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【摘要】： 第一部分:脂肪干細胞的分離、培養(yǎng)、鑒定和向表皮細胞、成骨細胞及脂肪細胞分化 目的:探討人脂肪干細胞(ADSCs)體外誘導分化為表皮樣細胞、成骨細胞及脂肪細胞的可能性。 方法:在一例年齡為30歲的行吸脂手術的女性吸脂者中,通過電動負壓吸引獲取腹部脂肪組織,酶消化法獲取ADSCs,體外培養(yǎng)擴增。經(jīng)傳代、擴增、純化至第3-5代。觀察細胞生長特性并記錄ADSCs生長曲線,通過流式細胞儀及免疫細胞化學檢測表面抗原表達。取生長良好的第3代人ADSCs,分別應用成表皮誘導培養(yǎng)液B[70%培養(yǎng)液A(90% L-DMEM,10%FBS,100U/ml青霉素, 100U/ml鏈霉素,2mmol/L谷氨酰胺, pH 7. 2)+30%成纖維細胞培養(yǎng)基上清液+10ng/LEGF],成骨誘導培養(yǎng)基C(DMEM/10%FBS,0.1μmol/L地塞米松, 50μmol/L維生素C, 10mmol/Lβ-甘油磷酸鈉, 100U/ml青霉素, 100U/ml鏈霉素)成脂肪細胞誘導培養(yǎng)基D(DMEM + 10% FBS + 500μmol/L IBMX +1μmol/L吲哚美辛),倒置顯微鏡觀察細胞形態(tài)變化,誘導20d后分別對成表皮誘導組進行免疫組化檢測CK19表達,成骨誘導組進行堿性磷酸酶檢測,成脂誘導組進行油紅“O”檢測。 結果:細胞接種時含有大量的脂滴、少量紅細胞和內皮細胞等。接種24h后可見少量較大的細胞開始貼壁,倒置顯微鏡下見細胞為大而扁平的單層細胞,有的細胞體細長,似成纖維細胞。48h后大多數(shù)細胞貼壁,開始伸展、分裂,呈梭形,有粗大突起。5-7天后,細胞逐漸分裂、融合成單層,成簇分布。流式細胞儀及免疫細胞化學鑒定結果均示人ADSCs的表面抗原CD44、CD49d呈陽性表現(xiàn),CD34呈陰性表現(xiàn)。誘導20d后,成表皮誘導組示:免疫組織化學鑒定結構顯示有CK19的表達。成骨誘導組示:細胞堿性磷酸酶染色陽性。成脂肪細胞誘導組示:油紅O染色,胞質內脂滴均被染成紅色,證實為脂性液體。 1本實驗通過酶消化法從人脂肪組織中分離出脂肪干細胞,并成功的在體外進行細胞擴增、培養(yǎng)和傳代,通過形態(tài)學觀察、細胞化學檢測及流式細胞儀鑒定證實所分離培養(yǎng)細胞為脂肪干細胞。 2 10代以內的脂肪干細胞隨著傳代次數(shù)的增加其增殖能力未現(xiàn)明顯降低的趨勢。 3脂肪干細胞在常規(guī)成骨培養(yǎng)基及成脂培養(yǎng)基的誘導下,可分別向成骨細胞和脂肪細胞方向分化。 4脂肪干細胞在成纖維細胞培養(yǎng)上清液聯(lián)合EGF的體外誘導條件下可向表皮樣細胞分化。 第二部分:鹽酸法舒地爾對脂肪干細胞向表皮細胞誘導分化的影響 目的:觀察Rho分子信號通路阻斷劑鹽酸法舒地爾(HA1077)對脂肪干細胞向表皮細胞分化的影響。 方法:按照實驗一的方法獲取生長良好達到90%融合的第3代人ADSCs分為四組,第1組持續(xù)用培養(yǎng)液1(培養(yǎng)液A)培養(yǎng)、傳代;將第2、3、4組,棄培養(yǎng)液A,無菌PBS沖洗,分別換用培養(yǎng)液2(培養(yǎng)基1+20umol/L HA1077)、培養(yǎng)液3(70%培養(yǎng)液1+30%成纖維細胞培養(yǎng)基上清液+10ng/LEGF)、培養(yǎng)液4(20μmol/L HA1077+培養(yǎng)液3),置培養(yǎng)箱中培養(yǎng),每隔2-3天更換各自培養(yǎng)液1次,細胞生長達到90%融合時用0.25%胰蛋白酶-1mmolEDTA消化,常規(guī)培養(yǎng)。第1組為空白對照組,第2組為HA1077單純對照組,第3組為單純誘導組,第4組為HA1077誘導組。用倒置顯微鏡定期觀察各組細胞形態(tài)及其生長情況。誘導20d時1~4組均用流式細胞儀檢測CK19抗體表達情況,所得數(shù)據(jù)均以均數(shù)±標準差表示,采用SPSS13.0統(tǒng)計分析軟件,運用t檢驗進行各實驗組與空白對照組陽性率的比較。誘導20d時將1~4組進行免疫組化檢測CK19、CK10抗體表達情況。 結果: 20d時流式細胞儀檢測各實驗組CK19的表達陽性率分別為(0.23±0.010)%、(0.35±0.020)%、(9.73±0.800)%、(17.65±0.998)%(n=3),第2組即經(jīng)HA1077單純對照組培養(yǎng)的細胞,20d時細胞形態(tài)仍結論:為原來的長梭形成纖維細胞狀結構,細胞緊密排列成片,與空白對照組細胞形態(tài)變化差別不大。第4組即20umol/L HA1077誘導組,細胞形態(tài)由原來的長梭形逐漸變粗變短,其細胞形態(tài)變化較其它組出現(xiàn)早,對人ADSCs向表皮樣細胞分化有促進趨勢。免疫組化檢測示第3、4組有CK19、CK10表達,第4組較第3組陽性率增高,對照組為陰性結果。 結論: 1單純的鹽酸法舒地爾不具有誘導功能,在常規(guī)培養(yǎng)基中加入一定濃度的鹽酸法舒地爾不能誘導人脂肪干細胞向表皮細胞分化。 2在誘導培養(yǎng)基中加入一定濃度的鹽酸法舒地爾對人脂肪干細胞向表皮細胞分化能起到明顯的促進作用。
[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,
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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