CN110898254B - 一种用于修复子宫内膜并提高生育力的生物活性支架 - Google Patents
一种用于修复子宫内膜并提高生育力的生物活性支架 Download PDFInfo
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Abstract
本发明涉及一种用于修复子宫内膜并提高生育力的生物活性支架,该支架包含基底及内含的生物活性因子,通过生物活性因子募集并捕获内源性干细胞。干细胞自身具有多能性,且在创面发挥免疫调控作用,能有效促进子宫内膜修复,显著提高生育率。本发明针对宫腔镜手术所导致的宫腔粘连、生育率降低等问题,采用集募集、捕获为一体的内源性干细胞归巢手段,对受损的子宫内膜进行修复,避免了传统修复材料中使用外源性细胞带来的安全隐患。本发明所采用的原料具有良好的生物相容性,制备工艺简单,不含外源细胞,生物安全性高。
Description
技术领域
本发明涉及组织工程支架领域,特别涉及一种用于修复子宫内膜并提高生育力的生物活性支架。
背景技术
宫腔粘连是女性继发不孕的第二大病因,主要由创伤、感染等引起;子宫内膜受损伴发再生修复障碍,内膜纤维化,最终导致宫腔粘连和内膜萎缩。子宫内膜纤维化作为宫腔粘连的主要病理特征,不仅会形成纤维疤痕导致宫腔形态异常致不孕或流产,而且会加重缺血缺氧造成再生障碍-纤维化的恶性循环。宫腔镜手术治疗去除纤维疤痕虽然能恢复宫腔形态,但往往难以恢复子宫内膜生理功能。中重度宫腔粘连术后极易再粘连,术后再粘连几率高达62.5%,预防术后再粘连目前仍无行之有效的方法,致使术后妊娠率仅为22.5%~33.3%。因此,研发一种具有预防粘连同时促进子宫内膜修复再生的生物活性支架,对宫腔操作后的预防、难治性宫腔粘连的治疗都有重要意义。
传统组织工程支架是指能与组织活体细胞结合并能植入生物体的支架材料,替代目标组织功能或帮助其修复再生。传统组织工程具有细胞、生物活性因子及支架三要素。为了使种子细胞增殖和分化,需要提供一个由生物材料所构成的细胞支架充当人工细胞外基质。治愈宫腔粘连的关键在于子宫内膜的修复再生,目前有研究表明,运用传统组织工程支架,植入脐带间充质干细胞的策略能有效提高内膜的再生,并同时提高生育率。然而由于干细胞的来源有限,体外培养扩增极易使其丢失干性,干细胞植入体内过程中存活率低,且植入外源性干细胞存在未知的安全隐患,本发明将采用不含种子细胞的支架材料,运用生物活性因子,募集并捕获内源性干细胞,改善受损子宫内膜创面微环境,进而促进内膜修复并显著提高生育力。
发明内容
本发明的目的是提供一种用于修复子宫内膜并提高生育力的生物活性支架,该支架所采用的原料具有良好的生物相容性,制备工艺简单,可操作性强。多肽修饰的支架及其内含的生物活性因子,可募集并捕获内源性干细胞。干细胞自身具有多能性,且在创面发挥免疫调控作用,能有效促进子宫内膜修复,显著提高生育率。内源性干细胞的募集与捕获,避免了传统组织工程支架负载外源性细胞所带来的安全隐患。
本发明的技术方案如下:
一种生物活性支架,包含胶原支架、E7多肽序列和SDF-1α缓释粒子,E7多肽序列修饰于胶原支架上,SDF-1α缓释粒子注射于E7多肽修饰的胶原支架中。
所述的SDF-1α缓释粒子具有募集内源性干细胞的功能,E7多肽可以捕获募集而来的内源性干细胞,胶原支架可为干细胞提供生长环境及力学支撑。
上述技术方案中,所述的E7多肽修饰的胶原支架,采用如下方法制得:
在酸性条件下配制质量浓度为0.5%的胶原溶液,37℃搅拌均匀,将其浇筑于模具中冻干,再置于真空烘箱中105℃干热交联12小时,制得胶原支架;
E7多肽序列为谷氨酸-脯氨酸-亮氨酸-谷氨酰胺-亮氨酸-赖氨酸-蛋氨酸(Glu-Pro-Leu-Gln-Leu-Lys-Met,EPLQLKM),为方便后续修饰到胶原支架,谷氨酸端额外修饰了半胱氨酸(Cys,C);
采用偶联剂4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐,通过其琥珀酰亚胺端与胶原支架上的氨基反应,及其马来酰亚胺端与E7多肽上半胱氨酸的巯基发生加成反应,将E7多肽修饰到胶原支架表面,将修饰后的支架再次冻干,得到E7多肽修饰的胶原支架。
所述的SDF-1α为基质细胞衍生因子-1的一种亚型,属于趋化因子蛋白家族,为保护其生物活性,且其本身带有微正电,采用带负电的磺化壳聚糖与带正电的聚赖氨酸静电络合包裹,形成SDF-1α缓释粒子。其中磺化壳聚糖可以以壳聚糖为基材,以氯磺酸为磺化试剂制得。
以注射器将SDF-1α缓释粒子注射进入E7多肽修饰的胶原支架,SDF-1α缓释粒子可以募集内源性干细胞,E7多肽促进干细胞粘附,即募集后的内源性干细胞被E7多肽修饰的胶原支架捕获,该生物活性支架作为药物被植入到受损SD大鼠子宫后,支架发挥内源性干细胞募集及捕获作用,干细胞的自身多能性及免疫调控作用可促进子宫内膜的修复,显著提高生育力,实验表明,利用该生物活性支架可将子宫受损大鼠的生育率提高约50%。
本发明的生物活性支架主要可用于受损子宫内膜,其对于内源性干细胞的募集与捕获,能有效修复内膜结构,显著提高生育率。
附图说明
图1为生物活性支架的构建示意图;
图2为生物活性支架的宏观及SEM图;
图3为HE染色图;
图4为Masson染色图;
图5为生育率实验结果及新生小鼠图。
具体实施方式
下面结合具体实例对本发明作详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明的阐述内容后,本领域技术人员可对本发明做修改,这些等价形式同样落于本申请所附权利要求书限定范围。
实例1
(a)具备募集功能的SDF-1α缓释粒子的制备方法
所述的SDF-1α缓释粒子采用带负电的磺化壳聚糖、带微正电的SDF-1α与带正电的聚赖氨酸静电络合而成。
磺化壳聚糖是以壳聚糖为基材,氯磺酸为磺化试剂制得。首先,将9g壳聚糖、15mL二氯乙酸和150mL甲酰胺在烧杯中充分混合,使壳聚糖充分溶胀。之后,将150mL无水DMF置于500mL三口烧瓶中,冰浴搅拌,并向其中缓慢滴加30mL氯磺酸。之后,将充分溶胀后的壳聚糖加入三口烧瓶中,60℃反应2小时。反应结束后,加入少量去离子水中和氯磺酸,离心分离沉淀,并将上清液用冷无水乙醇沉淀,将沉淀用冷无水乙醇反复清洗。之后,将沉淀溶于水中,加碳酸氢钠中和至无气泡产生且pH接近7。最后,用截留分子量为3500的透析袋透析3天,冻干获得磺化壳聚糖。
首先,配置质量浓度3%的磺化壳聚糖溶液、100μg/mL SDF-1α溶液与质量浓度0.3%的聚赖氨酸溶液。之后,将10μL磺化壳聚糖溶液、4μL SDF-1α溶液及10μL聚赖氨酸溶液依次加入离心管中,澄清溶液快速络合形成粒子(浑浊)。
(b)具备捕获功能的E7多肽修饰的胶原支架的构建方法
在酸性条件下配制质量浓度为0.5%的胶原溶液,37℃搅拌均匀,得到胶原溶液。将其浇筑于模具中冻干,再置于真空烘箱中105℃交联12小时制得胶原支架。
E7多肽序列为谷氨酸-脯氨酸-亮氨酸-谷氨酰胺-亮氨酸-赖氨酸-蛋氨酸(Glu-Pro-Leu-Gln-Leu-Lys-Met,EPLQLKM),为方便后续修饰到胶原支架,谷氨酸端额外修饰了半胱氨酸(Cys,C),可购买获得。
将胶原支架浸泡于1mg/mL的偶联剂4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐溶液中,通过琥珀酰亚胺端与胶原支架上的氨基反应。半小时后,用去离子水清洗胶原支架,并迅速转移至1mg/mL的E7多肽溶液中于4℃浸泡过夜,偶联剂的马来酰亚胺端与E7多肽上半胱氨酸的巯基发生加成反应,将E7多肽修饰到胶原表面,修饰后的支架再次冻干,制得E7多肽修饰的胶原支架。
(c)构建本发明生物活性支架
用注射器将SDF-1α缓释粒子注射进入E7多肽修饰的胶原支架,即制得生物活性支架。
动物实验过程如下:1、机械性损伤法损伤大鼠双侧子宫构建子宫内膜损伤模型,评估模型效果;2、将所有子宫内膜损伤大鼠分为6组:自然修复组(NR),胶原支架组(CS),胶原E7支架组(CS-E7),胶原E7支架/SDF-1α缓释粒子200ng组(CS-E7/SDF200),胶原E7支架/SDF-1α缓释粒子400ng组(CS-E7/SDF400),胶原E7支架/SDF-1α缓释粒子600ng组(CS-E7/SDF600)。所有的支架材料组在子宫内膜损伤模型大鼠双侧宫腔原位植入2×0.5cm2大小的生物材料;自然修复组即对子宫内膜损伤模型大鼠不做特殊处理。每组大鼠、每个时间点各6只,比较不同组子宫内膜修复情况。
我们发现,相较于NR、CS、CS-E7组,CS-E7/SDF200组,CS-E7/SDF400组,CS-E7/SDF600组明显促进了子宫内膜增厚,增加了子宫内膜处腺体的数目(图3);减少了子宫内膜处胶原的沉积(图4);提高了大鼠的生育力,其中,以CS-E7/SDF400组对大鼠生育力的提高尤为明显(图5)。
Claims (4)
1.一种生物活性支架,其特征在于,该生物活性支架包含胶原支架、E7多肽序列和SDF-1α缓释粒子,E7多肽序列修饰于胶原支架上,SDF-1α缓释粒子注射于E7多肽修饰的胶原支架中;
所述SDF-1α缓释粒子是通过将10μL质量浓度3%的磺化壳聚糖溶液、4μL浓度为100μg/mL SDF-1α溶液及10μL质量浓度0.3%的聚赖氨酸溶液混合静电络合而成;
所述E7多肽序列修饰于胶原支架上的方法如下:采用1mg/mL的偶联剂4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐溶液,通过其琥珀酰亚胺端与胶原支架上的氨基反应,以及其马来酰亚胺端与E7多肽序列上半胱氨酸的巯基发生加成反应,将E7多肽修饰到胶原支架表面,修饰后的支架再次冻干,制得E7多肽修饰的胶原支架;
所述的胶原支架是采用一次冻干法制成:在酸性条件下配制质量浓度为0.5%的胶原溶液,37℃搅拌均匀,将其浇筑于模具中冻干,再置于真空烘箱中105℃干热交联12小时制得。
2.根据权利要求1所述的生物活性支架,其特征在于,所述的E7多肽序列为谷氨酸-脯氨酸-亮氨酸-谷氨酰胺-亮氨酸-赖氨酸-蛋氨酸,即Glu-Pro-Leu-Gln-Leu-Lys-Met,EPLQLKM,为方便后续修饰到胶原支架,谷氨酸端额外修饰了半胱氨酸(Cys,C)。
3.根据权利要求1所述的生物活性支架,其特征在于,所述的磺化壳聚糖是以壳聚糖为基材,经氯磺酸修饰而得的,方法如下:向壳聚糖、二氯乙酸和甲酰胺的混合溶液中逐滴加入氯磺酸,反应结束后,经离心、沉淀、中和、透析及冻干,获得产物磺化壳聚糖,由于修饰了磺酸基团,磺化壳聚糖在水溶液中带负电。
4.如权利要求1-3任一项所述的生物活性支架的用途,其特征在于,该生物活性支架可用于制备促进损伤子宫内膜修复或提高生育力潜能的药物。
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