生长抑素诱导猕猴外周血中性粒细胞凋亡

摘要

目的: 观察生长抑素对猕猴外周血中性粒细胞的作用.

方法: 用生长抑素与猕猴外周血中性粒细胞共同孵育一定时间后, 采用流式细胞仪、DNA电泳、电镜观察猕猴外周血中性粒细胞的形态变化, 细胞DNA含量分布变化.

结果: 生长抑素能诱导猕猴外周血中性粒细胞凋亡, 在形态学上表现为细胞膜起泡、染色质固缩、核碎裂、凋亡小体形成; 电泳呈凋亡特征性Ladder带; 流式细胞仪分析显示, PMN凋亡比例增高(22.1% vs 50.5%. P<0.01).

结论: 生长抑素能诱导猕猴外周血中性粒细胞凋亡.

关键词: 猕猴; 生长抑素; 中性粒细胞; 凋亡; 多器官功能衰竭

Effect of somstostatin on apoptosis of polymorphonuclear neutrophils of rhesus monkey

Lan-Tao Xu, Cheng-Wei Tang

Lan-Tao Xu, Cheng-Wei Tang, Department of Digestology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China

Supported by National Science Foundation of China, No.30170875.

Correspondence to: Cheng-Wei Tang, Department of Digestology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China. sccdlx@sohu.com

Received: December 22, 2004
Revised: January 10, 2005
Accepted: January 20, 2005
Published online: March 1, 2005

Abstract

AIM: To study the effect of somstostatin on the apoptosis of polymorphonuclear neutrophils (PMNs) in rhesus monkey.

METHODS: PMNs were isolated from whole blood of normal rhesus monkey using one-step discontinuous Percoll gradient centrifugation technique. PMNs were cultured with 5×10^-5mol/L somstostatin. Cell morphology was examined by light and electronic microscopy. Apoptosis was detected by flow cytometry and DNA agarose electrophoresis.

RESULTS: Somstostatin at 5×10^-5mol/L induced significant apoptosis in PMNs. The cells manifested typical apoptotic morphology: cell membrane blebbing, chromatin condensation, nucleic fragmentation and apoptotic body formation. Agarose electrophoresis revealed specific DNA ladder. Flow cytometry analysis showed that the ratio of PMNs undergoing apoptosis was remarkably increased from 22.1% to 50.5% following somstostatin treatment (P<0.01).

CONCLUSION: Somstostatin induces apoptosis of PMNs in rhesus monkey. It may be a new target for the treatment of multiple organ dysfunction syndrome (MODS).

Key Words: PMN; Apoptosis; Somatostatin; Rhesus monkey; MODS

0 引言

中性粒细胞(polymorphonuclear neutrophil, PMN)是白细胞中功能最活跃、寿命最短的一类[1-2]. PMN不仅可以释放氧自由基、蛋白酶和炎性递质[3-4], 并且可以与刺激因素相互作用引起瀑布效应(cascade reaction)[5-6] , 诱发多器官功能衰竭(MODS)[7-8].由于PMN生存周期很短, 除了从黏膜表面脱落外, 通常以凋亡和坏死的方式被清除. 当PMN发生坏死时, 炎症反应扩大, 造成周围组织损伤. 如PMN发生凋亡, 则可被巨噬细胞或组织细胞识别并吞噬, 这是生理条件下PMN快速更替的方式. 而在病理条件下, 如炎症、感染、MODS时PMN清除方式变化, PMN凋亡与疾病的发生发展及转归的关系密切[9-17], PMN凋亡的分子机制及调控正是目前科研领域的一个研究热点. 对这些问题的解答将揭示MODS过程中PMN的作用地位, 及提出针对MODS早期组织损伤的防治措施. 生长抑素(somatostatin, ss)是一种脑肠肽, 他不仅广泛存在于中枢神经系统和胃肠道, 在淋巴器官中也有分布. 近3 a来发现, 生长抑素除抑制脑垂体中生长激素的释放外对免疫系统也有广泛的作用, 研究表明生长抑素能减少组胺及肿瘤坏死因子等细胞因子的释放, 从而逆转MODS的发展, 恢复各脏器功能[18-21]. 我们研究生长抑素对中性粒细胞的影响.

1 材料和方法

1.1 材料

Percoll分离液, 右旋糖酐Dextran, Hanks液、RPMI 1640培养液、苏木素(Merk, Germany)、台盼蓝、瑞氏一姬姆萨染液、裂解液、细胞凋亡试剂盒购自博士德公司, Annexin V FITC购自晶美公司. 从健康猕猴外周静脉取血5 mL, 肝素抗凝, 低速离心弃去富含血小板血浆, 余血用右旋糖酐Dextran 500沉降红细胞, 将上层细胞悬液置于Percoll分离液, 以 630 g/L Percoll和720 g/L percoll液密度梯度离心收集PMN, 2 500 r/min, 10 min, 用Hanks液洗涤两次, 2 500 r/min, 10 min, 残留红细胞以冰冷等渗氯化铵溶液(NH₄Cl 155 mmol/L, KHCO3 10 mmol/L, EDTA 0. l mmol/L)按9: l溶解, 再用Hanks液清洗2遍, 悬浮于含100 mL/L的小牛血清的RPMI 1640液中, 并调至5108/L, 分类计数PMN为95%以上, 细胞完整, 台盼蓝试验表明99%为活细胞.

1.2 方法

在培养状态下, 用倒置显微镜观察细胞生长状况及形态学改变, 并将5×10^-5 mol/L的生长抑素作用30 min的细胞在显微镜下观察凋亡细胞的形态. 流式细胞仪检测, 正常情况下分离出PMN, 取两管5×10⁸/L PMN细胞分别与终浓度为5×10^-5 mol/L、5×10^-6 mol/L的生长抑素作用30 min, 另取5×10⁸/L细胞不加药作为对照. 中性粒细胞用PBS洗涤二次; 离心后除去上清液, 加入100 μL 缓冲液; 拿另一只试管加入5 μL Annexin v; 再加入10 μL碘化丙锭; 加100 μL 标本, 在避光处孵育15 min; 加入400 μL PBS; 上机检测. 将5×10⁸/L PMN和5×10^-5 mol/L生长抑素作用细胞离心, 然后抽提DNA并电泳. PMN加入 DNA消化液500 μL, 蛋白酶K 5 μL; 37℃水浴摇床摇动2 h, 取出后过夜; 加等体积苯酚、氯仿、异戊醇(25: 24: 1)震荡混匀, 形成乳浊液, 各抽提一次; 10 000 g离心15 min, 离心后上层为清亮水相, DNA溶解在其中, 若混有下层液体, 则按上述方法再抽提一次; 将上层为清亮水相吸取移至另一新管, 用大口吸管, 以免损伤DNA. 加入1/10 体积 3 mol/L醋酸钠约40 μL, 2.5倍体积冷无水乙醇, 上下倒置混匀, 放入-20℃冰箱30 min; 12 000 g离心20 min, 弃上清; 加入冷乙醇1 mL; 12 000 g离心5 min. 弃上清, 将沉淀溶于40 μL TE液中; 制胶, 用TAE 100 mL溶解2 g琼脂糖, 制成凝胶, 在80V电压条件下, 电泳30 min, 紫外光透射仪下观察电泳条带并拍照. 将5×10⁸/L PMN细胞与5×10^-5mol/L生长抑素作用30 min, PBS清洗2次, 25 g/L戊二醛固定24 h, PBS 缓冲液漂洗3次, 每次15 min, 10 g/L锇酸固定1 h, 漂洗后经梯度丙酮逐级脱水, Epon 812包埋, LKP-2088型超薄切片机切片, 醋酸铀-枸橼酸铝染色后, 透射电镜下观察摄片.

统计学处理 数据以均数±标准差(mean±SD)表示, 应用方差分析处理数据, 比较各实验组与对照组, 确定结果差异的显著性.

2 结果

2.1 细胞形态学变化

未经生长抑素作用的中性粒细胞未见凋亡细胞和凋亡小体, (图1A)而猕猴外周血经生长抑素作用后显微镜观察见中性粒细胞体积变小, 膜起泡, 核浓缩, 凋亡小体形成(图1B).

图1 中性粒细胞. A: 无凋亡图; B: 凋亡.

2.2 流式细胞仪检测(表1)

表1 不同浓度生长抑素对PMN凋亡率的影响.

	组别	凋亡率(%)
1	对照组	22.1±0.28
2	ss (5×10-6 mol/L)	33.6±0.52
3	ss (5×10-5 mol/L)	50.5±0.56

P<0.01 vs对照组.

不同浓度生长抑素对PMN凋亡率的影响结果见表1.

2.3 DNA电泳结果

将5×10⁸/L PMN细胞与5×10^-5 mol/L生长抑素作用30 min, DNA电泳呈明显凋亡带, 对照组则无凋亡带(图2).

图2 DNA电泳Ladder带.

2.4 电镜观察

将5×10⁸/L PMN细胞与5×10^-5 mol/L生长抑素作用30 min, 电镜下可见染色质固缩, 聚集于核膜下成块状, 胞核固缩, 染色质凝集, 细胞质浓缩, 部分细胞碎裂成片, 形成质膜包被的大小不等的凋亡小体(图3).

图3 中性粒细包超微结构TEM×8000. A: 正常; B: 凋亡; C: 凋亡小体.

3 讨论

由于分子生物学发展, 尤其是抗炎症递质治疗在临床应用中并不理想后, 国内外学者转向炎症递质调控凋亡的研究, 就凋亡和MODS发生机制作了新的探讨. Haslett[22-26]提出炎症反应的失调与中性粒细胞凋亡比率有关. Keel[27-30]在MODS研究中发现外周血中中性粒细胞(PMN)凋亡延迟, 并且血中有抗凋亡因子存在. 认为凋亡延迟引起PMN寿命延长可能与MODS发生有关. Goris[31]提出经过特异性细胞因子触发凋亡可以解释进行性过度炎症反应可以发展到MODS的最后假说.

目前已经明确MODS是过度炎症反应的结果, 在创伤后有MODS高风险的患者中, PMN凋亡延迟, 血浆中的递质使PMN凋亡延迟, 并且PMN的作用引发过度炎症反应及器官损伤, 创伤后PMN的凋亡在MODS的发生发展中可能具有重要作用. 凋亡区别于坏死的重要之处是凋亡的细胞能被巨噬细胞或周围组织细胞平稳识别、清除而不引发炎症反应, 因此有研究认为通过凋亡方式清除炎症部位的PMN是限制组织损伤, 促进炎症吸收的重要机制. 识别清除凋亡PMN的能力降低, 大量凋亡的PMN不能被及时有效地处理而裂解坏死, 炎症不易控制, 因此这种损伤很容易导致MODS. 本实验研究表明: 生长抑素能诱导中性粒细胞凋亡, 可以减少氧自由基、蛋白酶和炎性递质的释放, 有利于巨噬细胞的吞噬, 必将为治疗MODS开辟新的领域.

备注

电编: 潘伯荣

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