脂肪来源干细胞（ADSCs）在周围神经损伤修复中的研究

发布时间：2018-09-17 12:00

【摘要】： 目的:采用酶预消化后连续组织块法进行大鼠脂肪来源干细胞的体外培养,并与胶原酶消化法的培养效果相比较,研究脂肪来源干细胞的获取途径并为其体外增殖培养方法提供参考依据。方法:取一月龄SD大鼠腹股沟处脂肪组织,分A、B两组。A组使用D-Hank's液冲洗净后剪成8mm~3左右组织块。将组织块置于尼龙网上放至培养皿中,0.25%胰蛋白酶消化5min,0.1%Ⅰ型胶原酶消化组织块20min,弃去消化液体,加入培养基后放于孵化箱中继续培养。2～3天后将组织块连同尼龙网一起拎除放至下一培养皿中以连续组织块法培养3～4次。B组使用胶原酶消化法培养。采用MTT检测方法对细胞增殖活性进行比较,流式细胞仪检测脂肪干细胞表面标志物。结果:A、B两组培养的细胞均具有典型的干细胞形态特征,生长曲线相近,脂肪干细胞标志物CD29、CD44、CD54、CD105呈阳性表达,造血干细胞标志物CD34、CD45阴性表达,骨髓来源干细胞标志物CD106阴性表达。A组的阳性表达率略高于B组,两组间的阳性表达率无统计学上的差异(P＞0.05)。结论:酶预消化连续组织块法是进行脂肪来源干细胞体外培养的另一种途径,获得脂肪来源干细胞的纯度略高于酶原消化法。优点是能短时间内获得多个脂肪干细胞样本,对细胞损伤较小,具有一定的实验应用价值。不足之处是细胞起始浓度较低,生长周期略长。目的:探讨SD大鼠脂肪来源干细胞(ADSCs)和雪旺细胞(Schwann,SC)利用Transwell培养板共培养时,ADSCs的生长增殖特性以及其向神经细胞分化的能力,并和使用试剂诱导(BME+BHA)下的ADSCs生长结果相比较,为构建组织工程人工神经提供种子细胞来源的理论和依据。方法:按实验第一部分获取ADSCs,同时取大鼠坐骨神经按植块法获取雪旺细胞。取P3代脂肪来源干细胞分三组,A组使用ADSCs和雪旺细胞置于Transwell培养板中构建共培养体系,B组利用β-巯基乙醇(BME)+丁酸酯羟基茴香醚(BHA)作为ADSCs诱导因子使其向神经细胞方向诱导,C组为空白对照组。比较各组细胞形态,观察脂肪来源干细胞生长、轴突长度、NF-M和GFAP表达情况,并做统计学分析。结果:A、B两组ADSCs均有部分细胞分化出细长突起,细胞生长代谢旺盛,GFAP免疫荧光染色阴性、NF-M免疫荧光染色阳性。在细胞数量上,A组与C组无差异,但明显高于B组(P＜0.05);在突起长度上,A组与B组无差异,但明显高于C组(P＜0.05)。结论:雪旺细胞与ADSCs共培养能促进ADSCs向神经细胞方向分化,诱导效果与使用BME+BHA试剂诱导的结果相近,并能有效防止ADSCs损伤和死亡,提示雪旺细胞和ADSCs共培养可以作为一种将ADSCs向神经细胞方向诱导的方法在组织工程领域加以利用。
[Abstract]:OBJECTIVE: To compare the effect of collagenase digestion on adipose-derived stem cells (ADSCs) cultured in vitro by enzymatic pre-digestion and continuous tissue block method, and to study the ways of obtaining ADSCs and provide reference for the methods of proliferation and culture in vitro.
Methods: One month old SD rats were divided into two groups: group A and group B. Group A was washed with D-Hank's solution and cut into about 8 mm~3 pieces of tissues. The pieces were placed on nylon mesh and digested with 0.25% trypsin for 5 min and 0.1% collagenase type I for 20 min. The digested tissues were discarded and put into incubator after adding the medium. After 2-3 days, the tissues were removed together with nylon mesh and placed in the next culture dish for 3-4 times. Group B was cultured with collagenase digestion method. Cell proliferation activity was compared by MTT assay, and surface markers of adipose-derived stem cells were detected by flow cytometry.
Results: The cells cultured in A and B groups had typical morphological characteristics of stem cells, and the growth curves were similar. Adipose stem cell markers CD29, CD44, CD54 and CD105 were positive, hematopoietic stem cell markers CD34 and CD45 were negative, and bone marrow stem cell markers CD106 were negative. There was no statistical difference in the expression rate (P > 0.05).
CONCLUSION: Enzymatic pre-digestion of continuous tissue block is another way to culture adipose-derived stem cells in vitro, and the purity of adipose-derived stem cells is slightly higher than that of enzymatic digestion. The initial concentration is low and the growth cycle is slightly longer.
AIM: To investigate the growth and proliferation characteristics of adipose-derived stem cells (ADSCs) and Schwann cells (SC) from SD rats co-cultured with Transwell plate and their ability to differentiate into neural cells, and to compare the growth results of ADSCs induced by reagents (BME + BHA), so as to provide seed cells for the construction of tissue-engineered artificial nerves. The theory and basis of source.
METHODS: ADSCs were obtained from the first part of the experiment, and Schwann cells were obtained from the sciatic nerve of rats by grafting. P3 adipose-derived stem cells were divided into three groups. ADSCs and Schwann cells in group A were used to construct a co-culture system in Transwell culture plate. In group B, beta-mercaptoethanol (BME) and butyrate hydroxyanisole (BHA) were used as inducing factors of ADSCs. Cell morphology, growth of adipose-derived stem cells, axon length, expression of NF-M and GFAP were observed and analyzed statistically.
Results: Some cells of ADSCs in group A and group B differentiated into elongated processes, the growth and metabolism of ADSCs were vigorous, GFAP immunofluorescence staining was negative, and NF-M immunofluorescence staining was positive.
CONCLUSION: Co-culture of Schwann cells and ADSCs can promote the differentiation of ADSCs toward neural cells. The induction effect is similar to that induced by BME+BHA reagent, and can effectively prevent the injury and death of ADSCs. It suggests that co-culture of Schwann cells and ADSCs can be used as a method to induce ADSCs toward neural cells in the field of tissue engineering. Make use of.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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