CN114479126B - 一种制备可模拟体内ecm刚度微环境的水凝胶的方法和应用 - Google Patents
一种制备可模拟体内ecm刚度微环境的水凝胶的方法和应用 Download PDFInfo
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Abstract
本发明公开了一种制备可模拟体内ECM刚度微环境的水凝胶的方法和应用，该方法结合PEG及GelMA的特性，合成PEG‑GelMA复合水凝胶，通过调节各自比例，使复合水凝胶具有不同的弹性模量。该模型制备简单，经济可靠，可广泛应用于细胞对体内不同刚度微环境相应的体外研究，在细胞培养、肿瘤微环境和再生医学应用中具有较大潜力。
Description
技术领域
本发明涉及细胞培养材料技术领域，尤其涉及一种制备可模拟体内ECM刚度微环境的水凝胶的方法和应用。
背景技术
细胞外基质(Extracellular matrix，ECM)是一种复杂的网状结构，是由多种细胞如成纤维细胞、脂肪细胞、干细胞等分泌，并经与细胞之间的相互作用产生，是组织和器官形成和发挥功能的基础。它不仅为细胞提供了必要的物理支架，而且为组织发育提供了所需的生化因子和物理微环境。ECM影响着细胞的行为和功能，与各种疾病的发生息息相关(例如癌症、纤维化等)。利用生物材料模拟微环境的物理性质，探索微环境物理因素对细胞的影响，对组织再生医学、疾病的治疗具有重要意义(Nat Rev Mol Cell Biol.2014,15(12):802-12)。
细胞微环境的物理特性包括刚度、形貌、粘附蛋白配体分布等。刚度，又称为弹性，是生物界面材料的重要特性之一，可以被细胞直接感知。不同弹性模量的生物基质不仅影响细胞铺展粘附，还能诱导细胞命运抉择(Science.2009,24(5935):1673-7)。如小模量(<5kPa)的生物界面会诱导干细胞向神经细胞与脂肪细胞的分化，适中的(5-20kPa)模量诱导向肌细胞与软骨细胞分化，较大的(>20kPa)模量利于向成骨细胞的分化(Cell,2006,126(4):677-89)。模拟体内微环境，制备不同刚度的生物材料模型，是研究刚度对细胞行为及功能的影响的必要前提。
由胶原蛋白、纤维蛋白、壳聚糖、海藻酸盐、明胶或透明质酸等天然材料合成的水凝胶，由于其良好的生物相容性以及具有天然细胞结合位点，而被广泛用于细胞，但它们的凝胶化过程及稳定性相对难以控制(Chem Rev.2001,101(7):1869–80)。合成聚合物可以在分子水平上根据分子量、嵌段结构和功能位点进行改造，其机械性能更容易调控、稳定性良好，被广泛应用于组织工程中。聚(乙二醇)(PEG)水凝胶在细胞培养和组织工程应用中很受欢迎，因为它们无毒并具有良好的水合作用。然而，细胞并不能粘附、重塑、或降解PEG水凝胶,PEG需要与RGD等生物分子联合使用，然而RGD的储存条件较为严格，且价格较为昂贵。(Acta Biomater.2021,128:42-59)。甲基丙烯酸酰化明胶(GelMA)源自变性胶原蛋白，可产生可酶降解、可光交联的水凝胶，细胞可以在其中降解、粘附和扩散。然而，GelMA水凝胶的稳定性较差(Biomaterials,2010,31(27):6941-51)。因此，亟需一种制备简单，经济可靠，可广泛应用于不同细胞、不同刚度微环境相应的体外细胞培养材料。
发明内容
有鉴于此，本发明的目的之一在于提供一种制备可模拟体内ECM刚度微环境的水凝胶的方法，该方法对使用的材料及设备要求简单，制备方法快速简便，制备的水凝胶生物相容性好，是一种刚度范围广的生物材料界面；本发明的目的之二在于提供所述方法制备的水凝胶在促进THP-1细胞分化中的应用；本发明的目的之三在于提供所述方法制备的水凝胶在培养上皮细胞中的应用；本发明的目的之四在于提供所述方法制备的水凝胶在在培养肿瘤细胞或肿瘤相关间质/免疫细胞中的应用。
为达到上述目的，本发明提供如下技术方案：
1、一种制备可模拟体内ECM刚度微环境的水凝胶的方法，包含如下步骤：
将甲基丙烯酸酰化明胶GelMA溶液与聚乙二醇PEG溶液按体积比1：1混匀后，加入蓝光引发剂LAP，混合均匀后，滴于防黏板上，盖上硅烷偶联剂修饰双键的盖玻片，紫外照射使发生化学交联，即得所述可模拟体内ECM刚度的水凝胶。
本发明优选的，制得的水凝胶中，GelMA终浓度的质量体积分数为4％，PEG终浓度的质量体积分数为0.5％-30％。
本发明优选的，制得的水凝胶中，GelMA终浓度的质量体积分数为4％，PEG终浓度的质量体积分数为16％。
本发明优选的，制得的水凝胶中，GelMA终浓度的质量体积分数为4％，PEG终浓度的质量体积分数为4％。
本发明优选的，所述蓝光引发剂LAP加入量为甲基丙烯酸酰化明胶GelMA溶液与聚乙二醇PEG溶液混合液质量的1％。
本发明优选的，所述紫外照射条件为365nm照射1-2min。
2、所述方法制备的水凝胶在促进THP-1细胞分化中的应用。
本发明优选的，分化后的THP-1细胞呈带突起的不规则形态，具有粘附功能，贴壁生长，且高表达CD11b和CD14分子。
3、所述方法制备的水凝胶在培养上皮细胞中的应用。
本发明优选的，所述上皮细胞为胰腺细胞。
4、所述方法制备的水凝胶在培养肿瘤细胞或肿瘤相关间质/免疫细胞中的应用。
本发明优选的，所述肿瘤细胞为胰腺癌细胞，所述肿瘤相关间质/免疫细胞为THP-1细胞。
本发明的有益效果在于：本发明公开了一种制备可模拟体内ECM刚度微环境的水凝胶的方法和应用，该方法结合PEG及GelMA的特性，合成PEG-GelMA复合水凝胶，通过调节各自比例，使复合水凝胶具有不同的弹性模量。该模型制备简单，经济可靠，可广泛应用于细胞对体内不同刚度微环境相应的体外研究，在细胞培养、肿瘤微环境和再生医学应用中具有较大潜力。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为PEG+GelMA复合水凝胶的制备流程；
图2为THP-1细胞在含不同浓度Gelma的PEG水凝胶的铺展及情况(明场10x)；
图3为制备的PEG+GelMA复合水凝胶的模量范围；
图4为人胰腺和胰腺癌组织测出的弹性模量范围。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明中PEG、LAP购自于Sigma，货号分别为455008、900889。
实施例1
制备含不同GelMA浓度的PEG水凝胶，探索利于细胞粘附的GelMA的含量，具体步骤如下：
一、利用硅烷偶联剂在盖玻片表面修饰上可光聚合的双键
按体积比30mL 30％(W/V)H2O2+70mLH2SO4配制食人鱼洗液，将直径为12mm盖玻片置于食人鱼洗液2-3h后，取出，用去离子水洗至中性，乙醇超声清洗两遍，每次超声5分钟。配制反应溶液，将3mL 10％(W/V)乙酸和1ml 3-(甲基丙烯酰氧)丙基三甲氧基硅烷加入到100mL乙醇中。将盖璃片置于上述反应液中，80℃反应5h后，乙醇超声清洗3次后，4℃冰箱密封保存。
二、PEG+GelMA复合水凝胶的制备
制备甲基丙烯酸酰化明胶(GelMA)溶液：使用磷酸盐缓冲液(PBS)配制1％、2％、4％、8％、16％、32％(W/V，g/ml)的GelMA溶液，37℃孵育溶解。
制备PEG溶液：PBS配制32％(W/V，g/ml)的聚乙二醇(PEG)溶液，涡旋震荡溶解。
制备LAP溶液：配制30％(W/V)的蓝光引发剂LAP溶液，超声30s使溶解，避光冰上保存，现用现配。
合成水凝胶：PEG溶液与不同浓度的GelMA溶液按体积比1：1混合均匀，加入LAP，使与LAP溶液与PEG+GelMA混合溶液的质量比为1：100(1％)的LAP溶液，涡旋仪震荡20s使混合均匀。取LAP、PEG、GelMA混合物80μL滴于防黏板上，盖上步骤一制备的修饰双键的盖玻片，紫外365nm照射1-2min使化学交联，使用镊子将水凝胶从防粘片取下来，水凝胶至于PBS中，4℃浸泡过夜。使用流变仪测定复合水凝胶的弹性模量。
三、细胞培养
水凝胶无菌处理：于生物安全柜中，水凝胶面朝上转移入所需的细胞培养板中(如6-孔板、12-孔板等)，用紫外灯照射10min灭菌消毒，PBS洗2遍，4℃保存备用。
准备THP-1细胞悬液：人单核细胞系THP-1的细胞浓度调整为3×105cells/mL，加入豆蔻佛波醇乙酯(phorbol 12-myristate 13-acetate，PMA)，使PMA终浓度为25ng/mL。PMA是蛋白激酶C(PKC)和SphK的激活剂，可诱导THP-1细胞向巨噬细胞方向分化。
接种细胞：将1mL THP-1细胞悬液加入含水凝胶的孔板中，置于细胞培养箱培养。24h后，观察细胞铺展情况；收集细胞，流式细胞仪分析细胞CD11b和CD14的表达情况。
PEG+GelMA复合水凝胶的制备流程如图1所示。
THP-1细胞被PMA刺激后，通常情况下，如果分化后的细胞在液体培养基上会从悬浮生长变为贴壁生长，而且高表达单核巨噬细胞特有的CD11b和CD14分子，则说明单核细胞已经成功分化为巨噬细胞。但是此时细胞大部分呈圆形或椭圆形，并没有形成明显的突触，不具备吞噬病原体的功能，故细胞摄取抗原的能力较弱，当继续分化至细胞表面出现非常明显的突起，形成多突起的不规则形态细胞时，此时的巨噬细胞才能对病原体进行有效的吞噬。
THP-1细胞在含不同浓度GelMA的PEG水凝胶的铺展及情况，结果如图2所示，图中圆圈处表示带有突起的巨噬细胞，A为液体培养基中的细胞；B为仅含16％PEG的水凝胶培养的细胞；C为16％PEG+0.5％GelMA复合水凝胶培养的细胞；D为16％PEG+1％GelMA复合水凝胶培养的细胞；E为16％PEG+2％GelMA复合水凝胶培养的细胞；F为16％PEG+4％GelMA复合水凝胶培养的细胞；G为16％PEG+8％GelMA复合水凝胶培养的细胞；H为16％PEG+16％GelMA复合水凝胶培养的细胞；I为THP-1细胞在含不同浓度GelMA的PEG水凝胶中CD11b的表达量；J为THP-1细胞在含不同浓度GelMA的PEG水凝胶中CD14的表达量。THP-1在16％PEG+4％GelMA的复合水凝胶中培养，细胞生长状态最好，带突起的不规则形状细胞数量最多，且铺展效果也较好，CD11b和CD14的表达量也较高(图2，F)。
实施例2
制备含4％GelMA的不同PEG浓度的水凝胶，测定复合水凝胶的弹性模量范围，具体步骤如下：
A.使用PBS配制8％的GelMA，37℃孵育溶解。PBS配制60％、50％、40％、36％、32％、28％、24％、20％、18％、16％、14％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％PEG溶液。不同浓度的PEG与8％的GelMA体积比1:1混合，加入1％的LAP溶液，混合均匀。80μL复合水凝胶于防粘板，盖上带双键的盖玻片，UV照射1min，水凝胶面朝上转移入24孔板，4℃过夜。
B.流变仪测定含4％GelMA和不同浓度PEG复合水凝胶的弹性模量。
结果如图3所示，随着PEG浓度的变化(0.5％-30％)，弹性模量可从260Pa升至60kPa，复合水凝胶的模量范围涵盖机体绝大多数器官组织的模量范围。其中，16％PEG+4％GelMA复合水凝胶中的弹性模量为20.53kPa，推断该浓度是最接近正常体内肿瘤微环境内的真实模量。
实施例3
测定人正常胰腺和胰腺癌组织测出的弹性模量范围结果如图4所示，模拟正常胰腺和胰腺癌体内微环境的最佳模量分别为1kPa和20kPa，其中弹性模量1kPa对应的复合水凝胶浓度为4％PEG+4％GelMA，弹性模量20kPa对应的复合水凝胶浓度为16％PEG+4％GelMA。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (3)
1.一种非疾病诊断和治疗方法的可模拟人胰腺癌细胞外基质ECM刚度微环境的水凝胶在促进单核巨噬细胞分化中的应用，其特征在于，分化后的单核巨噬细胞呈带突起的不规则形态，具有粘附功能，贴壁生长，且高表达CD11b和CD14分子；
所述可模拟人胰腺癌细胞外基质ECM刚度微环境的水凝胶的制备方法包含如下步骤：将甲基丙烯酸酰化明胶GelMA溶液与聚乙二醇PEG溶液按体积比1：1混匀后，加入蓝光引发剂LAP，混合均匀后，滴于防黏板上，盖上硅烷偶联剂修饰双键的盖玻片，紫外照射使发生化学交联，即得所述可模拟体内ECM刚度的水凝胶；制得的水凝胶中，GelMA终浓度为4%（W/V，g/ml），PEG终浓度为16%（W/V，g/ml），水凝胶的弹性模量为20kPa，用以模拟人胰腺癌细胞外基质ECM刚度微环境。
2.根据权利要求1所述的应用，其特征在于，所述可模拟人胰腺癌细胞外基质ECM刚度微环境的水凝胶的制备方法中，所述蓝光引发剂LAP加入量为甲基丙烯酸酰化明胶GelMA溶液与聚乙二醇PEG溶液混合液质量的1%。
3.根据权利要求1所述的应用，其特征在于，所述可模拟人胰腺癌细胞外基质ECM刚度微环境的水凝胶的制备方法中，紫外照射条件为365nm照射1-2 min。
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