CN111363716A - 去细胞羊膜载体复合自体子宫内膜干细胞的制备 - Google Patents
去细胞羊膜载体复合自体子宫内膜干细胞的制备 Download PDFInfo
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Abstract
本发明涉及去细胞羊膜载体复合自体子宫内膜干细胞的制备，具体包括以下步骤：自体子宫内膜干细胞的分离培养，去细胞羊膜载体的制备和保存以及去细胞羊膜载体复合自体子宫内膜干细胞的制备。本发明克服了中重度宫腔粘连患者宫腔镜手术机械性分离宫腔粘连，术后放置宫内节育器或防粘连材料，于术后给予雌激素促进内膜生长所不能实现的内膜功能性修复，且极易发再次粘连等缺陷。本发明中去细胞羊膜载体具有良好的抗粘连能力、生物相容性、可降解性及安全性，自体子宫内膜干细胞作为治疗严重子宫内膜损伤的活性成分，可分泌生长因子改善局部微环境及免疫调节，促进受损子宫内膜修复，增加内膜厚度及局部血管密度。
Description
技术领域
本发明属于生物医学领域，具体涉及去细胞羊膜支架复合自体子宫内膜干细胞的制备。
背景技术
子宫内膜由内到外分为功能层和基底层，在性激素作用下发生周期性增殖、剥脱，同时，在雌激素作用下，基底层腺体断端处上皮细胞增生形成新的功能层，覆盖子宫体腔达到修复创面的作用。因此，即使每个月经周期都有子宫内膜的坏死剥脱，子宫前后壁互相紧贴也不会形成粘连。而在重度宫腔粘连患者中，基底层损伤、腺体消失，导致上皮细胞、新生血管形成受阻；同时，在创面修复过程中，成纤维细胞生成增加，细胞外基质堆积，导致局部纤维结缔组织增殖，子宫前后壁相互贴附，导致粘连形成。因此，抑制创面渗出，避免创面互相接触、促进子宫内膜生长修复成为术后预防宫腔再粘连形成的主要手段。
羊膜是一种半透明膜，其独有的结构可阻止某些物质通过，保证包裹内组织正常营养供应，而且具有抗粘连能力、组织相容性好及可降解等特性，作为一种生物屏障广泛应用于烧伤、外周神经修复等外科领域。特别是在眼科领域，羊膜移植已成为一种较为成熟的治疗眼表损伤、预防术后粘连形成的常见术式。最近有研究表明，宫腔粘连术后用人羊膜覆盖创面能够显著降低宫腔再粘连的发生率，但无法促进受损的基底层再生。
干细胞是一类具有自我更新及多向分化潜能的细胞，干细胞移植已经应用于多种疾病的治疗。大量研究表明，子宫内膜存在干细胞，深入子宫基底层的损伤，会引起干细胞的丢失或功能异常，导致子宫内膜再生失败，因此子宫内膜干细胞移植治疗有望成为宫腔粘连患者更好的选择。近来有研究证实，子宫内膜干细胞宫腔注射后给与激素替代治疗，能增加子宫内膜厚度，但局部注射后很难在损伤局部定位，影响疗效。
发明内容
本发明的目的就在于克服上述缺陷，提供去细胞羊膜载体复合自体子宫内膜干细胞的制备。
本发明的技术方案是：
一种去细胞羊膜载体负载自体子宫内膜干细胞的制备方法，该方法包括以下步骤：
（1）自体子宫内膜干细胞的分离培养；
（2）去细胞羊膜载体的制备和保存；
（3）去细胞羊膜载体复合自体子宫内膜干细胞的制备。
所述步骤（1）中自体子宫内膜干细胞的分离培养主要是：搔刮采集少量子宫内膜组织，在无菌的环境下进行操作，用含1%青链霉素的生理盐水反复冲洗去除血污，剪碎至直径＜1毫米；加入约组织的3倍体积的0.1%Ⅰ型胶原酶，混匀，置37℃恒温摇床中振荡消化30分钟；加入含有10%FBS的DMEM/F12培养基终止消化；吹打细胞悬液后过滤400目筛网，收集子宫内膜干细胞；加入含有10%FBS的DMEM/F12培养基重悬，置于37℃、5% CO2培养箱中培养；24小时后换液，每隔2天换液，待细胞融合度达到90%左右时，行传代培养。
所述步骤（2）中去细胞羊膜载体的制备和保存主要是：收集术前四项阴性的剖腹产妇的胎盘组织，无菌操作下，冲洗胎盘上的血迹及污物，在羊膜与绒毛膜之间钝性分离；获得光滑、半透明的羊膜后，将羊膜上皮细胞面朝上平铺于培养皿中，用含1%青链霉素的生理盐水反复冲洗去除血污；将羊膜剪裁为10cm培养皿大小的羊膜片，上皮细胞面朝上放置在10cm培养皿中；每皿加入10ml 0.25%胰蛋白酶-EDTA消化液，于37℃消化2小时，无菌生理盐水冲洗至镜下观察无上皮细胞残留，制得去细胞羊膜载体；于－60℃下预冻3小时，然后在－70℃~－80℃下冷阱干燥24小时，取出于阴凉干燥处保存。
所述步骤（3）中去细胞羊膜载体复合自体子宫内膜干细胞的制备：取冻干去细胞羊膜载体置于紫外线照射0.5小时消毒，加入5毫升 DMEM/F12基础培养基，放置在37°恒温培养箱中预培养1小时，镜下观察羊膜无杂质；生理盐水浸洗3遍，吸弃旧液，将培养皿置于超净工作台风干0.5小时，使羊膜完全贴附于皿底；按照1*105/cm2的密度接种子宫内膜干细胞，37℃、5% CO2培养箱中培养24小时。
本发明治疗中重度宫腔粘连与以往治疗方法相比，具有以下优点：自体来源的子宫内膜干细胞具有间充质干细胞的特征，易于体外扩增，可作为治疗中重度宫腔粘连的活性成分，补充局部干细胞的数量，并分泌生长因子改善局部微环境及免疫调节等作用。去细胞羊膜载体具有抗粘连能力、可降解性及良好的生物相容性，可以防治宫腔粘连分离术后的再粘连，为自体子宫内膜干细胞提供支持位点，提高局部自体子宫内膜干细胞浓度，延长干细胞的作用时间，促进受损子宫内膜的生长修复。
附图说明
图1 自体子宫内膜干细胞分离培养形态图（100×）。细胞以细长、梭形、成纤维细胞样生长。
图2 自体子宫内膜干细胞的鉴定。其中，CD90阳性率为99.3%，CD29阳性率为0.3%，CD44阳性率达98.7%，CD34阳性率为0.6%，CD45阳性率为0.5%，HLA-DR阳性率为0.3%。
图3 去细胞羊膜载体制备示意图（100×）。其中，图A为新鲜羊膜；图B为新鲜羊膜经过0.25%胰酶-EDTA消化60分钟的形态；图C为新鲜羊膜经过0.25%胰酶-EDTA消化120分钟的形态图；图D为新鲜羊膜经胰酶消化120分钟后盐水冲洗干净的形态图。
图4 冻干去细胞羊膜形态图。
图5去细胞羊膜载体复合自体子宫内膜干细胞示意图（100×）。自体子宫内膜干细胞接种于去细胞羊膜载体上培养24小时，细胞完全贴壁，细胞形态逐渐变成细长梭形。
具体实施方式
下面结合具体实施例对本发明的内容进一步说明。
实施例1：
一、一种去细胞羊膜载体复合自体子宫内膜干细胞制备方法。
1. 所用设备，材料，试剂：超净工作台、CO2培养箱、倒置显微镜、离心机、真空冷冻干燥仪、恒温水浴箱、移液枪、细胞培养皿、离心管、移液管、DMEM/F12培养基、胎牛血清、青霉素、链霉素、0.25%胰酶-EDTA、生理盐水。
2. 自体子宫内膜干细胞的原代分离、培养、传代：搔刮采集子宫内膜组织约0.1ml，在无菌的环境下，立即用含有100 IU/ml青霉素、链霉素的PBS缓冲液洗涤5次，去除血污，剪碎至直径＜1mm；加入约组织的0.3ml的0.1%Ⅰ型胶原酶，混匀，置37℃恒温摇床中振荡消化60分钟。然后加入含有10%FBS的DMEM/F12培养基终止消化；吹打细胞悬液后过滤400目筛网，收集滤液，1500r/min离心5分钟，弃上清，加入适量PBS，重悬沉淀，再次离心并弃掉上清，加入含有10%FBS的DMEM/F12培养基重悬，置于37℃、5% CO2培养箱中培养。24小时后换液，每隔2天换液，每日用倒置显微镜观察细胞形态和细胞生长情况，并拍摄照片，可见细胞以细长、梭形、成纤维细胞样生长（结果见图1）。
3. 自体子宫内膜干细胞的鉴定：取90%融合度的P3代子宫内膜细胞，采用0.25%胰酶-EDTA消化后制备成单细胞悬液，以1500r/min离心5分钟，弃上清，加入相应抗体工作液，FITC-CD45、FITC-CD105、PE-CD29、PE-CD34、APC-CD90和APC-HLA-DR，阴性对照分别选FITC-IgG1k、PE-IgG1k和APC-IgG1k，4℃条件下避光孵育30分钟后，1500r/min离心5分钟，弃去上清，加入0.5ml PBS重悬，经200目细胞筛过滤后，避光，流式细胞仪分析（结果见图2）。
4. 自体子宫内膜干细胞的传代：待细胞融合度达到90%左右时，行传代培养。培养至贴壁细胞90％融合时，进行传代；吸除旧培养液，加入PBS 洗去残留培养液，吸除PBS ；加入1ml 0.25%胰酶-EDTA，轻轻摇动培养瓶；倒置显微镜下观察，发现细胞质回缩、细胞间隙增大，加入完全培养液终止消化；吹打瓶壁细胞，使其形成细胞悬液；将细胞悬液吸入离心管，1500r/min离心5分钟，弃上清；用含10%胎牛血清的DMEM/F12完全培养液调整细胞浓度后接种于培养瓶中，37℃、5％CO2 培养箱培养；隔天换液，继续培养。
5. 去细胞羊膜载体的制备：收集术前四项阴性的剖腹产妇的胎盘组织，无菌操作下，冲洗胎盘上的血迹及污物，在羊膜与绒毛膜之间钝性分离；获得光滑、半透明的羊膜后，将羊膜上皮细胞面朝上平铺于培养皿中，用含1%青链霉素的生理盐水反复浸洗去除血污；将羊膜剪裁为10cm培养皿大小的羊膜片，上皮细胞面朝上放置在10cm培养皿中；每皿加入10ml 0.25%胰蛋白酶-EDTA消化液，37°消化90分钟，每30分钟更换一次消化液，无菌生理盐水冲洗至镜下观察无上皮细胞残留（结果见图3）；置于－60℃下预冻3小时，然后在－70℃~－80℃下冷阱干燥24小时，制得冻干去细胞羊膜（结果见图4）。
6. 自体子宫内膜干细胞复合去细胞羊膜载体的制备：取冻干去细胞羊膜载体置于紫外线照射0.5小时消毒，加入5ml DMEM/F12基础培养基，放置在37℃恒温培养箱中预培养1小时。镜下观察羊膜无杂质；生理盐水浸洗3遍，吸弃旧液，将培养皿置于超净工作台风干0.5小时，使羊膜完全贴附于皿底；按照1*105/cm2的密度接种子宫内膜干细胞，37℃、5%CO2培养箱中培养24小时，可见大部分子宫内膜干细胞由圆形变为梭形，粘附于去细胞羊膜载体表面，并且增殖进入羊膜载体内部，保持良好的生物活性（结果见图5）。
Claims (5)
1.去细胞羊膜载体复合自体子宫内膜干细胞的制备，该方法包括以下步骤：
（1）自体子宫内膜干细胞的分离培养；
（2）去细胞羊膜载体的制备和保存；
（3）去细胞羊膜载体复合自体子宫内膜干细胞的制备。
2.所述步骤（1）中自体子宫内膜干细胞的分离培养主要是：搔刮采集少量子宫内膜组织，在无菌的环境下进行操作，用含1%青链霉素的生理盐水反复冲洗去除血污，剪碎至直径＜1毫米；加入约组织的3倍体积的0.1%Ⅰ型胶原酶，混匀，置37℃恒温摇床中振荡消化30分钟；加入含有10%FBS的DMEM/F12培养基终止消化；吹打细胞悬液后过滤400目筛网，收集子宫内膜干细胞；加入含有10%FBS的DMEM/F12培养基重悬，置于37℃、5% CO2培养箱中培养；24小时后换液，每隔2天换液，待细胞融合度达到90%左右时，行传代培养。
3.所述步骤（2）中去细胞羊膜载体的制备和保存主要是：收集术前四项阴性的剖腹产妇的胎盘组织，无菌操作下，冲洗胎盘上的血迹及污物，在羊膜与绒毛膜之间钝性分离；获得光滑、半透明的羊膜后，将羊膜上皮细胞面朝上平铺于培养皿中，用含1%青链霉素的生理盐水反复冲洗去除血污；将羊膜剪裁为10cm培养皿大小的羊膜片，上皮细胞面朝上放置在10cm培养皿中；每皿加入10ml 0.25%胰蛋白酶-EDTA消化液，于37℃消化2小时，无菌生理盐水冲洗至镜下观察无上皮细胞残留，制得去细胞羊膜载体；于－60℃下预冻3小时，然后在－70℃~－80℃下冷阱干燥24小时，取出于阴凉干燥处保存。
4.所述步骤（3）中去细胞羊膜载体复合自体子宫内膜干细胞的制备：取冻干去细胞羊膜载体置于紫外线照射0.5小时消毒，加入5毫升 DMEM/F12基础培养基，放置在37°恒温培养箱中预培养1小时，镜下观察羊膜无杂质；生理盐水浸洗3遍，吸弃旧液，将培养皿置于超净工作台风干0.5小时，使羊膜完全贴附于皿底；按照1*105/cm2的密度接种子宫内膜干细胞，37℃、5% CO2培养箱中培养24小时。
5.根据权利要求1得到的去细胞羊膜载体复合自体子宫内膜干细胞的应用，其特征在于去细胞羊膜载体复合自体子宫内膜干细胞贴附固定于宫腔粘连分离术后的创伤内膜表面，防治宫腔再粘连，促进受损子宫内膜的生长修复。
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| CN111701082A (zh) * | 2020-07-20 | 2020-09-25 | 浙江大学医学院附属妇产科医院 | 载雌二醇微球的脱细胞羊膜宫腔支架的制备方法 |
| CN112717203A (zh) * | 2021-01-15 | 2021-04-30 | 肖雁冰 | 一种仿生子宫内膜及其制备方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111701082A (zh) * | 2020-07-20 | 2020-09-25 | 浙江大学医学院附属妇产科医院 | 载雌二醇微球的脱细胞羊膜宫腔支架的制备方法 |
| CN111701082B (zh) * | 2020-07-20 | 2022-03-18 | 浙江大学医学院附属妇产科医院 | 载雌二醇微球的脱细胞羊膜宫腔支架的制备方法 |
| CN112717203A (zh) * | 2021-01-15 | 2021-04-30 | 肖雁冰 | 一种仿生子宫内膜及其制备方法 |
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