【作者】 左云龙；

【导师】 匡凤梧；

【作者基本信息】 重庆医科大学 ， 儿科学， 2007， 博士

【摘要】 内毒素血症是由于血中细菌或病灶内细菌释放出大量内毒素至血液,或输入大量内毒素污染的液体而引起,引起炎症的激活及细胞因子的释放,致炎和抗炎失衡,导致全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)、多器官功能不全综合征(multipal organ disfunction syndrome,MODS)甚至多脏器功能衰竭(multipal organ failure,MOF),是儿科创伤和感染患者死亡的主要原因之一。目前在国内外针对内毒素血症的治疗措施有:常规抗生素使用、抗内毒素抗体、抗细胞因子抗体等免疫疗法、肠道屏障的保护等。尽管治疗措施不断更新进展,内毒素血症的支持、对症治疗已取得很大进步,但由于随着耐药菌的不断产生及对症支持、辅助疗法效果有限,内毒素血症依然是医务工作者久攻不克但又必须攻克的堡垒。防治内毒素血症的根本问题是如何有效的控制由内毒素血症诱导的炎症因子的级联放大的“瀑布”样效应。锌指蛋白A20是一种存在胞浆中的泛素修饰蛋白,针对其研究已成为抗炎、抗凋亡研究的热点问题。研究指出:不需要N端,仅含4个锌指结构的A20就可以得到与7个锌指结构的野生型A20同样的对NF-κB的抑制效果;也有研究指出A20通过其N端及C端的双泛素修饰功能抑制NF-κB的活化。A20是通过特定的信号通路抑制NF-κB活化,从而抑制炎症介质和细胞因子的释放,参与对机体炎症反应调节和凋亡调控。本研究旨在通过动物实验,在前期工作的基础上,从基因水平采用“基于流体动力学基因转染体方法”和脂质体介导,体外给予编码mA20及其突变体mA20-ZnF4-7的真核表达载体pCAGGS- flagmA20及pCAGGS-flagmA20ZnF4-7,以抑制内毒素血症时过多的炎症介质的产生,中断炎症的“瀑布”样效应,纠正炎性失衡,阻止内毒素血症的恶化的发展。该研究为mA20及其突变体的临床应用提供实验基础。同时本课题拟采用分子设计思想,对人类锌指蛋白hA20基因进一步设计:保留部分N端及完整的4-7个锌指结构,删除hA20中的第332位到第1778位基因,构建hA20突变体质粒pcDNA3-Flagh 20N-ZnF4-7;采用编码hA20及其突变体的真核表达载体pcDNA3- laghA20及pcDNA3-FlaghA 20N-ZnF4-7,通过细胞表达初步实验,验证其是否具有抗炎活性,为探索体外人工合成这种结构新型的具有抗炎作用的蛋白提供前期基础,为生产新型治疗内毒素血症的药物铺平道路。第一部分基于流体动力学转染mA20及其突变体对内毒素血症的治疗作用目的研究mA20及其突变体mA20-ZnF4-7质粒对脂多糖(LPS)攻击所致实验性内毒素血症的治疗作用。方法用LPS攻击小白鼠制备内毒血症动物模型。将实验动物随机分为:生理盐水组、单纯内毒素组、空质粒组、mA20质粒治疗组和mA20-ZnF4-7质粒治疗组。按10μg质粒:1.6ml生理盐水比例把质粒溶于生理盐水中,采用基于流体动力学基因转染方法对实验组小鼠进行质粒治疗性注射。以ELISA检测各组血浆TNF-α、IL-1β表达;以逆转录-聚合酶链式反应(RT-PCR)检测白细胞中mA20及其突变体mRNA的表达;12小时观察组织病理变化。结果(1) mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的TNF-α在各时间点的表达(pg/ml)分别为: 6h : (201.797±4.789)、(235.710±6.108) , 12h : (236.580±7.497)、(255.130±7.827) , 24h :(154.551±16.460)、(223.246±11.316) ;该两组之间无显著性差异(p>0.05);与单纯内毒素组、空质粒组的TNF-α表达比较有显著性降低(p<0.05)。mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的IL-1β在各时间点的表达(pg/ml)分别为:6h:(87.103±3.840)、(101.586±2.486),12h : (83.655±4.973)、(94.460±3.110) , 24h : (77.678±6.555) ,(87.103±4.826);该两组之间无显著性差异(p>0.05);与单纯内毒素组、空质粒组的IL-1β表达比较有显著性降低(p<0.05);各时间点之间比较无明显差别(p>0.05)。(2) mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的mA20及其突变体mRNA表达与空质粒组、单纯内毒素组、生理盐水组比较有显著性升高(p<0.01)。空质粒组、单纯内毒素组的mA20及其突变体mRNA表达与生理盐水组比较有显著性升高(p<0.05);各时间点之间比较无明显差别(p>0.05)。(3)病理学观察结果显示:mA20治疗组、mA20-ZnF4-7治疗组的肝、肺损伤均较单纯内毒素组、空质粒组轻;与生理盐水对照组相比,肝、肺损伤无明显差别。结论“基于流体动力学的基因转染方法”能有效地把目的质粒DNA转入肝、肺;对实验性类毒素血症小鼠体外转染mA20-ZnF4-7和mA20有相似的治疗作用,这为其临床应用提供一定的实验基础。第二部分基于流体动力学转染mA20及其突变体对内毒素血症的预防作用目的研究mA20及其突变体mA20-ZnF4-7质粒转染对LPS攻击所致实验性内毒素血症的预防作用。方法用LPS攻击小白鼠制备内毒血症动物模型。按10μg质粒:1.6ml生理盐水比例把质粒溶于生理盐水中,采用“基于流体动力学基因转染方法”对实验组小鼠进行质粒预防性注射。以ELISA检测各组血浆TNF-α、IL-1β表达;以逆转录-聚合酶链式反应(RT-PCR)检测白细胞中mA20及其突变体mRNA的表达;12小时观察组织病理变化。结果(1) mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的TNF-α在各时间点表达(pg/ml)分别为:6h:(211.942±9.133)、(163.536±0.502),12h:(195.130±26.318)、(206.446±34.797),24h:(182.963±14.527)、(158.029±19.176);与空质粒组、单纯内毒素组的TNF-α表达比较有显著性降低(p<0.05)。mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的IL-1β在各时间点表达(pg/ml)分别为: 6h : (137.908±4.487)、(138.598±8.733) , 12h : (137.678±3.539)、(137.448±7.953) , 24h :(129.862±11.602)、(126.873±6.942);与空质粒组、单纯内毒素组的IL-1β表达比较有显著性降低(p<0.05);各时间点之间无显著性差异(p>0.05);(2) mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的mA20及其突变体mRNA表达与空质粒组、单纯内毒素组、生理盐水组比较有显著性升高。空质粒组、单纯内毒素组的mA20及其突变体mRNA表达与生理盐水组比较有显著性升高;各时间点之间无显著性差异(p>0.05);(3)病理观察结果显示,mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的肝、肺损伤均较空质粒组、单纯内毒素组轻;与生理盐水组相比,肝、肺损伤无明显差别。结论“基于血流动力学的基因转染方法”能有效果地把目的质粒DNA转入肝、肺,对实验性内毒素血症小鼠体外转染mA20-ZnF4-7和mA20有相似的预防作用,这也为临床应用进一步提供一定的实验基础。第三部分脂质体包裹mA20及其突变体对内毒素血症的治疗作用目的研究脂质体包裹A20及其突变体mA20-ZnF4-7质粒对LPS攻击所致实验性内毒素血症的治疗作用。方法用LPS攻击小白鼠制备内毒素血症动物模型。随机分为:生理盐水对照组(NS组)、单纯内毒素组、空质粒组、脂质体包裹mA20质粒治疗组(L-mA20组)、脂质体包裹mA20?质粒治疗组(L-mA20?组)。采用旋转蒸发法制备脂质体,并按200μl脂质体:10μg质粒的比例作为每只小鼠的使用量。采用尾静脉注射脂质体包裹质粒的溶液,提取血浆、分离白细胞、留取肝肺;以ELISA检测各组血浆TNF-α、IL-1β表达;以逆转录-聚合酶链式反应(RT-PCR)检测白细胞中mA20极其突变体mRNA的表达;并于12小时点观察组织病理变化。结果(1) L-mA20组和L-mA20?组的TNF-α在各时间点表达(pg/ml)分别为: 6h : (304.696±6.087)、(304.985±6.291) , 12h :(321.217±10.579)、(256.580±33.524) , 24h : (307.594±11.181)、(280.348±11.698);与单纯内毒素组、空质粒组的TNF-α表达比较有显著性降低(p<0.05)。L-mA20组和L-mA20?组的IL-1β在各时间点表达(pg/ml)分别为: 6h : (256.989±3.921 )、(263.655±5.645), 12h :(205.724±4.827)、(154.690±1.380),24h:(165.724±7.772)、(101.816±3.259);依时间有显著性下降趋势;与单纯内毒素组、空质粒组的IL-1β表达比较有显著性降低(p<0.05);L-mA20?组24h点的IL-1β与6h点比较有显著性降低(p<0.05);(2) mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的mA20及其突变体mRNA表达与空质粒组、单纯内毒素组、生理盐水组比较有显著性升高。空质粒组、单纯内毒素组的mA20及其突变体mRNA表达与生理盐水组比较有显著性升高;(3)病理观察结果显示:L-mA20组和L-mA20?组的肝、肺损伤均较单纯内毒素组、空质粒组轻;与NS相比,肝、肺损伤无明显差别。结论尾静脉注射脂质体包裹mA20及其突变体mA20-ZnF4-7质粒对内毒素血症小鼠有相似的治疗作用,为临床用脂质体包裹质粒DNA治疗内毒素血症作好铺垫,为临床应用结构更简单的A20蛋白提供了一定实验基础。第四部分人锌指蛋白A20基因突变提构建及真核表达初步研究目的采用分子设计软件设计并构建人锌指蛋白A20基因突变体,转染小鼠RAW264.7真核表达细胞,检测其对LPS刺激的RAW264.7细胞的抗炎活性。方法采用分子设计软件对hA20基因进行分析,把第332位到第1778位基因剔除。使用核酸内切酶AccⅢ酶切真核表达质粒pcDNA3- FlaghA20,使其成线性后。再采用DNA连接酶对质粒进行自连反应,将重组子转染JM109,挑取菌落,用PCR和酶切方法鉴定真假阳性克隆。使用小鼠RAW264.7巨噬细胞作为研究对象,并完全随机分为:单纯LPS刺激组、hA20全长质粒组和hA20N-ZnF4-7质粒组。应用Lipofectamine2000真核转染系统转染LPS刺激的RAW264.7细胞,用ELISA检测细胞培养上清的TNF-α、IL-1β表达变化。结果通过PCR、酶切可见部分克隆片段只为923bp,即全长hA20经AccⅢ酶切后的片段;送检测序,显示构建成功;RAW264.7细胞培养上清的TNF-α、IL-1β表达(pg/ml)分别为:单纯LPS刺激组:(250.10±60.18)、(221.33±30.02);hA20全长质粒组:(120.87±33.75)、(69.21±13.18);hA20N-ZnF4-7质粒组:(116.51±22.82)、(63.45±12.73)。结论:我们成功的构建了hA20突变体质粒pcDNA3.0- Flag-hA20N-ZnF4-7,其对LPS攻击的RAW264.7细胞有明显的抗炎活性,为后续的进一步真核表达研究作好铺垫。更多还原

【Abstract】 Endotoxemia is the presence of endotoxins in the blood which derived from gram-negative rod-shaped bacteria in the blood or in focal infection or from lipopolysaccharide(LPS)-contaminant fluid infusion. The endotoxins in blood may lead to inflammatory factors actvation and cytokines release, inducing inflammation and anti-inflammation unbalance, resulting in systemic inflammatory response syndrome(SIRS), multi-organ disfunction syndrome ( MODS) and even multi-organ failure(MOF). Endotoxemia is the one of the major cause resulting in the death of pediatrics trauma and infectious diseases. At present, the therapeutic measures directed to endotoxemia include: antibiotics administration, immunotherapy of antiendotoxin antibodies and anticytokin antibodies, intestinal tract barria protection and so on so forth in domestic and abroard. Even though therapeutic measures continue to be up-to-dated and much improvement of support and symptomatic treatment of endotoxemia have been made, endotoxemia is still a stronghold to overcome for medical workers. The fundamental measures of endotoxemia treatment is to effectively control the waterfall-like effect of endotoxemia induced cytokins cascade amplification.Zinc finger protein A20 is a ubiquitin-editing protein that present in the endochylema. It has been already a hot topic of anti-inflammatory and anti-apoptotic research. A study showed that the inhibition effect on TNF-induced nuclear factor-κB(NF-κB) activation of A20 protein with only four zinc fingers was compatible to that of wild-type A20 protein with 7 zinc fingers. Studies showed no strict requirement for a particular zinc finger structure, since the first four zinc fingers mutant A20 protein is as potent as the last four zinc fingers mutant protein. Another study showed that A20 inhibited NF-κB activation by dual ubiquitin-editing (de-ubiquitination and ubiquitination) of its N-terminal end and C-terminal end. Zinc finger protein A20 inhibits NF-κB activation through specific signal pathways, then suppress the inflammatory mediators and cytokines releasing , and participates in the regulation of inflammatory reaction and cell apoptosis.The aim of this study is to provide an animal experiment data for clinical application through inhibiting excess endotoxemia-induced inflammatory mediators, terminating“waterfall-like”effect of cascade amplification of inflammation, correcting inflammatory reaction imbalance, and preventing endotoxemia from aggravated development by eukaryotic expression vector pCAGGS-flagmA20 and pCAGGS-flagmA20-ZnF4-7 coding mA20 and its mutant mA20-ZnF4-7 in gene level. To explore the feasibility of artificial synthesizing this neotype anti-inflammatory protein, we are planning to apply the idea of molecular designing to make a further design for A20. Successful construction of hA20 mutant plasmid pcDNA3Flag-hA20N-ZnF4-7 may achieved by protecting large portion of N-terminal end and whole C-terminal end, and deleting the gene between number 332 and 1778, and may identified through cells expression of lipsome-mediated pcDNA3hA20 and pcDNA3-FlaghA20N-ZnF4-7. It is prospected to pave a nicely path for producing anti-endotoxemia medicine.PARTⅠEFFECT OF HYDRODYNAMICS-BASED DELIVERY OF MA20 AND ITS MUTANTS PLASMID ON EXPERIMENTAL ENDOTOXEMIA IN MICEObjective To study the therapeutic effect of mA20 and its mutant mA20-ZnF4-7 plasmid in endotoxemia mice of lipopolysaccharide (LPS)- assaulting.Methods The endotoxemia animal model was established by injection of 2.5mg/kg LPS via the tail vein. The animals were randomly divided into 5 groups: group A: normal saline(NS) control, group B: LPS-assaulting, group C: naked plasmid treatment, group D: mA20 plasmid-treatment, group E: mA20-ZnF4-7 plasmid-treatment. Hydro- dynamics-based gene transfection method was achieved by intravenous injection of 10μg plasmid DNA in 1.6ml saline 30 minutes after LPS injection. The contents of plasma TNF-αand IL-1βwere detected by ELISA. The contents of WBC mA20 and its mutant mA20-ZnF4-7 mRNA were detected by Reverse transcription and polymerase chain reaction (RT-PCR). The tissue inflammatory histopathologic changes were observed under microscope.Results (1) The TNF-αexpressions of mA20 and mA20-ZnF4-7 in plasmid-treatment group at the time point of 6h, 12h and 24h were(pg/ml): (201.797±4.789), (236.580±7.497), (154.551±16.460) and(235.710±6.108), (255.130±7.827), (223.246±11.316) respectively, which were significantly lower than those in LPS- assaulting group and naked plasmid group (p<0.05). The IL-1βexpressions of mA20 and mA20-ZnF4-7 in plasmid-treatment group at the time point of 6h, 12h, 24h were(pg/ml): (87.103±3.84), (83.655±4.973), (77.678±6.555 and (101.586±2.486), (94.460±3.110), (87.103±4.826) respectively, which were significantly lower than those in LPS-assaulting group and naked plasmid group (p<0.05). (2) The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in mA20 plasmid-treatment group and mA20-ZnF4-7 plasmid-treatment group were significantly higher than those in LPS-assaulting group, naked plasmid group and normal saline group (p<0.05). The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in LPS-assaulting group and naked plasmid group were significantly higher than those values in normal saline group (p<0.05). No significant difference was found between every time point.(3) Pathological changes under microscope showed that the inflammatory damages in mA20 plasmid-treatment group and in mA20-ZnF4-7 plasmid-treatment group were lighter than those in LPS-assaulting group and naked plasmid group. No significant differences were found between mA20 plasmid-treatment group, mA20-ZnF4-7 plasmid- treatment group and NS group.Conclusions Hydrodynamics-based gene transfection can efficiently deliver plasmid DNA into livers and lungs. Transfection of mA20-ZnF4-7 plasmid showed similar therapeutic effect to mA20 plasmid on experimental endotoxemia in mice, which may provide valuable experimental base for clinical application. PARTⅡPREVENTIIVE EFFECT OF EXPERIMENTAL ENDOTOXEMIA BY HYDROD-YNAMICS-BASED DELIVERY OF MA20 AND ITS MUTANTS PLASMIDObjective To study the preventive effect of zinc finger protein mA20 and its mutant mA20-ZnF4-7 on endotoxemia mice of lipopolysaccharide (LPS)-assaulting.Methods The endotoxemia animal model was established by injection of 2.5 mg/kg LPS via the tail vein. The animals were randomly divided into 5 groups: group A: normal saline(NS) control, group B: LPS-assaulting, group C: naked plasmid treatment, group D: mA20 plasmid-prevention, group E: mA20-ZnF4-7 plasmid-prevention. Hydro- dynamics-based gene transfection method was achieved by intravenous injection of 10μg plasmid DNA in 1.6ml saline. The contents of plasma TNF-αand IL-1βwere detected by ELISA. The contents of WBC mA20 and its mutant mA20-ZnF4-7 mRNA were detected by RT-PCR. The tissue inflammatory histopathologic changes were observed under microscope.Results (1) The TNF-αexpressions in group D and group E at the time point of 6h, 12h, and 24h were(pg/ml): (201.797±4.789), (236.580±7.497), (154.551±16.460),and(235.710±6.108), (255.130±7.827), 223.246±11.316, respectively, which were significantly lower than those in group B and group C. The IL-1βexpressions in group D and group E at the time point of 6h, 12h, and 24h were(pg/ml): (87.103±3.84), (83.655±4.973), (77.678±6.555), and (101.586±2.486), (94.460±3.110), (87.103±4.826)respectively, which were significantly lower than those in group B and group C. (2) The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in group D and group E were significantly higher than those in group A, group B and group C (p<0.05). The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in group B and group C were significantly higher than those in normal saline group(p<0.05). No significant difference was found between each time point. (3) Microscope observation of pathological section The inflammatory damage under microscope showed milder changes in group D and group E than those in group B and group C. No significant differences were found between group D, group E and group A.Conclusions (1) Hydrodynamics-based gene transfection is a simple, convenient, economic and efficient method in delivering plasmid DNA into liver and lung. (2) Transfection of mA20-ZnF4-7 plasmid in vitro shows similar preventive effect to mA20 plasmid on experimental endotoxemia in mice, which may provide valuable experimental base for clinical application. PARTⅢTHEAPEUTIC EFFICACY OF LIPOSOME ENCAPSULATING MA20 AND ITSMUTANTS PLASMID TRANSFECTION IN EXPERIMENTAL ENDOTOXEMIA IN MICEObjective To study the therapeutic effect of liposome encapsulating mA20 and its mutant mA20-ZnF4-7 plasmid in mice with lipopolysaccharides (LPS) induced endotoxemia.Methods Forty-five healthy mice weighing 18-20g were randomized into five groups: normal saline control group (NS group), LPS-assaulting group, naked plasmid treatment group, liposome encap-sulating mA20 plasmid treatment group (L-mA20 group) and liposome encapsulating mA20-ZnF4-7 plasmid treatment group (L-mA20? group). The contents of plasma TNF-αand IL-1βwere detected by ELISA. The contents of WBC mA20 and its mutant mA20-ZnF4-7mRNA were detected by RT-PCR. The tissue inflammatory histopathologic changes were observed under microscope.Results (1)The TNF-αexpressions of L-mA20 and L-mA20? group at the time point of 6h, 12h and 24h were(pg/ml): (304.696±6.087), (321.217±10.579), (307.594±11.181) and (304.985±6.291), (256.580± 33.524), (280.348±11.698)respectively, which were significantly lower than those in LPS-assulting group and in naked plasmid treatment group (p<0.05). The IL-1βexpressions in L-mA20 treatment group and in L-mA20? treatment group at the time point of 6h, 12h, 24h were(pg/ml): (256.989±3.921), (205.724±4.827), (165.724±7.772) and (263.655±5.645), (154.690±1.380), (101.816±3.259) respectively, which were significantly lower than those in LPS-assulting group and in naked plasmid treatment group (p<0.05). (2) The expressions of mA20 and its mutant mA20-ZnF4-7 mRNA in mA20 plasmid-treatment group and mA20- ZnF4-7 plasmid-treatment group were significantly higher than those in LPS-assulting group, naked plasmid group and normal saline group (p<0.05). The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in LPS-assulting group and in naked plasmid group were significantly higher than those in normal saline group (p<0.05). No significant differences were found between each time point. (3) Severe inflammatory damages were found in LPS-assulting group and naked plasmid treatment group, but only mild inflammatory changes were found in L-mA20 treatment group and in L-mA20? treatment group. There were no significant histopathological differences among groups of L-mA20 treatment group, L-mA20? treatment group and NS group.Conclusions mA20-ZnF4-7 plasmid transfection with liposome in vitro can achieve similar therapeutic efficacy to mA20 plasmid on experimental endotoxemia, which may provide valuable experimental base for clinical application of more simple structure A20 protein.PARTⅣMUTANT CONSTRUCTION OF HUMAN ZINC FINGER PROTEIN A20 AND PRIMARY STUDY OF EUKARYOTIC EXPRESSIONObjectives: to design and construct human zinc finger protein A20 gene mutants plasmid by molecular designing software, to transfect human zinc finger protein A20 gene mutants plasmid into mouse RAW264.7 macrophages and to detect their anti-inflammatory activities in mouse RAW264.7 macrophages stimulated by LPS.Methods: Human zinc finger protein A20 gene was identified by sequencing. The hA20 gene were analyzed by molecular designing software and the gene fragment between no. 332 to no. 1778 were deleted. The eukaryotic expression plasmid pcDNA3.0-Flag hA20 was digested linearitely by Acc III nucleate endonuclease. Then, a coupled reaction for this linearity fragment was made by DNA ligase. The recon was transfected into JM109 overnight. The real positive clone was identified from false clone by PCR and enzyme cutting. mouse RAW264.7 macrophages were randomized into three groups: LPS-treatment group, hA20 plasmid group and hA20N-ZnF4-7 plasmid group.The LPS-stimulated RAW264.7 macrophages were transfected through Lipofectamine 2000 eukaryotic transfection system. The contents of TNF-αand IL-1βin the cell culture supernatant were detected by ELISA.Results: The sequence of human zinc finger protein A20 gene was identified to be correct by sequencing. The electropherogram of PCR confirmed that fragment length deleted was 915bp. The expressions of TNF-α, IL-1βof mouse RAW264.7 cell culture supernatant in LPS- assaulting group, hA20 plasmid-treatment group and hA20N-ZnF4-7 plasmid-treatment group respectively were: 250.10±60.18, 221.33±30.02; 120.87±33.75, 69.21±13.18 and 116.51±22.82, 63.45±12.73.Conclusions: We have successfully constructed hA20 mutant plasmid, pcDNA3.0-Fflag-hA20N-ZnF4-7 which posses significant anti-inflam- matory effects on LPS-assulting mouse RAW264.7 macrophages. The Results provide valuable base in future eukaryotic expression research.更多还原