |
| Y6 | 108.12％ |
从表2结果可知，S2,S4,S5,Y2,Y4,Y5培养基培养下的细胞增值率最高。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (10)
1.一种高效扩增人表皮成纤维细胞的培养基，其特征在于，所述细胞培养基中含有miRNA。
2.根据权利要求1所述培养基，其特征在于，所述细胞培养基中还含有细胞生长因子。
3.根据权利要求1所述培养基，其特征在于，所述细胞培养基中还含有特制载体。
4.根据权利要求1所述培养基，其特征在于，所述miRNA选自hsa-miR-214、miR-UL112-3p、hsa-miR-168a、hsa-miR-156a、miR-1260b。
5.根据权利要求2所述培养基，其特征在于，所述细胞生长因子为转化生长因子FGF9。
6.根据权利要求3所述培养基，其特征在于，所述特制载体的制备方法包括：用多聚甲醛和二硫化物还原剂二硫苏糖醇封闭巯基以促进细胞产生细胞外囊泡。
7.根据权利要求6所述培养基，其特征在于，所述miRNA包含于特制载体中。
8.根据权利要求1所述培养基，其特征在于，所述细胞培养基含有转铁蛋白、转化生长因子FGF9、地塞米松、烟酸肌醇酯、装载有miRNA的载体、DMEM培养基和胎牛血清。
9.根据权利要求8所述培养基，其特征在于，所述细胞培养基含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有hsa-miR-214的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
10.根据权利要求8所述培养基，其特征在于，所述细胞培养基含终浓度为3.5mg/L转铁蛋白、5μg/L转化生长因子FGF9、0.15μmol/L地塞米松、2.0mg/L烟酸肌醇酯、0.48mg/L装载有has-miR-UL112-3p的载体、DMEM培养基和体积分数为8.5％的胎牛血清。
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