II 摘要 目的： 以飞秒激光脉冲技术辅助制备的超薄脱细胞猪角膜基质片(Femtosecond laser-Acellular Porcine Cornea Matrix，FS-APCM )为支架，收集人脐带血间充质 干细胞（human umbilical mesenchymal stem cells，HUMSCs）调节培养基促进兔 角膜内皮细胞的体外增殖为种子细胞，体外模拟构建组织工程生物兔角膜内皮 层植片，从而探讨组织工程角膜内皮层构建的可行性研究。 方法： 将成年新西兰大白兔完整的角膜在无菌条件下取出后将带有内皮细胞的后 弹力层面完整分离，体外消化、纯化后得到兔角膜内皮细胞（Rabbit corneal endothelial cell，RCECs ）后进行培养。选用 P3-P7 代 HUMSCs，以 5×10 5 /cm2 接种传代、重悬，分别加入 10％、30％、50％、70％、100％不同浓度 HUMSCs 调 节 培 养 基 （ human umbilical mesenchymal stem cells condition medium ， HUMSCs-CM）培养，观察不同浓度 HUMSCs-CM 对 RCECs 细胞增殖能力影响。 通过传统手工剖切技术和飞秒激光脉冲技术对比以寻找和优化支架条件，利用 飞秒激光脉冲技术获得带有后弹力层的超薄猪角膜基质支架（厚度约 100μm、 直径约 8.5mm），于 0.5％SDS 溶液中 4℃脱细胞 24 h 后，获取 FS-APCM。选 用 70% 浓度 HUMSCs-CM 培养下 RCECs 的进行 Dil 荧光标记追踪，悬架培养 法将 RCECs 细胞悬液小心滴加在经过预处理的于 FS-APCM 支架上，倒置显微 镜下分别在接种后 2h，3d，5d，1w 分别观察体外重建后内皮细胞形态变化。 结果： 1. 体外 RCECs 原代培养活性良好。RCECs 在体外生长状态良好，6 天可基 本长满皿底形成紧密单层，细胞大小均一，形态基本一致，多为近似鹅卵石内 皮样形态。 2 . 70% HUMSCs-CM能够有效促进RCECs体外增殖。RCECs形态学及MTT 法显示，在同一作用时间（24h）、不同浓度梯度（10%CEC-CM、30%HUMSCs-CM、 50%HUMSCs-CM、70%HUMSCs-CM、100%HUMSCs-CM）作用下均能促进 RCECs 增殖，其中 70%HUMSCs-CM 作用最为显著，P1 至 P4 代具有一致性结 果。平板克隆实验结果显示，70%HUMSCs-CM 能有效促进 RCECs 细胞体外增摘要 III 殖。 3 .与传统手工剖切支架方式相比，飞秒激光脉冲技术辅助下超薄脱细胞猪 角膜支架（厚度约 100μm、直径约 8.5mm）具有较好的重复性和可操作性。组 织学检测发现传统手工环钻剖切猪角膜组（T-PC 组）角膜厚度最厚，飞秒激光 脉冲技术剖切猪角膜支架组（FS-PC）组织切片十分微薄，类似于一薄片。含水 量检测结果显示 NPC 组溶胀后含水量最高，T-PC 次之，FS-PC 组含水量最低。 HE 染色、DAPI 染色及 DNA 含量检测均显示 0.5%SDS 脱细胞猪角膜基质脱细 胞彻底无细胞残留。 4 .体外模拟构建人工生物角膜内皮植片证实内皮细胞存活状态良好。将 RCECs 种子细胞接种于 FS-APCM 支架后悬架培养 1w，茜素红染色、Dil 荧光 追踪观察显示，RCECs 可在 FS-APCM 上均可形成连续的单细胞层，胞间连接 紧密，细胞计数密度大于 2000cells/mm 2 。 结论： 1 . 70%人脐带血间充质干细胞调节培养基（HUMSCs-CM）能有效刺激兔角 膜内皮细胞体外增殖，为今后角膜内皮细胞体外培养提供一种简单、高效的方 法； 2 . 飞秒激光脉冲技术辅助制备的超薄脱细胞猪角膜基质支架（FS-APCM） 能对角膜基质进行精准切割，为组织工程支架材料优化提供更佳的选择。 关键词：飞秒激光脉冲技术，人脐带血间充质干细胞，调节培养基，组织工程 角膜Abstract IV ABSTRACT Purpose: Femtosecond laser-Acellular Porcine Cornea Matrix (FS-APCM) was used as a scaffold to utilize human umbilical mesenchymal stem cells (HUMSCs). The rabbit cultured rabbit corneal endothelial cells were promoted to be seed cells, and artificial corneal endothelial grafts were constructed in vitro to investigate the feasibility of tissue engineering corneal endothelium construction. Method： The intact cornea of adult New Zealand white rabbits was taken out under aseptic conditions, and the post-elastic layer with endothelial cells was completely separated. After in vitro digestion and purification, rabbit corneal endothelial cells (RCECs) were obtained and cultured. P3-P7 generation HUMSCs were selected, subcultured and resuspended at 5×10 5 /cm2, and 10%, 30%, 50%, 70%, 100% different concentrations of HUMSCs conditioned medium (human umbilical mesenchymal stem cells condition medium, HUMSCs-CM) cultured to observe the effects of different concentrations of HUMSCs-CM on the proliferation of RCECs. By comparing traditional manual cutting technology with femtosecond laser pulse technology to find suitable conditions for optimizing the stent, ultra-thin porcine corneal stroma scaffold with a post-elastic layer (thickness of about 100 μm and diameter of about 8.5 mm) was obtained by femtosecond laser pulse technique. After deactivating at 4 °C for 24 h in 0.5% SDS solution, FS-APCM was obtained. The RCECs cultured in 70% concentration of HUMSCs-CM were used for Dil fluorescent labeling. The suspension of RCECs was carefully added to the pretreated FS-APCM scaffold by suspension culture. Under inverted microscope, 2 h after inoculation, respectively. 3d, 5d, 1w were observed to observe the morphological changes of endothelial cells after in vitro reconstruction. Result： 1. The primary culture of RCECs in vitro has good activity. RCECs grow well in vitro, and can form a tight monolayer at the bottom of the dish in 6 days. The cell sizeAbstract V is uniform and the morphology is basically the same, mostly similar to the cobble- stone endothelium morphology. 2. 70% HUMSCs-CM can effectively promote the proliferation of RCECs in vitro. RCECs morphology and MTT assay showed that at the same time (24h), different concentration gradients (10% CEC-CM, 30% HUMSCs-CM, 50% HUMSCs-CM, 70% HUMSCs-CM, 100% HUMSCs-CM) Under the action, it can promote the proliferation of RCECs, and 70% of HUMSCs-CM has the most significant effect, and P1 to P4 have consistent results. The results of plate cloning experiments showed that 70% of HUMSCs-CM could effectively promote the proliferation of RCECs in vitro. 3. Compared with the traditional manual cutting stent, the femtosecond laser-assisted ultra-thin decellularization scaffold with a post-elastic layer (thickness about 100μm, diameter about 8.5mm) has good repeatability and operability. Histological examination found that the traditional manual circumcision cut porcine corneal group (T-PC group) had the thickest corneal thickness, femtosecond laser-assisted porcine corneal deep-layer matrix slice group (FS-PC) tissue section was very thin, similar A thin sheet. The water content test results showed that the NPC group had the highest water content after swelling, followed by T-PC, and the FS-PC group had the lowest water content. HE staining, DAPI staining and DNA content detection showed that there was no cell residue in the decellularized 0.5% SDS acellular porcine corneal stroma. 4. In vitro simulation of artificial corneal endothelial grafts confirmed that endothelial cells survived well. The RCECs seed cells were inoculated into the FS-APCM scaffold and cultured for 1 w. The anisin red staining and Dil fluorescence tracking showed that the RCECs could form a continuous single cell layer on the FS-APCM. Compact, cell count density is over 2000/mm 2. conclusion: 1. 70% human umbilical cord blood mesenchymal stem cell condition medium (HUMSCs-CM) can effectively stimulate the proliferation of rabbit corneal endothelial cells in vitro, providing a simple and efficient method for in vitro culture of corneal endothelial cells.Abstract VI 2. Femtosecond laser-assisted ultra-thin acellular porcine corneal stroma (FS-APCM) can accurately cut the corneal stroma and provide a better choice for tissue engineering scaffold material optimization. Keywords: femtosecond laser technology, human umbilical cord blood mesenchymal stem cells, condition medium, tissue engineering cornea目录 VII 目 录 第 1 章 引言...............................................................................................................1 第 2 章 人脐带血间充质干细胞调节培养基促进兔角膜内皮细胞体外增殖.........4 2.1 实验动物及细胞株.........................................................................................4 2.2 试剂及仪器.....................................................................................................4 2.3 实验方法.........................................................................................................6 2.3.1 体外兔角膜内皮细胞原代培养及鉴定...............................................6 2.3.2 人脐带血间充质干细胞调节培养基（HUMSCs-CM）的制备........7 2.3.3 MTT法检测不同浓度梯度HUMSCs-CM对RCECs增殖能力的影响 ..