CN112717203A - 一种仿生子宫内膜及其制备方法 - Google Patents
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Abstract
本发明公开一种仿生子宫内膜及其制备方法,包括两组呈镜面对称的BAM(仿生羊膜),两组所述BAM(仿生羊膜)之间设置有ESC(子宫内膜间质细胞),该种仿生子宫内膜及其制备方法通过在两组呈镜面对称的BAM之间设置有ESC,有助于增加最终成膜后的力学强度和膜转化为子宫内膜的性能,且BAM以“夹心三明治结构”模式构成,两个外层的hAAM中具备双层的基底膜,其内含有丰富的天然纤维组织,从前后两方面加固能够形成与新鲜羊膜力学特性较为接近的充分的生物力学强度,另外,在双层hAAM的夹持和hAMSCs定植融合的固定作用下,具有更好的抗拉强度、弹性和延展特性。
Description
技术领域
本发明涉及生物工程技术领域,尤其涉及一种仿生子宫内膜及其制备方法。
背景技术
宫腔粘连(Intrauterine Adhesions,IUA)是指由于损伤或感染等原因引起子宫内膜基底层损伤并发生瘢痕愈合,导致子宫腔、子宫峡部和宫颈管腔的宫壁发生部分或全部的粘连,引起宫腔缩小、有效内膜免疫减少、经量减少甚至闭经等症状的临床疾病。随着无痛人流、无痛清宫手术的增加,IUA发病呈逐年上升趋势,且粘连程度逐渐加重,严重影响女性身心健康和生育能力;
而人羊膜及其制品的宫腔移植已较广泛用于IUA的临床治疗,但最新的荟萃分析显示,在宫腔镜下粘连松解术后单纯使用人羊膜及其制品仅能增加患者的月经血容量,并不能改善粘连的复发率、再次妊娠率和自然流产率,产生上述现象的主要原因与人新鲜羊膜独特的结构有关,因表面的上皮层光滑,缺乏网状结构使外源性的细胞不易与其发生粘附、融合,因此本发明提出一种仿生子宫内膜及其制备方法以解决现有技术中存在的问题。
发明内容
针对上述问题,本发明的目的在于提出一种仿生子宫内膜及其制备方法,该种仿生子宫内膜及其制备方法通过在两组呈镜面对称的BAM之间设置有ESC,有助于增加最终成膜后的力学强度和膜转化为子宫内膜的性能,且BAM以“夹心三明治结构”模式构成,两个外层的hAAM中具备双层的基底膜,其内含有丰富的天然纤维组织,从前后两方面加固能够形成与新鲜羊膜力学特性较为接近的充分的生物力学强度,而且,双层hAAM中均具备完整的基质层,其内含有大量的胶原蛋白,保证了BAM的弹性和抗拉力,另外,双层hAAM的夹持和hAMSCs定植融合的固定作用下,具有更好的抗拉强度、弹性和延展特性。
为实现本发明的目的,本发明通过以下技术方案实现:一种仿生子宫内膜,包括两组呈镜面对称的BAM(仿生羊膜),两组所述BAM(仿生羊膜)之间设置有ESC(子宫内膜间质细胞)。
一种仿生子宫内膜的制备方法,其特征在于:包括以下步骤:
步骤一:制备hAAM(人脱细胞羊膜)
从正常孕妇妇剖宫产手术中获取人新鲜羊膜组织,再经盐水充分漂洗除去血迹和表面可能粘附的其他杂质后与戊二醛交联过夜,再使用胰蛋白酶充分消化2次,每次的时间为30min-40min,将新鲜羊膜表皮细胞消化至松解状态,之后再用生理盐水轻柔漂洗3次,去除多余的胰蛋白酶,即得到hAAM,然后将制成的hAAM冷冻干燥、分装,将其用环氧乙烷灭菌后置于-20℃条件下冷冻备用;
步骤二:提取hAMSCs(人羊膜间充质干细胞)
将新鲜羊膜剪碎至1mm大小碎片后,加入胰蛋白酶消化2次,每次的时间为30min,之后加入Ⅱ型胶原酶消化1h,收集细胞滤液并离心,重悬于含10%胎牛血清的DMEM/F12培养基中,之后在37℃孵箱中恒温孵育,培养液每2d更换1次,当细胞融合达到80%时,胰蛋白酶消化,1:3传代;
步骤三:制备AAM(人工羊膜)
用无血清的DMEM/F12培养基按1:3稀释BD基质胶后分装入冻存管中保存。实验前在冰浴中融化预先分装的BD基质胶,于冰上将步骤二中提取的hAMSCs与基质胶按1:1混合注入24孔板中,制成底面积为2cm2、厚度约3-5mm、细胞浓度为3×105/ml的圆形薄膜,即得到AAM;
步骤四:制备BAM(仿生羊膜)
先将一片所述步骤一中得到的hAAM(网孔面向上)置于Transwell小室底部,在其上加一片所述步骤三中得到的AAM,再将另一片所述步骤一中得到的hAAM(网孔面向下)覆盖于AAM上,即得到BAM;
步骤五:制备ESC(子宫内膜间质细胞)
刮取少许全子宫切除术后子宫的内模组织,用含有1%青链霉素的基础培养基反复洗涤内膜组织,每次5min,共洗涤3次,随后用不含有青链霉素的培养基洗涤1次,再用长组织剪将内膜组织剪成2mm3左右的小碎块,并1000r/min离心1次,之后在含有组织块的离心管中加入5倍体积含1%I型胶原酶的基础培养基,吹打混匀,置于37℃、5%CO2培养箱中进行首次消化,每隔10min震荡1次,1小时后取出,之后将首次消化液经100um孔径的细胞滤网过滤一次,过滤所得滤液经40um孔径的细胞滤网二次过滤,滤出的液体(主要为ESC)移入新的离心管,1200r/min离心5分钟,去上清,用完全培养基漂洗1次后接种于细胞培养瓶。
进一步改进在于:所述步骤一中和步骤二中胰蛋白酶均为0.25%质量分数的胰蛋白酶。
进一步改进在于:所述步骤一中新鲜羊膜表皮细胞消化的过程中,须在倒置显微镜下观察,当上皮层细胞松解、脱落率达80%以上时停止消化。
进一步改进在于:所述步骤二中DMEM/F12培养基内还含有100U/mL青链霉素。
进一步改进在于:所述步骤三中BD基质胶的浓度为50ul/cm2生长面积。
进一步改进在于:所述步骤五中过滤后滤网上残留的组织块放入原离心管内,加入含1%I型胶原酶的基础培养基进行二次消化,二次消化时间可延长至90min,且二次消化所得滤液中ESC提取方式与首次相同。
本发明的有益效果为:该种仿生子宫内膜及其制备方法通过在两组呈镜面对称的BAM之间设置有ESC,有助于增加最终成膜后的力学强度和膜转化为子宫内膜的性能,且BAM以“夹心三明治结构”模式构成,两个外层的hAAM中具备双层的基底膜,其内含有丰富的天然纤维组织,从前后两方面加固能够形成与新鲜羊膜力学特性较为接近的充分的生物力学强度,而且,双层hAAM中均具备完整的基质层,其内含有大量的胶原蛋白,保证了BAM的弹性和抗拉力,另外,双层hAAM的夹持和hAMSCs定植融合的固定作用下,具有更好的抗拉强度、弹性和延展特性,此外本发明以“仿生学”角度选择来源广泛、无伦理学限制、低免疫原性、无致瘤风险的hAMSCs作为干细胞成份;以低免疫原性、富含纤维组织、结缔组织和胶原蛋白的人脱细胞羊膜为双层结构支架;且两者的体外制备技术的完善、成熟;以无细胞和可溶性成分、无免疫活性、已商品化的生物材料BD基质胶作为hAMSCs的三维细胞支架;使本项目从材料来源、伦理许可、实验操作、预期结果等方面均得到充分的保障,极大地方便未来的产品转化、进行规模化生产和后续的临床使用。
附图说明
图1是本发明制备流程示意图。
具体实施方式
为了加深对本发明的理解,下面将结合实施例对本发明做进一步详述,本实施例仅用于解释本发明,并不构成对本发明保护范围的限定。
根据图1所示,本实施例提出了一种仿生子宫内膜,包括两组呈镜面对称的BAM(仿生羊膜),两组所述BAM(仿生羊膜)之间设置有ESC(子宫内膜间质细胞)。
一种仿生子宫内膜的制备方法,其特征在于:包括以下步骤:
步骤一:制备hAAM(人脱细胞羊膜)
从正常孕妇妇剖宫产手术中获取人新鲜羊膜组织,再经盐水充分漂洗除去血迹和表面可能粘附的其他杂质后与戊二醛交联过夜,经过戊二醛的交联和胰蛋白酶的消化,可以将新鲜羊膜表层的上皮细胞松解,容易与下面的基底层分离,在生理盐水漂洗后很容易脱落,再使用胰蛋白酶充分消化2次,每次的时间为30min-40min,将新鲜羊膜表皮细胞消化至松解状态,之后再用生理盐水轻柔漂洗3次,去除多余的胰蛋白酶,即得到hAAM,然后将制成的hAAM冷冻干燥、分装,将其用环氧乙烷灭菌后置于-20℃条件下冷冻备用,所述步骤一中胰蛋白酶为0.25%质量分数的胰蛋白酶,所述步骤一中新鲜羊膜表皮细胞消化的过程中,须在倒置显微镜下观察,当上皮层细胞松解、脱落率达80%以上时停止消化;
步骤二:提取hAMSCs(人羊膜间充质干细胞)
将新鲜羊膜剪碎至1mm大小碎片后,加入胰蛋白酶消化2次,每次的时间为30min,之后加入Ⅱ型胶原酶消化1h,收集细胞滤液并离心,重悬于含10%胎牛血清的DMEM/F12培养基中,之后在37℃孵箱中恒温孵育,培养液每2d更换1次,当细胞融合达到80%时,胰蛋白酶消化,1:3传代,所述步骤二中胰蛋白酶为0.25%质量分数的胰蛋白酶,步骤二中DMEM/F12培养基内还含有100U/mL青链霉素,经过胰蛋白酶和Ⅱ型胶原酶的消化,可以成功分离新鲜羊膜中的间充质干细胞,在加有抗生素的培养基中进行培养可以促进细胞分裂和增值,同时有效抗菌减少细胞被污染的概率,而细胞传代培养可以获得更多的细胞数量;
步骤三:制备AAM(人工羊膜)
用无血清的DMEM/F12培养基按1:3稀释BD基质胶后分装入冻存管中保存。实验前在冰浴中融化预先分装的BD基质胶,于冰上将步骤二中提取的hAMSCs与基质胶按1:1混合注入24孔板中,制成底面积为2cm2、厚度约3-5mm、细胞浓度为3×105/ml的圆形薄膜,即得到AAM,所述步骤三中BD基质胶的浓度为50ul/cm2生长面积,BD基质胶在零度以下呈液体状态,可以与hAMSCs相互混合且细胞密度均匀,当温度升高至37℃以后,BD基质胶呈现固态,成为hAMSCs生长的三维基质框架,hAMSCs与BD基质胶在常温条件下静置后最终形成“果冻状”AAM;
步骤四:制备BAM(仿生羊膜)
先将一片所述步骤一中得到的hAAM(网孔面向上)置于Transwell小室底部,在其上加一片所述步骤三中得到的AAM,再将另一片所述步骤一中得到的hAAM(网孔面向下)覆盖于AAM上,即得到BAM,Transwell小室为带有微孔的细胞培养装置,通常置于加有培养基的24孔板中,用于细胞培养和细胞生物特性观察。由于底部的微孔不影响细胞培养液的透过,能够保证BAM上下两面均接受相同的营养物质,保证两层hAAM和中间AAM在培养期间有充分的营养条件。两层hAAM中含有丰富的天然纤维组织,能够形成与新鲜羊膜力学特性较为接近的充分的生物力学强度,从根本上解决既往研究和发明技术中羊膜抗拉强度不足、延展性差和弹性不足的缺陷。同时,中间层AAM中含有大量的具有生物活性的hAMSCs,可以充分发挥其生物学功能,达到真正的“仿生羊膜”效果;
步骤五:制备ESC(子宫内膜间质细胞)
刮取少许全子宫切除术后子宫的内模组织,用含有1%青链霉素的基础培养基反复洗涤内膜组织,每次5min,共洗涤3次,随后用不含有青链霉素的培养基洗涤1次,再用长组织剪将内膜组织剪成2mm3左右的小碎块,并1000r/min离心1次,之后在含有组织块的离心管中加入5倍体积含1%I型胶原酶的基础培养基,吹打混匀,置于37℃、5%CO2培养箱中进行首次消化,每隔10min震荡1次,1小时后取出,之后将首次消化液经100um孔径的细胞滤网过滤一次,过滤所得滤液经40um孔径的细胞滤网二次过滤,滤出的液体(主要为ESC)移入新的离心管,1200r/min离心5分钟,去上清,用完全培养基漂洗1次后接种于细胞培养瓶,用于后续的细胞传代,以获得更多的细胞数量,所述步骤五中过滤后滤网上残留的组织块放入原离心管内,加入含1%I型胶原酶的基础培养基进行二次消化,二次消化时间可延长至90min,且二次消化所得滤液中ESC提取方式与首次相同。
该种仿生子宫内膜及其制备方法通过在两组呈镜面对称的BAM之间设置有ESC,有助于增加最终成膜后的力学强度和膜转化为子宫内膜的性能,且BAM以“夹心三明治结构”模式构成,两个外层的hAAM中具备双层的基底膜,其内含有丰富的天然纤维组织,从前后两方面加固能够形成与新鲜羊膜力学特性较为接近的充分的生物力学强度,而且,双层hAAM中均具备完整的基质层,其内含有大量的胶原蛋白,保证了BAM的弹性和抗拉力,另外,双层hAAM的夹持和hAMSCs定植融合的固定作用下,具有更好的抗拉强度、弹性和延展特性,此外本发明以“仿生学”角度选择来源广泛、无伦理学限制、低免疫原性、无致瘤风险的hAMSCs作为干细胞成份;以低免疫原性、富含纤维组织、结缔组织和胶原蛋白的人脱细胞羊膜为双层结构支架;且两者的体外制备技术的完善、成熟;以无细胞和可溶性成分、无免疫活性、已商品化的生物材料BD基质胶作为hAMSCs的三维细胞支架;使本项目从材料来源、伦理许可、实验操作、预期结果等方面均得到充分的保障,极大地方便未来的产品转化、进行规模化生产和后续的临床使用。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (7)
1.一种仿生子宫内膜,其特征在于:包括两组呈镜面对称的BAM(仿生羊膜),两组所述BAM(仿生羊膜)之间设置有ESC(子宫内膜间质细胞)。
2.应用于权利要求1的一种仿生子宫内膜的制备方法,其特征在于:包括以下步骤:
步骤一:制备hAAM(人脱细胞羊膜)
从正常孕妇妇剖宫产手术中获取人新鲜羊膜组织,再经盐水充分漂洗除去血迹和表面可能粘附的其他杂质后与戊二醛交联过夜,再使用胰蛋白酶充分消化2次,每次的时间为30min-40min,将新鲜羊膜表皮细胞消化至松解状态,之后再用生理盐水轻柔漂洗3次,去除多余的胰蛋白酶,即得到hAAM,然后将制成的hAAM冷冻干燥、分装,将其用环氧乙烷灭菌后置于-20℃条件下冷冻备用;
步骤二:提取hAMSCs(人羊膜间充质干细胞)
将新鲜羊膜剪碎至1mm大小碎片后,加入胰蛋白酶消化2次,每次的时间为30min,之后加入Ⅱ型胶原酶消化1h,收集细胞滤液并离心,重悬于含10%胎牛血清的DMEM/F12培养基中,之后在37℃孵箱中恒温孵育,培养液每2d更换1次,当细胞融合达到80%时,胰蛋白酶消化,1:3传代;
步骤三:制备AAM(人工羊膜)
用无血清的DMEM/F12培养基按1:3稀释BD基质胶后分装入冻存管中保存。实验前在冰浴中融化预先分装的BD基质胶,于冰上将步骤二中提取的hAMSCs与基质胶按1:1混合注入24孔板中,制成底面积为2cm2、厚度约3-5mm、细胞浓度为3×105/ml的圆形薄膜,即得到AAM;
步骤四:制备BAM(仿生羊膜)
先将一片所述步骤一中得到的hAAM(网孔面向上)置于Transwell小室底部,在其上加一片所述步骤三中得到的AAM,再将另一片所述步骤一中得到的hAAM(网孔面向下)覆盖于AAM上,即得到BAM;
步骤五:制备ESC(子宫内膜间质细胞)
刮取少许全子宫切除术后子宫的内模组织,用含有1%青链霉素的基础培养基反复洗涤内膜组织,每次5min,共洗涤3次,随后用不含有青链霉素的培养基洗涤1次,再用长组织剪将内膜组织剪成2mm3左右的小碎块,并1000r/min离心1次,之后在含有组织块的离心管中加入5倍体积含1%I型胶原酶的基础培养基,吹打混匀,置于37℃、5%CO2培养箱中进行首次消化,每隔10min震荡1次,1小时后取出,之后将首次消化液经100um孔径的细胞滤网过滤一次,过滤所得滤液经40um孔径的细胞滤网二次过滤,滤出的液体(主要为ESC)移入新的离心管,1200r/min离心5分钟,去上清,用完全培养基漂洗1次后接种于细胞培养瓶。
3.根据权利要求2所述的一种仿生子宫内膜的制备方法,其特征在于:所述步骤一中和步骤二中胰蛋白酶均为0.25%质量分数的胰蛋白酶。
4.根据权利要求2所述的一种仿生子宫内膜的制备方法,其特征在于:所述步骤一中新鲜羊膜表皮细胞消化的过程中,须在倒置显微镜下观察,当上皮层细胞松解、脱落率达80%以上时停止消化。
5.根据权利要求2所述的一种仿生子宫内膜的制备方法,其特征在于:所述步骤二中DMEM/F12培养基内还含有100U/mL青链霉素。
6.根据权利要求2所述的一种仿生子宫内膜的制备方法,其特征在于:所述步骤三中BD基质胶的浓度为50ul/cm2生长面积。
7.根据权利要求2所述的一种仿生子宫内膜的制备方法,其特征在于:所述步骤五中过滤后滤网上残留的组织块放入原离心管内,加入含1%I型胶原酶的基础培养基进行二次消化,二次消化时间可延长至90min,且二次消化所得滤液中ESC提取方式与首次相同。
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