大鼠脂肪干细胞的分离培养及向软骨表型分化的实验研究

发布时间：2018-05-24 18:49

  本文选题：干细胞 + 脂肪组织　； 参考：《中国医科大学》2008年硕士论文

【摘要】： 实验目的 探讨从大鼠的脂肪组织中分离、培养脂肪干细胞(ADSCs)的方法及鉴定方法。并对在单层培养条件下用外源性转化生长因子TGF-β1定向诱导ADSCs分化为软骨细胞的可行性进行初步的探讨研究。为软骨组织工程研究中的种子细胞的选择提供一个新的思路。 实验方法 Wistar雄性大鼠处死,取腹股沟区及睾丸脂肪垫等处脂肪组织,清除小血管和结缔组织充分剪碎,加入0.15%的Ⅰ型胶原酶消化,离心后收集细胞置于孵箱中培养。2-3天换液一次去除未贴壁细胞,待贴壁细胞生长融合至80-90%时传代培养。应用倒置相差显微镜观察各代ADSCs形态学的改变,并用免疫荧光法检测传代ADSCs表面抗原CD29表达。取第3代生长良好的脂肪干细胞,分成两组:一组用含有TGF-β_1的培养基定向诱导培养,另一组为对照组(空白组)用普通培养基继续培养,诱导两周后行Ⅱ型胶原免疫组化染色进行检测鉴定。 结果 1、脂肪干细胞的形态特点:原代培养的脂肪干细胞24小时左右即可见细胞贴壁,多数贴壁细胞呈小圆形,折光性强,其中散在少量的梭形细胞或多角细胞。3d后梭形细胞逐渐增多,可见核分裂相。5d后细胞呈团簇状生长,并形成集落。约7d左右细胞融合超过90%时进行传代,传代后细胞形态较为均一,呈长梭形,排列紧密,类似成纤维细胞。 2、脂肪干细胞的鉴定:免疫荧光法检测脂肪干细胞(ADSCs)。结果显示细胞表面抗原CD29呈阳性表达,从而证实所培养细胞为干细胞来源。 3、ADSCs向软骨细胞的定向分化:实验组细胞生长速度变慢,诱导第2天即可见少数单个细胞由梭形变为多边形,多角形,可见细长伪足,核周出现黑色颗粒,随诱导时间延长,形态发生如前变化的细胞逐渐增多。诱导第14天左右部分细胞开始变圆,细胞边界不清,核略呈偏位,核周颗粒密集,细胞逐渐聚集成团。 4、诱导后细胞Ⅱ型胶原免疫组化检测:实验组在TGF-β1向软骨表型定向诱导后14d,Ⅱ型胶原免疫组化染色结果显示有部分细胞呈阳性,可见棕黄色颗粒分布于细胞胞浆内而对照组则为阴性。 结论 1、表面抗原CD29表达阳性,证实大鼠脂肪组织中存在干细胞来源细胞。 2、从大鼠脂肪组织中可以分离出脂肪干细胞(ADSCs),并且细胞在体外培养条件下生物学性状保持稳定。 3、外源性转化生长因子TGF-β定向分化、定向诱导连续培养14d后,Ⅱ型胶原免疫组织化学染色呈阳性反应,表现出来软骨细胞的部分特性。证实脂肪干细胞具有向软骨细胞定向分化的能力。
[Abstract]:Experimental purpose To study the method and identification of adipose stem cells isolated and cultured from adipose tissue of rats. The feasibility of inducing ADSCs to differentiate into chondrocytes by transforming growth factor TGF- 尾 1 in monolayer culture was studied. It provides a new idea for the selection of seed cells in cartilage tissue engineering. Experimental method The male Wistar rats were killed, the fat tissues in the inguinal region and testis fat pad were removed, the small vessels and connective tissues were cut and digested with 0.15% type I collagenase. After centrifugation, the collected cells were cultured in incubators for 2 to 3 days. The unadherent cells were removed once for 2-3 days. When the adherent cells were fused to 80-90%, the cells were subcultured. The morphologic changes of ADSCs were observed by inverted phase contrast microscope and the expression of ADSCs surface antigen CD29 was detected by immunofluorescence. The third generation of adipose stem cells with good growth was divided into two groups: one group was cultured on the medium containing TGF- 尾 1, the other group was the control group (blank group) and the control group (blank group). Two weeks after induction, type 鈪，

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