CN115025287A - 一种用于子宫内膜修复的长效缓释细胞支架及其制备方法和应用 - Google Patents
一种用于子宫内膜修复的长效缓释细胞支架及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种用于子宫内膜修复的长效缓释细胞支架,所述长效缓释细胞支架内部包含细胞因子缓释微球,所述细胞因子缓释微球中包覆有复合水凝胶/TGF‑β1蛋白与丝素蛋白的混合物;所述细胞因子缓释微球外部还包覆有复合蛋白;所述复合水凝胶/TGF‑β1蛋白为复合水凝胶/TGF‑β1蛋白。所述长效缓释细胞支架的制备方法,包括制备复合水凝胶‑TGF/beta蛋白、制备丝素蛋白混合溶液、制备细胞因子缓释微球、制备长效缓释细胞支架四步。所述长效缓释细胞支架的应用,通过注射方式进入子宫内对子宫内膜进行治疗。本发明实现原位注射水凝胶改善子宫内膜,加快了受损组织的生长和修复,实现了子宫内膜长效地抗菌抗炎和再生修复作用。
Description
技术领域
本发明涉及细胞支架技术领域,特别涉及一种用于子宫内膜修复的长效缓释细胞支架。
背景技术
子宫内膜发育不良,是不孕症患者试管婴儿(辅助生殖技术)助孕失败的重要原因,子宫内膜发育不良的机制复杂,可能由机械损伤、炎症、免疫、内分泌等综合因素导致,目前尚无有效的治疗方法。如何解决现有治疗手段不力的情况,使子宫内膜发育不良的患者也能获得良好的治疗效果、增加受孕几率,成为目前亟需解决的一个问题。
当前,在药物传输领域,原位水凝胶的研究引起人们广泛的兴趣。水凝胶材料作为药物控制释放载体,可以包载化多种治疗性药物,具有多种生物应用形式,应用广泛。作为释药载体,因水凝胶自身结构特性而具有独特的优势:一方面,水凝胶可作为抗肿瘤药物或组织修复生长因子的载体,通过体内靶向给药介入治疗癌症或者修复缺损的组织或器官,这类给药系统可以选择性地作用于靶器官、靶组织、靶细胞内,从而在临床治疗疾病的过程中能够最大限度地增强药物的疗效,同时将药物的不良反应降至最低,也减轻了病人多次用药的痛苦;另一方面,因为凝胶自身为连通多孔结构,为创伤细胞提供气体交换通道,并由于凝胶高溶胀率可以吸收伤口多余渗液并保持创面的湿润、减少炎症反应,还能够包埋抗菌消炎药物构建成缓释体系,从而控制药物缓慢释放以使伤口局部达到适宜的药物浓度,达到了提高药物疗效、降低毒副作用的目的。
细胞支架,也称为组织工程支架材料,是指能与组织活体细胞结合并能植入生物体的不同组织,并根据具体替代组织具备的功能的材料。为了使种子细胞增殖和分化,需要提供一个由生物可溶性材料所构成的细胞支架,细胞支架材料相当于人工细胞外基质。组织工程支架材料包括:骨、软骨、血管、神经、皮肤和人工器官,如肝、脾、肾、膀胱等的组织支架材料,是治疗各种慢性软组织疾病的有效手段,其应用前景广阔。
发明内容
本发明的第一个目的是,针对现有技术中的不足,提供一种用于子宫内膜修复的长效缓释细胞支架,稳定、持续地在体内起到机械支持、指导细胞分化的作用,有效改善子宫内膜发育不良状况。。
本发明的第二个目的是,提供一种用于子宫内膜修复的长效缓释细胞支架的制备方法。
本发明的第三个目的是,提供一种长效缓释细胞支架的应用。
为实现上述第一个目的,本发明采取的技术方案是:
一种用于子宫内膜修复的长效缓释细胞支架,所述长效缓释细胞支架内部包含细胞因子缓释微球,所述细胞因子缓释微球中包覆有复合水凝胶/TGF-β1蛋白与丝素蛋白的混合物;所述细胞因子缓释微球外部还包覆有复合蛋白;所述复合水凝胶/TGF-β1蛋白为复合水凝胶/TGF-β1蛋白。
本发明的进一步改就在于:所述丝素蛋白为家蚕丝、柞蚕丝或野蚕丝中的一种或多种蚕丝素纤维经氯化钙处理得到的再生丝素蛋白;所述复合蛋白包括明胶、透明质酸。
本发明的进一步改就在于:所述复合水凝胶/TGF-β1蛋白中,水凝胶为ODex/PLL水凝胶;所述复合水凝胶/TGF-β1蛋白包含浓度为0.2~1.8mg/ml的过氧化物酶CAT、浓度为0.2~1.8mg/ml的转化生长因子β1。
为实现上述第二个目的,本发明采取的技术方案是:
所述一种用于子宫内膜修复的长效缓释细胞支架的制备方法,包括以下步骤:
S1、制备复合水凝胶-TGF/beta蛋白
利用高碘酸钠对具有良好粘附性和促细胞生长的葡聚糖进行氧化处理,制备氧化葡聚糖;将氧化葡聚糖与具良好抑菌性的ε-聚赖氨酸为原料,通过席夫碱反应制备ODex/PLL水凝胶;
将过氧化物酶CAT和转化生长因子-β1装载进ODex/PLL水凝胶中,制备得复合水凝胶/TGF-β1蛋白;
S2、制备丝素蛋白混合溶液
将脱胶的蚕丝素纤维加入氯化钙/乙醇/水三元溶液中,70℃~74℃的条件下搅拌溶解1h,得到浓度为20mg/mL的丝素蛋白混合溶液,备用;
S3、制备细胞因子缓释微球
将复合水凝胶/TGF-β1蛋白溶解于Tris-HCl缓冲液,制得预制液A;
将步骤S1制得的浓度为20mg/mL的丝素蛋白混合溶液,与浓度为4mM的葡萄糖酸钙溶液或浓度为4mM的CaCl2溶液按照体积比为1:1的比例混合,制得预制液B;
将1mL溶液A加入到40ml预制液B中,然后置于37℃水浴环境中处理40min,得到细胞因子缓释微球溶液;将制得的细胞因子缓释微球溶液在12000r/min下离心处理10min,取沉淀;用去离子水反复洗涤、除去未被包被的复合水凝胶/TGF-β1蛋白、葡萄糖酸钙或CaCl2,洗净后再冷冻干燥、并于4℃保存,即得固体微粒状态的纳米级细胞因子缓释微球;
S4、制备长效缓释细胞支架
在步骤S3制得的细胞因子缓释微球外再包被一层可被细胞利用的复合蛋白,经冷却、离心后制得冻干微球,即完成长效缓释细胞支架的制备。
所述步骤S1的具体步骤包括:
S1.1、制备氧化葡聚糖ODex
向单口烧瓶中加入葡聚糖Dex,用超纯水完全溶解,置于37℃水浴中,滴加NaIO4水溶液,避光搅拌反应8h;向反应液中加入乙二醇,继续反应1h,反应液滴入至冰乙醇沉降,离心收集沉淀,将沉淀溶于去离子水并透析5天后冷冻干燥,得到氧化葡聚糖ODex;
S1.2、制备酰胺化ε-聚赖氨酸
配制质量分数为15%~30%的ε-聚赖氨酸水溶液、体积浓度为1%~10%的酸酐水溶液,将ε-聚赖氨酸水溶液、酸酐水溶液混合,在30℃~60℃条件下搅拌反应45min~90min;反应结束后,用去离子水对反应液进行透析纯化15h~30h,冷冻干燥后即得酰胺化ε-聚赖氨酸;
S1.3、制备ODex/PLL水凝胶
配制质量分数为10%~30%的氧化葡聚糖水溶液、质量分数为5%~25%的酰胺化ε-聚赖氨酸水溶液,将氧化葡聚糖水溶液投入反应器中,在25℃~30℃条件下,加入酰胺化ε-聚赖氨酸水溶液,搅拌5min,即得ODex/PLL水凝胶;
S1.4、制备复合水凝胶-TGF/beta蛋白
将过氧化物酶CAT和转化生长因子-β1溶于水中,制备预制液D,将预制液D步骤1.3制备的ODex/PLL水凝胶混合,搅拌10min,即得复合水凝胶-TGF/beta蛋白。
本发明的进一步改进在于:
所述步骤1.1中,葡聚糖超纯水溶液的质量分数为2%-4%;NaIO4与葡聚糖重复单元的投料摩尔比为1:(2~6);乙二醇的投料量为NaIO4摩尔量的0.6~0.8倍;
所述步骤1.2中,ε-聚赖氨酸水溶液、酸酐水溶液的混合体积为(1~3):1;
所述步骤1.3中,氧化葡聚糖水溶液与酰胺化ε-聚赖氨酸水溶液的混合体积比为2~3:1;
所述步骤1.4中,所述复合水凝胶/TGF-β1蛋白包含浓度为0.2mg/ml~1.8mg/ml的过氧化物酶CAT、浓度为0.2mg/ml~1.8mg/ml的转化生长因子β1。
所述步骤S2中,蚕丝素纤维与氯化钙/乙醇/水三元溶液的浴比为1:5,氯化钙/乙醇/水三元溶液中三者的摩尔比为1:2:8。
所述步骤S4的具体步骤包括:
S4.1、取2.5g的明胶粉剂、25mg的透明质酸钠加入40mL双蒸水中,于30℃条件下搅拌至完全溶解,制得预制液C;用0.2μm针式滤器对预制液C进行过滤除菌处理,得到复合蛋白溶液;
S4.2、将60mL的无菌植物油和1mL的Span80混合并预热至60℃,然后缓慢滴入预热至60℃的所述复合蛋白溶液,并采用电动搅拌机以500r/min~900r/min的转速搅拌均匀,乳化时间10~15min,形成复合蛋白的稳定乳液;
S4.3:将步骤S3中制得的细胞因子缓释微球与制得的复合蛋白的稳定乳液按照混合比1:3v/v的比例混合,得到混合乳液;
S4.4:将步骤S4.3得到混合乳液冰浴冷却至5℃以下,此过程持续电动搅拌机以500-900r/min的转速搅拌,然后加入30mL预冷丙酮搅拌40min后电动搅拌机停止搅拌,再经高速离心机分离处理出微球后用蒸馏水反复清洗微球,最后将微球冻干处理,即完成长效缓释细胞支架的制备。
为实现上述第三个目的,本发明采取的技术方案是:
所述用于子宫内膜修复的长效缓释细胞支架的应用,所述长效缓释细胞支架用于子宫发育不良情况下子宫内膜的修复。具体来说,所述长效缓释细胞支架通过宫腔涂抹或注射方式作用于子宫内膜。
本发明优点在于:
本发明提供了一种用于子宫内膜修复的长效缓释细胞支架,通过长效缓释细胞支架,实现原位注射水凝胶改善子宫内膜,结合两者优点,稳定、持续地在体内起到机械支持、指导细胞分化、缓释药用成分等作用,加快了受损组织的生长和修复,实现了子宫内膜长效地抗菌抗炎和再生修复作用;同时原位局部治疗可避免注射等医疗方式伴随的副作用,为临床治疗提供更好的技术支持。
本发明的复合水凝胶/TGF-β1蛋白采用葡聚糖和ε-聚赖氨酸之别水凝胶,再装载过氧化物酶CAT和转化生长因子-β1。葡聚糖和ε-聚赖氨酸均低细胞毒性,葡聚糖是由肠系膜明串珠菌生物合成,是一类有良好的生物相容性和可降解性的葡萄糖聚合物,而ε-聚赖氨酸是一种由链霉菌属生物合成的均聚物,是一种生物性优良的合成多肽,易溶干水,且其降解产物为赖氨酸,是身体所必需的8种氨基酸之一,并具有良好的抑菌性,生物相容性好;采用低细胞毒性的葡聚糖和ε-聚赖氨酸制备水凝胶,有效降低长效缓释细胞支架的刺激性和毒性,可在体内长期使用、缓慢释放药效。所述葡聚糖的糖环含有大量-OH,通过将部分-OH氧化为-CHO,作为化学结合位点,与酰胺化ε-聚赖氨酸中-NH2键合形成席夫碱键,具有反应活性高、交联度精细可控的特点,确保ODex/PLL水凝胶成为合适的缓释药载体。
过氧化物酶CAT为人身存在的酶之一,其主要作用是催化过氧化氢分解为水和氧气,清除体内的过氧化氢,从而使细胞免于遭受H2O2的毒害,是生物防御体系的关键酶之一,为机体提供了抗氧化防御机理。转化生长因子-β1,不仅在炎症治疗、组织修复和胚胎发育等方面具有其独特作用,而且其对间充质起源的细胞起刺激作用、对上皮或神经外胚层来源的细胞起抑制作用,能够调节细胞生长、分化和免疫。通过过氧化物酶CAT和转化生长因子-β1的缓慢释放,能够刺激子宫内膜细胞的修复和再生,有效改善子宫内膜状况。
本发明先通过丝素蛋白包被复合水凝胶-TGF/beta蛋白形成细胞大子缓释微球,再在其外层用可被细胞降解利用的含有明胶的复合登蛋白进行包被,制得的长效缓释细胞支架可现实药物3~5个月的缓释。在应用时,复合蛋自形成水凝胶的杂合体比单纯的胶原蛋白具有更强的机械耐受性;采用的复合蛋白中的胶原蛋白可以保护细胞子缓释微球避免机体免疫系线流和酶系统的更多攻击。
附图说明
图1为长效缓释细胞支架的结构示意图;
图2为复合水凝胶/TGF-β1蛋白的合成步骤示意图;
图3为动物实验过程示意图;
图4为动物实验中大鼠机械造模的示意图;
图5为动物试验中大鼠化学造模的示意图;
图6为化学造模后大鼠子宫内膜的状态示意图;
图7为机械损伤实验组和机械损伤对照组大鼠子宫内膜的腺体数目检测;
图8为机械损伤实验组和机械损伤对照组大鼠子宫内膜纤维化程度的检测图谱;
图中,1、复合水凝胶/TGF-β1蛋白,2、丝素蛋白,3、细胞因子缓释微球,4、复合蛋白,5、长效缓释细胞支架。
具体实施方式
下面通过结合附图和实施例对本发明的技术方案做进一步详细描述。
结合图1、图2所示,本实施例提供了一种用于子宫内膜修复的长效缓释细胞支架,所述长效缓释细胞支架5内部包覆有细胞因子缓释微球3,所述细胞因子缓释微球3中包覆有有若干复合水凝胶/TGF-β1蛋白1与丝素蛋白2的混合物;所述细胞因子缓释微球3外部还包覆有复合蛋白4。载药的复合水凝胶/TGF-β1蛋白外部分别包被丝素蛋白和复合蛋白,使得细胞支架具有双重缓释效果,药物分层缓释,有效延长了药物作用期。
所述复合水凝胶/TGF-β1蛋白1为复合水凝胶/TGF-β1蛋白。在复合水凝胶/TGF-β1蛋白中,所用水凝胶为ODex(葡聚糖)/PLL(ε-聚赖氨酸)水凝胶;所述复合水凝胶/TGF-β1蛋白中的药物包括浓度为0.2mg/ml~1.8mg/ml的过氧化物酶CAT、浓度为0.2~1.8mg/ml的转化生长因子β1。
所述丝素蛋白为家蚕丝、柞蚕丝或野蚕丝中的一种或多种蚕丝素纤维经氯化钙处理得到的再生丝素蛋白。
所述复合蛋白包括明胶、透明质酸,明胶的保护可使细胞因子缓释微球逃逸机体的免疫攻击,并最终被细胞降解代谢,对身体无副作用。出于特殊情况的需求,复合蛋白还可以用I型胶原、II型胶原、III型胶原聚乳酸及其衍生物中一种或多种来补充或替代。
在本实施例中,所述复合水凝胶/TGF-β1蛋白1在经所述丝素蛋白3包被后或经所述经复合蛋白4包被后,可再经交联处理或者不经交联处理,用于所述交联处理的交联剂包括但不限于TG酶、聚乳酸、聚乙酸、环氧化合物和戊二醛中的一种或多种。
本发明还提供了一种用于子宫内膜修复的长效缓释细胞支架的制备方法,包括以下步骤:
S1、制备复合水凝胶-TGF/beta蛋白
利用高碘酸钠对具有良好粘附性和促细胞生长的葡聚糖进行氧化处理,制备氧化葡聚糖;将氧化葡聚糖与具良好抑菌性的ε-聚赖氨酸为原料,通过席夫碱反应制备ODex/PLL水凝胶;
将过氧化物酶CAT和转化生长因子-β1装载进ODex/PLL水凝胶中,制备得复合水凝胶/TGF-β1蛋白;
S2、制备丝素蛋白混合溶液
将脱胶的蚕丝素纤维加入氯化钙/乙醇/水三元溶液中,70℃~74℃的条件下搅拌溶解1h,得到浓度为20mg/mL的丝素蛋白混合溶液,备用;
本步骤中,蚕丝素纤维与氯化钙/乙醇/水三元溶液的浴比为1:5,氯化钙/乙醇/水三元溶液中三者的摩尔比为1:2:8;
S3、制备细胞因子缓释微球
将复合水凝胶/TGF-β1蛋白溶解于Tris-HCl缓冲液,制得预制液A;
将步骤S1制得的浓度为20mg/mL的丝素蛋白混合溶液,与浓度为4mM的葡萄糖酸钙溶液或浓度为4mM的CaCl2溶液按照体积比为1:1的比例混合,制得预制液B;
将1mL溶液A加入到40ml预制液B中,然后置于37℃水浴环境中处理40min,得到细胞因子缓释微球溶液;将制得的细胞因子缓释微球溶液在12000r/min下离心处理10min,取沉淀;用去离子水反复洗涤、除去未被包被的复合水凝胶/TGF-β1蛋白、葡萄糖酸钙或CaCl2,洗净后再冷冻干燥、并于4℃保存,即得固体微粒状态的纳米级细胞因子缓释微球;
S4、制备长效缓释细胞支架
在步骤S3制得的细胞因子缓释微球外再包被一层可被细胞利用的复合蛋白,经冷却、离心后制得冻干微球,即完成长效缓释细胞支架的制备。
所述步骤S1的具体步骤包括:
S1.1、制备氧化葡聚糖ODex
向单口烧瓶中加入葡聚糖Dex,用超纯水完全溶解,置于37℃水浴中,滴加NaIO4水溶液,避光搅拌反应8h;向反应液中加入乙二醇,继续反应1h,反应液滴入至冰乙醇沉降,离心收集沉淀,将沉淀溶于去离子水并透析5天后冷冻干燥,得到氧化葡聚糖ODex;
本步骤中,葡聚糖超纯水溶液的质量分数为2%-4%,优选为3%;NaIO4与葡聚糖重复单元的投料摩尔比为1:(2~6),优选为1:5;乙二醇的投料量为NaIO4摩尔量的0.6~0.8倍,优选为0.7倍;
S1.2、制备酰胺化ε-聚赖氨酸
配制质量分数为15%~30%的ε-聚赖氨酸水溶液、体积浓度为1%~10%的酸酐水溶液,将ε-聚赖氨酸水溶液、酸酐水溶液混合,在30℃~60℃条件下搅拌反应45min~90min;反应结束后,用去离子水对反应液进行透析纯化15h~30h,冷冻干燥后即得酰胺化ε-聚赖氨酸;
本步骤中,ε-聚赖氨酸水溶液、酸酐水溶液的混合体积为(1~3):1,优选为2:1;
S1.3、制备ODex/PLL水凝胶
配制质量分数为10%~30%的氧化葡聚糖水溶液、质量分数为5%~25%的酰胺化ε-聚赖氨酸水溶液,将氧化葡聚糖水溶液投入反应器中,在25℃~30℃条件下,加入酰胺化ε-聚赖氨酸水溶液,搅拌5min,即得ODex/PLL水凝胶;
本步骤中,氧化葡聚糖水溶液与酰胺化ε-聚赖氨酸水溶液的混合体积比为2~3:1,优选为2.5:1;
S1.4、制备复合水凝胶-TGF/beta蛋白
将过氧化物酶CAT和转化生长因子-β1溶于水中,制备预制液D,将预制液D步骤1.3制备的ODex/PLL水凝胶混合,搅拌10min,即得复合水凝胶-TGF/beta蛋白;
本步骤中,所述复合水凝胶/TGF-β1蛋白包含浓度为0.2mg/ml~1.8mg/ml的过氧化物酶CAT、浓度为0.2mg/ml~1.8mg/ml的转化生长因子β1,优选为包含浓度为0.8mg/ml~1.5mg/ml的过氧化物酶CAT、浓度为0.8mg/ml~1.5mg/ml的转化生长因子β1。
所述步骤S4的具体步骤包括:
S4.1、取2.5g的明胶粉剂、25mg的透明质酸钠加入40mL双蒸水中,于30℃条件下搅拌至完全溶解,制得预制液C;用0.2μm针式滤器对预制液C进行过滤除菌处理,得到复合蛋白溶液;
S4.2、将60mL的无菌植物油和1mL的Span80混合并预热至60℃,然后缓慢滴入预热至60℃的所述复合蛋白溶液,并采用电动搅拌机以500r/min~900r/min的转速搅拌均匀,乳化时间10~15min,形成复合蛋白的稳定乳液;
S4.3、将步骤S3中制得的细胞因子缓释微球与制得的复合蛋白的稳定乳液按照混合比1:3v/v的比例混合,得到混合乳液;
S4.4、将步骤S4.3得到混合乳液冰浴冷却至5℃以下,此过程持续电动搅拌机以500-900r/min的转速搅拌,然后加入30mL预冷丙酮搅拌40min后电动搅拌机停止搅拌,再经高速离心机分离处理出微球后用蒸馏水反复清洗微球,最后将微球冻干处理,即完成长效缓释细胞支架的制备。
本发明还提供了所述长效缓释细胞支架的应用,通过注射方式进入子宫内膜处。
通过动物实验来证明本发明长效缓释细胞支架的治疗效果,动物实验的具体过程为:
1、采用8~10周龄,体重为220~260g且动情周期正常的雌性SD大鼠进行建模,实验前每日予大鼠阴道涂片观察,连续监测2个动情周期;
2、将SD大鼠随机分为四组,分别为化学损伤实验组、化学损伤对照组、机械损伤实验组、机械损伤对照组;
3、给予开关腹手术,使用无水乙醇化学损伤化学损伤实验组、化学损伤对照组大鼠的双侧子宫内膜造模;
4、给予开关腹手术,刮匙物理损伤机械损伤实验组、机械损伤对照组大鼠的双侧子宫内膜造模;
5、向化学损伤实验组、机械损伤实验组的大鼠宫腔内注射药载支架,每日登记四组造模鼠的进食及一般情况;
6、1~2周后处死机械损伤实验组大鼠、机械损伤对照组大鼠,取子宫标本,观测子宫内膜厚度、纤维度,并分别对免疫化学、分子生物学指标HOXA10/CD38/CD138等的检测;
7、1~2周后,将化学损伤实验组大鼠、化学损伤对照组大鼠与雄鼠合笼,观察受孕及子鼠情况。
经对比发现,与机械损伤对照组大鼠相比,使用本发明细胞支架的机械损伤实验组明显促进了子宫内膜的增厚,子宫内膜处腺体的数目增加,子宫内膜的纤维化程度降低,由50.66%降低至22.20%,证明受到机械损伤的大鼠子宫内膜得到了有效治疗和改善,生育能力得以提高和加强。
CD38/CD138检测结果见下表:
CD38阳性率 | CD138阳性率 | |
机械损伤实验组 | 0.5% | 0.6% |
机械损伤对照组 | 98% | 100% |
通过上述数据可以看出,本发明长效缓释细胞支架可对受损的子宫内膜组织进行有效消炎,并促进内膜组织修复,减少子宫内膜慢性炎症的发病率,为提高胚胎着床率提供了基础。
通过检测机械损伤实验组、机械损伤对照组子宫内膜中HOXA10基因mRNA表达,发现两组均有HOXA表达,机械损伤实验组的HOXA10基因mRNA表达显著高于机械损伤对照组。HOXA10基因作为胚胎期决定子宫发育的转录因子参与调节子宫内膜腺上皮及间质细胞周期性的增殖与分化,与众多因子相互作用,使子宫内膜进入容受状态,介导胚泡成功着床,并参与子宫内膜的蜕膜化从而维持妊娠状态,因此,通过两组大鼠HOXA10基因mRNA表达检测结果,可以确定采用本发明细胞支架的机械损伤实验组大鼠的子宫内膜容受性高,有利于胚胎着床。
与化学损伤对照组大鼠相比,使用本发明细胞支架的化学损伤实验组的受孕成功率显著提高,受孕率比未治疗的化学损伤对照组高出35%以上,子鼠健康率100%;而化学损伤对照组大鼠孕育的子鼠,有约15%出现腺体或血液异常。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.一种用于子宫内膜修复的长效缓释细胞支架,其特征在于:所述长效缓释细胞支架内部包含细胞因子缓释微球,所述细胞因子缓释微球中包覆有复合水凝胶/TGF-β1蛋白与丝素蛋白的混合物;所述细胞因子缓释微球外部还包覆有复合蛋白;所述复合水凝胶/TGF-β1蛋白为复合水凝胶/TGF-β1蛋白。
2.根据权利要求1所述的一种用于子宫内膜修复的长效缓释细胞支架,其特征在于:所述丝素蛋白为家蚕丝、柞蚕丝或野蚕丝中的一种或多种蚕丝素纤维经氯化钙处理得到的再生丝素蛋白;所述复合蛋白包括明胶、透明质酸。
3.根据权利要求1所述的一种用于子宫内膜修复的长效缓释细胞支架,其特征在于:所述复合水凝胶/TGF-β1蛋白中,水凝胶为ODex/PLL水凝胶;所述复合水凝胶/TGF-β1蛋白包含浓度为0.2~1.8mg/ml的过氧化物酶CAT、浓度为0.2~1.8mg/ml的转化生长因子β1。
4.如权利要求1~3任一项所述的一种用于子宫内膜修复的长效缓释细胞支架的制备方法,其特征在于包括以下步骤:
S1、制备复合水凝胶-TGF/beta蛋白
利用高碘酸钠对具有良好粘附性和促细胞生长的葡聚糖进行氧化处理,制备氧化葡聚糖;将氧化葡聚糖与具良好抑菌性的ε-聚赖氨酸为原料,通过席夫碱反应制备ODex/PLL水凝胶;
将过氧化物酶CAT和转化生长因子-β1装载进ODex/PLL水凝胶中,制备得复合水凝胶/TGF-β1蛋白;
S2、制备丝素蛋白混合溶液
将脱胶的蚕丝素纤维加入氯化钙/乙醇/水三元溶液中,70℃~74℃的条件下搅拌溶解1h,得到浓度为20mg/mL的丝素蛋白混合溶液,备用;
S3、制备细胞因子缓释微球
将复合水凝胶/TGF-β1蛋白溶解于Tris-HCl缓冲液,制得预制液A;
将步骤S1制得的浓度为20mg/mL的丝素蛋白混合溶液,与浓度为4mM的葡萄糖酸钙溶液或浓度为4mM的CaCl2溶液按照体积比为1:1的比例混合,制得预制液B;
将1mL溶液A加入到40ml预制液B中,然后置于37℃水浴环境中处理40min,得到细胞因子缓释微球溶液;将制得的细胞因子缓释微球溶液在12000r/min下离心处理10min,取沉淀;用去离子水反复洗涤、除去未被包被的复合水凝胶/TGF-β1蛋白、葡萄糖酸钙或CaCl2,洗净后再冷冻干燥、并于4℃保存,即得固体微粒状态的纳米级细胞因子缓释微球;
S4、制备长效缓释细胞支架
在步骤S3制得的细胞因子缓释微球外再包被一层可被细胞利用的复合蛋白,经冷却、离心后制得冻干微球,即完成长效缓释细胞支架的制备。
5.根据权利要求4所述的一种用于子宫内膜修复的长效缓释细胞支架的制备方法,其特征在于所述步骤S1的具体步骤包括:
S1.1、制备氧化葡聚糖ODex
向单口烧瓶中加入葡聚糖Dex,用超纯水完全溶解,置于37℃水浴中,滴加NaIO4水溶液,避光搅拌反应8h;向反应液中加入乙二醇,继续反应1h,反应液滴入至冰乙醇沉降,离心收集沉淀,将沉淀溶于去离子水并透析5天后冷冻干燥,得到氧化葡聚糖ODex;
S1.2、制备酰胺化ε-聚赖氨酸
配制质量分数为15%~30%的ε-聚赖氨酸水溶液、体积浓度为1%~10%的酸酐水溶液,将ε-聚赖氨酸水溶液、酸酐水溶液混合,在30℃~60℃条件下搅拌反应45min~90min;反应结束后,用去离子水对反应液进行透析纯化15h~30h,冷冻干燥后即得酰胺化ε-聚赖氨酸;
S1.3、制备ODex/PLL水凝胶
配制质量分数为10%~30%的氧化葡聚糖水溶液、质量分数为5%~25%的酰胺化ε-聚赖氨酸水溶液,将氧化葡聚糖水溶液投入反应器中,在25℃~30℃条件下,加入酰胺化ε-聚赖氨酸水溶液,搅拌5min,即得ODex/PLL水凝胶;
S1.4、制备复合水凝胶-TGF/beta蛋白
将过氧化物酶CAT和转化生长因子-β1溶于水中,制备预制液D,将预制液D步骤1.3制备的ODex/PLL水凝胶混合,搅拌10min,即得复合水凝胶-TGF/beta蛋白。
6.根据权利要求5所述的一种用于子宫内膜修复的长效缓释细胞支架的制备方法,其特征在于:
所述步骤1.1中,葡聚糖超纯水溶液的质量分数为2%~4%;NaIO4与葡聚糖重复单元的投料摩尔比为1:(2~6);乙二醇的投料量为NaIO4摩尔量的0.6~0.8倍;
所述步骤1.2中,ε-聚赖氨酸水溶液、酸酐水溶液的混合体积为(1~3):1;
所述步骤1.3中,氧化葡聚糖水溶液与酰胺化ε-聚赖氨酸水溶液的混合体积比为2~3:1;
所述步骤1.4中,所述复合水凝胶/TGF-β1蛋白包含浓度为0.2mg/ml~1.8mg/ml的过氧化物酶CAT、浓度为0.2mg/ml~1.8mg/ml的转化生长因子β1。
7.根据权利要求4所述的一种用于子宫内膜修复的长效缓释细胞支架的制备方法,其特征在于:所述步骤S2中,蚕丝素纤维与氯化钙/乙醇/水三元溶液的浴比为1:5,氯化钙/乙醇/水三元溶液中三者的摩尔比为1:2:8。
8.根据权利要求5所述的一种用于子宫内膜修复的长效缓释细胞支架的制备方法,其特征在于所述步骤S4的具体步骤包括:
S4.1、取2.5g的明胶粉剂、25mg的透明质酸钠加入40mL双蒸水中,于30℃条件下搅拌至完全溶解,制得预制液C;用0.2μm针式滤器对预制液C进行过滤除菌处理,得到复合蛋白溶液;
S4.2、将60mL的无菌植物油和1mL的Span80混合并预热至60℃,然后缓慢滴入预热至60℃的所述复合蛋白溶液,并采用电动搅拌机以500r/min~900r/min的转速搅拌均匀,乳化时间10~15min,形成复合蛋白的稳定乳液;
S4.3、将步骤S3中制得的细胞因子缓释微球与制得的复合蛋白的稳定乳液按照混合比1:3v/v的比例混合,得到混合乳液;
S4.4、将步骤S4.3得到混合乳液冰浴冷却至5℃以下,此过程持续电动搅拌机以500-900r/min的转速搅拌,然后加入30mL预冷丙酮搅拌40min后电动搅拌机停止搅拌,再经高速离心机分离处理出微球后用蒸馏水反复清洗微球,最后将微球冻干处理,即完成长效缓释细胞支架的制备。
9.如权利要求1~权利要求8任一项所述的用于子宫内膜修复的长效缓释细胞支架的应用,其特征在于:所述长效缓释细胞支架用于子宫发育不良情况下子宫内膜的修复。
10.根据权利要求9所述的用于子宫内膜修复的长效缓释细胞支架的应用,其特征在于:所述长效缓释细胞支架通过宫腔涂抹或注射方式作用于子宫内膜。
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