【作者】 沙德潜；

【导师】 白晓东；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 硕士

【摘要】 第一部分:脂肪干细胞的分离、培养、鉴定和向表皮细胞、成骨细胞及脂肪细胞分化目的:探讨人脂肪干细胞(ADSCs)体外诱导分化为表皮样细胞、成骨细胞及脂肪细胞的可能性。方法:在一例年龄为30岁的行吸脂手术的女性吸脂者中,通过电动负压吸引获取腹部脂肪组织,酶消化法获取ADSCs,体外培养扩增。经传代、扩增、纯化至第3-5代。观察细胞生长特性并记录ADSCs生长曲线,通过流式细胞仪及免疫细胞化学检测表面抗原表达。取生长良好的第3代人ADSCs,分别应用成表皮诱导培养液B[70%培养液A(90% L-DMEM,10%FBS,100U/ml青霉素, 100U/ml链霉素,2mmol/L谷氨酰胺, pH 7. 2)+30%成纤维细胞培养基上清液+10ng/LEGF],成骨诱导培养基C(DMEM/10%FBS,0.1μmol/L地塞米松, 50μmol/L维生素C, 10mmol/Lβ-甘油磷酸钠, 100U/ml青霉素, 100U/ml链霉素)成脂肪细胞诱导培养基D(DMEM + 10% FBS + 500μmol/L IBMX +1μmol/L吲哚美辛),倒置显微镜观察细胞形态变化,诱导20d后分别对成表皮诱导组进行免疫组化检测CK19表达,成骨诱导组进行碱性磷酸酶检测,成脂诱导组进行油红“O”检测。结果:细胞接种时含有大量的脂滴、少量红细胞和内皮细胞等。接种24h后可见少量较大的细胞开始贴壁,倒置显微镜下见细胞为大而扁平的单层细胞,有的细胞体细长,似成纤维细胞。48h后大多数细胞贴壁,开始伸展、分裂,呈梭形,有粗大突起。5-7天后,细胞逐渐分裂、融合成单层,成簇分布。流式细胞仪及免疫细胞化学鉴定结果均示人ADSCs的表面抗原CD44、CD49d呈阳性表现,CD34呈阴性表现。诱导20d后,成表皮诱导组示:免疫组织化学鉴定结构显示有CK19的表达。成骨诱导组示:细胞碱性磷酸酶染色阳性。成脂肪细胞诱导组示:油红O染色,胞质内脂滴均被染成红色,证实为脂性液体。1本实验通过酶消化法从人脂肪组织中分离出脂肪干细胞,并成功的在体外进行细胞扩增、培养和传代,通过形态学观察、细胞化学检测及流式细胞仪鉴定证实所分离培养细胞为脂肪干细胞。2 10代以内的脂肪干细胞随着传代次数的增加其增殖能力未现明显降低的趋势。3脂肪干细胞在常规成骨培养基及成脂培养基的诱导下,可分别向成骨细胞和脂肪细胞方向分化。4脂肪干细胞在成纤维细胞培养上清液联合EGF的体外诱导条件下可向表皮样细胞分化。第二部分:盐酸法舒地尔对脂肪干细胞向表皮细胞诱导分化的影响目的:观察Rho分子信号通路阻断剂盐酸法舒地尔(HA1077)对脂肪干细胞向表皮细胞分化的影响。方法:按照实验一的方法获取生长良好达到90%融合的第3代人ADSCs分为四组,第1组持续用培养液1(培养液A)培养、传代;将第2、3、4组,弃培养液A,无菌PBS冲洗,分别换用培养液2(培养基1+20umol/L HA1077)、培养液3(70%培养液1+30%成纤维细胞培养基上清液+10ng/LEGF)、培养液4(20μmol/L HA1077+培养液3),置培养箱中培养,每隔2-3天更换各自培养液1次,细胞生长达到90%融合时用0.25%胰蛋白酶-1mmolEDTA消化,常规培养。第1组为空白对照组,第2组为HA1077单纯对照组,第3组为单纯诱导组,第4组为HA1077诱导组。用倒置显微镜定期观察各组细胞形态及其生长情况。诱导20d时1~4组均用流式细胞仪检测CK19抗体表达情况,所得数据均以均数±标准差表示,采用SPSS13.0统计分析软件,运用t检验进行各实验组与空白对照组阳性率的比较。诱导20d时将1~4组进行免疫组化检测CK19、CK10抗体表达情况。结果: 20d时流式细胞仪检测各实验组CK19的表达阳性率分别为(0.23±0.010)%、(0.35±0.020)%、(9.73±0.800)%、(17.65±0.998)%(n=3),第2组即经HA1077单纯对照组培养的细胞,20d时细胞形态仍结论:为原来的长梭形成纤维细胞状结构,细胞紧密排列成片,与空白对照组细胞形态变化差别不大。第4组即20umol/L HA1077诱导组,细胞形态由原来的长梭形逐渐变粗变短,其细胞形态变化较其它组出现早,对人ADSCs向表皮样细胞分化有促进趋势。免疫组化检测示第3、4组有CK19、CK10表达,第4组较第3组阳性率增高,对照组为阴性结果。结论:1单纯的盐酸法舒地尔不具有诱导功能,在常规培养基中加入一定浓度的盐酸法舒地尔不能诱导人脂肪干细胞向表皮细胞分化。2在诱导培养基中加入一定浓度的盐酸法舒地尔对人脂肪干细胞向表皮细胞分化能起到明显的促进作用。更多还原

【Abstract】 PartⅠ: The Separation, Evaluation and Culture of Adipose derived Stem Cells (ADSCs) and the Differentiation of the ADSCsObjective: To observe the possibility of the induction of ADSCs into epidermic cells,skeletogenous cells and adipose cells.Method: Human ventral adipose tissues were obtained by electric vacuum aspiration technique from a thirty-year old woman who underwent suction lipectomy sugery. The ADSCs were obtained by using enzyme digestion and then the cells were cultured in vitro. After generating, amplifying and purifying, the 3rd-5th generation of ADSCs were obtained. The growth character of ADSCs was observed and the growth curve was recorded accordingly. Then the express of surface antigen was detected by flow cytometry and immunohistochemistry assay. The 3rd generation of ADSCs was selected for different induction as follows: Culture solution B:[70% culture solution A(90% L-DMEM,10%FBS,enicillin100 U/ml, phytomycin100μg/ml,2mmol/L glutamine, pH=7.2) +30% supernatant fluid from fibroblast medium+10 ng/LEGF] to induce human ADSCs differentiated into epidermic cells; Culture solution C: ( DMEM/10%FBS, 0.1μmol/L desacort, 50μmol/L vitamin C, 10mmol/L sodiumβ-glycerophosphate, 100U/ml penicilin, 100U/ml phytomycin ) to induce human ADSCs differentiated into skeletogenous cells; Culture solution D:(DMEM +10%FBS + 500μmol/L IBMX +1μmol/L antinfan) to induce human ADSCs differentiated into adipose cells. After 20d, the changes of cell morphology after induction were observed through inverted phase contrast microscope, the express of CK19 was detected by immunohistochemistry assay in group B, the ALP assay in group C and the Oil red O staining in group D.Result: Many lipid droplets, a few red blood cells and endothelial cells were observed when the cells are cultured. After 24h, we could see a few larger cells attached and most of the monolayer cells were large and applanate, few were slender as fibroblast through inverted phase contrast microscope. Most of them are attached and almost shuttle-shaped or needle tip-shaped after 48h. 5 to 7 days later, the cells were fused to monolayer gradually and distributed into clustering. The express of CD44 and CD49d were examined to identify human ADSCs by flow cytometry and immunohistochemistry assay, and the express of CD44 and CD49d were positive while the express of CD34 was negative. After 20d-induction, CK19 of the group which is induced into epidermic cells was detected to be positive by immunohistochemistry, ALP of the group which is induced into osteoblast cells is positive and the Oil red O staining of the group which is induced into adipose cells is positive.Conclusion:1. We obtained human ADSCs from human adipose tissue, amplified and cultivated them in vitro and then identified them by cell morphology, cytochemistry and flow cytometry assay. It confirmed that what we isolated were human adipose derived stem cells.2. The proliferative activity of ADSCs was not declined obviously within the 10 passages.3. ADSCs can be induced to differentiate into osteoblast cells and adipose cells with the osteoinductive culture and adipoinductive culture.4. ADSCs can be induced to differentiate into epidermis cells in medium with supernatant fluid from fibroblast and EGF in vitro.PartⅡ: The effect of Hydrochloric Fasudil (HA1077) on the differentiation of Epidermic Cells from hunam ADSCsObjective: To observe the effect of HA1077(a blocking agent against Rho molecular signal path)on the differentiation of ADSCs into epidermic cells. Method: According to the method above, we obtained and cultured human ADSCs in vitro. The 3rd generation of ADSCs was chosen from the cells which grew well with a 90% fusion rate, then divided into 4 groups. Group 1 was continuously cultured with culture solution 1:(culture solution A);In group 2,3,and 4, solution 1 were washed with sterile PBS and the cells were cultured with the different medium respectively as follows: Culture solution 2: (culture solution1+20μmol/L HA1077); Culture solution 3:( 70%culture solution 1 +30%clear supernatant liquid from fibroblast nutritive medium +10 ng/LEGF); Culture solution 4: (20μmol/L HA1077+ culture solution 3). The culture solution was changed every 2-3 days until the fusion rate rising up to 90%. We observed their morphology and growth condition regularly through inverted phase contrast microscope. After 20 day-induction, flow cytometry assay (CK19)and immunohistochemistry (CK10, CK19) were investigating the express of the related antibody in group 1~4.Result: The positive rate of CK19 with flow cytometry in group 1~4 were as follows:(0.23±0.010)%,(0.35±0.020)%,(9.73±0.800)% and(17.65±0.998)%(n=3). We observed the 2rd group which was induced respectively with solution 2 on 20d. The cells are still fusiform fibroblast and rank tightly, and have no difference with the blank control group. We then observed the 4th group which was induced respectively with solution 4. The cells which are fusiform fibroblas become short and coarse gradually, and rank tightly, the morphous changes of cells in this group are earlier than others, so this group has a promote effect on differentiation of epidermic cells from hunam ADSCs. We found a positive expression of CK19 and CK10 in group 3 and group 4, and a negative expression of CK19 and CK10 in group 1 and group 2.Conclusion:1. The application of certain concentration of HA1077 in the conditioned medium can not induce the differentiation of human ADSCs into epidermic cells. 2. The application of certain concentration of HA1077 in the induced medium can promote the differentiation of human ADSCs into epidermic cells obviously.更多还原