游離亞油酸對(duì)胰腺腺泡細(xì)胞的影響及其機(jī)制的探討

1、游離亞油酸對(duì)胰腺腺泡細(xì)胞的影響及其機(jī)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(duì)胰腺腺泡細(xì)胞的影響及其可能的機(jī)制。方法 分離胰腺腺泡細(xì)胞，分別與0.1、0.5、1.0 mmol/L亞油酸共同孵育30、60、90 min，測(cè)定細(xì)胞的L

2、DH釋放率，DNA片段梯度分析腺泡細(xì)胞凋亡，流式細(xì)胞儀測(cè)定細(xì)胞凋亡率，Western blot檢測(cè)腺泡細(xì)胞PKC的膜轉(zhuǎn)位，激光共聚焦測(cè)定細(xì)胞內(nèi)鈣的變化。結(jié)果 腺泡細(xì)胞與亞油酸共同孵育后，臺(tái)盼藍(lán)染色顯示存在細(xì)胞損傷，LDH釋放率與亞油酸呈時(shí)間和劑量依賴(lài)關(guān)系。細(xì)胞總DNA凝膠電泳結(jié)果顯示1.0 mmol/L亞油酸可出現(xiàn)DNA梯狀條帶，證明存在細(xì)胞凋亡。同時(shí)，腺泡細(xì)胞凋亡率及PKC的膜轉(zhuǎn)位率，均與亞油酸呈劑量和時(shí)間依賴(lài)關(guān)系。另外，鈣離子是參與細(xì)胞損傷和PKC激活的因子之一，采用激光共聚焦方法檢測(cè)腺泡細(xì)胞內(nèi)鈣濃度，發(fā)現(xiàn)其升高與亞油酸呈濃度依賴(lài)性。結(jié)論 亞油酸對(duì)胰腺腺泡細(xì)胞損傷存在劑量和時(shí)間依賴(lài)關(guān)系。游

3、離亞油酸刺激腺泡細(xì)胞內(nèi)鈣升高和活化PKC誘導(dǎo)細(xì)胞凋亡可能是高脂血癥性胰腺炎的發(fā)病機(jī)制之一。 【關(guān)鍵詞】  胰腺腺泡細(xì)胞 游離亞油酸 細(xì)胞凋亡 蛋白激酶C 細(xì)胞內(nèi)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(jiàn)病因。目前還沒(méi)有可以用來(lái)研究高脂血癥誘導(dǎo)AP理想的動(dòng)物模型， 高脂血癥引起AP的機(jī)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(chǎn)物游離脂肪酸(Free fatty acid, FFA)的毒性。近年來(lái)，已有FFA通過(guò)激活細(xì)胞內(nèi)蛋白激酶C(Protein kinase C,PKC)調(diào)控胰島素的分泌或誘導(dǎo)胰腺內(nèi)分泌細(xì)胞凋亡造成2型糖尿病的報(bào)道1。亞油酸是TG中常見(jiàn)不飽和FFA，因此，探討亞油酸對(duì)胰腺腺泡細(xì)胞損傷的可能機(jī)制，對(duì)AP的診治有著重要的意義。

11、0;   1  材料與方法    1.1  動(dòng)物及主要試劑    雄性SD大鼠(200250 g)購(gòu)自軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心；大豆蛋白酶抑制劑、膠原酶、透明質(zhì)酸酶、亞油酸、PKC抗體等均為美國(guó)Sigma公司產(chǎn)品；Fluo3/AM購(gòu)自美國(guó)BioRad公司。    1.2  方法與步驟    王亞軍，等. 游離亞油酸對(duì)胰腺腺泡細(xì)胞的影響及其機(jī)制的探討      釋放率(%)=上清液LDH細(xì)胞裂解液LDH+上清液LDH×100%    1.3  統(tǒng)計(jì)學(xué)方法    實(shí)驗(yàn)結(jié)果數(shù)據(jù)以±s表示，采用多因素方差分析，在