CN114949322A - 石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法 - Google Patents
石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法 Download PDFInfo
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Abstract
石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，它涉及诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法。方法：一、制备GQDs溶液；二、合成纳米水凝胶液；三、纯化及固态；四、静电纺丝溶液；五、构建组织诱导性材料；六、静电纺丝、干燥、紫外交联和灭菌。本发明制备所得材料，具有生物安全性，能够自主捕获MSC归巢到糖尿病性局部组织，利用石墨烯量子点稳定释放CXCL‑8招募MSC归巢的特性，主动定点捕获组织内或血管中MSC等细胞，有效提高MSC归巢，促进生物材料血管化，增加了生物材料与机体相互作用，进而促进组织损伤修复。本发明材料适用于可调控的缓释性医用生物材料和医用敷料。
Description
技术领域
本发明涉及诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法。
背景技术
根据国际糖尿病联盟组织（IDF）预测：2015年全球约有4.15亿糖尿病患者，2045年全球患糖尿病的人数将达到6.42亿，因糖尿病而导致的年死亡人数为500万/年，全球每年因糖尿病而消耗的医疗费用至少6730亿美元，患者家庭经济负担非常沉重。研究发现，糖尿病患者小（微）动脉常发生微血栓而导致组织缺血缺氧，皮肤溃疡是糖尿病常见的并发症，尽管多种方法被用于治疗糖尿病皮肤溃疡，但是糖尿病皮肤溃疡的治愈率依然很低，促进糖尿病足等并发症修复是国内外致力攻克的医学难题。
慢性损伤会破坏皮肤的完整性，基于纳米和超细纤维的生物材料可以作为细菌感染的屏障，保持适当的湿度并吸附渗出液，通过模仿细胞外基质加速组织损伤的愈合过程，并支持受损组织的重建。聚已内酯（PCL）等高分子化合物具有优异的生物相容性和生物降解性，PCL通过提供防止水分流失和环境的疏水屏障，对药物相容性和药物释放动力学产生积极影响。静电纺丝PCL纳米生物材料能充分模拟细胞外基质，发挥较好的细胞攀爬、血管新生、抗菌排毒等支架作用，加速伤口的愈合过程。
石墨烯量子点（Graphene quantum dots，GQD）颗粒直径＜100.0nm，具备优异光学性能和高度生物相容性、低细胞毒性等特点，在生物制药、药物输送系统、组织工程等领域具有广泛应用价值。添加GQD可用于改善静电纺丝聚合物的机械张力和物理性能。GQDs作为增强剂，能够提高静电纺丝纳米纤维的机械性能和拉伸力。石墨烯良好的载药功能，静电纺丝优异的三维纳米纤维网络很好模拟细胞外基质，有利于细胞攀附、血管新生、神经纤维生长，促进伤口愈合。
人体内含有间充质干细胞（MSC）、血管内皮细胞等许多与伤口修复有关的细胞，若激发机体细胞活性，招募细胞归巢到伤口周围，可以分化为皮肤组织、血管等结构，加速伤口愈合，并对降低治疗成本起到积极作用。诱导血管内或者组织间MSC迁移至目标组织的过程是MSC进行再生修复和血管新生的关键。高糖环境下，捕获组织内MSC能有效促进MSC生物材料血管化和组织再生修复。但是，如何促进生物材料血管化，发挥自主捕获、招募细胞的效力还需要深入研究。石墨烯敷料支架具有良好的抗菌、调节免疫、血管生成、基质重塑作用，以及很好的细胞相容性，石墨烯联合MSC将有效促进皮肤溃疡修复过程，是非常有前景的皮肤替代物。粒径＜100.0nm的GQDs对细胞没有明显的毒性，且容易被细胞内吞。因此石墨烯纳米复合材料在生物医学、载药给药体系、细胞内分子分析和临床基因治疗等的广阔应用前景。
趋化因子是对细胞归巢有重要作用的一类细胞因子，其家族成员趋化因子-8（CXCL-8），具有招募细胞归巢，促进血管新生的重要作用，是正常伤口愈合过程的关键因子。糖尿病组织区域，CXCL-8等细胞因子大量减少，细胞归巢能力降低，伤口迁延愈合。
虽然静电纺丝纳米纤维、量子点、趋化因子和间充质干细胞分别在构建生物材料、招募细胞归巢、促进组织损伤修复分别有各自的效果，但是，以静电纺丝纳米纤维富载GQDs/CXCL-8颗粒，构建成GQDs/CXCL-8诱导性生物材料，在自主捕获招募间充质干细胞归巢、促进生物材料血管化等相关研究尚无报道。
发明内容
本发明目的是制备具有高效模拟细胞外基质、有效生物力学、良好生物学活性、稳定递送CXCL-8的石墨烯载药体系、适合细胞生存和诱导宿主细胞归巢分化的石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，为深入研发具有捕获细胞功能，促血管新生作用的生物缓释敷料奠定研究基础，为糖尿病足等缺血性溃疡的治疗寻求解决途径，开发其治疗价值和商业价值。
石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，它按以下步骤实现：
一、制备GQDs溶液：将5-10g柠檬酸（CA）粉末放入微波炉，1000W功率下热解80-90min，得到深棕色液体，然后倒入10-20 ml的1mol/L NaOH溶液中，于4℃下搅拌30min，获得GQDs溶液；
二、合成GQDs/CXCL-8纳米水凝胶液：28℃下将100mmol-200mmol CXCL-8、8.717μmol-17.434mmol N,N二乙基丙烯酰胺、129.7μmol-259.4μmol N,N'-甲基双丙烯酰胺和87.67μmol-175.34μmol过二硫酸铵溶解于5mL-10mL去离子水中，超声处理器处理4-8h，然后转移到含有环己烷的圆底烧瓶中并加入span80均匀化，获得GQDs/CXCL-8纳米水凝胶液；
三、纯化及固态：磁力搅拌器连续搅拌上述GQDs/CXCL-8纳米水凝胶液，加入50-100μL N,N,N',N'-四甲基乙二胺，于氮气下聚合反应6-12h，所得沉淀产物经洗涤离心后置于透析袋中，然后在磁力搅拌器上常温下透析3天，获得纯化的GQDs/CXCL-8纳米水凝胶液，经真空冷冻干燥，获得GQDs/CXCL-8纳米水凝胶颗粒；
四、配置静电纺丝溶液：将135.0-270.0mg聚已内酯和45.0-90.0mg胶原蛋白溶解于1ml六氟异丙醇中，于室温下磁力搅拌2-4h，然后加入30-60μlTween80并磁力搅拌1-2h，再以的0.05-0.08mg/min速度加入2.0-3.0mg GQDs/CXCL-8纳米水凝胶颗粒并磁力搅拌4-5h，然后超声分散，获得静电纺丝溶液；
五、构建组织诱导性材料：上述静电纺丝溶液置于27号针头注射器中，以承载ADM（脱细胞真皮基质）的铝箔纸作为接收载体，采用静电纺丝机进行包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料的制备；
六、静电纺丝、干燥、紫外交联和灭菌：上述包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料，经真空冷冻干燥、紫外交联和灭菌后，获得石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，完成所述的制备方法。
本发明解决了“如何仿真细胞外基质设计纳米级自主捕获招募MSC、促进血管化的载供体系，推进基础与应用的并行研究，增加生物材料与机体相互作用”的问题。将高分子化学、生物材料学、量子医学、组织工程学、干细胞生物学、再生医学、生物信息学等多学科的理论和技术交叉融合；本发明制备的石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，具有生物安全性，能够通过“量子点镊”自主捕获MSC归巢到糖尿病性局部组织，利用石墨烯量子点稳定释放CXCL-8招募MSC归巢的特性，主动定点捕获组织内或血管中MSC等细胞，有效提高MSC归巢，促进生物材料血管化，增加了生物材料与机体相互作用，进而促进组织损伤修复。
本发明中石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料适用于可调控的缓释性医用生物材料和医用敷料。
附图说明
图1为实施例中包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料的扫描电子显微图，标尺=3.0µm；
图2为实施例中石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的扫描电子显微图，标尺=10µm，箭头指示为GQDs/CXCL-8量子点；
图3为实施例中CXCL-8刺激MSC的基因表达的热图；
图4为实施例中CXCL-8刺激MSC的表达基因的Biological process (BP)的富集分析柱状图；
图5为实施例中CXCL-8刺激MSC的表达基因的cellular component (CC) 的富集分析柱状图；
图6为实施例中CXCL-8刺激MSC的表达基因的molecular function (MF) 的富集分析柱状图；
图7为实施例中CXCL-8刺激MSC的表达基因与BP的chord图；
图8为实施例中CXCL-8刺激MSC的表达基因与CC的chord图；
图9为实施例中CXCL-8刺激MSC的表达基因与MF的chord图；
图10为实施例中CXCL-8刺激MSC的基因的KRGG Pathway富集分析柱状图；
图11为实施例中CXCL-8刺激MSC的基因与KRGG Pathway的chord图；
图12为实施例中MTT法检测HUVEC增殖的柱状图；* P<0.05，# P<0.01，△ P<0.01；
图13为实施例中各组HUVEC凋亡率比较的柱状图；* P<0.01，# P<0.01，△ P<0.01；
图14为实施例中Annexin V-PI流式细胞术检测各组HUVEC凋亡情况图；
图15为实施例中脱细胞兔真皮基质HE染色图；
图16为实施例中各组HUVEC的LC3-GFP平均荧光强度比较的柱状图；* P<0.01，# P<0.05，△ P<0.05；
图17为实施例中各组培养24h的HUVECs划痕图，其中黑线代表0h时的细胞划痕面积，比例尺=100μm；
图18为实施例中各组HUVEC划痕区闭合率比较的柱状图；* P<0.05，# P<0.05，△P<0.05；
图19为实施例中各组培养12h的HUVECs迁移图，比例尺=100μm。
具体实施方式
具体实施方式一：本实施方式石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，它按以下步骤实现：
一、制备GQDs溶液：将5-10g柠檬酸粉末放入微波炉，1000W功率下热解80-90 min，得到深棕色液体，然后倒入10-20 ml的1mol/L NaOH溶液中，于4℃下搅拌30min，获得GQDs溶液；
二、合成GQDs/CXCL-8纳米水凝胶液：28℃下将100mmol-200mmol CXCL-8、8.717μmol-17.434mmol N,N二乙基丙烯酰胺、129.7μmol-259.4μmol N,N'-甲基双丙烯酰胺和87.67μmol-175.34μmol过二硫酸铵溶解于5mL-10mL去离子水中，超声处理器处理4-8h，然后转移到含有环己烷的圆底烧瓶中并加入span80均匀化，获得GQDs/CXCL-8纳米水凝胶液；
三、纯化及固态：磁力搅拌器连续搅拌上述GQDs/CXCL-8纳米水凝胶液，加入50-100μL N,N,N',N'-四甲基乙二胺，于氮气下聚合反应6-12h，所得沉淀产物经洗涤离心后置于透析袋中，然后在磁力搅拌器上常温下透析3天，获得纯化的GQDs/CXCL-8纳米水凝胶液，经真空冷冻干燥，获得GQDs/CXCL-8纳米水凝胶颗粒；
四、配置静电纺丝溶液：将135.0-270.0mg聚已内酯和45.0-90.0mg胶原蛋白溶解于1ml六氟异丙醇中，于室温下磁力搅拌2-4h，然后加入30-60μlTween80并磁力搅拌1-2h，再以的0.05-0.08mg/min速度加入2.0-3.0mg GQDs/CXCL-8纳米水凝胶颗粒并磁力搅拌4-5h，然后超声分散，获得静电纺丝溶液；
五、构建组织诱导性材料：上述静电纺丝溶液置于27号针头注射器中，以承载ADM的铝箔纸作为接收载体，采用静电纺丝机进行包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料的制备；
六、静电纺丝、干燥、紫外交联和灭菌：上述包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料，经真空冷冻干燥、紫外交联和灭菌后，获得石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，完成所述的制备方法。
本实施方式步骤一中热解过程中柠檬酸粉末全部被液化，液体颜色从无色至深棕色。
本实施方式步骤二中N,N'-甲基双丙烯酰胺作为交联剂使用。
本实施方式步骤二中过二硫酸铵作为引发剂使用。
本实施方式步骤二中均匀化后水相乳化成连续的有机相。
本实施方式步骤三中洗涤离心目的是去除span 80和未反应的化学品。
本实施方式步骤三中透析目的是除去GQDs/CXCL-8纳米水凝胶液中过量的未反应的化学品。
本实施方式步骤三中所得GQDs/CXCL-8纳米水凝胶颗粒为固态粉末，于-80℃储存备用。
本实施方式步骤四中超声分散目的是使GQDs/CXCL-8纳米水凝胶颗粒均匀分散在溶液中。
本实施方式步骤六中所得石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，于-80℃下封装保存备用。
具体实施方式二：本实施方式与具体实施方式一的不同是，步骤二中所述均匀化：室温下10000rpm均匀化1h。其它步骤及参数与具体实施方式一相同。
具体实施方式三：本实施方式与具体实施方式一的不同是，步骤三中所述洗涤离心：采用环己烷洗涤并于12000 rpm离心30min，重复3次，所得离心产物分散在mili-Q水中，于12000rpm离心30min，重复3次。其它步骤及参数与具体实施方式一相同。
具体实施方式四：本实施方式与具体实施方式一的不同是，步骤四中所述超声分散：90W功率下超声30min。其它步骤及参数与具体实施方式一相同。
具体实施方式五：本实施方式与具体实施方式一的不同是，步骤五中所述ADM采用脱细胞兔真皮基质，其制备如下：取雄性新西兰白兔，麻醉，剃毛后，切取雄性新西兰白兔背部15-20cm圆形全层皮肤，37℃，1mol/L氯化钠溶液震荡孵育12h，去除表皮层，然后用0.125%胰酶，37℃震荡孵育24h后，0.5%十二烷基硫酸钠溶液在37℃震荡孵育4h，真空冷冻干燥机干燥6-8h，钴60灭菌，-80℃封装。其它步骤及参数与具体实施方式一相同。
本实施方式氯化钠能够改变细胞渗透压；胰酶能够消化细胞；十二烷基硫酸钠能够破碎细胞，达到脱细胞的目的。
具体实施方式六：本实施方式与具体实施方式一的不同是，步骤五中所述静电纺丝机：设置参数为正负极电压21kv，接收距离15cm，喷丝孔径0.41mm，转速为2800r/min，纺丝速度0.5mL/h，温度22.0±0.5℃，湿度42.4±0.5%。其它步骤及参数与具体实施方式一相同。
具体实施方式七：本实施方式与具体实施方式一的不同是，步骤六中所述紫外交联：于6KW紫外线下交联2h；所述灭菌：采用25kGy钴60辐照灭菌15~20min。其它步骤及参数与具体实施方式一相同。
实施例：
石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，它按以下步骤实现：
一、制备GQDs溶液：将5g柠檬酸粉末放入微波炉，1000W功率下热解80min，得到深棕色液体，然后倒入10ml的1mol/L NaOH溶液中，于4℃下搅拌30min，获得GQDs溶液；
二、合成GQDs/CXCL-8纳米水凝胶液：28℃下将100mmol CXCL-8、8.717μmol N,N二乙基丙烯酰胺、129.7μmol N,N'-甲基双丙烯酰胺和87.67μmol过二硫酸铵溶解于5mL-10mL去离子水中，超声处理器处理4h，然后转移到含有环己烷的圆底烧瓶中并加入span80均匀化，获得GQDs/CXCL-8纳米水凝胶液；
三、纯化及固态：磁力搅拌器连续搅拌上述GQDs/CXCL-8纳米水凝胶液，加入50μLN,N,N',N'-四甲基乙二胺，于氮气下聚合反应6h，所得沉淀产物经洗涤离心后置于透析袋中，然后在磁力搅拌器上常温下透析3天，获得纯化的GQDs/CXCL-8纳米水凝胶液，经真空冷冻干燥，获得GQDs/CXCL-8纳米水凝胶颗粒；
四、配置静电纺丝溶液：将135.0mg聚已内酯和45.0mg胶原蛋白溶解于1ml六氟异丙醇中，于室温下磁力搅拌2h，然后加入30μl Tween80并磁力搅拌1h，再以的0.05mg/min速度加入2.0mg GQDs/CXCL-8纳米水凝胶颗粒并磁力搅拌4h，然后超声分散，获得静电纺丝溶液；
五、构建组织诱导性材料：上述静电纺丝溶液置于27号针头注射器中，以承载ADM的铝箔纸作为接收载体，采用静电纺丝机进行包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料的制备；
六、静电纺丝、干燥、紫外交联和灭菌：上述包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料，经真空冷冻干燥、紫外交联和灭菌后，获得石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，完成所述的制备方法。
本实施例步骤二中所述均匀化：室温下10000rpm均匀化1h。
本实施例步骤三中所述洗涤离心：采用环己烷洗涤并于12000 rpm离心30min，重复3次，所得离心产物分散在mili-Q水中，于12000rpm离心30min，重复3次。
本实施例步骤四中所述超声分散：90W功率下超声30min。
本实施例步骤五中所述ADM采用脱细胞兔真皮基质，其制备如下：取4只体重为2-2.5kg的雄性新西兰白兔，麻醉，剃毛后，切取雄性新西兰白兔背部15-20cm圆形全层皮肤，37℃，1mol/L氯化钠溶液震荡孵育12h，去除表皮层，然后用0.125%胰酶，37℃震荡孵育24h后，0.5%十二烷基硫酸钠溶液在37℃震荡孵育4h，真空冷冻干燥机干燥6h，钴60灭菌，-80℃封装。
本实施例步骤五中所述静电纺丝机：设置参数为正负极电压21kv，接收距离15cm，喷丝孔径0.41mm，转速为2800r/min，纺丝速度0.5mL/h，温度22.0±0.5℃，湿度42.4±0.5%。
本实施例步骤六中所述紫外交联：于6KW紫外线下交联2h；所述灭菌：采用25kGy钴60辐照灭菌15min。
发明人在国家自然科学委员会项目（81541137）和黑龙江省自然科学基金项目（LH2021H121）的支持下发现：
① 静电纺丝纳米生物材料有效模拟细胞外基质微环境，适合MSC三维生长。
②脱细胞真皮基质无生物致病性、脱细胞非常彻底。
③ GQDs/CXCL-8水凝胶能够经内吞作用进入MSC内，调节PH值可实现稳定释放CXCL-8。
④ 高糖或低氧细胞环境中，GQDs/CXCL-8水凝胶、CXCL-8通过Akt-mTOR信号通路使MSC归巢，并发挥保护MSC抗高糖缺氧损伤的重要效果。
⑤ GQDs/CXCL-8水凝胶可提高MSC线粒体荧光强度的表达，对细胞能量代谢具有积极作用。
（1）显微观察：
本实施例中所得包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料，如图1所示，支架直径和孔径分别为1.38±0.12µm、10.15±1.08µm。
本实施例中所得石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，如图2所示，GQDs/CXCL-8水凝胶直径6.5±0.09μm。
（2）高通量细胞测序技术检测CXCL-8刺激MSC的基因表达：
① CXCL-8刺激MSC的基因表达谱
CXCL-8刺激MSC共发现40个差异性基因，如图3所示，包括EDNRA, TACR2, CCKAR,GHSR, SSTR2, FNTA, SSTR, ITGA2B, ITGB1, NRP1,SSTR5, NTSR1, NCOR2, HDAC1,AVPR1A, ITGAV, CAPN1, HDAC2, MMP1, MMP2, PPARG, ECE1, MMP7, BACE1,EPHX2,PTGS2, GRB2, ITGA3,MEN1, CTSC, CTSD, MLNR, CASP1, OPRL1, PLG, ITGA4, ITGAL,ITGB2, ICAM1, ITGB3。
② CXCL-8刺激MSC的基因表达谱的基因本体论（GO）富集分析
CXCL-8刺激MSC表达基因的cellular component (CC), Biological process(BP), molecular function (MF)分别为53、495、41条（P＜0.05），图4-图6。这些基因分布在cell-substrate adherens junction、extracellular matrix等细胞部位，图8。与血管发育、促进血管内皮细胞增殖、迁移或间充质干细胞分化相关的主要生物学过程包括cell-matrix adhesion, positive regulation of cell motility, blood vesseldevelopment, positive regulation of cellular component movement,angiogenesis, blood vessel morphogenesis, blood circulation, positiveregulation of cell death, endothelial cell migration, endothelial cellproliferation, positive regulation of vasculature development, regulation ofangiogenesis, regulation of vasculature development, vascular endothelialcell proliferation, positive regulation of sprouting angiogenesis, regulationof nitric oxide biosynthetic process, blood vessel endothelial cellmigration, sprouting angiogenesis, vascular endothelial growth factorreceptor signaling pathway, cell migration involved in sproutingangiogenesis, positive regulation of endothelial cell migration, regulationof sprouting angiogenesis, mesenchymal cell differentiation等70条。共37个基因，其中出现频次在中位数以上的基因包括17个，分别是PTGS2，NRP1，ITGB2，ITGB1，ICAM1，ITGB3，PPARG，ITGA4，GHSR，ITGAV，ITGA3，EDNRA，AVPR1A，ECE1，PLG，GRB2，MEN1，图7。与血管发育有关的分子功能包括G protein-coupled peptide receptor activity, C-X3-Cchemokine binding, extracellular matrix binding, cell adhesion moleculebinding, cytokine binding, growth factor binding。共38个基因，其中出现频次在中位数以上的基因包括17个，分别是ITGAV，ITGB1，ITGB3，CTSC，ECE1，AVPR1A，MMP1，MMP2，MMP7，PLG，PPARG，SSTR2，CASP1，HDAC1，ITGA4，SSTR3，SSTR5，图9。
③ CXCL-8刺激MSC的基因表达谱的KRGG Pathway富集分析
CXCL-8刺激MSC表达基因的KEGG Pathway为47条（P＜0.05），图10。与血管新生或血管内皮细胞增殖等有关的KEGG Pathway主要为：PI3K-Akt signaling pathway,Pathways in cancer, Cell adhesion molecules, ECM-receptor interaction, Focaladhesion, cAMP signaling pathway, Calcium signaling pathway, Rap1 signalingpathway等26个，共37个基因，其中出现频次在中位数以上的基因包括18个，分别是ITGB1,ITGB3, ITGAV, GRB2, ITGA2B, ITGB2, HDAC1, ITGA4, HDAC2, ICAM1, ITGA3, ITGAL,PTGS2, MMP2, PPARG, EDNRA, MMP1, PLG，图11。
（3）GQDs/CXCL-8（IL-8）水凝胶刺激的MSC条件培养液对人脐静脉血管内皮细胞（HUVEC）的影响
① 实验分组
高糖环境（HG）下，4×106 MSCs为高糖对照组（HG-control组）；以50ng/mL或100ng/mL的GQDs/CXCL-8（IL-8）水凝胶分别刺激MSCs为CXCL-8刺激组（分别为HG-IL-850组和HG-IL-8100组）；若预先在HG-control组添加10μmol/L Triciribine，37˚C，5%CO2培养30min后，0.01mmol/L PBS冲洗3次，再以50ng/mL或100ng/mL的IL-8刺激MSCs，则分别为Akt抑制剂组（HG-AI50组和HG-AI100组）。正常环境下培养的MSCs为正常对照组（control组）。
分别收集各组MSCs细胞上清液，用HUVEC高糖培养基按1:3比例稀释为条件培养基（CM），培养HUVECs为相应的条件培养基组HUVEC，分别为HG-control CM, HG-CXCL-8 CM和HG-Hh inhibitor CM group；高糖条件下，无任何刺激的HUVEC为非条件培养基组（NCM）。
② GQDs/CXCL-8（IL-8）水凝胶刺激的MSC条件培养液对HUVEC增殖的影响
结果见图12~图19，在高糖环境下，GQDs/CXCL-8（IL-8）水凝胶刺激的MSC条件培养液提高HUVEC增殖、自噬和运动，抑制HUVEC的凋亡；
为了确定在高糖条件下，GQDs/CXCL-8水凝胶刺激的MSC条件培养基（CM）对人脐静脉血管内皮细胞（HUVEC）生长的影响，我们用MTT法检测HUVEC的增殖情况。与HG-NCM group和HG-control CM组相比，HG-IL-850CM组和HG-IL-8100CM组HUVEC增殖A值逐渐升高（P<0.05），图12。此外，HG-IL-8100CM组HUVEC增殖A值是HG-IL-850CM组的1.465倍（P<0.01），图12。相比之下，HG-AI50 CM和HG-AI100CM组的HUVEC增殖A值分别是HG-IL-850CM和HG-IL-8100CM组的0.532倍和0.310倍（P<0.01），图12。
③ 各组条件培养液对HUVEC凋亡的影响
采用AnnexinV-PI细胞凋亡实验，探讨条件培养液对HUVECs凋亡的影响。我们发现HG-IL-850CM组和HG-IL-8100CM组的HUVECs凋亡率较HG-NCM组和HG-control CM组降低（P<0.01），图13，图14。同时，HG-IL-8100CM组HUVECs凋亡率是HG-IL-850CM组的0.395倍（P<0.01），图13，图14。相比之下，HG-AI50 CM组和HG-AI100 CM组的HUVECs凋亡率分别是HG-IL-850 CM组和HG-IL-8100 CM组的1.372倍和1.135倍（P<0.01），图13，图14。这些结果表明，在高糖环境下，IL-8刺激的MSCs条件培养基中含有抑制HUVEC凋亡的细胞因子。
④脱细胞真皮基质
扫描电子显微镜发现正常皮肤真皮组织表面比较光滑，脱细胞真皮基质的细胞被完全去除，图15。
⑤ 各组条件培养基对HUVEC迁移的影响
为了确定GQDs/CXCL-8（IL-8）水凝胶刺激的MSC条件培养基对HUVEC运动的影响，我们进行了HUVEC划痕实验和Transwell室实验。细胞划痕实验发现，与HG-NCM组和HG-control CM组相比，HG-IL-850 CM组和HG-IL-8100 CM组的HUVEC划痕闭合率逐渐增加（P<0.05），图16，图18。但与HG-IL-850 CM组和HG-IL-8100 CM组相比，HG-AI50 CM组和HG-AI100 CM组的HUVEC划痕闭合率分别下降了47.162%和38.014%（P<0.05），图16，图17。
接下来，在Transwell实验中，我们发现与HG-NCM组和HG-control CM组相比，HG-IL-850 CM组和HG-IL-8100 CM组的HUVEC迁移率逐渐增加（P<0.01），图18，图19。与HG-IL-850CM组相比，HG-IL-8100 CM组的迁移率增加了29.021%（P<0.01）。相比之下，与HG-IL-850 CM组和HG-IL-8100 CM组相比，HG-AI50 CM组和HG-AI100 CM组的HUVEC迁移率分别下降了41.204%和30.994%（P<0.01），图18，图19。这些结果提示IL-8可能通过Akt信号通路调控细胞因子的表达来促进HUVEC的迁移。
经实验证明量子点能够稳定释放CXCL-8等细胞因子，CXCL-8能够积极招募细胞归巢等特性，利用石墨烯量子点稳定释放CXCL-8招募MSC归巢的特性，主动定点捕获组织内或血管中MSC等细胞，有效提高MSC归巢，促进生物材料血管化，增加了生物材料与机体相互作用，进而促进组织损伤修复。
Claims (7)
1.石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，其特征在于它按以下步骤实现：
一、制备GQDs溶液：将5-10g柠檬酸粉末放入微波炉，1000W功率下热解80-90 min，得到深棕色液体，然后倒入10-20 ml的1mol/L NaOH溶液中，于4℃下搅拌30min，获得GQDs溶液；
二、合成GQDs/CXCL-8纳米水凝胶液：28℃下将100mmol-200mmol CXCL-8、8.717μmol-17.434mmol N,N二乙基丙烯酰胺、129.7μmol-259.4μmol N,N'-甲基双丙烯酰胺和87.67μmol-175.34μmol过二硫酸铵溶解于5mL-10mL去离子水中，超声处理器处理4-8h，然后转移到含有环己烷的圆底烧瓶中并加入span80均匀化，获得GQDs/CXCL-8纳米水凝胶液；
三、纯化及固态：磁力搅拌器连续搅拌上述GQDs/CXCL-8纳米水凝胶液，加入50-100μLN,N,N',N'-四甲基乙二胺，于氮气下聚合反应6-12h，所得沉淀产物经洗涤离心后置于透析袋中，然后在磁力搅拌器上常温下透析3天，获得纯化的GQDs/CXCL-8纳米水凝胶液，经真空冷冻干燥，获得GQDs/CXCL-8纳米水凝胶颗粒；
四、配置静电纺丝溶液：将135.0-270.0mg聚已内酯和45.0-90.0mg胶原蛋白溶解于1ml六氟异丙醇中，于室温下磁力搅拌2-4h，然后加入30-60μlTween80并磁力搅拌1-2h，再以的0.05-0.08mg/min速度加入2.0-3.0mg GQDs/CXCL-8纳米水凝胶颗粒并磁力搅拌4-5h，然后超声分散，获得静电纺丝溶液；
五、构建组织诱导性材料：上述静电纺丝溶液置于27号针头注射器中，以承载ADM的铝箔纸作为接收载体，采用静电纺丝机进行包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料的制备；
六、静电纺丝、干燥、紫外交联和灭菌：上述包裹ADM的GQDs/CXCL-8水凝胶-聚已内酯-胶原蛋白纳米纤维生物材料，经真空冷冻干燥、紫外交联和灭菌后，获得石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料，完成所述的制备方法。
2.根据权利要求1所述的石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，其特征在于步骤二中所述均匀化：室温下10000rpm均匀化1h。
3.根据权利要求1所述的石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，其特征在于步骤三中所述洗涤离心：采用环己烷洗涤并于12000 rpm离心30min，重复3次，所得离心产物分散在去离子水中，于12000rpm离心30min，重复3次。
4.根据权利要求1所述的石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，其特征在于步骤四中所述超声分散：90W功率下超声30min。
5.根据权利要求1所述的脱细胞真皮基质的制备方法，其特征在于步骤五中所述ADM采用脱细胞兔真皮基质，其制备如下：取雄性新西兰白兔，麻醉，剃毛后，切取雄性新西兰白兔背部15-20cm圆形全层皮肤，37℃，1mol/L氯化钠溶液震荡孵育12h，去除表皮层，然后用0.125%胰酶，37℃震荡孵育24h后，0.5%十二烷基硫酸钠溶液在37℃震荡孵育4h，真空冷冻干燥机干燥6-8h，钴60灭菌，-80℃封装。
6.根据权利要求1所述的石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，其特征在于步骤五中所述静电纺丝机：设置参数为正负极电压21kv，接收距离15cm，喷丝孔径0.41mm，转速为2800r/min，纺丝速度0.5mL/h，温度22.0±0.5℃，湿度42.4±0.5%。
7.根据权利要求1所述的石墨烯量子点镊诱捕宿主细胞归巢的组织诱导性纳米纤维生物材料的制备方法，其特征在于步骤六中所述紫外交联：于6KW紫外线下交联2h；所述灭菌：采用25kGy钴60辐照灭菌15~20min。
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