NF-κB/p65基因在TNF-α诱导肺泡上皮细胞凋亡中的作用及可能机制
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摘要
研究背景和目的
重症社区获得性肺炎(severe community-acquired pneumonia, SCAP)是呼吸内科重症监护病房最常见的危重病之一,常导致全身炎症反应综合征(systemic inflammatory response syndrome, SIRS),病情进展快,处理不及时,可发展为多器官功能障碍综合征(multiple system organ dysfunction, MODS)。急性肺损伤(acute lung injury, ALI)是SCAP最常见而严重的病理生理过程,是肺部炎症致肺泡上皮细胞受损和肺微血管通透性增加而导致的急性、进行性缺氧性呼吸衰竭,其最终阶段即为急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)。传统认为,肺泡上皮细胞是肺部炎症反应的靶细胞,具有调节炎症发展及预后的能力。在细菌所致肺部急性炎症反应中,革兰阴性菌细胞壁的糖脂成分脂多糖(LPS)发挥了关键作用,LPS可以直接募集血液中性粒细胞进入肺泡进而促进细胞因子,如肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)和白细胞介素-1β(interleukin-1β, IL-1β)等的释放,通过调控炎症通路的活化以及诱导其它细胞因子和粘附分子的产生,进而造成更严重的肺部损伤。
细胞凋亡是细胞在一定生理或病理条件下,遵循自身程序,自己结束生命的过程,对于机体稳定和生长发育具有重要的意义。大量研究显示,肺泡Ⅱ型上皮细胞(alveolar typeⅡepithelial cell, AT-Ⅱ)的凋亡在肺部重症感染所致ALI中起了至关重要的作用,AT-Ⅱ细胞的过度凋亡将导致结构细胞数量减少和肺组织病理变化加重；另外在ALI病理变化过程中需将受损的AT-Ⅱ细胞清除,此过程可能引起炎性反应而加重已存在肺组织的损伤,因此肺泡上皮细胞凋亡在调节炎症对肺损伤的反应方面发挥着重要的作用。细胞凋亡是有一序列酶参与、由基因控制的一个主动的、高度有序的过程,其中Bcl-2家族成员在细胞凋亡的基因调控过程中起着十分重要的作用,根据作用不同分为两类：①抑制性亚族,如Bcl-2、Bcl-X、Mcl-1,可在多个环节上阻断细胞凋亡；②促进性亚族,如Bax、Bak、Bad、Bik、Bid等,与抑制性亚族结合而封闭其活性域。
核因子-KB(nuclear factor-κB, NF-κB)是由Sen等从B淋巴细胞核提取物中发现的一种能与免疫球蛋白κ链基因增强子κB序列特异结合的核蛋白因子,因此命名为NF-κB。目前所知NF-κB家族成员包括RelA(p65)、RelB、c-Rel、p50和p52五个亚单位。通常NF-κB以p65／p50二聚体形式存在胞浆中,其中p65含有转录活化区域,在静息状态下,胞浆中的NF-κB与抑制性蛋白IκB-α结合,不能发挥转录调节作用,当细胞受到如TNF-α等细胞外信号刺激时,IκB-α被磷酸化并从NF-κB二聚体上解离下来,p65／p50二聚体立即转移入核,与其靶基因启动子／增强子上的KB位点结合,启动相关基因的转录。NF-κB信号通路参与了感染、炎症反应和肿瘤等病理过程以及细胞周期调控等,近年来研究表明,NF-κB家族及其介导的细胞信号转导在细胞凋亡中起到了重要作用,它参与了多种凋亡相关基因的转录调控,具有抑制细胞凋亡和促进细胞凋亡的双向作用。
NF-κB与细胞凋亡的关系最早在肿瘤生长等方面研究,以往研究显示NF-κB可诱导细胞表达TNFR1结合因子(TFAF)和细胞凋亡蛋白抑制物,抑制caspase的级联反应,从而阻断死亡受体TNFR/Fas介导的凋亡信号转导,而抑制细胞凋亡,所以NF-κB在肿瘤中的主要作用表现为抗凋亡作用。但在SCAP中NF-κB与细胞凋亡的关系却复杂得多,是抑制凋亡还是促进凋亡?目前还有争论。一方面,在多形核中性粒细胞(PMNs)等炎症细胞,NF-κB活化使抗凋亡蛋白合成增加,而使其凋亡延迟,Kupfner J G等在内毒素引起的ARDS中发现NF-κB活化继而引起A1、A20及Bcl-X的转录增加,并引起凋亡抑制,在应用NF-κB抑制剂后PMNs的凋亡加快；另一方面,对于肺泡上皮细胞和内皮细胞等肺结构细胞,有报道认为,NF-κB调控失常使促凋亡蛋白如IL-8、TNF、FasL等的转录表达增强,引起细胞凋亡的加强,但其凋亡的调控机制还不是很清楚。
鉴此,本研究拟采用重组的人细胞因子TNF-α刺激AT-Ⅱ细胞A549,探讨炎症环境下肺泡上皮细胞凋亡的基因调控机制。首先,在TNF-α刺激的不同时间点检测细胞凋亡率的变化及凋亡基因Bcl-2、Bax的表达情况,了解TNF-α对肺泡上皮细胞凋亡的影响；其次,利用Bax小干扰RNA(small interfering RNA, siRNA)沉默凋亡基因Bax的表达,明确Bax基因与TNF-α诱导肺泡上皮细胞凋亡的关系；最后,再利用NF-κB/p65 siRNA沉默NF-κB家族亚单位p65基因的表达,初步探讨NF-κB/p65基因在TNF-α诱导肺泡上皮细胞凋亡中的作用及可能机制。
方法
1、培养AT-Ⅱ细胞(来源于人肺泡上皮细胞株A549),在细胞密度为80%～90%时采用低浓度(10ng/ml)的TNF-α刺激A549细胞,分别在刺激细胞0h、6h、12h、24和48h五个时间点收集细胞,流式细胞仪(Annexin V-FITC法)检测细胞凋亡率,RT-PCR检测Bcl-2和Bax mRNA, Western blotting和免疫组化检测Bcl-2和Bax蛋白表达；
2、利用脂质体转染试剂LipofectamineTM2000将Bax siRNA转染入A549细胞,6h后给予TNF-α(10ng/ml)刺激24h收集细胞,实验分4组：空白对照组、TNF-α组、TNF-α+Bax siRNA组(干扰组)、TNF-α+scramble siRNA组(阴性对照组)。RT-PCR检测Bax mRNA, Western blotting和免疫组化检测Bax蛋白表达,流式细胞仪(Annexin V-FITC法)检测细胞凋亡率；
3、利用脂质体转染试剂LipofectamineTM2000将NF-κB/p65 siRNA转染入A549细胞,6h后给予TNF-α(10ng/ml)刺激24h收集细胞,实验分4组：空白对照组、TNF-α组、TNF-α+NF-κB/p65 siRNA组(干扰组)、TNF-α+scramble siRNA组(阴性对照组)。RT-PCR检测NF-κB/p65、Bcl-2、Bax mRNA,免疫组化检测NF-κB/p65的活化,Western blotting检测Bcl-2、Bax蛋白表达,流式细胞仪(Annexin V-FITC法)检测细胞凋亡率。
结果
第一部分TNF-α诱导肺泡上皮细胞凋亡及对Bcl-2和Bax的影响
(1)、流式细胞仪结果显示：TNF-α0h组有少量细胞凋亡(0.09±0.05)%,6h组(4.38±0.40)%、12h组(5.31±0.17)%、24h组(5.84±0.14)%和48h组(6.37±0.32)%均明显高于TNF-α0h组,差异有统计学意义(P<0.05)。
(2)、RT-PCR结果显示：TNF-α0h组Bcl-2 mRNA相对含量最高(0.24±0.03),6h组(0.21±0.02)高于TNF-α0h组,但差异无统计学意义(P=0.139),12h组(0.16±0.02)、24h组(0.15±0.01)和48h组(0.11±0.02)均高于TNF-α0h组,差异有统计学意义(P<0.05); TNF-α0h组Bax mRNA相对含量较低(0.15±0.03),6h组(0.26±0.05)、12h组(0.33±0.03)、24h组(0.38±0.03)和48h组(0.41±0.12)均高于TNF-α0h组,差异有统计学意义(P<0.05)。
(3)、Bcl-2/Bax蛋白Western blotting结果显示：胞质内Bcl-2蛋白相对含量随着时间延长表达减少,6h组(0.64±0.04)、12h组(0.63±0.03)、24h组(0.40±0.06)、48h组(0.18±0.05)均低于TNF-α0h组(0.76±0.07),差异有统计学意义(P<0.05); Bax蛋白相对含量随时间延长而表达增加,6h组(0.42±0.04)、12h组(0.59±0.03)、24h组(0.75±0.04)、48h组(0.97±0.05)均高于TNF-α0h组(0.34±0.05),差异有统计学意义(P<0.05)。
(4)、免疫组化结果显示：Bcl-2蛋白在TNF-α0h组即有较强的阳性表达,IOD值(9.89±0.75),6h组(4.89±0.26)、12h组(1.94±0.11)、24h组(1.44±0.06)和48h组(1.25±0.12)阳性表达均低于TNF-α0h组,差异有统计学意义(P<0.05); Bax蛋白在TNF-α0h组阳性表达较少,IOD值(1.58±0.38),6h组(6.19±0.70)、12h组(16.82±2.37)、24h组(36.04±2.45)和48h组(38.02±2.84)阳性表达均高于TNF-α0h组,差异有统计学意义(P<0.05)。
第二部分沉默Bax基因对TNF-α诱导肺泡上皮细胞凋亡的影响
(1)、RT-PCR结果显示：Bax siRNA组Bax mRNA相对含量(0.19±0.03)均低于其它三组,差异有统计学意义(P<0.05)。
(2)、Western blotting结果显示：Bax siRNA组Bax蛋白相对含量(0.05±0.01)均低于其它三组,差异有统计学意义(P<0.05)。
(3)、免疫组化结果显示：Bax siRNA组胞质内Bax蛋白阳性表达(5.00±0.79)低于TNF-α组(35.15±5.33)和scramble siRNA组(39.16±4.53),差异有统计学意义(P<0.05)。
(4)、流式细胞仪结果显示：Bax siRNA组细胞凋亡率(1.55±0.28)%低于TNF-α组(5.21±0.53)%和scramble siRNA组(4.84±0.47)%,差异有统计学意义(P<0.05)。
第三部分沉默NF-κB/p65基因对TNF-α诱导肺泡上皮细胞凋亡的影响
(1)、RT-PCR结果显示：NF-κB/p65 siRNA组NF-κB/p65 mRNA相对含量(0.04±0.01)均低于其它三组,差异有统计学意义(P<00.05); NF-κB/p65 siRNA组Bcl-2 mRNA相对含量(0.41±0.06)低于TNF-α组(0.21±0.02)和scramble siRNA组(0.17±0.03),差异有统计学意义(P<0.05)；相反NF-κB/p65 siRNA组Bax mRNA相对含量(0.10±0.03)高于TNF-α组(0.27±0.02)和scramble siRNA组(0.28±0.02),差异有统计学意义(P<0.05)。
(2)、免疫组化结果显示：NF-κB/p65 siRNA组胞质内NF-κB/p65蛋白阳性表达(7.00±1.06)均低于TNF-α组(38.41±1.67)和scramble siRNA组(7.00±1.06),差异有统计学意义(P<0.05),而且TNF-α组和scramble siRNA组NF-κB/p65蛋白阳性表达主要定位于胞核。
(3)、Western blotting结果显示：NF-κB/p65 siRNA组Bcl-2蛋白相对含量(0.32±0.06)高于TNF-α组(0.09±0.08)和scramble siRNA组(0.13±0.04),差异有统计学意义(P<0.05)；相反NF-κB/p65 siRNA组Bax蛋白相对含量(0.08±0.04)低于TNF-α组(1.14±0.09)和scramble siRNA组(1.11±0.10),差异有统计学意义(P<0.05)。(4)、流式细胞仪结果显示：NF-κB/p65 siRNA组细胞凋亡率(3.52±1.11)%明显低于TNF-α组(12.66±1.07)%和scramble siRNA组(12.10±1.15)%,差异有统计学意义(P<0.05)。
结论
本研究发现：
1、低浓度(10ng/ml)TNF-α通过上调凋亡相关基因Bax/Bcl-2表达比例诱导AT-Ⅱ细胞(A549)凋亡；
2、Bax基因沉默能明显减少TNF-α诱导A549细胞凋亡；
3、NF-κB/p65基因沉默能明显减少TNF-α诱导A549细胞凋亡,其机制可能是通过下调凋亡相关基因Bax/Bcl-2表达比例从而减少A549细胞凋亡；
4、NF-κB家族亚单位p65的活化参与了肺泡上皮细胞凋亡信号转导,提示NF-κB/p65基因在炎症细胞因子如TNF-α诱导的肺泡上皮细胞凋亡中起了重要作用。调控NF-κB活性,减少肺泡上皮细胞过度凋亡,可能成为治疗炎症失衡所致急性肺损伤的新靶点。
Objective
The apoptosis of alveolar epithelial cells plays a key role in acute lung injury (ALI) by inflammatory mediators such as TNF-α. Apoptosis-related molecules, NF-κB and Bcl-2 family are important apoptosis regulators in alveolar epithelial cells. Clarity the apoptosis mechanism of alveolar epithelial cells in ALI is conducive to the treatment of ALI.
In this study, we used recombinant human TNF-αto stimulate alveolar typeⅡcells A549. The rate of apoptosis was investigated by flow cytometry, the expression of Bcl-2 and Bax was tested by RT-PCR, immunohistochemisty and Western blotting at different times in order to understand the effect of apoptosis and the expression of Bcl-2/Bax in alveolar typeⅡepithelial cells by TNF-α.
We employed RNA interference (RNAi) to inhibit the expression of apoptotic gene Bax to investigate the effect of TNF-αon the apoptosis in alveolar typeⅡepithelial cells. Then we also use RNAi to inhibit the expression of NF-κB/p65 to clarify the function and mechanism of NF-KB/p65 in TNF-αinduced apoptosis of alveolar typeⅡepithelial cells.
Methods
Part 1:Alveolar typeⅡepithelial cells A549 were stimulated by TNF-α(10ng/ml) in vitro when the cell density at 80%-90%. The rate of apoptosis was tested by flow cytometry, the level of the Bcl-2 and Bax mRNA were tested by RT-PCR, the expression of Bcl-2 and Bax protein were tested by Western blotting and immunohistochemisty.
Part 2:A549 cells were divided into four groups according to RNAi treatment: control group, TNF-a treated group, Bax siRNA group and control siRNA group. Chemically synthesized small interfering RNA (siRNA) directed against human Bax gene was transfected into A549 cells by cationic liposome. RNAi effect was investigated by RT-PCR, Western blotting and immunohistochemisty, and the rate of apoptosis was investigated by flow cytometry.
Part 3:A549 cells were divided into four groups according to RNAi treatment: control group, TNF-a treated group, NF-κB/p65 siRNA group and control siRNA group. Chemically synthesized siRNA directed against human NF-κB/p65 gene was transfected into A549 cells by cationic liposome. RNAi effect was investigated by RT-PCR and immunohistochemisty, and the rate of apoptosis was investigated by flow cytometry. Furthermore, the expression of Bcl-2, Bax was investigated by RT-PCR and Western blotting.
Results
Part 1:The analysis of flow cytometry revealed that the rate of apoptosis of A549 cells was gradually increased in a time-dependent manner (P<0.05). The mRNA and protein expression of Bcl-2 mRNA and protein were significantly decreased compared with TNF-a Oh group (P<0.05). In contrast, the level of mRNA and protein of Bax were significantly increased (P<0.05).
Part 2:Bax gene was knocked down significantly in Bax RNAi group(P<0.05). At the same time, apoptosis was induced by TNF-αtreated group and control siRNA group. However, it was abolished in Bax siRNA group by the down-regulation of Bax gene compared to TNF-αtreated group and control siRNA group(P<0.05).
Part 3:Compared with normal control group, TNF-αsignificantly increased the level of NF-κB/p65 mRNA and protein in A549 cells (P<0.05). Chemically synthesized siRNA significantly inhibited the expression of NF-κB/p65mRNA and protein in A549 cells (P<0.05). The rate of apoptosis in NF-KB/p65 siRNA group was significantly lower than other groups (P<0.05). The level of Bcl-2 protein decreased significantly, oppositely the level of Bax protein increased.
Conclusion
1. Low concentration of TNF-αin 10ng/ml induces apoptosis in A549 and promotes the expression of Bax and inhibits the expression of Bcl-2;
2. Bax gene silencing significantly reduced TNF-αinduced apoptosis in A549 cells;
3. Silencing NF-κB/p65 gene inhibits TNF-α-mediated apoptosis in alveolarⅡepithelial cells via regulating the expression of Bcl-2 and Bax;
4. NF-κB/p65 plays an important role on apoptosis of alveolar epithelial cells.
重症社区获得性肺炎(severe community-acquired pneumonia, SCAP)是呼吸内科重症监护病房最常见的危重病之一,常导致全身炎症反应综合征(systemic inflammatory response syndrome, SIRS),病情进展快,处理不及时,可发展为多器官功能障碍综合征(multiple system organ dysfunction, MODS)。急性肺损伤(acute lung injury, ALI)是SCAP最常见而严重的病理生理过程,是肺部炎症致肺泡上皮细胞受损和肺微血管通透性增加而导致的急性、进行性缺氧性呼吸衰竭,其最终阶段即为急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)。传统认为,肺泡上皮细胞是肺部炎症反应的靶细胞,具有调节炎症发展及预后的能力。在细菌所致肺部急性炎症反应中,革兰阴性菌细胞壁的糖脂成分脂多糖(LPS)发挥了关键作用,LPS可以直接募集血液中性粒细胞进入肺泡进而促进细胞因子,如肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)和白细胞介素-1β(interleukin-1β, IL-1β)等的释放,通过调控炎症通路的活化以及诱导其它细胞因子和粘附分子的产生,进而造成更严重的肺部损伤。
细胞凋亡是细胞在一定生理或病理条件下,遵循自身程序,自己结束生命的过程,对于机体稳定和生长发育具有重要的意义。大量研究显示,肺泡Ⅱ型上皮细胞(alveolar typeⅡepithelial cell, AT-Ⅱ)的凋亡在肺部重症感染所致ALI中起了至关重要的作用,AT-Ⅱ细胞的过度凋亡将导致结构细胞数量减少和肺组织病理变化加重；另外在ALI病理变化过程中需将受损的AT-Ⅱ细胞清除,此过程可能引起炎性反应而加重已存在肺组织的损伤,因此肺泡上皮细胞凋亡在调节炎症对肺损伤的反应方面发挥着重要的作用。细胞凋亡是有一序列酶参与、由基因控制的一个主动的、高度有序的过程,其中Bcl-2家族成员在细胞凋亡的基因调控过程中起着十分重要的作用,根据作用不同分为两类：①抑制性亚族,如Bcl-2、Bcl-X、Mcl-1,可在多个环节上阻断细胞凋亡；②促进性亚族,如Bax、Bak、Bad、Bik、Bid等,与抑制性亚族结合而封闭其活性域。
核因子-KB(nuclear factor-κB, NF-κB)是由Sen等从B淋巴细胞核提取物中发现的一种能与免疫球蛋白κ链基因增强子κB序列特异结合的核蛋白因子,因此命名为NF-κB。目前所知NF-κB家族成员包括RelA(p65)、RelB、c-Rel、p50和p52五个亚单位。通常NF-κB以p65／p50二聚体形式存在胞浆中,其中p65含有转录活化区域,在静息状态下,胞浆中的NF-κB与抑制性蛋白IκB-α结合,不能发挥转录调节作用,当细胞受到如TNF-α等细胞外信号刺激时,IκB-α被磷酸化并从NF-κB二聚体上解离下来,p65／p50二聚体立即转移入核,与其靶基因启动子／增强子上的KB位点结合,启动相关基因的转录。NF-κB信号通路参与了感染、炎症反应和肿瘤等病理过程以及细胞周期调控等,近年来研究表明,NF-κB家族及其介导的细胞信号转导在细胞凋亡中起到了重要作用,它参与了多种凋亡相关基因的转录调控,具有抑制细胞凋亡和促进细胞凋亡的双向作用。
NF-κB与细胞凋亡的关系最早在肿瘤生长等方面研究,以往研究显示NF-κB可诱导细胞表达TNFR1结合因子(TFAF)和细胞凋亡蛋白抑制物,抑制caspase的级联反应,从而阻断死亡受体TNFR/Fas介导的凋亡信号转导,而抑制细胞凋亡,所以NF-κB在肿瘤中的主要作用表现为抗凋亡作用。但在SCAP中NF-κB与细胞凋亡的关系却复杂得多,是抑制凋亡还是促进凋亡?目前还有争论。一方面,在多形核中性粒细胞(PMNs)等炎症细胞,NF-κB活化使抗凋亡蛋白合成增加,而使其凋亡延迟,Kupfner J G等在内毒素引起的ARDS中发现NF-κB活化继而引起A1、A20及Bcl-X的转录增加,并引起凋亡抑制,在应用NF-κB抑制剂后PMNs的凋亡加快；另一方面,对于肺泡上皮细胞和内皮细胞等肺结构细胞,有报道认为,NF-κB调控失常使促凋亡蛋白如IL-8、TNF、FasL等的转录表达增强,引起细胞凋亡的加强,但其凋亡的调控机制还不是很清楚。
鉴此,本研究拟采用重组的人细胞因子TNF-α刺激AT-Ⅱ细胞A549,探讨炎症环境下肺泡上皮细胞凋亡的基因调控机制。首先,在TNF-α刺激的不同时间点检测细胞凋亡率的变化及凋亡基因Bcl-2、Bax的表达情况,了解TNF-α对肺泡上皮细胞凋亡的影响；其次,利用Bax小干扰RNA(small interfering RNA, siRNA)沉默凋亡基因Bax的表达,明确Bax基因与TNF-α诱导肺泡上皮细胞凋亡的关系；最后,再利用NF-κB/p65 siRNA沉默NF-κB家族亚单位p65基因的表达,初步探讨NF-κB/p65基因在TNF-α诱导肺泡上皮细胞凋亡中的作用及可能机制。
方法
1、培养AT-Ⅱ细胞(来源于人肺泡上皮细胞株A549),在细胞密度为80%～90%时采用低浓度(10ng/ml)的TNF-α刺激A549细胞,分别在刺激细胞0h、6h、12h、24和48h五个时间点收集细胞,流式细胞仪(Annexin V-FITC法)检测细胞凋亡率,RT-PCR检测Bcl-2和Bax mRNA, Western blotting和免疫组化检测Bcl-2和Bax蛋白表达；
2、利用脂质体转染试剂LipofectamineTM2000将Bax siRNA转染入A549细胞,6h后给予TNF-α(10ng/ml)刺激24h收集细胞,实验分4组：空白对照组、TNF-α组、TNF-α+Bax siRNA组(干扰组)、TNF-α+scramble siRNA组(阴性对照组)。RT-PCR检测Bax mRNA, Western blotting和免疫组化检测Bax蛋白表达,流式细胞仪(Annexin V-FITC法)检测细胞凋亡率；
3、利用脂质体转染试剂LipofectamineTM2000将NF-κB/p65 siRNA转染入A549细胞,6h后给予TNF-α(10ng/ml)刺激24h收集细胞,实验分4组：空白对照组、TNF-α组、TNF-α+NF-κB/p65 siRNA组(干扰组)、TNF-α+scramble siRNA组(阴性对照组)。RT-PCR检测NF-κB/p65、Bcl-2、Bax mRNA,免疫组化检测NF-κB/p65的活化,Western blotting检测Bcl-2、Bax蛋白表达,流式细胞仪(Annexin V-FITC法)检测细胞凋亡率。
结果
第一部分TNF-α诱导肺泡上皮细胞凋亡及对Bcl-2和Bax的影响
(1)、流式细胞仪结果显示：TNF-α0h组有少量细胞凋亡(0.09±0.05)%,6h组(4.38±0.40)%、12h组(5.31±0.17)%、24h组(5.84±0.14)%和48h组(6.37±0.32)%均明显高于TNF-α0h组,差异有统计学意义(P<0.05)。
(2)、RT-PCR结果显示：TNF-α0h组Bcl-2 mRNA相对含量最高(0.24±0.03),6h组(0.21±0.02)高于TNF-α0h组,但差异无统计学意义(P=0.139),12h组(0.16±0.02)、24h组(0.15±0.01)和48h组(0.11±0.02)均高于TNF-α0h组,差异有统计学意义(P<0.05); TNF-α0h组Bax mRNA相对含量较低(0.15±0.03),6h组(0.26±0.05)、12h组(0.33±0.03)、24h组(0.38±0.03)和48h组(0.41±0.12)均高于TNF-α0h组,差异有统计学意义(P<0.05)。
(3)、Bcl-2/Bax蛋白Western blotting结果显示：胞质内Bcl-2蛋白相对含量随着时间延长表达减少,6h组(0.64±0.04)、12h组(0.63±0.03)、24h组(0.40±0.06)、48h组(0.18±0.05)均低于TNF-α0h组(0.76±0.07),差异有统计学意义(P<0.05); Bax蛋白相对含量随时间延长而表达增加,6h组(0.42±0.04)、12h组(0.59±0.03)、24h组(0.75±0.04)、48h组(0.97±0.05)均高于TNF-α0h组(0.34±0.05),差异有统计学意义(P<0.05)。
(4)、免疫组化结果显示：Bcl-2蛋白在TNF-α0h组即有较强的阳性表达,IOD值(9.89±0.75),6h组(4.89±0.26)、12h组(1.94±0.11)、24h组(1.44±0.06)和48h组(1.25±0.12)阳性表达均低于TNF-α0h组,差异有统计学意义(P<0.05); Bax蛋白在TNF-α0h组阳性表达较少,IOD值(1.58±0.38),6h组(6.19±0.70)、12h组(16.82±2.37)、24h组(36.04±2.45)和48h组(38.02±2.84)阳性表达均高于TNF-α0h组,差异有统计学意义(P<0.05)。
第二部分沉默Bax基因对TNF-α诱导肺泡上皮细胞凋亡的影响
(1)、RT-PCR结果显示：Bax siRNA组Bax mRNA相对含量(0.19±0.03)均低于其它三组,差异有统计学意义(P<0.05)。
(2)、Western blotting结果显示：Bax siRNA组Bax蛋白相对含量(0.05±0.01)均低于其它三组,差异有统计学意义(P<0.05)。
(3)、免疫组化结果显示：Bax siRNA组胞质内Bax蛋白阳性表达(5.00±0.79)低于TNF-α组(35.15±5.33)和scramble siRNA组(39.16±4.53),差异有统计学意义(P<0.05)。
(4)、流式细胞仪结果显示：Bax siRNA组细胞凋亡率(1.55±0.28)%低于TNF-α组(5.21±0.53)%和scramble siRNA组(4.84±0.47)%,差异有统计学意义(P<0.05)。
第三部分沉默NF-κB/p65基因对TNF-α诱导肺泡上皮细胞凋亡的影响
(1)、RT-PCR结果显示：NF-κB/p65 siRNA组NF-κB/p65 mRNA相对含量(0.04±0.01)均低于其它三组,差异有统计学意义(P<00.05); NF-κB/p65 siRNA组Bcl-2 mRNA相对含量(0.41±0.06)低于TNF-α组(0.21±0.02)和scramble siRNA组(0.17±0.03),差异有统计学意义(P<0.05)；相反NF-κB/p65 siRNA组Bax mRNA相对含量(0.10±0.03)高于TNF-α组(0.27±0.02)和scramble siRNA组(0.28±0.02),差异有统计学意义(P<0.05)。
(2)、免疫组化结果显示：NF-κB/p65 siRNA组胞质内NF-κB/p65蛋白阳性表达(7.00±1.06)均低于TNF-α组(38.41±1.67)和scramble siRNA组(7.00±1.06),差异有统计学意义(P<0.05),而且TNF-α组和scramble siRNA组NF-κB/p65蛋白阳性表达主要定位于胞核。
(3)、Western blotting结果显示：NF-κB/p65 siRNA组Bcl-2蛋白相对含量(0.32±0.06)高于TNF-α组(0.09±0.08)和scramble siRNA组(0.13±0.04),差异有统计学意义(P<0.05)；相反NF-κB/p65 siRNA组Bax蛋白相对含量(0.08±0.04)低于TNF-α组(1.14±0.09)和scramble siRNA组(1.11±0.10),差异有统计学意义(P<0.05)。(4)、流式细胞仪结果显示：NF-κB/p65 siRNA组细胞凋亡率(3.52±1.11)%明显低于TNF-α组(12.66±1.07)%和scramble siRNA组(12.10±1.15)%,差异有统计学意义(P<0.05)。
结论
本研究发现：
1、低浓度(10ng/ml)TNF-α通过上调凋亡相关基因Bax/Bcl-2表达比例诱导AT-Ⅱ细胞(A549)凋亡；
2、Bax基因沉默能明显减少TNF-α诱导A549细胞凋亡；
3、NF-κB/p65基因沉默能明显减少TNF-α诱导A549细胞凋亡,其机制可能是通过下调凋亡相关基因Bax/Bcl-2表达比例从而减少A549细胞凋亡；
4、NF-κB家族亚单位p65的活化参与了肺泡上皮细胞凋亡信号转导,提示NF-κB/p65基因在炎症细胞因子如TNF-α诱导的肺泡上皮细胞凋亡中起了重要作用。调控NF-κB活性,减少肺泡上皮细胞过度凋亡,可能成为治疗炎症失衡所致急性肺损伤的新靶点。
Objective
The apoptosis of alveolar epithelial cells plays a key role in acute lung injury (ALI) by inflammatory mediators such as TNF-α. Apoptosis-related molecules, NF-κB and Bcl-2 family are important apoptosis regulators in alveolar epithelial cells. Clarity the apoptosis mechanism of alveolar epithelial cells in ALI is conducive to the treatment of ALI.
In this study, we used recombinant human TNF-αto stimulate alveolar typeⅡcells A549. The rate of apoptosis was investigated by flow cytometry, the expression of Bcl-2 and Bax was tested by RT-PCR, immunohistochemisty and Western blotting at different times in order to understand the effect of apoptosis and the expression of Bcl-2/Bax in alveolar typeⅡepithelial cells by TNF-α.
We employed RNA interference (RNAi) to inhibit the expression of apoptotic gene Bax to investigate the effect of TNF-αon the apoptosis in alveolar typeⅡepithelial cells. Then we also use RNAi to inhibit the expression of NF-κB/p65 to clarify the function and mechanism of NF-KB/p65 in TNF-αinduced apoptosis of alveolar typeⅡepithelial cells.
Methods
Part 1:Alveolar typeⅡepithelial cells A549 were stimulated by TNF-α(10ng/ml) in vitro when the cell density at 80%-90%. The rate of apoptosis was tested by flow cytometry, the level of the Bcl-2 and Bax mRNA were tested by RT-PCR, the expression of Bcl-2 and Bax protein were tested by Western blotting and immunohistochemisty.
Part 2:A549 cells were divided into four groups according to RNAi treatment: control group, TNF-a treated group, Bax siRNA group and control siRNA group. Chemically synthesized small interfering RNA (siRNA) directed against human Bax gene was transfected into A549 cells by cationic liposome. RNAi effect was investigated by RT-PCR, Western blotting and immunohistochemisty, and the rate of apoptosis was investigated by flow cytometry.
Part 3:A549 cells were divided into four groups according to RNAi treatment: control group, TNF-a treated group, NF-κB/p65 siRNA group and control siRNA group. Chemically synthesized siRNA directed against human NF-κB/p65 gene was transfected into A549 cells by cationic liposome. RNAi effect was investigated by RT-PCR and immunohistochemisty, and the rate of apoptosis was investigated by flow cytometry. Furthermore, the expression of Bcl-2, Bax was investigated by RT-PCR and Western blotting.
Results
Part 1:The analysis of flow cytometry revealed that the rate of apoptosis of A549 cells was gradually increased in a time-dependent manner (P<0.05). The mRNA and protein expression of Bcl-2 mRNA and protein were significantly decreased compared with TNF-a Oh group (P<0.05). In contrast, the level of mRNA and protein of Bax were significantly increased (P<0.05).
Part 2:Bax gene was knocked down significantly in Bax RNAi group(P<0.05). At the same time, apoptosis was induced by TNF-αtreated group and control siRNA group. However, it was abolished in Bax siRNA group by the down-regulation of Bax gene compared to TNF-αtreated group and control siRNA group(P<0.05).
Part 3:Compared with normal control group, TNF-αsignificantly increased the level of NF-κB/p65 mRNA and protein in A549 cells (P<0.05). Chemically synthesized siRNA significantly inhibited the expression of NF-κB/p65mRNA and protein in A549 cells (P<0.05). The rate of apoptosis in NF-KB/p65 siRNA group was significantly lower than other groups (P<0.05). The level of Bcl-2 protein decreased significantly, oppositely the level of Bax protein increased.
Conclusion
1. Low concentration of TNF-αin 10ng/ml induces apoptosis in A549 and promotes the expression of Bax and inhibits the expression of Bcl-2;
2. Bax gene silencing significantly reduced TNF-αinduced apoptosis in A549 cells;
3. Silencing NF-κB/p65 gene inhibits TNF-α-mediated apoptosis in alveolarⅡepithelial cells via regulating the expression of Bcl-2 and Bax;
4. NF-κB/p65 plays an important role on apoptosis of alveolar epithelial cells.
引文
[1]方怡,冉丕鑫.重症社区获得性肺炎炎性机制研究进展[J].国外医学呼吸系统分册,2005,25(1)：23.
[2]Robert M. A brief overview of acute respiratory distress syndrome[J]. World J Surg,2006,30(8):1829-1834.
[3]Gentry M, Taormina J, Pyles R B, et al. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection[J]. Infect Immun, 2007,75(8):3969-3978.
[4]Oyoshi M K, Barthel R, Tsitsikov E N. TRAF1 regulates recruitment of lymphocytes and, to a lesser extent, neutrophils, myeloid dendritic cells and monocytes to the lung airways following lipopolysaccharide inhalation[J]. Immunology,2007,120(3):303-314.
[5]Chopra M, Reuben J S, Sharma A C. Acute lung injury:apoptosis and signaling mechanisms[J]. Exp Biol Med (Maywood),2009,234(4):361-371.
[6]Martin T R, Nakamura M, Matute-Bello G. The role of apoptosis in acute lung injury[J]. Crit Care Med,2003,31(4):184-188.
[7]Xu G, Gong Z, Yu W, et al. Increased expression ratio of Bcl-2/Bax is associated with crocin-mediated apoptosis in bovine aortic endothelial cells[J]. Basic Clin Pharmacol Toxicol,2007,100(1):31-35.
[8]Daher S, Guimaraes A J, Mattar R, et al. Bcl-2 and Bax expressions in pre-term, term and post-term placentas[J]. Am J Reprod Immunol,2008,60(2):172-178.
[9]Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences[J]. Cell,1986,46(5):705-716.
[10]Vallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system[J]. Annu Rev Immunol,2009,27(4):693-733.
[11]Dutta J, Fan Y, Gupta N, et al. Current insights into the regulation of programmed cell death by NF-kappaB[J]. Oncogene,2006,25(51):6800-6816.
[12]Wang CY, M ayo MW, Komeluk RG, et al. NF-kappaB antiapoptosis:induction of TRAF1 and TRAF2 and c-IAPl and c-IAP2 to suppress caspase-8 activation[J]. Science,1998,281(5383):1680-1683.
[13]Kupfner J G, Arcaroli J J, Yum H K, et al. Role of NF-kappaB in endotoxemia-induced alterations of lung neutrophil apoptosis[J]. J Immunol, 2001,167(12):7044-7051.
[14]M atute-Bello G, lilesWC, Steinberg KP et al. Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury(ARDS)[J]. J Immunol, 1999,163(4):2217-2225.
[15]Serrao KL, Fo rtenberry JD, OwensML et al. Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand[J]. Am J Physiol Lung CellMol Physiol,2001,280(2):298-305.
[16]张秋金,李银平,黎檀实.肺泡上皮细胞功能特性与内毒素性急性肺损伤[J].中国危重病急救医学,2005,17(6)：382-384.
[17]蒋建新,主编.细菌内毒素基础与临床[M].第2版.北京：人民军医出版社,2004，265.
[18]Tesfaigzi J, Wood M, Johnson N F, et al. Apoptosis is a pathway responsible for the resolution of endotoxin induced alveolar type II cell hyperplasia in the rat[J]. Int J Exp Pathol,1998,79(5):303-311.
[19]Fujita M, Kuwano K, Kunitake R, et al. Endothelial cell apoptosis in lipopo lysaccharide induced lung injury in mice[J]. Int Arch A leergy Immunol,1998, 117(3):202-208.
[20]Albertine K H, Soulier M F, Wang Z, et al. Fas and fas ligand are up regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the acute respiratory distress syndrome[J]. Am J Pathol,2002,161(5):1783-1796.
[21]Kalia S, Bansal M P. Regulation of apoptosis by Caspases under oxidative stress conditions in mice testicular cells:in vitro molecular mechanism [J]. Mol Cell Biochem,2009,322(1-2):43-52.
[22]Degterev A, Boyce M, Yuan J. A decade of caspases [J]. Oncogene,2003,22(53): 8543-8567.
[23]De Souza P M, Lindsay M A. Apoptosis as a therapeutic target for the treatment of lung disease[J]. Curr Opin Pharmacol,2005,5(3):232-237.
[24]Kupfner J G, Arcaroli J J, Yum H K, et al. Role of NF-kappaB in endotoxemia-induced alterations of lung neutrophil apoptosis[J]. J Immunol, 2001,167(12):7044-7051.
[25]Parsey M V, Kaneko D, Shenkar R, et al. Neutrophil apoptosis in the lung after hemorrhage or endotoxemia:apoptosis and migration are independent of IL-1beta[J]. Clin Immunol,1999,91(2):219-225.
[26]Aggarwal A, Baker C S, Evans T W, et al. G-CSF and IL-8 but not GM-CSF correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome[J]. Eur Respir J,2000,15(5):895-901.
[27]Lesur O, Kokis A, Hermans C, et al. Interleukin-2 involvement in early acute respiratory distress syndrome:relationship with polymorphonuclear neutrophil apoptosis and patient survival[J]. Crit Care Med,2000,28(12):3814-3822.
[28]Lehner B, Fraser A G., Sanderson C M. Technique review:how to use RNA interference[J]. Brief Funct Genomic Proteomic,2004,3(1):68-83.
[29]贾明旺,黄文杰.Caspase-3基因沉默对肺炎链球菌感染的肺泡上皮细胞凋亡的影响[J].中国病理生理杂志,2010,26(1)：132-136.
[30]Marriott H M, Dockrell D H. Streptococcus pneumoniae:the role of apoptosis in host defense and pathogenesis[J]. Int J Biochem Cell Biol,2006,38(11): 1848-1854.
[31]Locksley R M, Killeen N, Lenardo M J. The TNF and TNF receptor superfamilies:integrating mammalian biology[J]. Cell,2001,104(4):487-501.
[32]Crisafulli C, Galuppo M, Cuzzocrea S. Effects of genetic and pharmacological inhibition of TNF-alpha in the regulation of inflammation in macrophages[J]. Pharmacol Res,2009,60(4):332-340.
[33]Herold S, Steinmueller M, Von Wulffen W, et al. Lung epithelial apoptosis in influenza virus pneumonia:the role of macrophage-expressed TNF-related apoptosis-inducing ligand[J]. J Exp Med,2008,205(13):3065-3077.
[34]Gong Q, Yin H, Fang M, et al. Heme oxygenase-1 upregulation significantly inhibitsTNF-alpha and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice[J]. Int Immunopharmacol, 2008,8(6):792-798.
[35]Lykouras D, Sampsonas F, Kaparianos A, et al. Role and pharmacogenomics of TNF-alpha in asthma[J]. Mini Rev Med Chem,2008,8(9):934-942.
[36]Kupeli E, Karnak D, Beder S, et al. Diagnostic accuracy of cytokine levels (TNF-alpha,IL-2 and IFN-gamma) in bronchoalveolar lavage fluid of smear-negative pulmonary tuberculosis patients[J]. Respiration,2008,75(1): 73-78.
[37]顾俭勇,黄培志.脂多糖致急性肺损伤后Bax/Bcl-2变化[J].中国基础医药,2006,13(10)：1585-1586.
[38]Oltvai Z N, Milliman C L, Korsmeyer S J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[J]. Cell,1993, 74(4):609-619.
[39]Buccellato L J, Tso M, Akinci O I, et al. Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells[J]. J Biol Chem,2004,279(8):6753-6760.
[40]符跃强,卢仲毅,方芳,等.氧化应激诱导肺泡Ⅱ型上皮细胞凋亡及Bax和p53的表达变化[J].中国危重病急救医学,2008,20(2)：76-79,130.
[41]Manfredini R, Capobianco M L, Trevisan F, et al. Antisense inhibition of Bax mRNA increases survival of terminally differentiated HL60 cells [J]. Antisense Nucleic Acid Drug Dev,1998,8(4):341-350.
[42]Isenmann S, Engel S, Gillardon F, et al. Bax antisense oligonucleotides reduce axotomy-induced retinal ganglion cell death in vivo by reduction of Bax protein expression[J]. Cell Death Differ,1999,6(7):673-682.
[43]Pepper C, Thomas A, Hoy T, et al. Antisense oligonucleotides complementary to Bax transcripts reduce the susceptibility of B-cell chronic lymphocytic leukaemia cells to apoptosis in a bcl-2 independent manner[J]. Leuk Lymphoma, 2002,43(10):2003-2009.
[44]Okada M, Sugita K, Inukai T, et al. Hepatocyte growth factor protects small airway epithelial cells from apoptosis induced by tumor necrosis facor-a or oxidative stress[J]. Pedia Res,2004,56(3):330-334.
[45]Cregan SP, MacLaurin JG, Craig CG, et al. Bax-dependent caspase 3 activation is a key determinant in p53 induced apoptosis in neurons[J]. J Neurosci, 1999,19(18):7860-7869.
[46]Pepper C, Thomas A, Hoy T, et al. Antisense oligonucleotides complementary to Bax transcripts reduce the susceptibility of B cell chronic lymphocytic leukaemia cells to apoptosis in a Bcl-2 independent manner[J]. Leuk Lymphoma, 2002,43(10):2003-2009.
[47]吴朝晖,金惠铭.NF-κB与细胞凋亡[J].中国病理生理杂志,1999,15(11)：1045-1048.
[48]Quillet-Mary A, Jaffrezou JP, Mansat V, et al. Implication of mitochondial hydrogen peroxide generation in ceramide-induced apoptosis[J]. J Biol Chem, 1997,272(34):21388-21399.
[49]Hunot S, Brugg B, Ricard D, et al. Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease[J]. Proc Natl Acad Sci USA,1997,94(14):7531-7536.
[50]Grimm S, Bauer MKA, Baeuerle PA, et al. Bcl-2 down-regulates the activity of transcription factor NF-κB induced upon apoptosis[J]. J Cell Biol,1996,134(1): 13-23.
[51]Wang CY, Mayo MW, Baldwin AS, et al. TNF-and cancer therapy-induced apoptosis:potentiation by inhibition of NF-kappaB[J]. Science,1996,274(5288): 784-787.
[52]Emmanuelle Faure, Lisa Thomas, Helen Xu, et al. Bacterial lipopolysaccharide and IFN-y induce toll-like receptor 2 and toll-like receptor 4 expression in human endothelial cells:role of NF-κB Activation[J]. J Immunol,2001,166(3): 2018-2024.
[53]侯慧芳,高建芝,魏林郁,等.核因子-κB对感染性休克小鼠心肌细胞凋亡的影响[J].实用儿科临床杂志,2008,23(18)：1456-1457.
[54]李磊,汤耀卿,纪玉宝,等.NF-κB对内毒素诱导的血管内皮细胞凋亡的影响[J].中国微循环杂志,2009,13(1)：22-24.
[55]Brown GC, Borutaite V. Regulation of apoptosis by the redox state of cytochrome c[J]. Biochim Biophys Acta,2008,1777(7-8):877-881.
[2]Robert M. A brief overview of acute respiratory distress syndrome[J]. World J Surg,2006,30(8):1829-1834.
[3]Gentry M, Taormina J, Pyles R B, et al. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection[J]. Infect Immun, 2007,75(8):3969-3978.
[4]Oyoshi M K, Barthel R, Tsitsikov E N. TRAF1 regulates recruitment of lymphocytes and, to a lesser extent, neutrophils, myeloid dendritic cells and monocytes to the lung airways following lipopolysaccharide inhalation[J]. Immunology,2007,120(3):303-314.
[5]Chopra M, Reuben J S, Sharma A C. Acute lung injury:apoptosis and signaling mechanisms[J]. Exp Biol Med (Maywood),2009,234(4):361-371.
[6]Martin T R, Nakamura M, Matute-Bello G. The role of apoptosis in acute lung injury[J]. Crit Care Med,2003,31(4):184-188.
[7]Xu G, Gong Z, Yu W, et al. Increased expression ratio of Bcl-2/Bax is associated with crocin-mediated apoptosis in bovine aortic endothelial cells[J]. Basic Clin Pharmacol Toxicol,2007,100(1):31-35.
[8]Daher S, Guimaraes A J, Mattar R, et al. Bcl-2 and Bax expressions in pre-term, term and post-term placentas[J]. Am J Reprod Immunol,2008,60(2):172-178.
[9]Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences[J]. Cell,1986,46(5):705-716.
[10]Vallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system[J]. Annu Rev Immunol,2009,27(4):693-733.
[11]Dutta J, Fan Y, Gupta N, et al. Current insights into the regulation of programmed cell death by NF-kappaB[J]. Oncogene,2006,25(51):6800-6816.
[12]Wang CY, M ayo MW, Komeluk RG, et al. NF-kappaB antiapoptosis:induction of TRAF1 and TRAF2 and c-IAPl and c-IAP2 to suppress caspase-8 activation[J]. Science,1998,281(5383):1680-1683.
[13]Kupfner J G, Arcaroli J J, Yum H K, et al. Role of NF-kappaB in endotoxemia-induced alterations of lung neutrophil apoptosis[J]. J Immunol, 2001,167(12):7044-7051.
[14]M atute-Bello G, lilesWC, Steinberg KP et al. Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury(ARDS)[J]. J Immunol, 1999,163(4):2217-2225.
[15]Serrao KL, Fo rtenberry JD, OwensML et al. Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand[J]. Am J Physiol Lung CellMol Physiol,2001,280(2):298-305.
[16]张秋金,李银平,黎檀实.肺泡上皮细胞功能特性与内毒素性急性肺损伤[J].中国危重病急救医学,2005,17(6)：382-384.
[17]蒋建新,主编.细菌内毒素基础与临床[M].第2版.北京：人民军医出版社,2004，265.
[18]Tesfaigzi J, Wood M, Johnson N F, et al. Apoptosis is a pathway responsible for the resolution of endotoxin induced alveolar type II cell hyperplasia in the rat[J]. Int J Exp Pathol,1998,79(5):303-311.
[19]Fujita M, Kuwano K, Kunitake R, et al. Endothelial cell apoptosis in lipopo lysaccharide induced lung injury in mice[J]. Int Arch A leergy Immunol,1998, 117(3):202-208.
[20]Albertine K H, Soulier M F, Wang Z, et al. Fas and fas ligand are up regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the acute respiratory distress syndrome[J]. Am J Pathol,2002,161(5):1783-1796.
[21]Kalia S, Bansal M P. Regulation of apoptosis by Caspases under oxidative stress conditions in mice testicular cells:in vitro molecular mechanism [J]. Mol Cell Biochem,2009,322(1-2):43-52.
[22]Degterev A, Boyce M, Yuan J. A decade of caspases [J]. Oncogene,2003,22(53): 8543-8567.
[23]De Souza P M, Lindsay M A. Apoptosis as a therapeutic target for the treatment of lung disease[J]. Curr Opin Pharmacol,2005,5(3):232-237.
[24]Kupfner J G, Arcaroli J J, Yum H K, et al. Role of NF-kappaB in endotoxemia-induced alterations of lung neutrophil apoptosis[J]. J Immunol, 2001,167(12):7044-7051.
[25]Parsey M V, Kaneko D, Shenkar R, et al. Neutrophil apoptosis in the lung after hemorrhage or endotoxemia:apoptosis and migration are independent of IL-1beta[J]. Clin Immunol,1999,91(2):219-225.
[26]Aggarwal A, Baker C S, Evans T W, et al. G-CSF and IL-8 but not GM-CSF correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome[J]. Eur Respir J,2000,15(5):895-901.
[27]Lesur O, Kokis A, Hermans C, et al. Interleukin-2 involvement in early acute respiratory distress syndrome:relationship with polymorphonuclear neutrophil apoptosis and patient survival[J]. Crit Care Med,2000,28(12):3814-3822.
[28]Lehner B, Fraser A G., Sanderson C M. Technique review:how to use RNA interference[J]. Brief Funct Genomic Proteomic,2004,3(1):68-83.
[29]贾明旺,黄文杰.Caspase-3基因沉默对肺炎链球菌感染的肺泡上皮细胞凋亡的影响[J].中国病理生理杂志,2010,26(1)：132-136.
[30]Marriott H M, Dockrell D H. Streptococcus pneumoniae:the role of apoptosis in host defense and pathogenesis[J]. Int J Biochem Cell Biol,2006,38(11): 1848-1854.
[31]Locksley R M, Killeen N, Lenardo M J. The TNF and TNF receptor superfamilies:integrating mammalian biology[J]. Cell,2001,104(4):487-501.
[32]Crisafulli C, Galuppo M, Cuzzocrea S. Effects of genetic and pharmacological inhibition of TNF-alpha in the regulation of inflammation in macrophages[J]. Pharmacol Res,2009,60(4):332-340.
[33]Herold S, Steinmueller M, Von Wulffen W, et al. Lung epithelial apoptosis in influenza virus pneumonia:the role of macrophage-expressed TNF-related apoptosis-inducing ligand[J]. J Exp Med,2008,205(13):3065-3077.
[34]Gong Q, Yin H, Fang M, et al. Heme oxygenase-1 upregulation significantly inhibitsTNF-alpha and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice[J]. Int Immunopharmacol, 2008,8(6):792-798.
[35]Lykouras D, Sampsonas F, Kaparianos A, et al. Role and pharmacogenomics of TNF-alpha in asthma[J]. Mini Rev Med Chem,2008,8(9):934-942.
[36]Kupeli E, Karnak D, Beder S, et al. Diagnostic accuracy of cytokine levels (TNF-alpha,IL-2 and IFN-gamma) in bronchoalveolar lavage fluid of smear-negative pulmonary tuberculosis patients[J]. Respiration,2008,75(1): 73-78.
[37]顾俭勇,黄培志.脂多糖致急性肺损伤后Bax/Bcl-2变化[J].中国基础医药,2006,13(10)：1585-1586.
[38]Oltvai Z N, Milliman C L, Korsmeyer S J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[J]. Cell,1993, 74(4):609-619.
[39]Buccellato L J, Tso M, Akinci O I, et al. Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells[J]. J Biol Chem,2004,279(8):6753-6760.
[40]符跃强,卢仲毅,方芳,等.氧化应激诱导肺泡Ⅱ型上皮细胞凋亡及Bax和p53的表达变化[J].中国危重病急救医学,2008,20(2)：76-79,130.
[41]Manfredini R, Capobianco M L, Trevisan F, et al. Antisense inhibition of Bax mRNA increases survival of terminally differentiated HL60 cells [J]. Antisense Nucleic Acid Drug Dev,1998,8(4):341-350.
[42]Isenmann S, Engel S, Gillardon F, et al. Bax antisense oligonucleotides reduce axotomy-induced retinal ganglion cell death in vivo by reduction of Bax protein expression[J]. Cell Death Differ,1999,6(7):673-682.
[43]Pepper C, Thomas A, Hoy T, et al. Antisense oligonucleotides complementary to Bax transcripts reduce the susceptibility of B-cell chronic lymphocytic leukaemia cells to apoptosis in a bcl-2 independent manner[J]. Leuk Lymphoma, 2002,43(10):2003-2009.
[44]Okada M, Sugita K, Inukai T, et al. Hepatocyte growth factor protects small airway epithelial cells from apoptosis induced by tumor necrosis facor-a or oxidative stress[J]. Pedia Res,2004,56(3):330-334.
[45]Cregan SP, MacLaurin JG, Craig CG, et al. Bax-dependent caspase 3 activation is a key determinant in p53 induced apoptosis in neurons[J]. J Neurosci, 1999,19(18):7860-7869.
[46]Pepper C, Thomas A, Hoy T, et al. Antisense oligonucleotides complementary to Bax transcripts reduce the susceptibility of B cell chronic lymphocytic leukaemia cells to apoptosis in a Bcl-2 independent manner[J]. Leuk Lymphoma, 2002,43(10):2003-2009.
[47]吴朝晖,金惠铭.NF-κB与细胞凋亡[J].中国病理生理杂志,1999,15(11)：1045-1048.
[48]Quillet-Mary A, Jaffrezou JP, Mansat V, et al. Implication of mitochondial hydrogen peroxide generation in ceramide-induced apoptosis[J]. J Biol Chem, 1997,272(34):21388-21399.
[49]Hunot S, Brugg B, Ricard D, et al. Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease[J]. Proc Natl Acad Sci USA,1997,94(14):7531-7536.
[50]Grimm S, Bauer MKA, Baeuerle PA, et al. Bcl-2 down-regulates the activity of transcription factor NF-κB induced upon apoptosis[J]. J Cell Biol,1996,134(1): 13-23.
[51]Wang CY, Mayo MW, Baldwin AS, et al. TNF-and cancer therapy-induced apoptosis:potentiation by inhibition of NF-kappaB[J]. Science,1996,274(5288): 784-787.
[52]Emmanuelle Faure, Lisa Thomas, Helen Xu, et al. Bacterial lipopolysaccharide and IFN-y induce toll-like receptor 2 and toll-like receptor 4 expression in human endothelial cells:role of NF-κB Activation[J]. J Immunol,2001,166(3): 2018-2024.
[53]侯慧芳,高建芝,魏林郁,等.核因子-κB对感染性休克小鼠心肌细胞凋亡的影响[J].实用儿科临床杂志,2008,23(18)：1456-1457.
[54]李磊,汤耀卿,纪玉宝,等.NF-κB对内毒素诱导的血管内皮细胞凋亡的影响[J].中国微循环杂志,2009,13(1)：22-24.
[55]Brown GC, Borutaite V. Regulation of apoptosis by the redox state of cytochrome c[J]. Biochim Biophys Acta,2008,1777(7-8):877-881.