盐酸法舒地尔促进脂肪干细胞体外诱导为表皮样细胞的实验研究

发布时间：2019-04-18 09:43

【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。
[Abstract]:Part I: Isolation, Culture, Identification and Differentiation of Adipose Stem Cells to Epidermal Cells, Osteoblasts and Adipocytes Objective: To study the effect of human adipose-derived stem cells (ADSCs) on the differentiation of human adipose-derived stem cells (ADSCs) into epidermal-like cells, osteoblasts and adipocytes. Method: In the case of a 30-year-old female liposuction, the abdominal fat tissue was obtained by electric negative pressure suction, and the ADSCs were obtained by enzyme digestion. external culture and amplification, transacting, amplification, and purification to Generation 3-5. Observe the cell growth characteristics and record the ADSCs growth curve by flow cytometry and immunocytochemical test Surface antigen expression. The third generation ADSCs with good growth were respectively applied to epidermal induced culture solution B[70% culture solution A (90% L-DMEM,10% FBS,100 U/ ml penicillin,100 U/ ml streptomycin,2 mmol/ L glutamate). Amine, pH 7.2) + 30% Fibroblast Medium Supernatant + 10 ng/ LEGF], Osteogenic Induction Medium C (DMEM/10% FBS, 0.1. mu.mol/ L Dexamethasone,50 & mu; mol/ L Vitamin C,10 mmol/ L)-Sodium glycerophosphate,100 U/ Ml of penicillin and 100 U/ ml of streptomycin were used to induce the culture medium D (DMEM + 10% FBS + 500. mu.mol/ L IBMX + 1. mu.mol/ L), and the morphological changes of the cells were observed by an inverted microscope. After 20 days, the expression of CK19 and the formation of the osteogenic induction group were detected by immunohistochemistry. Phosphatase detection, lipid-forming induction group, oil red

"O" Test. Results: The cells were seeded with a large amount of lipid droplets and a small amount of red Cells and endothelial cells, etc. After 24 h inoculation, a small amount of the larger cells began to adhere, and the cells were found to be large, flat, single-layer cells under the inverted microscope, and some of the cell bodies were elongated and fibroblast-like. After 48 h, the majority of the cells were attached, started to stretch, split, in the form of a shuttle, and had a thick protrusion.5-7 days After that, the cells are gradually split, fused, The results of flow cytometry and immunocytochemical identification showed that the surface antigen CD44 and CD49d of human ADSCs were positive. D34 showed a negative expression. After 20 days of induction, the skin-inducing group indicated that the structure of the immunohistochemical method showed The expression of CK19. The osteogenic induction group indicated that the cell base Aliphatic cell-induced group showed that the oil and red O were stained, and the intracytoplasmic lipid droplets were stained with red color. In this experiment, adipose-derived stem cells were isolated from human adipose tissue by enzyme digestion, and the cells were successfully amplified, cultured and passaged in vitro. The results were confirmed by morphological observation, cell chemical detection and flow cytometry. adipose-derived stem cells are isolated from the cultured cells. The increase in the number of passage times of the adipose-derived stem cells within 10 generations the proliferation ability of the 3-fat stem cells is not obviously reduced, and the 3-fat stem cells are induced under the induction of a conventional osteogenic medium and a fat-forming medium, Can be respectively differentiated into the direction of the osteoblast and the fat cell, and the 4-fat stem cells are combined with the culture supernatant of the fibroblast. EGF can differentiate into epidermal-like cells under in vitro induction conditions. The effect of fasudil hydrochloride on the differentiation of adipose-derived stem cells to epidermal cells: a study of the effect of the Rho molecular signal pathway blocking agent on the differentiation of epidermal cells The effect of diltiazem (HA1077) on the differentiation of the adipose-derived stem cells to the epidermal cells was carried out. Methods: The third generation ADSCs with good growth achieved by 90% were divided into four groups according to the experimental method. The first group was continuously cultured and passaged with the culture solution 1 (culture solution A), and the second, the third, the fourth group, the disposable culture solution A and the sterile PBS were washed. The culture solution 2 (culture medium 1 + 20 umol/ L HA1077), culture solution 3 (70% culture solution 1 + 30% fibroblast culture medium supernatant + 10 ng/ LEGF), culture solution 4 (20. mu.mol/ L HA1077 + culture solution 3) were used to culture, and each culture solution was replaced every 2-3 days, and the cell growth reached 90%. The conventional culture was performed with 0.25% trypsin-1 mmol EDTA, and the first group was the blank control group, and the second group was HA1077. In the control group, the third group was the pure induction group, and the fourth group was HA1. 077 induction group. The morphology and the growth of each group were observed on a regular basis with an inverted microscope. The expression of CK19 antibody was detected by flow cytometry in 1-4 groups at 20 days. The data obtained were expressed in the standard deviation of mean number, and the SPSS13.0 series was used. The positive rate of each experimental group and the blank control group was compared by using t-test. The expression of CK19 and CK10 was detected by immunohistochemistry in 1-4 groups. Results: The positive rates of CK19 expression in each experimental group were (0.23-0.010)%, (0.35-0.020)%, (9.73-0.800)%, (17.65-0.998)% (n = 3), respectively. In the second group, the cells cultured in the control group of HA1077 were cultured in the control group, and the morphology of the cells at the time of 20d was still the conclusion: to form the fiber for the original long shuttle. In group 4,20 umol/ L HA1077 induction group, the cell morphology was gradually shortened by the original long shed, and its cells The change of morphology was earlier in the other groups, and the differentiation of human ADSCs to the epidermoid cells was promoted. K19 The positive rate of CK10 was higher in group 4 than in group 3, and negative result in the control group. adding a certain concentration of fasudil hydrochloride in a conventional culture medium can not induce the differentiation of human adipose-derived stem cells into the epidermal cells,
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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