组蛋白是细胞核染色质中核小体的主要组份，在细胞死亡或形成胞外陷阱的过程中被释放到胞外，进入血液循环。检测循环血组蛋白含量，发现各种重症病人循环血组蛋白水平显著升高，指征不良预后。文献报道胞外组蛋白参与调控多种细胞功能。重症急性胰腺炎是临床常见急腹症，发病时循环血组蛋白显著升高。尽管如此，胞外组蛋白是否调控胰腺腺泡细胞的信号转导，没有系统文献报道。故而本文系统研究了组蛋白在新鲜分离正常大鼠胰腺腺泡，及培养大鼠胰腺腺泡肿瘤细胞AR4-2J，对腺泡细胞钙信号的调控作用。 本文试验所用组蛋白浓度范围，参见文献报道重症病人循环血组蛋白浓度。试验发现，组蛋白对新鲜分离正常大鼠胰腺腺泡细胞基础钙离子浓度没有影响，但混合组蛋白Hx浓度(50，150，200 mg·L-1，30 min)、时间(200 mg·L-1，3，10，30 min)依赖性抑制胆囊收缩素(cholecystokinin CCK 20 pM)和乙酰胆碱(acetylcholine ACh 30 nM)所引发钙振荡。与正常大鼠胰腺腺泡不同，混合组蛋白Hx浓度(2 - 200 mg·L-1)依赖性刺激培养大鼠胰腺腺泡肿瘤细胞AR4-2J引发规则胞浆钙振荡，其浓度效应曲线显示为半梯形 - Hx 50 mg·L-1产生最大钙反应。提纯的单一重组组蛋白H1, 2A, 2B, 3, 4均可浓度(2 - 20 mg·L-1) 依赖性刺激AR4-2J细胞引发胞浆钙振荡，有效性为H3 / H4 > H1 / Hx > H2A / H2B。由于组蛋白刺激免疫细胞主要激活Toll-like receptors (TLR) 2, 4, 9,因此检测了大鼠胰腺腺泡中TLR2, 4, 9的表达和亚细胞分布。RT-PCR实验mRNA检测和免疫印迹实验蛋白质水平检测证实TLR2, 4, 9 在正常大鼠胰腺腺泡和培养AR4-2J均显著表达。细胞免疫化学发现TLR2和TLR4在正常胰腺腺泡细胞顶端质膜表达，TLR9在酶原颗粒区域有低水平表达。AR4-2J细胞团块，TLR2在相邻细胞旁侧面质膜显著表达，TLR4细胞核表达，TLR9在细胞团块外周基底质膜表达。TLR受体药理学试验发现，TLR2受体激动剂酵母多糖和TLR4受体激动剂脂多糖，对AR4-2J细胞基础钙离子浓度没有影响，但TLR9受体激动剂ODN1826刺激AR4-2J细胞引发规则胞浆钙振荡。TLR9受体拮抗剂ODN2088可完全抑制组蛋白H4刺激AR4-2J细胞所引发的钙振荡。说明组蛋白激活AR4-2J细胞的TLR9触发胞浆钙振荡。进一步试验发现，糖皮质激素地塞米松(dexamethasone Dex) 处理过的AR4-2J细胞，胞外组蛋白不再有刺激作用。同时糖皮质激素Dex 处理后，TLR9从AR4-2J细胞团块外周基底部质膜，转移到细胞内部，胞外组蛋白与TLR9脱离接触。TLR9表达的siRNA干扰试验发现，在TLR9特异性siRNA处理后的AR4-2J，组蛋白刺激所引发钙振荡被显著抑制，对照试验没有显著变化。以上相互独立的几组证据，说明胞外组蛋白通过刺激TLR9引发AR4-2J细胞胞浆钙振荡。 关于胞外组蛋白激活AR4-2J细胞基底部质膜TLR9受体，引发胞浆钙振荡的分子机制，主要结果如下： 1 胞外组蛋白刺激AR4-2J所引发钙振荡依赖于外钙内流。多模态抑制剂2-APB、ORAI抑制剂GSK-7975A均显著抑制或阻断H4 (5 mg·L-1)所引发AR4-2J细胞钙振荡。磷脂水解酶PLC抑制剂、IP3R抑制剂没有作用。 2 小牛胸腺DNA浓度依赖性抑制组蛋白所引发胞浆钙振荡。 3 组蛋白增强CCK刺激或光动力作用对CCK1受体的激活。 4 组蛋白浓度依赖性刺激AR4-2J细胞活性氧的产生。Hx 2 mg·L-1 刺激AR4-2J不引发胞浆钙振荡，不诱导AR4-2J细胞的凋亡和坏死。Hx ≥ 5 mg·L-1 刺激AR4-2J细胞引发钙振荡，也诱导细胞凋亡。 综上所述，胞外组蛋白在大鼠胰腺腺泡细胞具有双重调控作用：在新鲜分离的正常大鼠胰腺腺泡细胞，湮灭CCK及ACh刺激所引发胞浆钙振荡；在培养大鼠胰腺腺泡肿瘤细胞AR4-2J，激活细胞基底部质膜TLR9受体，触发胞浆钙振荡。胞外组蛋白刺激AR4-2J细胞所产生胞浆钙振荡依赖于ORAI所介导的钙内流，也可被胞外DNA所阻断。

The nuclear protein histones are major components of the nucleosome, but can escape from the nucleus and be released in the extracellular fluid when epithelial cells die or when neutrophil extracellular traps are formed. The released histones can enter the general circulation to be delivered to distant organs. The circulating histones have been reported to be transiently enhanced in patients with severe trauma, sepsis or other severe diseases. In vitro works have found that extracellular histones can modulate cellular activities, especially in vascular endothelial cells. One particular case of inflammatory diseases we have been working on in the laboratory is acute pancreatitis. It has been reported that the circulating histones increase up to 33.8 mg·L-1 in patients with severe acute pancreatitis. Although extracellular histones may be rather high in acute pancreatitis, whether histones would have any direct effect on the pancreatic acinar cell has not been investigated in detail. Therefore in the present work I examined histone modulation of calcium signaling in rat pancreatic acinar cells. Both freshly isolated rat pancreatic acini and cultured rat pancreatic acinar tumor cell AR4-2J were used. Mixed histones were found to have no effect on basal calcium in isolated rat pancreatic acini, but markedly blocked concentration- (50, 150, 200 mg·L-1, 30 min) and time-dependently (200 mg·L-1, 3, 10, 30 min) calcium oscillations induced by cholecystokinin (CCK 20 pM) or acetylcholine (ACh 30 nM). In contrast, mixed histones dose (2-200 mg·L-1)-dependently induced calcium oscillations in AR4-2J cells, with maximal effect at ≥ 50 mg·L-1. Individual recombinant histones (H1, 2A, 2B, 3, 4) induced concentration (2-20 mg·L-1)-dependently calcium oscillations in AR4-2J, with varied efficacies of H3 / H4 > H1 / Hx > H2A / H2B. Since in immune cells histones have been reported to mainly target the Toll-like receptors TLR2, 4, 9, I investigated TLR2, 4, 9 expression in rat pancreatic acinar cells. RT-PCR and Western blot experiments confirmed TLR2, 4, 9 expression in both isolated rat pancreatic acini and in cultured AR4-2J cells. Immunocytochemistry revealed TLR2 > TLR4 expression on apical plasma membrane, and low TLR9 expression in the zymogen granule regions in isolated rat pancreatic acini. TLR2 was found on lateral / neighboring, TLR9 on basal / peripheral plasma membranes in AR4-2J cell clusters, but TLR4 was exclusively found in the nucleus. TLR2, 4, 9 receptor pharmacology revealed that neither TLR2 agonist Zymosan nor TLR4 agonist lipopolysaccharide had any effect on basal calcium concentration, but TLR9 agonist ODN1826 induced robust calcium oscillations in AR4-2J. In addition, TLR9 antagonist ODN2088 blocked H4-induced calcium oscillations. After treatment with glucocorticoid dexamethasone (Dex, 10 nM) for 3 - 7 days, H4 (5 mg·L-1) no longer induced calcium oscillations in AR4-2J cells. Significantly, the basal / peripheral plasma membrane TLR9 after AR4-2J differentiation with Dex was found to have transferred to the cell interiors. Therefore after Dex treatment, the spatial separation of intracellular TLR9 from extracellular H4 led to the phenomenon that H4 no longer induced any calcium oscillations. When TLR9 expression in AR4-2J was down regulated with TLR9-specific siRNA, H4 also no longer induced calcium oscillations. The above independent lines of evidence firmly establish that histones activate basal plasma membrane TLR9 in AR4-2J cells to trigger calcium oscillations. Further experiments revealed the molecular details of TLR9 activation-triggered calcium oscillations in AR4-2J cells. 1/ Histone-induced calcium oscillations in AR4-2J were dependent on calcium influx, not involving Cav. Both the multimodal calcium entry inhibitor 2-APB and ORAI inhibitor GSK-7975A blocked completely calcium oscillations induced by H4 (5 mg·L-1). Phospholipase C / IP3R inhibitors were without any effect. 2/ Calf thymus DNA blocked histone-induced calcium oscillations in AR4-2J. 3/ Histone pre-treatment of AR4-2J enhanced CCK stimulation and photodynamic activation of CCK1 receptors. 4/ Extracellular histones stimulated the generation of reactive oxygen species. Hx 2 mg·L-1 failed to induce calcium oscillation and apoptosis / necrosis in AR4-2J. Hx ≥ 5 mg·L-1 induced both calcium oscillations and apoptosis. In conclusion, extracellular histones have dual and contrasting effects on calcium signaling in rat pancreatic acinar cells: histones quenched calcium oscillations induced by CCK and ACh in the freshly isolated rat pancreatic acini, but triggered calcium oscillations in cultured rat pancreatic acinar tumor cell AR4-2J. Extracellular histones activate basal plasma membrane TLR9 to trigger calcium oscillations in AR4-2J cells, such calcium oscillations were supported by ORAI-mediated calcium influx but inhibited by extracellular DNA.