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[Ca~(2+)]_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A_2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[Ca~(2+)]_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[Ca~(2+)]_i缓慢、微弱的升高，而再次恢复培养液中Ca~(2+)至生理浓度，LPS可以引发一个快速、幅度更高而持久的[Ca~(2+)]_i变化；在细胞外无钙的环境中SDOC未见引起胞浆[Ca~(2+)]_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[Ca~(2+)]_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。
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 and
remarkable 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
引文
1． 张红，李永渝．急性胰腺炎发病机制研究进展．中国危重病急救医学杂志 2000：12：121-124．
2． Trump BF, Berezesky IK. Calcium-mediated cell injury and cell death. FASEB J 1995; 87: 2466-2470．
3． Hanne KS, Rainer S, Michael G, Hans-ulrich S, Dieter H, Claus N. Effect of oxidative stress on cellular functions and cytosolic free calcium of rat pancreatic acinar cells. Am J Physiol 1997; 272: G1489-1498．
4． Lake-bakaar G, Lyubsky S. Dose-dependent effect of continous subcutaneous verapamil infusion on experimental acute pancreatitis in mice. Dig Dis Sci 1995; 40: 2349-2354．
5． Hughes CB, Eldin AB, Kotb M. Calcium channel blockade inhibits release of TNF alpha and improves survival in rat model of pancreatitis. Pancreas 1996; 13:22-29．
6. Ratter D W, Napolitano L M, Corsetti J. Hypocalcemia in experimental pancreatitis occurs independently of change in serum nonesterified fatty acid level, Int J Pancretol 1990;6:249-262.
7. Zhou W, Shen F. Evidence for altered cellular calcium in the pathogenetic mechanism of acute pancreatitis in rats. J Surg Res, 1996;60:147-155.
8．李冬冬，贾玉杰，王冬梅．茵陈蒿汤及其组分对急性出血坏死他胰腺炎防治作用的正交设计研究．中国中西医结合脾胃杂志1996；4：163-165．
9. Raetz CRH. Bacterial endotoxins, extraordinary lipid that activate eucaryotic signal transduction. J Bacteriol 1993; 175: 5745-5753.
10. Wig JD, Kochhar R, Ray JD, Krishna RDV, Gupta NM, Ganguly NK. Endotoxaemia predicts outcome in acute pancreatitis. J Clin Gastroenterol 1998; 26: 121-124.
11. Exley AR, Leese T, Holliday MP, Swann RA, Cohen J. Endotoxaemia and serum tumour necrosis factor as prognostic markers in severe acute pancreatitis. Gut 1992; 33:1126-1128.
12. Laine VJO, Nyman KM, Peuravouri HJ, Henriksen K, Parvinen M, Nevalainen TJ. Lipopolysaccharide incuced apoptosis of rat pancreatic acinar cells. Gut 1996; 38:747-759
13. Kitagawa M, Williams JA, Lisle RC. Amylase release from streptolysin Opermeabilized pancreatic acini. Am J Physiol 1990; 259: G157-164.
14. Wang LZ, Zhang QZ, Hu XZ. Verapamil, cyproheptadine, and anisodamine antagonized [Ca~(2+)]_i elevation induced by TNT α in a single endothelial cell. Acta Pharmacol Sin 2001; 22:918-922.
15．陈思锋，吴中立．体液和组织匀浆磷脂酶A_2的简便快速测定法．第二军医大学学报1989；10：254-256
16． Gukovsky I, Gukovskya AS, Blinman TA, Zaninvic V, Pandol SJ. Early NF-κ B activation is associated with hormonr-induced pancreatitis. Am J Physiol 1998; 275:G1402-1414．
17． Luo WB, WangYP. Magnesium lithospermate B inhibits hypoxia-induced calcium influx and nitric oxide release in endothelia cells. Acta Pharmacol Sin 2001; 22: 1135-1142．
18． Li HW, Han QD. Mechanism of calcium influx from extracellular fluid. Trends Physiol 1996; 27: 307-310．
19． Wojcikiewicz R J, Ernst S A, Yule D I. Secretagogues cause ubiquitination and down-regulation of inositol 1,4,5-trisphosphate receptors in rat pancreatic acinar cells. Gastroenterology 1999;116:1194-1201
20． Li YY, Li XL, Yang CX, Zhang H, Yao H, Zhu L. Effects of Tetrandrine and QYT on ICAM-1 and SOD gene expression in pancreas and liver of rats with acute pancreatitis. World J Gastroenterol 2003;9:154-159
21． 范学良，李永渝，葛树培．大鼠实验性急性胰腺炎时观察血浆和胰腺LPO 的变化．中国病理生理学杂志 1991：7：430-432
22． 张红，李永渝．汉防己甲素对急性胰腺炎的保护作用．遵义医学院学报 2000：23：91-95
23． Luthen R, Niederauc C, Kotb M. Intrapancreatic zymogene activation and level of ATP and glutathione during caerulein pancreatitis in rats. Am Jphysiol 1995,268:G592-604
24． Aufenanger J, Samman M, Quintel M, Fassbender K, Zimmer W, Bertsch T. Pancreatic phospholipase A2 activity in acute pancreatitis: a prognostic marker for early identification of patients at risk.Clin Chem Lab Med 2002;40:293-297
25． Weber H, Roesner JP, Nebe B. Increased cytosolic Ca2+ amplifies oxygen radical induced alterations of the ultrastructure and the energy metabolism of isolated rat pancreatitis. Digestion 1998;59:175-185
26． Vaccaro MI, Calvo EL, Suburo AM, Sordelli DO, Lanosa G, lovanna JL. Lipopo-lysaccharide directly affects pancreatic acinar cells. Dig Dis Sci 2000; 45: 915-926
27． Bhatia M, Brady M, Kang YK, Costello E, Newton DJ, Christmas SE, Neoptolemos JP, Slavin J. MCP-1 but not CINC synthesis is increased in rat pancreatic acini in response to cerulein hyperstimulation. Am J Physiol Gastrointest Liver Physiol 2002;282:G77-85
28． Pu QF, Yan LN, Liu XB, Shen J, Tan JS, Zuo FQ. Disturbance of calcium homeostasis and PG during the development of acute pancreatitis from edema to necrosis. Chin J Exp Surg 1998; 15: 391-392．