游离亚油酸对胰腺腺泡细胞的影响及其机制的探讨

1、游离亚油酸对胰腺腺泡细胞的影响及其机制的探讨         08-07-21 11:03:00     编辑：studa20           作者：王亚军 孙家邦 李非 孙海晨 【摘要】  目的 探讨游离亚油酸对胰腺腺泡细胞的影响及其可能的机制。方法 分离胰腺腺泡细胞，分别与0.1、0.5、1.0 mmol/L亚油酸共同孵育30、60、90 min，测定细胞的L

2、DH释放率，DNA片段梯度分析腺泡细胞凋亡，流式细胞仪测定细胞凋亡率，Western blot检测腺泡细胞PKC的膜转位，激光共聚焦测定细胞内钙的变化。结果 腺泡细胞与亚油酸共同孵育后，台盼蓝染色显示存在细胞损伤，LDH释放率与亚油酸呈时间和剂量依赖关系。细胞总DNA凝胶电泳结果显示1.0 mmol/L亚油酸可出现DNA梯状条带，证明存在细胞凋亡。同时，腺泡细胞凋亡率及PKC的膜转位率，均与亚油酸呈剂量和时间依赖关系。另外，钙离子是参与细胞损伤和PKC激活的因子之一，采用激光共聚焦方法检测腺泡细胞内钙浓度，发现其升高与亚油酸呈浓度依赖性。结论 亚油酸对胰腺腺泡细胞损伤存在剂量和时间依赖关系。游

3、离亚油酸刺激腺泡细胞内钙升高和活化PKC诱导细胞凋亡可能是高脂血症性胰腺炎的发病机制之一。 【关键词】  胰腺腺泡细胞 游离亚油酸 细胞凋亡 蛋白激酶C 细胞内钙离子    Exploration on free fatty acid affecting pancreatic acinar cell and its possible mechanism  WANG Yajun, SUN Jiabang, LI Fei, SUN Haichen. Department of General Surgery, Xuanwu Hospital of

4、Capital University of Medical Science, Beijing 100053, China    【Abstract】  Objective  To investigate the effects of free fatty acid on pancreatic acinar cell and explore possible mechanism. Methods  Pancreatic acinar cells from adult rats were incubated with linoleic a

5、cid at concentrations of 0.1 mmol/L, 0.5 mmol/L and 1.0 mmol/L for 30, 60 and 90 minutes respectively to measure the LDH release rate of acinar cells. DNA ladder analysis was used to analyze apoptosis of acinar cells, flow cytometry to determine apoptosis rate, Western blot to detect PKC membrane tr

6、anslocation and laser scanning confocal microscopy (LSCM) to observe intracellular calcium changes. Results  After incubation with linoleic acid, pancreatic acinar cells stained with trypan blue were damaged, with LDH release rate at time and dosedependent fashion with linoleic acid. DNA ladder

7、 was formed by agarose gel electrophoresis when acinar cells were incubated with 1.0 mmol/L linoleic acid, as proved apoptosis of acinar cells. Also, acinar cell apoptosis rate and PKC membrane translocation level were related with linoleic acid at a time and dosedependent fashion. In addition, intr

8、acellular calcium was one of the factors leading to cell injury and PKC activation. LSCM showed that the rising of intracellular calcium existed at a dosedependent fashion with linoleic acid. Conclusions  Linoleic acid damaging acinar cell is dependently related to dose and time. Free fatty aci

9、d stimulating increase of intracellular calcium and activation of PKC inducing apoptosis is one of pathogenetic mechanisms of hyperlipaemic pancreatitis.    【Key words】  Pancreatic acinar cells; Free fatty scid; Cell apoptosis; Protein Kinase C; Intracellular calcium  &

10、#160; 高脂血症是继胆石和酒精之后急性胰腺炎(acute pancreatitis，AP)的又一常见病因。目前还没有可以用来研究高脂血症诱导AP理想的动物模型， 高脂血症引起AP的机制仍不清楚。高脂血症的胰腺毒性主要源于甘油三酯(Triglyceride,TG)分解产物游离脂肪酸(Free fatty acid, FFA)的毒性。近年来，已有FFA通过激活细胞内蛋白激酶C(Protein kinase C,PKC)调控胰岛素的分泌或诱导胰腺内分泌细胞凋亡造成2型糖尿病的报道1。亚油酸是TG中常见不饱和FFA，因此，探讨亚油酸对胰腺腺泡细胞损伤的可能机制，对AP的诊治有着重要的意义。

11、0;   1  材料与方法    1.1  动物及主要试剂    雄性SD大鼠(200250 g)购自军事医学科学院实验动物中心；大豆蛋白酶抑制剂、胶原酶、透明质酸酶、亚油酸、PKC抗体等均为美国Sigma公司产品；Fluo3/AM购自美国BioRad公司。    1.2  方法与步骤    王亚军，等. 游离亚油酸对胰腺腺泡细胞的影响及其机制的探讨      释放率(%)=上清液LDH细胞裂解液LDH+上清液LDH×100%    1.3  统计学方法    实验结果数据以±s表示，采用多因素方差分析，在