【作者】 孙小闵；

【导师】 伍津津；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2010， 硕士

【摘要】 目的:利用磁力搅拌培养器微载体搅拌培养人皮肤成纤维细胞,观察细胞的生长增殖情况;检测细胞增殖达到平台期后不同保存条件下细胞的活力及保存时间;观察搅拌培养微载体上细胞接种到普通培养瓶中移行、增殖和分泌Ⅰ型胶原含量的情况;观察搅拌培养的人皮肤成纤维细胞用于制备复方壳多糖人工皮肤后的增殖情况及细胞形态。方法:利用酶消化法分别培养出人包皮成纤维细胞和包皮表皮细胞,选取6～10代的人皮肤成纤维细胞(细胞量为8×10~6,密度为4×10~4/ml)接种到浓度为2.5g/L的微载体cytodex-3搅拌培养瓶中,用200ml改良培养基按20rpm/min,连续搅拌50min,暂停5min,循环模式进行搅拌培养,以静态培养为对照,利用CASY细胞计数仪检测两种方法培养每隔24h后细胞各自的增殖情况,倒置相差显微镜定时观察和扫描电镜选时相点观察细胞形态并照相;检测细胞培养增殖达到平台期后的保存时间和MTT法检测在室温和4℃保存条件下细胞混悬液的OD值,据此计算出活细胞量制作活细胞量曲线;在搅拌培养第9天取微载体成纤维细胞混悬液15ml进行移行培养观察,并做细胞爬片的vimentin免疫组化染色鉴定人皮肤成纤维细胞;用ELISA方法测定移入方瓶培养后培养液中Ⅰ型胶原的含量;按我科方法制备复方壳多糖组织工程皮肤,对比观察搅拌培养和普通培养的成纤维细胞植入复方壳多糖组织工程皮肤后的增殖情况,进行HE染色观察。结果:人皮肤成纤维细胞接种在微载体上搅拌培养具有正常的形态,与静态培养比较,需要的微载体量少(2.5g/L),以此微载体浓度进行细胞扩增培养,细胞总产量与接种量之间呈数量级扩增,大多数微载体上能长满成纤维细胞,且需要的培养基量少(每瓶200ml改良培养基),能快速大规模扩增,从接种增殖到达平台期只需10～12天,且收获细胞量是接种量的5倍左右;在搅拌培养的人皮肤成纤维细胞达到增殖平台期后,室温和4℃条件下在微载体上保存的细胞活力比静态培养的人皮肤成纤维细胞保存的时间长,室温条件下能保存三周左右,在第3天时保存的细胞活力有较大一过性降低,随后又逐渐恢复,活力在第19天时最佳,可达到86.96%,到第23天时细胞活力明显下降只有64.36%,而4℃条件下相应保存的细胞活力到第19天时只有13.04%,细胞活力下降明显,均低于室温保存的细胞;搅拌培养后第9天移入方瓶中培养的细胞具有正常形态和增殖特性,收集不同时间点的细胞培养液测定Ⅰ型胶原含量结果显示,在一定时间范围内,Ⅰ型胶原含量前5天随时间的推移和细胞的增殖而分泌增加;观察混合成纤维细胞培养的复方壳多糖组织工程皮肤,结果显示,2周左右HE染色可见人皮肤成纤维细胞增殖布满真皮。结论:选取微载体浓度为2.5g/L搅拌培养HDF是一种较为简易、经济且迅速有效扩增细胞的方法;HDF经搅拌培养后保持了良好的生物学特性;搅拌培养后HDF保存的时间比静态的时间长,室温下能保存3周左右;该保存方法简单易行、经济实用,为种子细胞的运输和保存开辟了一条新的思路;微载体上培养的HDF制备复合壳多糖组织工程皮肤形态良好,细胞增殖快速均匀。更多还原

【Abstract】 Objective:To observe the growth and proliferation status of human dermal fibroblasts cultured on microcarriers in strring model by magnetic stirring apparatus; To detect the viability and preservative time of human dermal fibroblasts under difference preservative conditions after propagating to grown platform stage in stirring culture; To observe the condition of migrate and proliferation from stirring culture and microcarriers in keeping time to usual culture flask;To investigate the proliferated characteristics of human dermal fibroblasts by stirring culture after imbedding artificial skin and to identify the cell.Methods: Human foreskin fibroblast cells and foreskin epidermal cells were proliferated by enzyme digestion. The six to ten passage human dermal fibroblasts were cultured on microcarrier cytodex-3 at cell density 4×10~4/ml in the modified medium, the stirring mode was set as 20 rpm, 50 min running, and 5 min pause. The viability of human dermal fibroblasts were detected under difference culture methods by CASY hemocytometer in stirring culture every 24 hours, and the fibroblasts shape was observed with inverted phase contrast microscpope and SEM at different point time. Cell viability and preservative period were detected in the different culture methods by MTT assay. Fibroblast microbeads were observed and removed from the bioreactor and cultivated in tissue culture flasks at the nineth day. TheⅠcollagen contents in the culture medium was detected by ELISA at different time point. The composite chitosan skin equivalent was prepared in our laboratory, the fibroblasts proliferation status was observed in it with HE.Result: The human dermal fibroblasts have normal shap when seeded in microcarrier at stirring culture condition. It needs less microcarriers (2.5g/L) or culture media (improved culture media 200ml per bottle) and can be quickly amplified in large scale compared with the culture in static state. The proliferation period is only 10~12 days from seeding to platform stage. The harvesting cell is five times the size of inoculums; The human dermal fibroblasts can be preserved about three weeks in room temperature after it was cultured and proliferated to platform stage. The activity of cell which was preserved in the third day reduced largely, then gradually recovered. Its activity is the best in the nineteenth day, which retained to 86.96%. At the twenty-third day, the activity decreased obviously, and the activity of cells which were preserved in 4℃is only 13.04% at the nineteenth day and the cell activity rapidly decreased; Fibroblast microbeads were removed from the bioreactor and cultivated the cell microbeads in tissue culture flasks at the nineth day. The stirring cultured fibroblasts were able to migrate from the microcarriers and showed normal shape with the capability of large scale expansion, theⅠcollagen content in the culture medium was detected and showed theⅠcollagen increasing with the proliferation of the fibroblasts in the limit period; The result shows that the human dermal fibroblasts were imbed in corium of tissue engineering after 2 weeks proliferated to be covered with corium and that the cells were HDF by being identified with HE.Conclusion: The human dermal fibroblasts have normal shape when seeded in microcarriers at stirring culture. It was amplified rapidly and well. Higher activity and more amount cells were acquired than those were cultured in static state. At room temperature the cells cultured in stirring may be preserved long time and have better viability than at 4°C. This preservative method is easy, which makes the cells have good viability and has long preservative time, as well as the superiority that the cells cultured in static state are inimitable. There are good practical value for skin seed cells to transportation and preserve. After imbedding in the corium of tissue engineering, the human dermal fibroblasts retain good proliferative characteristics. The stirring culture cells are the normal human dermal fibroblasts by being identified with HE.更多还原