【作者】 张红；

【导师】 吴咸中； 李永渝；

【作者基本信息】 天津医科大学 ， 中西医结合临床， 2003， 博士

【摘要】 目的：观察脂多糖（lipopolysaccharide，LPS）和去氧胆酸钠（Sodium Deoxycholate，SDOC）是否对大鼠胰腺腺泡细胞有直接损伤作用，及这两种刺激剂作用的不同时段胰腺腺泡细胞自由基、胰酶生成释放量的变化，核因子κB（Nuclear Factor-κB，NF-κB）活化的情况以及单个胰腺腺泡细胞内[Ca²⁺]_i的变化。探究它们之间的先后次序和相互关系，以探讨脂多糖和胆盐是否可以引起胰腺腺泡细胞钙超载；钙超载是否乃AP病理过程中的早期事件，通过影响过氧化反应、胰酶及NF-κB的活化，从而在AP的发生、发展中居关键环节。另外，采用中药非特异性钙通道拮抗剂（calcium channel blocker，CCB）粉防己碱（Tetrandrine，Tet），观察其是否可以减轻LPS和胆盐所致的胰腺腺泡细胞损伤，Tet是否可以通过抑制钙超载而发挥对胰腺腺泡细胞的保护作用，探讨其保护胰腺的作用机制，为这类药物应用于临床治疗AP提供实验依据。方法：胶原酶法分离大鼠胰腺腺泡细胞，经不同浓度Tet、LPS、SDOC或正常培养液处理，在不同的时间点（30min，1h，4h，10h）采集上清液，检测其中丙二醛含量、超氧化物歧化酶、磷脂酶A₂的活性；采用MTT法检测胰腺腺泡细胞的活性；另外收集胰腺腺泡细胞、采用免疫荧光染色检测NF-κB亚单位p65核易位；提取核蛋白进行凝胶电泳迁移率检测NF-κB的DNA结合活性；部分胰腺腺泡细胞经Fluo-3／AM负载后，于相应的时间点采用灌流方式给予药物或刺激剂，激光共聚焦显微镜观察单个胰腺腺泡细胞[Ca²⁺]_i的变化。结果：（1）LPS和SDOC引起胰腺腺泡细胞的存活率降低、且呈时间及浓度依赖性（P＜0.05），1mmol／L依他酸（egtazic acid，EGTA）使LPS和SDOC诱发的腺泡细胞损伤程度明显减轻（P＜0.05）；在无钙且含EGTA的培养液中LPS引起胰腺腺泡细胞[Ca²⁺]_i缓慢、微弱的升高，而再次恢复培养液中Ca²⁺至生理浓度，LPS可以引发一个快速、幅度更高而持久的[Ca²⁺]_i变化；在细胞外无钙的环境中SDOC未见引起胞浆[Ca²⁺]_i的变化，再次恢复培养液中的钙离子浓度至生理水平，SDOC引发胞浆[Ca²⁺]_i迅速升高；LPS和SDOC致胰腺腺泡细胞上清液中磷天津医科大学博士研究生学位论文脂酶AZ的活性及丙二醛含量增加（尸<0 .05），超氧化物歧化酶活性明显降低（尸<0.05）。腺泡细胞内〔Ca灼‘的变化明显先于细胞培养上清液中的生化改变和腺泡细胞的损伤;（2）1009/L LPS及50umol/L SIX)C作用后30 oin，NF--‘B p65的胞浆内免疫荧光染色增强，并可见核易位，胞浆荧光染色强度及胞核阳染率于卜PS和SDOC作用后lh达高峰、4h仍维持较高水平;NF一KB的ONA结合活性变化趋势与免疫荧光表现相似;EGTA可以抑制LPs和SooC诱发的胰腺腺泡细胞内N卜KB活化。（3）Tet可减轻LPS和SDOC所致的细胞损伤（尸<0.05）;抑制LPS及SDOC诱发的胰腺腺泡细胞内NF一KB活化和细胞[Caz+〕‘升高（尸<（），05）:降低细胞培养上清液中丙二醛含量和磷脂酶A:的活性、增加超氧化物歧化酶的活性（尸<0 .05）。结论（1）:LPS和SooC可致胰腺腺泡细胞损伤，且该损伤对刺激时间、刺激剂浓度及胞浆钙离子具有依赖性;LPS和SD（）C均可导致胰腺腺泡细胞胞浆内钙超载，但C扩的来源方式不同，LPs既可引起细胞内贮CaZ’的释放，又可诱发胞外CaZ‘内流;而SDOC则仅通过胞外Ca2+的内流，引起胞浆钙超载。钙超载作为早期的病理事件通过过氧化反应和胰酶的过度活化参与腺泡细胞损伤的发生，钙稳态失衡是LPS和SD侃致胰腺细胞损伤的重要因素之一。（2）:NF一KB活化是LPS及SDOC诱发胰腺腺泡细胞损伤的一个重要的早期事件;C扩可能作为一个重要信使介导了胰腺腺泡细胞NF--K!、的活化。（3）: Tet对LPS和SDOC诱发的胰腺腺泡细胞损伤具有一定的保扩，作用，其保护作用的可能机制为:①通过钙拮抗作用，抑制胰腺腺泡细胞钙超载、FI1断其所引发的一系列连锁反应;②降低NF一KB活化，可能减少其下游炎症介质表达、减轻炎症反应;③增强胰腺腺泡细胞的抗氧化能力，减轻过氧化损伤;④降低磷脂酶A:活性，减少胰酶活化对胰腺腺泡细胞的损伤。更多还原

【Abstract】 AIM: To investigate the damage of pancreatic acinar cell induced by lipopolysaccharide (LPS) and sodium deoxycholate(SDOC), and the alteration of the content of maloidialdehyde (MDA), the activity of superoxide dismutase (SOD) and phospholipase (PLA2), the concentration of [Ca2+]i and the activity of NF- K B in pancreatic acinar cell, further to explore the possible mechanism of pancreatic acinar cell damage induced by LPS and SDOC. Moreover, to investigate the protective effect of tetrandrine (Tet) on LPS- or SDOC-induced pancreatic acinar cell damage and its mechanism. METHODS: Rat pancreatic acinar cells were isolated by collagenase digestion and were treated with varying concentration of LPS (from 1 mg/L to 20 mg/L), SDOC (from 10 umol/L to 100 umol/L), Tet (50 umol/L and 100 umol/L), or culture media , respectively. At different time points (30 min,1 h, 2 h, 4 h, 10 h) , cell viability was determined by MTT and the supernatant of cells was collected to measure the content of MDA, the activity of SOD and PLA2. Some cells were loaded with Fluo-3/AM, then exposed to varying doses of LPS and SDOC in the absence or presence of Ca2+ in extracellular fluid. The dynamic change of [Ca2+]i in single pancreatic acinar cell was determined by laser scanning confocal microscopy. The nuclear translocation of the subunit p6S of NF- K B was visualized by immunofluorescence staining and nuclei protein was extracted to perform electrophoretic mobility shift assay (EMSA) which was used to assay the activity of NF- K. B binding to the DNA sequence containing recognition site of NF- K B. RESULTS: (D LPS and SDOC initiated cell damage in a time- and concentration-dependent manner( P<0.05), and 1 mmol/L egtazic acid (EGTA) could decrease the cell mortality( P<0.05); LPS induced a slight rise of [Ca2+]i in the calcium-free extracellular fluid containing 1 mmol/L EGTA. Addition of extracellular calcium in the presence of LPS resulted in a more immediate andremarkable rise of [Ca2+]j, which reached the peak value within 150 s and maintained the value for 100 s. SDOC did not induce a rise of [Ca2+]i in the calcium-free extracellular fluid, but caused a rapid and remarkable rise of [Ca2+]j when adding calcium into the media to recover a normal concentration of [Ca2+]e. The increase in intracellular [Ca2+]; preceded the pathological and biological alteration of pancreatic acinar cells. In the supernatant of LPS and SDOC groups, the content of MDA and the activity of PLA2 increased ( /*<0.05) and the activity of SOD decreased (P<0.05). (2) NF- K B p65 immunofluorescence staining showed that the intensity in cytoplasma increased and nuclear translocation occurred at 30 min and its zenith was at 1 h after LPS (10 mg/L) or SDOC (50 umol/L) treatment and the intensity of immunofluorescence staining still kept a higher level at 4h . NF- K B DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF- K B activation preceded the pathological alteration of pancreatic acinar cells. A Ca2* chelator EGTA inhibited the LPS- or SDOC- induced NF- K B activation. (3) Tet attenuated LPS- or SDOC-induced cell damage (50umol/L, P<0.05; 100umol/L, P<0.01) and inhibited the elevation of cytoplasmic free calcium of rat pancreatic acinar cells. In the supernatant, let decreased the content of MDA and the activity of PLAa (P<0.05) and increased the activity of SOD (P<0.05). NF- K B p65 immunofluorescence staining in the cytoplasma or the nuclear, and NF- K B DNA binding activity were lessened by Tet. CONCLUSION: (1) LPS and SDOC initiated cell damage in a time-, concentration-and calcium-dependent manner. LPS and SDOC induced the calcium overload in the cytoplasm of pancreatic acinar cell in a different manner. LPS not only induced the discharge of Ca2+ from the inteacellular calcium store but also the influx of Ca2+ from the extracellular fluid. Whereas, SDOC only induced the influx of Ca2+ from the extracellular fluid. Calcium overload as an early pathogenetic event took part in the damage of pancreatic acinar cells by更多还原