体外诱导人脂肪组织来源的干细胞向角膜上皮样细胞分化的初步研究

发布时间：2019-04-21 16:36

【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。
[Abstract]:Purpose: 1. Isolation, culture and identification of stem cells (ADSCs) from human adipose tissue sources of different donor properties (surgical resection of the adipose tissue mass and liposuction fat suspension) ). Observation of its biological characteristics, the difference in the activity of stem cells 2. In vitro simulation of the microenvironment of the growth and differentiation of the corneal epithelium, the ADSCs extracted from different methods were individually/ jointly induced to explore the potential for the differentiation of the corneal epithelial-like cells and the ability to induce differentiation. line approach Methods:1. The improved method was used to separate and purify human ADSCs and to use modified in vitro amplification culture. in-vitro culture of liquid.2. MTT method ADSCs proliferation curve. Flow cytometry was used to detect CD29, CD34, CD49d and CD105 of 3 to 5 generations. And the expression rate of the CD106 is higher than the expression rate of the CD106. Identification and differentiation of two traits: the phenotype and proliferation of the cells extracted from the two traits 3. Application of CFD A SE-labeled human ADSCs was used to extract the rat corneal epithelial cells in vitro by a tissue block culture method, and a rat corneal stroma with no epithelium and endothelial cells was prepared. In combination with ADSCs.4. The human ADSCs labeled by the CFDA SE and the rat corneal epithelial cells were each crawled and cultured in the same system. 5. The migration of ADSCs was observed.5. The gradient concentration of 0-50 ng/ mL of EGF and the addition of 10,20 ng/ mL of EGF on the basis of 50 ng/ mL of EGF were added in different culture solution systems (the in-vitro expansion culture system of the cell culture system and the mesenchymal cells and the above-mentioned two-product mixed intermediate system). The changes of the morphology of ADSCs and the expression of the corneal-specific protein AE5 (CK3/ CK12) were observed by light microscopy, electron microscopy, immunohistochemical staining and immunofluorescent staining. The growth of ADSCs and the expression of the corneal-specific protein AE5 (CK3/ CK12) were observed. The difference in the effect of the factor on ADSCs.6. The rat corneal epithelial cells or the corneal stroma were placed on the upper layer of the Transwell system, and the lower layer of the ADSCs was placed, and the keratinocytes were selected not to add/ add 40 ng/ mL of EGF. The expression of the human ADSCs was induced by the culture system, and the expression of the keratin-spectrum AE1 and the corneal-specific protein AE5 in the ADSCs after the induction of 30 d was observed by the immunofluorescence staining. The difference of the induction rate of human ADSCs was induced.7. The ADSCs of 2 traits were induced separately, and the results were compared. After guide 3 The positive rate of AE1 and AE5 in both AE1 and AE5 was 1. The results were as follows:1. No matter whether the fat mass or the fat suspension A large number of high-purity ADSCs can be obtained by the above method, but the latter The operation process is more time-saving and labor-saving, and the amount of the obtained cells is relatively high. The flow cytometry is used to determine the ADSCs of 3 to 5 generations, and the cells of the two different donor characters are CD34 ~-, CD106 ~-- CD29 ~ +, CD49d ~ +, CD105 ~ +, CD29 ~ +, CD49d ~ + cells were 82.5% and 8.0%, respectively, and the number of CD29 ~ +, CD49d ~ + and CD49d ~ +, CD105 ~ +. The expression of CD29 ~ +, CD49d ~ +, CD105 ~ + in CD29 ~ +, CD49d ~ + and CD105 ~ + was 99%,40% and 80%, respectively. There was a significant difference in the positive rate of CD49d (P <0.01). MTT colorimetric method The results showed that the proliferation of ADSCs increased with the increase of the number of passages. In addition, ADSCs were induced respectively in vitro and in vitro. The positive results were found in 2 weeks after the staining of oil red O and alkaline phosphatase. The positive rate of the two groups in the subcutis was 81.6%, respectively. 26.5%, in the liposuction group 64.6%, 29.3%.2. The ADSCs marked by the CFDA SE were in the vegetative state. In addition, the positive rate of ADSCs in the culture of the cell culture system to the corneal epithelial-like cells has an EGF concentration-dependent manner; bFGF has an inhibitory effect on differentiation; and the other two systems are The effect of EGF and bFGF on the induction of ADSCs was negative.3. Different induction groups under the same culture system showed the highest proportion of AE5 ~ + ADSCs with the addition of EGF. The expression of AE1 in 7% and AE1 was 0, and it was suggested to differentiate into mature corneal epithelial-like cells. Second, the rate of AE5 ~ + was 75.4% (addition of EGF), 46.0% (no EGF) and the ratio of AE1 ~ + was 86.0%. (addition of EGF), 96.5% (EGF not added), differentiation under-mature, and combination induction of corneal stroma without EGF stimulation The positive rates of AE5 and AE1 in group AE5 and AE1 were 2.4% and 51.2% respectively. -0%, and no EGF are Conclusion:1. The cell differentiation ability of the group is stronger than that of the liposuction group. Conclusion:1. The present study successfully separates the human subcutaneous fat tissue and the liposuction waste liquid by an improved method, and the stem cells derived from high-purity adipose tissue are cultured. And can be proved to be multi-directional by in-vitro condition induction. The differentiation potential can be used as a good tissue engineering, cell therapy and gene therapy seed cell. In this study, a 40 ng/ mL EGF-containing cutin cell culture solution was added to the Transwell system. It is the best way to induce the differentiation of ADSCs to the corneal epithelial-like cells in vitro. In addition, the ADSCs derived from the subcutaneous fat tissue have better differentiation and are more suitable for future clinical treatment. and the cells extracted by liposuction are relatively simple in working procedures and are more abundant in sources and are more suitable for laboratory studies.3. CF
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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