1、游離亞油酸對胰腺腺泡細胞的影響及其機制的探討         08-07-21 11:03:00     編輯：studa20           作者：王亞軍 孫家邦 李非 孫海晨 【摘要】  目的 探討游離亞油酸對胰腺腺泡細胞的影響及其可能的機制。方法 分離胰腺腺泡細胞，分別與0.1、0.5、1.0 mmol/L亞油酸共同孵育30、60、90 min，測定細胞的L

2、DH釋放率，DNA片段梯度分析腺泡細胞凋亡，流式細胞儀測定細胞凋亡率，Western blot檢測腺泡細胞PKC的膜轉位，激光共聚焦測定細胞內鈣的變化。結果 腺泡細胞與亞油酸共同孵育后，臺盼藍染色顯示存在細胞損傷，LDH釋放率與亞油酸呈時間和劑量依賴關系。細胞總DNA凝膠電泳結果顯示1.0 mmol/L亞油酸可出現DNA梯狀條帶，證明存在細胞凋亡。同時，腺泡細胞凋亡率及PKC的膜轉位率，均與亞油酸呈劑量和時間依賴關系。另外，鈣離子是參與細胞損傷和PKC激活的因子之一，采用激光共聚焦方法檢測腺泡細胞內鈣濃度，發現其升高與亞油酸呈濃度依賴性。結論 亞油酸對胰腺腺泡細胞損傷存在劑量和時間依賴關系。游

3、離亞油酸刺激腺泡細胞內鈣升高和活化PKC誘導細胞凋亡可能是高脂血癥性胰腺炎的發病機制之一。 【關鍵詞】  胰腺腺泡細胞 游離亞油酸 細胞凋亡 蛋白激酶C 細胞內鈣離子    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; 高脂血癥是繼膽石和酒精之后急性胰腺炎(acute pancreatitis，AP)的又一常見病因。目前還沒有可以用來研究高脂血癥誘導AP理想的動物模型， 高脂血癥引起AP的機制仍不清楚。高脂血癥的胰腺毒性主要源于甘油三酯(Triglyceride,TG)分解產物游離脂肪酸(Free fatty acid, FFA)的毒性。近年來，已有FFA通過激活細胞內蛋白激酶C(Protein kinase C,PKC)調控胰島素的分泌或誘導胰腺內分泌細胞凋亡造成2型糖尿病的報道1。亞油酸是TG中常見不飽和FFA，因此，探討亞油酸對胰腺腺泡細胞損傷的可能機制，對AP的診治有著重要的意義。

11、0;   1  材料與方法    1.1  動物及主要試劑    雄性SD大鼠(200250 g)購自軍事醫學科學院實驗動物中心；大豆蛋白酶抑制劑、膠原酶、透明質酸酶、亞油酸、PKC抗體等均為美國Sigma公司產品；Fluo3/AM購自美國BioRad公司。    1.2  方法與步驟    王亞軍，等. 游離亞油酸對胰腺腺泡細胞的影響及其機制的探討      釋放率(%)=上清液LDH細胞裂解液LDH+上清液LDH×100%    1.3  統計學方法    實驗結果數據以±s表示，采用多因素方差分析，在