【作者】 任卫英；

【导师】 朱蕾；

【作者基本信息】 复旦大学 ， 内科学， 2010， 博士

【摘要】 RNA干扰沉默肺泡巨噬细胞MD-2对脂多糖所致大鼠急性肺损伤的影响背景和目的急性肺损伤(acute lung injury, ALI)的发病机制复杂,但多种细胞因子和炎症介质以及效应细胞共同参与的、过度失控的炎症反应是发病的关键环节。由于炎症反应的环节众多、过程复杂,难以选择有效的靶点,故缺乏有效的抗炎治疗方法,ALI的病死率仍相当高。致病因子参与的炎症反应的上游环节则简单得多,可能会成为防治过度炎症反应的靶点。脂多糖(lipopolysaccharide,LPS)是革兰阴性杆菌的主要致病成分,是导致ALI的重要致病因素。LPS由Toll样受体4(Toll like receptor 4, TLR4)及髓样分化蛋白-2(myeloid differentiation protein 2, MD-2)组成的复合受体介导跨膜信号转导,并通过激活效应细胞内的一系列下游信号分子,引起细胞因子和炎症介质的大量释放。MD-2在识别LPS和辅助TLR4将LPS信号转入细胞内的过程中起到核心作用。本研究探讨MD-2在LPS所致大鼠ALI中的作用,以及RNA干扰沉默肺泡巨噬细胞MD-2基因对LPS/TLR4-MD-2/MyD88信号通路及细胞因子分泌的影响。方法1)采用尾静脉注射LPS方法诱导大鼠急性肺损伤模型,20只SD大鼠随机分为对照组和LPS组,测定肺组织湿/干重比值(W/D),肺组织标本石蜡切片常规HE染色观察形态学改变,通过支气管肺泡灌洗获取肺泡巨噬细胞,采用半定量RT-PCR、Western Blot以及免疫组化等方法分别检测肺组织标本和肺泡巨噬细胞MD-2 mRNA及蛋白的表达情况,采用ELISA方法测定血清TNF-α水平。2)体外培养大鼠肺泡巨噬细胞系NR8383,用以下两种方法进行实验：用不同浓度(0.01μg/ml-10μg/ml)的LPS刺激细胞2h；用单一浓度(1μg/ml)的LPS刺激细胞2h-24h。采用半定量RT-PCR方法检测细胞TLR4 mRNA和MD-2mRNA的表达,采用ELISA方法测定细胞培养上清液中TNF-α、IL-1β和IL-6水平。3)设计合成5对针对MD-2的小干扰RNA片段(MD-2 siRNA),运用脂质体Lipofectamine 2000将MD-2 siRNA转染至NR8383细胞,采用实时荧光定量PCR和Western Blot方法分别检测细胞MD-2 mRNA和蛋白的表达,筛选出最佳MD-2 siRNA序列进行下一步实验。采用以下两种方法对NR8383细胞进行LPS刺激：细胞先经LPS刺激2h,然后转染MD-2 siRNA,称之为LPS+1组；用MD-2 siRNA沉默细胞MD-2基因,然后给予LPS刺激2h,称之为LPS+2组。设置未转染NR8383细胞作为对照组(Blank)。采用实时荧光定量PCR方法分别检测细胞MD-2 mRNA、TLR4 mRNA和MyD88 mRNA的表达,采用Western Blot方法检测细胞MD-2蛋白的表达,采用ELISA方法分别测定细胞培养上清液中TNF-αIL-1β和IL-6的水平。结果1)与对照组比较,LPS组大鼠的肺组织W/D增大,肺泡巨噬细胞和肺组织标本中MD-2 mRNA和蛋白的表达均显著上调(P<0.01),血清TNF-α水平也明显升高(P<0.01)。2)大鼠肺泡巨噬细胞NR8383表达MD-2 mRNA和TLR4mRNA,其中对照组的相对表达量分别为0.52±0.05和0.44±0.09；在0.01μg/mlLPS的刺激下,两者表达无明显变化；在0.1μg/ml时表达量增加；随着LPS浓度的升高表达量进一步增加；在10μg/ml刺激下两者的相对表达量分别为0.72±0.06和0.65±0.10 (P<0.01)。TNF-α、IL-6和IL-1β的浓度具有类似变化。在1μtg/mlLPS刺激下,TLR4和MD-2 mRNA的表达量在2h明显增加,至6h达高峰(0.67±0.05和0.59±0.05),8h开始回落,但24h仍高于基础值(P<0.01)。TNF-α、IL-6和IL-1p的浓度具有类似变化,其中TNF-α和IL-1p在6h达高峰,持续至8h,IL-6在8h达高峰,持续至12 h。TLR4 mRNA和MD-2 mRNA表达呈正相关(r=0.513,P<0.01)。3)通过检测MD-2 mRNA和蛋白的表达,筛选出最佳MD-2 siRNA序列(正义链：5’-CCA UAU UUA CUG AAU CUG ATT-3’；反义链：5’-UCA GAU UCA GUA AAU AUG GGA-3’),在mRNA水平的最佳干扰时间为24h,基因沉默效率为67%。对照组细胞经LPS刺激后MD-2、TLR4和MyD88 mRNA的表达均显著上调,MD-2蛋白合成显著增加,细胞培养上清液中TNF-αIL-1β和IL-6水平均显著升高(P<0.01)。在LPS+1实验中,干扰组细胞经LPS刺激后MD-2 mRNA的表达量与LPS未刺激组比较未见明显上调(P>0.05)；而TLR4 mRNA和MyD88 mRNA的表达量显著上调(0.69±0.10和0.82±0.09 vs 0.44±0.12和0.62±0.06,P<0.05),细胞培养上清液TNF-α,IL-1p和IL-6水平均显著升高(P<0.01),但mRNA上调幅度和细胞因子水平升高幅度均小于对照组(P<0.05)。在LPS+2实验中,干扰组细胞MD-2 mRNA、TLR4 mRNA和MyD88 mRNA的表达量与LPS未刺激组比较均未见上调,细胞培养上清液TNF-α、IL-1β和IL-6水平亦未见升高(P>0.05)。结论MD-2在LPS所致的大鼠ALI中起重要作用。LPS刺激可引起大鼠肺泡巨噬细胞MD-2和TLR4 mRNA表达上调及细胞因子分泌增加。早期应用或预防应用RNAi沉默MD-2基因可阻断大鼠肺泡巨噬细胞LPS/TLR4-MD-2/MyD88信号通路,减少细胞因子的分泌。更多还原

【Abstract】 Knockdown MD-2 gene in alveolar macrophages with RNA interference and its effect on rat acute lung injury induced by lipopolysaccharideBackground Excessive amount of proinflammation cytokines and effector cells as the core of pulmonary inflammation lead to acute lung injury (ALI). There is no effective anti-flammation therapy because of the complex courses of inflammation. The mortality of ALI is still very high. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, has been recognized as an important cause of ALI. Toll like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) are the main LPS receptor complex. MD-2 is required in the course of LPS recognization and signal transmembrane transduction through TLR4. These signal transduction intermediary molecules in turn up-regulate inflammatory mediators and cytokines synthesis and also trigger preformed mediator release. Here we explore the expreeions of MD-2 in lung tissues of ALI rat induced by LPS, and the effect of knockdown MD-2 gene of alveolar macrophages with RNA interference on the upper-stream course of LPS/TLR4- MD-2 /MyD88 signal transduction and proinflammation cytokines secretion during ALI.Methods 1) A rat model of ALI was established by vena caudalis injection of LPS. 20 SD rats were randomly divided into control group and LPS group. The wet/dry ratios of lung tissue were measured and the histology changes of lung tissue were observed under microscope. Rat alveolar macrophages were collected form bronchial alveolar lavage fluid (BALF). The MD-2 mRNA and protein expressions were detected by semi-quantitative revels transcription polymerase (RT-PCR)、Western Blot and immunohistochemistry, respectively. The levels of TNF-a in serum were detected by ELISA.2) Rat alveolar macrophage cell NR8383 was cultured with F-12K medium and stimulated with LPS. The expressions of TLR4 mRNA and MD-2 mRNA in cells were detected by RT-PCR. The contents of TNF-α、IL-6 and IL-1βin the cell cultured supernatant were tested by ELISA.3) Five MD-2 siRNA oligos were transfected into NR8383 by Lipofectamine 2000. The gene expression of MD-2 was detected by realtime PCR. The synthesis of MD-2 protein was analyzed by Western blot analyze. The MD-2 siRNA with highest gene knockdown effect was screened and been used in the following experiments. The cells treated with MD-2 siRNA were stimulated with LPS by two different methods, LPS+1 and LPS+2. In method LPS+1, the cells were stimulated by LPS and then treated with MD-2 siRNA. In method LPS+2, the cells were treated with MD-2 siRNA and then stimulated by LPS. The siRNA untreated cells were used as control group. The gene expressions of MD-2、TLR4 and MyD88 were detected by realtime PCR. The synthesis of MD-2 protein was analyzed by Western blot. The levels of TNF-αI1-1βand IL-6 in cell supernatants were measured by ELISA.Results 1) Compared with the control group, W/D ratio and the expressions of MD-2 mRNA and protein of both lung tissue and alveolar macrophages in LPS group increased significantly. The serum level of TNF-αin LPS group was also higher than that in control group (P<0.05).2) Both the TLR4 and MD-2 mRNA expressed in rat alveolar macrophages cell NR8383. The mRNA expression of TLR4 and MD-2 in control group were 0.52±0.05 and 0.44±0.09, respectively. There was no obviously change after O.01μg/ml LPS stimulation. The increase occurred in a dose dependent manner from 0.1μg/ml to 10μg/ml. The highest TLR4 and MD-2 mRNA expression were 0.72±0.06 and 0.65±0.10 (P＜0.01). The changes of TNF-αIL-6 and IL-1βcontents in cell cultured supernatant were similar with that of TLR4 and MD-2 gene expression (P<0.01). When stimulated with 1μg/ml LPS, the mRNA expressions of TLR4 and MD-2 in NR8383 cell were increased from hour 2. The highest mRNA expressions of TLR4 and MD-2 occurred at hour 6, and then decreased slowly from hour 8. The mRNA expressions at hour 24 were still higher than those in cells without LPS stimulation (P＜0.01). The changes of TNF-α、IL-6 and IL-1βin cell cultured supernatant were also similar with that of gene expression (P<0.01). The secretion peaks of TNF-αand IL-1βoccurred from hour 6 to hour 8, and the secretion peak of IL-6 occurred from hour 8 to hour12. The mRNA expression of MD-2 was related with that of TLR4 positively (r=0.513, P＜0.01).3) A pair of MD-2 siRNA (Sense: 5’- CCA UAU UUA CUG AAU CUG ATT-3’; antisense:5’- UCA GAU UCA GUA AAU AUG GGA-3’) was affirmed to be effective by detecting the expressions of MD-2 mRNA and protein in NR8383 cell. The best work time was 24-hour and the gene knockdown effect was 67% in mRNA level. The expression of MD-2 mRNA and MD-2 protein increased obviosly after LPS stimulation in MD-2 siRNA untreated cells, as well as the expressions of TLR4 mRNA and MyD88 mRNA. After simulated with LPS in LPS+1 style, the expressions of MD-2 mRNA and MD-2 protein in MD-2 siRNA treated groups didn’t increase (P＞0.05). The expressions of TLR4 mRNA and MyD88 mRNA increased obviously(0.69±0.10 and 0.82±0.09 vs 0.44±0.12 and 0.62±0.06,P<0.05), as well as the levels of TNF-αIL-1βand IL-6 in cell supernatants. After simulated with LPS in LPS+2 style, the expressions of MD-2 mRNA、MyD88 mRNA and TLR4 mRNA in MD-2 siRNA treated cells didn’t increase, as well as the levels of TNF-αI1-1βand IL-6 in cell supernatants (P>0.05).Conclusion MD-2 may play an important role in rat acute lung injury induced by LPS. Stimulation by LPS can upregulate the expressions of MD-2 and TLR4 mRNA in rat alveolar macrophage cell NR8383 and increase pro-inflammation cytokines secretion. Knockdown MD-2 gene in NR8383 by RNAi can blockade LPS/TLR4-MD-2/MyD88 signal transduction, and then reduce the secretion of pro-inflammation cytokines.更多还原