中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (51): 8234-8240.doi: 10.3969/j.issn.2095-4344.2015.51.007

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

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

毕晓娟1,2,张传山1,李 亮1,吕国栋1,林仁勇1,2   

  1. 1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,新疆维吾尔自治区乌鲁木齐市 830011;2新疆医科大学基础医学院生物化学与分子生物学教研室,新疆维吾尔自治区乌鲁木齐市 830011
  • 收稿日期:2015-11-02 出版日期:2015-12-10 发布日期:2015-12-10
  • 通讯作者: 林仁勇,法国Franche-Comte大学博士,研究员,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,新疆维吾尔自治区乌鲁木齐市 830011;新疆医科大学基础医学院生物化学与分子生物学教研室,新疆维吾尔自治区乌鲁木齐市 830011
  • 作者简介:毕晓娟,女,1983年生,新疆维吾尔族自治区人,汉族,2007年新疆医科大学毕业,助理研究员,主要从事干细胞相关研究。
  • 基金资助:

    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)

Transforming growth factor β1 stimulation improves activation of mouse primary hepatic stellate cells obtained using the improved culture method

Bi Xiao-juan1, 2, Zhang Chuan-shan1, Li Liang1, Lv Guo-dong1, Lin Ren-yong1, 2   

  1. 1Clinical Medicine Research Institute, State Key Laboratory Incubation Base of Xinjiang Major Diseases Research, Xinjiang Key Laboratory of Echinococcosis, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; 2Department of Biochemistry and Molecular Biology, Preclinical Medicine College, Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
  • Received:2015-11-02 Online:2015-12-10 Published:2015-12-10
  • Contact: Lin Ren-yong, M.D., Researcher, Clinical Medicine Research Institute, State Key Laboratory Incubation Base of Xinjiang Major Diseases Research, Xinjiang Key Laboratory of Echinococcosis, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Department of Biochemistry and Molecular Biology, Preclinical Medicine College, Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
  • About author:Bi Xiao-juan, Assistant researcher, Clinical Medicine Research Institute, State Key Laboratory Incubation Base of Xinjiang Major Diseases Research, Xinjiang Key Laboratory of Echinococcosis, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Department of Biochemistry and Molecular Biology, Preclinical Medicine College, Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81371838; the Natural Science Foundation of Xinjiang Uygur Autonomous Region, No. 2015211C084

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10 μg/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 mRNA表达水平明显升高(P < 0.05)。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。 

 

关键词: 组织构建, 组织工程, 肝星状细胞, 肝纤维化, 原代培养, 分离, 鉴定, 转化生长因子&beta, 1, 胶原蛋白, 平滑肌肌动蛋白&alpha, 国家自然科学基金

Abstract:

BACKGROUND: Hepatic stellate cells are the key cell type in the liver to secrete the extracellular matrix; therefore, it is important in the study of liver fibrosis.
OBJECTIVE: To improve the primary culture method of hepatic stellate cells so as to obtain an adequate amount of cells that are subjected to transforming growth factor β1 stimulation to study the expression of fibrogenic factors.
METHODS: Mice were anesthetized using ketamine to isolate the liver using in situ liver perfusion through puncture via the portal vein. Then, hepatic stellate cells were isolated using the density gradient centrifugation method. Primary hepatic stellate cells were identified by morphological observation and immunofluorescence staining. At 24 hours after culture, hepatic stellate cells were processed with 10 μg/L transforming growth factor 
β1 (experimental group) and PBS (control group) for 48 hours, respectively.
RESULTS AND CONCLUSION: Mouse primary hepatic stellate cells were successfully harvested by using in situ liver perfusion and enzymatic digestion methods. The positive rate of cells for trypan blue staining was (97.2±0.8)%, and the purity of cells for immunofluorescence staining was (90.4±1.2)%. The total cell count was about 2.5×106 per mouse. Real-time quantitative PCR assay showed that compared with the control group, the mRNA expressions of smooth muscle actin α, type I collagen, transforming growth factor β receptor 1 and transforming growth factor β receptor 2 were significantly increased in the experimental group (P < 0.05). These findings indicate that the use of improved culture method has more efficient access to high-quality primary hepatic stellate cells, and transforming growth factor β1 stimulation can lead to the activation of hepatic stellate cells to secrete fibrogenic factors. 

 

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