【作者】 钟刘学颖；

【导师】 董方田； 李莹； 钟勇；

【作者基本信息】 中国协和医科大学 ， 眼科学， 2009， 博士

【摘要】 目的:1.分离、培养和鉴定不同供体性状(手术切除脂肪组织块及吸脂术脂肪悬液)的人脂肪组织来源的干细胞(ADSCs)。观察其生物学特性、干细胞活性的差异。2.体外模拟角膜上皮生长、分化的微环境,对不同方法提取的人ADSCs进行单独/联合诱导,探讨其向角膜上皮样细胞分化的潜在能力,及诱导分化的可行途径。方法:1.改良方法分离、纯化人ADSCs,使用改良的体外扩增培养液进行体外培养。2.MTT法测定ADSCs增殖曲线。流式细胞仪检测传3～5代的CD29、CD34、CD49d、CD105、CD106表达率。进行成脂、成骨多向诱导,2周后用油红O及碱性磷酸酶法鉴定分化能力。对2种性状提取之细胞的表型及增殖、分化能力进行比较。3.应用CFDA SE标记人ADSCs。采用组织块培养法体外提取大鼠角膜上皮细胞,并制作无上皮、内皮细胞的大鼠角膜基质,用于ADSCs的联合诱导。4.CFDA SE标记的人ADSCs与大鼠角膜上皮细胞各自爬片,并列培养于同一体系,观察人ADSCs迁徙情况。5.在不同培养液体系(角质细胞培养体系、间充质细胞体外扩充培养体系及上述二者等积混合的中间体系)内添加梯度浓度0～50ng/mL EGF,及50ng/mL EGF基础上添加的10,20ng/mLbFGF,对ADSCs进行单独诱导。采取光镜、电镜、免疫组织化学染色、免疫荧光染色的手段,观察诱导21d后ADSCs形态学的改变及角膜特异性蛋白AE5(CK3/CK12)的表达情况,比较不同培养体系及不同浓度生长因子对ADSCs诱导作用的差异。6.在Transwell体系上层放置大鼠角膜上皮细胞或角膜基质,下层放置人ADSCs,选择不添加/添加40ng/mL EGF的角质细胞培养体系对人ADSCs进行联合诱导,采用免疫荧光染色法观察诱导30d后ADSCs细胞角蛋白谱AE1和角膜特异性蛋白AE5的表达情况,比较添加/不添加EGF、单独诱导/角膜上皮细胞联合诱导/角膜基质联合诱导对人ADSCs诱导成功率的差异。7.单独诱导2种性状提取的ADSCs,比较诱导后30d二者AE1、AE5阳性率差异。结果:1.不论脂肪块还是脂肪悬液,均可通过上述方法获取大量高纯度的ADSCs。但后者的操作流程更为省时、省力,获取的细胞量相对较多。流式细胞仪测定传3代至传5代的ADSCs,两个不同供体性状的细胞均为CD34~-、CD106~-、CD29~+、CD49d~+、CD105~+。皮下组织块来源的ADSCs传3代的表型CD29~+、CD49d~+细胞分别占82.5%和8.0%,传至5代逐渐增加为89.4%、70.1%;CD105~+细胞则稳定于80%左右。吸脂术提取的ADSCs传3～5代的表型较为稳定,CD29~+、CD49d~+、CD105~+表达分别在99%、40%、80%左右,与皮下组织块组比较,传3～5代的CD29和传5代的CD49d阳性率有统计学差异(P＜0.01)。MTT比色法显示,ADSCs增殖活性随传代次数增加而增加。另外,ADSCs分别进行成脂/成骨体外诱导,2周后油红O、碱性磷酸酶染色,均见阳性结果,皮下组织块组2者阳性率分别为81.6%、26.5%,吸脂术组为64.6%、29.3%。2.CFDA SE标记的ADSCs在营养状态欠佳的角膜上皮细胞诱导下迁徙、并逐渐包绕、替代后者。另外,角质细胞培养体系培养下的ADSCs向角膜上皮样细胞分化的阳性率具有EGF浓度依赖性;bFGF则对分化有抑制性作用。而另2个体系对各浓度EGF、bFGF对ADSCs诱导作用均为阴性。3.相同培养体系作用下的不同诱导组,以添加EGF刺激下的角膜基质联合诱导组AE5~+ ADSCs的比例最高,可达97.7%,AE1表达则为0,提示向成熟的角膜上皮样细胞分化。其次为单独诱导组,AE5~+比率为75.4%(添加EGF)、46.0%(未添加EGF),AE1~+比率为86.0%(添加EGF)、96.5%(未添加EGF),分化欠成熟。未添加EGF刺激下的角膜基质联合诱导组AE5、AE1阳性率最低,分别为2.4%、51.2%。4.角质细胞培养体系下,皮下组织块组在EGF的刺激下AE5~+100%、AE1~-0%,而无EGF的作用下则分别为16.6%、97.7%。该组细胞分化能力强于吸脂术组。结论:1.本研究通过改良的方法,成功地从人皮下脂肪组织及吸脂术废弃液中分离、培养了高纯度脂肪组织来源的干细胞,并经体外条件诱导证实具备多向分化潜能,可以作为一种优良的组织工程、细胞治疗和基因治疗的种子细胞。2.本研究中,在Transwell体系内,添加40ng/mL EGF的角质细胞培养液联合角膜基质进行诱导,是体外诱导ADSCs向角膜上皮样细胞分化的最佳方法。另外,皮下脂肪组织来源的ADSCs具备更好的分化能力,更适用于将来的临床治疗;而吸脂术提取的细胞由于工序相对简单、来源更为充足,更适用于实验室研究。3.CFDA SE是一种稳定的细胞示踪剂,可作为今后体内动物实验的细胞示踪的很好手段。更多还原

【Abstract】 Objective:1.To isolate,cultivate and identify human adipose-derived stem cells(ADSCs) which from adipose tissue masses and processed lipoaspirate.To observe their biological characteristics and activities as stem cells.2.To explore the possibility and the best method of ADSCs transdifferentiated into corneal epithelial-like cells by single inducing and co-culture inducing in simulating the vegetating microenvironment of corneal epithelial cells.Methods:1.The subcutaneous tissue masses-derived and processed lipoaspirate-derived ADSCs were isolated and purifed with improved methods,and cultured with modified medium in vitro.2.The growth curves were drawn by MTT colorimetry.Phenotypes including CD29、CD34、CD49d、CD105、CD106 were identified by flow cytometry at passage 3,4 and 5.Adipogenesis and osteogenesis tests were put into practice,and identified by Oil Red O stain and alkaline phosphatase activity tests.Contrasted the differences of phenotypes,proliferation and differentiation abilities were compared between 2 sources of ADSCs.3.The human ADSCs were marked with CFDA SE.And rat corneal epithelial cells (CECs) were isolated by tissue culture process,and made the corneal stroma tissue masses.They would be used into co-cultured induction in the following steps.4.The human ADSCs and rat CECs were placed together in the same plate which grown on slips,and co-cultured 3 weeks,observed the migration ability of human ADSCs. 5.Single induced group of ADSCs were cultured with several kinds of mediums, including Keratinocyte medium(KM),Dulbecco’s Modified Eagle Medium/F12(DMEM/F12) and KM+DMEM/F12(1:1),and stimulated with different concentrations of growth factors.Observed the morphological changes under phase contrast microscope and electron microscope.Immunohistochemical and immunofluorescent staining of AE5(CK3/CK12),the specific protein of cornea, were taken at the same time.Contrasted the transdifferentiated ability of ADSCs in different mediums and with different concentrations of growth factors.6.Placed rat CECs or corneal stroma tissue masses into the upper section of Transwell co-culture system,while human ADSCs into the under section,and co-culture with KM for 30 days,added 40ng/mL EGF in them or free.Observed the expressions of AE1(broad spectrum of CytoKeratins) and AE5 by immunofluorescent staining. Evaluated the expressing differences of AE1 and AE5 between the groups of EGF-added and free,single and co-cultured induction,and CECs co-culture induction and corneal stroma tissue masses co-culture induction.7 The 2 sources of single induced groups for 30 days,and contrasted their expressing differences of AE1 and AE5.Results:1.Large amounts of ADSCs were obtained from both 2 kinds of isolated processes, tissue masses and processed lipoaspirate,and with high purity.However,the operation of the latter process was more time- and strength-saving,while gained more cells.The phenotypes of ADSCs at passage 3～5 are CD34~-,CD106~-,CD29~+,CD49d~+ and CD105~+.CD29~+,CD49d~+ cells rates of subcutaneous tissue masses-derived ADSCs at passage 3 accounted for 82.5%and 8.0%,and gradually increased to 89.4%,70.1% at passage 5,while CD105~+ cells rates of each passage ADSCs were about 80%. Processed lipoaspirate-derived ADSCs at passage 3～5 expressed more stable phenotype,which spread approximately 99%、40%、80%of CD29~+、CD49d~+、CD105~+; And compared with the subcutaneous tissue-derived ADSCs,the positive rate of CD29 at passage 3～5 and CD49d at passage 5 had a significant difference(P＜0.01).MTT colorimetry showed,ADSCs proliferative activity improved gradually following the increased of passage.After two weeks’ adipogenic/osteogenic induction in vitro,the oil red O staining and alkaline phosphatase activity test showed positive results. Positive rates of subcutaneous tissue-derived ADSCs were 81.6%,26.5%,while another derivative were 64.6%,29.3%.2.CFDA SE-labeled ADSCs migrated obviously,and gradually enveloped and replaced the ECEs with induced by poor nutrition of CECs.The AE5 expression rate of ADSCs cultured in KM system presented a dose-dependence of EGF;while bFGF inhibited the differentiation.ADSCs cultured in the other two systems were negative either with or without growth factor.3.Among the different induced groups that cultured with KM,AE5-positive rate of the corneal stroma Transwell co-culture induction group which added EGF reached top to 97.7%,and AE1-positive expression was 0%,which prompted to mature corneal epithelial-like cells.The single induction group was the second top,that AE5~+ cells occupied 75.4%(add EGF),46.0%(EGF free),AE1~+ cells accounted for 86.0%(add EGF),96.5%(EGF free),premature.ADSCs co-culture induced by the corneal stroma with EGF free showed the least positive rate of AE5(2.4%),AE1(51.2%).4.subcutaneous tissue-derived ADSCs were single induced with KM and added 40ng/mL EGF,which expression was 100%AE5~+ rate,and 0%AE1~-,on the other hand,EGF free group were 16.6%,97.7%.It suggested that the differentiation capacity of this source was stronger than liposuction group.Conclusion:1.We could isolate more quantity and purer ADSCs from subcutaneous tissue and Processed lipoaspirate adipose liquid with improved process methods.And it was evidenced with multi-lineage differentiation potential abilities and could be used in engineering researches,cell and gene therapies in the future.2.Following the results of our study,the KM contained 40ng/mL EGF co-culture corneal stroma co-induction in Transwell system could be the best way to make ADSCs have a transdifferention to corneal epithelial-like cells.In addition,the subcutaneous adipose tissue-derived ADSCs have the better differential ability that could be more suitable for future clinical treatment than processed lipoaspirate-derived ADSCs.Howere, processed lipoaspirate-derived ADSCs have less simple isolated process and are more adequate,so it may be more suitable for laboratory research.3.CFDA SE is a stable cell tracer,and can be used as the tracer of in vivo animal experiments in future.更多还原