转化生长因子β1刺激改良培养小鼠原代肝星状细胞的活化

发布时间：2018-04-05 10:32

本文选题：组织工程　切入点：肝细胞　出处：《中国组织工程研究》2015年51期

【摘要】：背景:肝星状细胞是肝内分泌细胞外基质的关键细胞类型,因此在肝纤维化研究中很重要。目的:实验改良了肝星状细胞原代培养方法,获得充足的细胞量,用转化生长因子β1刺激活化,研究致纤维化的相关因子表达情况。方法:氯胺酮麻醉小鼠后采用门静脉穿刺原位灌注肝脏的方法分离肝脏,采用密度梯度离心获得肝星状细胞,利用形态学、免疫荧光染色等方法对原代肝星状细胞进行鉴定;将培养24 h肝星状细胞用质量浓度10μg/L转化生长因子β1刺激48 h,同时设PBS组进行对比。结果与结论:通过原位灌注肝脏并且酶消化的方法可以成功获得小鼠原代肝星状细胞,并且锥虫蓝染色活率为(97.2±0.8)%,免疫荧光染色纯度为(90.4±1.2)%,细胞计数总量约2.5×106/只。实时荧光定量聚合酶链式反应检测显示,与PBS组比较,转化生长因子β1刺激组的肝星状细胞平滑肌肌动蛋白α、Ⅰ型胶原蛋白、转化生长因子β受体1、转化生长因子β受体2 mR NA表达水平明显升高(P0.05)。结果证实,实验采用的改良的培养方法可更高效高质量的获得原代肝星状细胞,转化生长因子β1刺激可导致肝星状细胞活化,分泌致纤维化因子。
[Abstract]:Background: hepatic stellate cells (HSCs) are the key cell types of extracellular matrix (ECM) of liver, so they are very important in the study of hepatic fibrosis.Aim: to improve the primary culture method of hepatic stellate cells (HSC), obtain sufficient cells, stimulate activation with transforming growth factor 尾 1 (TGF- 尾 1), and study the expression of fibrosis related factors.Methods: after ketamine anesthetized mice liver was isolated by portal vein puncture in situ perfusion. Hepatic stellate cells were obtained by density gradient centrifugation. Primary hepatic stellate cells were identified by means of morphology and immunofluorescence staining.Hepatic stellate cells were cultured for 24 h and stimulated with 10 渭 g / L TGF- 尾 1 for 48 h, and compared with PBS group.Results and conclusion: primary hepatic stellate cells of mice could be successfully obtained by in situ perfusion of liver and enzyme digestion. The viability of trypanosome blue staining was 97.2 卤0.8. The purity of immunofluorescence staining was 90.4 卤1.2. The total number of cells was about 2.5 脳 106 per mouse.Real-time fluorescence quantitative polymerase chain reaction showed that the hepatic stellate cell smooth muscle actin 伪, type I collagen were stimulated by transforming growth factor 尾 1 (TGF- 尾 1) compared with PBS group.The expression of transforming growth factor 尾 receptor 1 and transforming growth factor 尾 receptor 2mR na increased significantly (P 0.05).The results showed that the improved culture method could obtain the primary hepatic stellate cells more efficiently and qualitatively, and the stimulation of transforming growth factor 尾 1 could lead to the activation of hepatic stellate cells and the secretion of fibrogenic factors.
【作者单位】：新疆医科大学第一附属医院/临床医学研究院新疆重大疾病医学重点实验室-省部共建国家重点实验室培育基地新疆包虫病基础医学重点实验室;新疆医科大学基础医学院生物化学与分子生物学教研室;
【基金】：国家自然科学基金面上项目(81371838) 新疆维吾尔自治区自然科学基金青年项目(2015211C084)~~
【分类号】：R575.2

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