【作者】 王静；

【导师】 陈蔚文；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 研究背景和目的中医理论认为脾主运化水谷精微和水湿,脾主统血,脾主升清,为气血生化之源、后天之本,脾主思,脾主四肢肌肉,开窍于口,其华在唇等。由此看出,脾的生理功能所涉及的广泛性,决定了它的病理也不会局限于一病一症一脏器。同时,脾与五脏六腑的关系,“内伤脾胃,百病由生”等,说明脾胃损伤会引起很多疾病的发生,脾病会引起很多脏器系统的病变,而很多疾病、脏器病变也会引起脾病的发生。从脾虚证入手进行脾本质的研究,长期以来一直是中西医结合证本质研究的重要课题。众多学者运用多种现代技术和方法,分别从生化、生理、超微结构等方面进行探讨,近年更有人从神经—内分泌—免疫网络方面来探讨脾虚证的实质,对其进行了各种客观指标的研究,取得了一定的成果。然而,这些研究都只反映了部分已知基因表达产物(受体、酶、细胞因子以及肽类等)的改变,远未揭示证的实质。基因组及后基因组时代的到来,为从基因水平切入证候的研究提供了新的思路与方法。多数学者认为,基因组学在整体观、阴阳学说、五脏学说、病因学说等方面与中医学具有相似性,中医“证”的内涵即基因表达的改变,因此提出了中医证候基因组学的概念。目前,主要是采用基因芯片技术分析证候的差异基因表达谱,来进行中医证候基因组学的研究,具有代表性的是关于脾虚证、肾阳虚证、虚寒证、心阳虚证、肺虚证等的研究。我们实验室前期先后对慢性浅表性胃炎脾气虚证者与正常志愿者、慢性浅表性胃炎脾气虚证患者与慢性浅表性胃炎脾胃湿热证患者、溃疡性结肠炎脾气虚证患者与正常志愿者、溃疡性结肠炎脾气虚证患者与溃疡性结肠炎脾胃湿热证患者进行基因表达谱的分析。证实了慢性浅表性胃炎脾气虚证者,具有差异表达基因,其中蛋白合成相关的差异基因大多下调;慢性浅表性胃炎脾气虚证患者与慢性浅表性胃炎湿热证患者比较,相关差异基因也表达下调,这与脾气虚时机体功能抑制,而湿热时机体功能亢进相符合;进行扩大样本量重复分析或进行同证异病分析时,趋势相同。通过对这些海量数据进行生物信息学的分析整合,我们认为脾气虚证具有蛋白质合成相关基因下调的趋势,其中以核糖体蛋白基因下调比较显著。核糖体蛋白基因是核糖体的重要组成部分,起到组装核糖体,促进蛋白质合成的作用。核糖体蛋白高度保守,但核糖体蛋白基因发生突变或缺失时,可以产生存活且异常的表型。核糖体蛋白基因突变或核糖体蛋白缺失或不恰当的化学修饰,一方面将影响核糖体的功能,降低多肽合成的活性,另一方面会影响RP(ribosomal protein)的生理功能,从而导致各种严重的后果。我们选择重复性表达下调的RPS20(ribosomal protein S20)对其进行生物功能鉴定。实验以大鼠小肠隐窝细胞株(Intestinal crypt cell line,IEC-6)为模型,采用RNAi(RNA interference)技术干扰RPS20,造成RPS20缺失表型,观察对细胞生理功能的影响,以初步探讨脾气虚证相关基因RPS20的生物功能。研究方法1.IEC-6细胞生长状况的观察采用相差显微镜技术观察细胞形态结构;台盼蓝排斥试验检测细胞活力;细胞计数法及MTT比色法绘制IEC-6细胞生长曲线。2.RNA干扰过程中IEC-6细胞转染条件的优化以24孔板为例,采用阳离子脂质体转染FAM-siRNA片段进入IEC-6细胞内,通过调整FAM-siRNA和Lipofectamine2000的剂量,分别采用荧光显微镜和流式细胞仪两种仪器检测转染后荧光强度。3.筛选针对RPS20基因的有效干扰片段3.1 RT-PCR检测RNAi后IEC-6细胞RPS20 mRNA的表达设置空白对照组、siRNA1组、siRNA2组、siRNA3组,6孔板每孔种植8×10~5个细胞,第2日转染时,按照每孔转染总体积2.5mL、20μM浓度的siRNA20μL(即siRNA终浓度160nM)、Lipofectamine2000 4.2μL的条件进行转染,然后采用RT-PCR检测干扰后24h、48h、72h后RPS20 mRNA表达。3.2荧光定量RT-PCR检测RNAi后IEC-6细胞RPS20 mRNA的表达设置空白对照组、转染试剂对照组、阴性对照组、siRNA1组、siRNA2组、siRNA3组,转染条件及方法同3.1,然后采用实时荧光定量RT-PCR检测干扰48h后RPS20mRNA表达。3.3 Western-Blot检测RNAi后IEC-6细胞RPS20蛋白的表达设置空白对照组、转染试剂对照组、阴性对照组、siRNA1组、siRNA2组、siRNA3组,转染条件及方法同3.1,然后采用Western-Blot检测干扰后24h、48h、72h,RPS20蛋白的表达。4.RNAi后IEC-6细胞形态功能的改变4.1 RNAi后IEC-6细胞形态的变化转染条件及方法同3.1,设置空白对照组、转染试剂对照组、siRNA1组、siRNA2组、siRNA3组,采用相差显微镜观察细胞的大体形态;设置空白对照组、转染试剂对照组、阴性对照组、siRNA1组、siRNA2组、siRNA3组,采用透射电镜观察干扰RPS20基因48h后,IEC-6细胞超微结构的变化。4.2 RNAi后对IEC-6细胞迁移能力的影响设置空白对照组、转染试剂对照组、阴性对照组、siRNA1组、siRNA2组、siRNA3组,转染条件及方法同3.1,转染后48h划痕法建立细胞迁移模型,划痕24h后,拍照,计算细胞迁移的数目,以观察干扰RPS20后,细胞迁移能力的变化。4.3 RNAi后IEC-6细胞增殖效率的变化设置空白对照组、siRNA1组、siRNA2组、siRNA3组,转染条件及方法同3.1,采用细胞计数法计数干扰后24h、48h、72h IEC-6细胞数目的变化;设置空白对照组、转染试剂对照组、siRNA1组、siRNA2组、siRNA3组,96孔板每孔种植2.5×10~4个细胞,第2日转染时,按照每孔转染总体积0.6mL、20pM浓度的siRNA20μL(即siRNA终浓度160nM)、Lipofectamine2000 4.2μL的条件进行转染,MTT法观察干扰RPS20后24h、48h、72h IEC-6细胞增殖效率的变化。4.4 RNAi后对IEC-6细胞凋亡的影响设置空白对照组、转染试剂对照组、阴性对照组、siRNA1组、siRNA2组、siRNA3组,转染条件及方法同2,采用Anexin V—FITC检测方法,观察干扰RPS20 48h后诱导IEC-6细胞凋亡的情况。结果1.IEC-6细胞生长状况的观察引进的IEC-6细胞具有典型的正常肠上皮隐窝细胞的形态特征;具有较高的生存活力;细胞在第3天时进入对数生长期,在第7～10天时达到平台期,之后又继续缓慢增长。2.RNA干扰过程中IEC-6细胞转染条件的优化荧光显微镜检测转染效率,以评分进行秩和检验:结果仅有转染试剂或仅有FAM-siRNA,或两者均无的组别,评分均为0;转染试剂加FAM-siRNA组别均有或强或弱的荧光表达。流式细胞仪检测转染效率,以Lipofectamine2000 1.0μL+FAM-siRNA 160nM组转染效率最高(平均76.3%);而空白对照组约为1%。3.筛选针对RPS20基因的有效干扰片段3.1 RT-PCR检测干扰后RPS20 mRNA的表达在干扰后24h,与空白组比较,siRNA1组RPS20的mRNA表达明显减少,差异具有显著性意义(P＜0.05),而siRNA2、siRNA3组都没有明显抑制其表达;在干扰后48h、72h,siRNA1、siRNA2、siRNA3组,都没有明显抑制RPS20 mRNA表达的趋势。3.2荧光定量RT-PCR检测干扰后RPS20 mRNA的表达相对于空白对照组来说,siRNA1组、siRNA2组、siRNA3组三组都在转染48h后成功地干扰了RPS20基因的表达,干扰效率在80%-90%左右。3.3 Western-Blot检测干扰后RPS20蛋白的表达与空白对照组比较,干扰后24h,siRNA1、siRNA2组蛋白表达量均降低,且差异具有显著性意义(P＜0.028,P＜0.049),siRNA3组蛋白表达量也趋向降低,但并不具有显著性差异(P＜0.065);与空白对照组比较,干扰后48h,siRNA2、siRNA3组蛋白表达量均降低,且差异具有显著性意义(P＜0.028,P＜0.006),siRNA1组蛋白表达量也趋向降低,但并不具有显著性差异(P＜0.619);与空白对照组比较,干扰后72h,siRNA2、siRNA3组蛋白表达量均降低,且差异具有显著性意义(P＜0.019,P＜0.015),siRNA1组蛋白表达量也趋向降低,但并不具有显著性差异(P＜0.056)。4.RNAi后IEC-6细胞形态功能的改变4.1 RNAi后IEC-6细胞形态的变化相差显微镜下观察,与空白组比较,siRNA1组、siRNA2组、siRNA3组三组干扰序列细胞数都有所减少,但细胞形态并没有明显改变。透射电镜检测干扰后48h细胞超微结构的变化,发现:空白对照组IEC-6细胞膜、细胞核完整,细胞器丰富;转染试剂对照组及阴性对照组,细胞核都比较完整,有少许空泡及脂肪颗粒;siRNA1组、siRNA2组、siRNA3组细胞膜不完整,胞浆内有大量空泡出现,溶酶体增加,siRNA2组、siRNA3组空泡内还有疑似高电子致密度内容物的生成;细胞器较少,部分细胞呈现细胞器模糊。4.2 RNAi后对IEC-6细胞迁移能力的影响与空白组比较,siRNA1组、siRNA2组、siRNA3组都能在干扰后48h抑制细胞的迁移(P＜0.01,P＜0.01,P＜0.01)。4.3 RNAi后IEC-6细胞增殖效率的变化细胞计数法:与空白对照组比较,转染后24h、48h、72h,siRNA1组、siRNA2组、siRNA3组都能明显抑制IEC-6细胞生长的数目(P＜0.01)。MTT法:与空白对照组比较,转染后24h,siRNA2组、siRNA3组都能明显地抑制细胞增殖(P＜0.05);转染后48h,siRNA2组能抑制细胞的增殖(P＜0.05);转染后72h,siRNA2组、siRNA3组能抑制细胞的增殖(P＜0.01,P＜0.05)。4.4 RNAi后对IEC-6细胞凋亡的影响与空白对照组比较,siRNA1组活力细胞减少,差异具有显著性意义(P＜0.05);siRNA1组早期凋亡细胞有所增多,但差异没有显著性意义;各组正常死亡细胞及晚期凋亡细胞比较,差异都没有显著性意义。结论1.IEC-6细胞生长曲线在第10后,从平台期又进入了缓慢增长阶段,由于在连续培养IEC-6细胞10天后,细胞会呈现分化状态,分析缓慢增长可能与分化有关;2.本课题设计并合成了3对针对大鼠RPS20的siRNAs,而且都能够有效地抑制IEC-6细胞RPS20mRNA与蛋白的表达,表明了序列的有效性;3 IEC-6细胞胞浆空泡化,并且迁移、增殖能力受到抑制,但并没有导致细胞早期凋亡的发生,该结果提示干扰RPS20可能影响IEC-6细胞的消化吸收功能和胃肠粘膜损伤修复功能,与前期收集的脾气虚证患者消化吸收功能下降、黏膜损伤修复能力下降等表现相符合。本结果支持由临床研究基因表达谱归纳出的特点,支持脾气虚证本质与RP相关的假说。更多还原

【Abstract】 Background and Objective:In Chinese Medicine,the physical function of Spleen involves many aspects:governs transport and transformation of nutrients from foodstuff,controls blood,is in charge of sending up essential substance,is the source of generating Qi and blood,provides the material basis for the acquired constitution,is in charge of thoughts,is responsible for the growth of muscles and four limbs,and mouth is the window of spleen,lip is the reflection of splenic conditions.Above its function determine that its pathology doesn’t involve one kind of disease or one kind of syndrome or one organ.Meanwhile,the relation between of spleen and other organs,indicates if hurting spleen and stomach in the body,every kind of diseases would onset,and vice versa.To study on the essence of spleen from spleen deficiency syndrome,always is an important topic to investigate the essence of syndrome with method of integrated traditional Chinese and western medicine.Many scholars approach the essence from biochemistry,physiology, ultra structure and so on by lots of modern technology,even someone go on research from the net of nerve-endocrine secretion-immunity recently,and required several outcome. However,these researches reflect some change of known gene expression product(such as receptor,enzyme,cytokine,and peptide),these keep away from the essence of syndrome. Genome era and post-genome era have been coming,this provides new method to study syndrome from gene.Most scholars presume that there are similarities between Chinese Medicine on holistic approach,yin-yang theory,theory of five organs,etiological theory and genome;they think that the connotation of syndrome is change of gene expression,so they put forward definition of Chinese Medicine syndrome genome.At present,scholars mostly analyze the spectrum of differentially expressed genes with cDNA chip technology to go on research of Chinese Medicine syndrome genome.The representative syndromes are kidney-yang deficiency syndrome,deficiency-cold syndrome,heart-yang deficiency syndrome,lung deficiency syndrome,spleen deficiency syndrome,and so on. We researched the spectrum of differentially expressed genes before on gastritis patients with spleen-qi deficiency syndrome and normal volunteers,chronic superficial gastritis patients with spleen-qi deficiency syndrome and chronic superficial gastritis patients with damp-heat syndrome,ulcerative colitis patients with spleen-qi deficiency syndrome and normal volunteers,ulcerative colitis patients with spleen-qi deficiency syndrome and ulcerative colitis patients with damp-heat syndrome.Our results prove that there are differentially expressed genes on chronic superficial gastritis patients with spleen-qi deficiency syndrome,and most of the genes are down regulation;compared with chronic superficial patients with damp-heat syndrome,differentially expressed genes of chronic superficial gastritis patients with spleen-qi deficiency syndrome are also down regulation, this status consistent with condition which body function behaves repressed under the condition of spleen-qi deficiency,and excited under the condition of damp-heat syndrome. The tendency is same when we made repeated analysis.After analysis lots of data by bioinformatics,we presume that spleen-qi deficiency syndrome has tendency of genes related protein synthesis to down regulation,especially ribosomal protein genes.Ribosomal protein constitutes ribosome,and plays an act to promote protein synthesis.Although ribosomal protein is highly conserved,it will produce live but abnormal phenotype when ribosomal protein gene mutable or absence or unfit chemical modification,this will impact ribosome’s function,lower activity of polypeptide, and impact physical function of ribosomal protein,lead to serious results.We choose RPS20,which expressed down regulation repeatedly,to identify its function.Our experiment observes effect on function of IEC-6 cells,after interference RPS20,in order to detect its function.Methods:1.Observation to growth conditions of IEC-6 cellsObserve cell morphology by phase contrast microscope;detect cell viability with trypan blue;draw growth curve by viable cell counts and MTT colorimetric assay.2.Optimization of transfect conditions of IEC-6 cells in the process of RNA interference For example,in 24 well plates,adjust the ratio between cationic liposome and FAM-siRNA, transfect FAM-siRNA into IEC-6 cells with cationic liposome,then detect fluorescence intensity after interference by fluorescence microscope and flow cytometry.3.Screen the best efficient siRNA to RPS203.1 Detect the expression of RPS20 mRNA by RT-PCRSet blank group,group siRNA1,group siRNA2,group siRNA3.As 6 well plate for an example,plant 8×10~5 cells every well,on the second day,transfect following this condition: whole transfection volume is 2.5mL,final concentration of siRNA is 160nM,the volume of Lipofectamine2000 is 4.2μL.Then detect the expression of RPS20 mRNA after transfection 24h,48h,72h by RT-PCR.3.2 Detect the expression of RPS20 mRNA by fluorescence quantitative RT-PCR Set blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1,Then detect the expression of RPS20 mRNA by fluorescence quantitative RT-PCR after transfection 48h.3.3 Detect the expression of RPS20 protein by western-blotSet blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1.Then detect the expression of RPS20 protein by western-blot after transfection 24h,48h,72h.4.Change of cellular morphology and function after RNA interference4.1 Change of cells morphology after RNA interferenceSet blank group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1.Observe cell morphology by phase contrast microscope after interference 24h, 48h,72h.Set blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Observe cell ultra structure with transmission electron microscope after interference 48h.4.2 Effect on migration of IEC-6 cells after RNA interferenceSet blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 3.1.Establish migration model by scratch after transfection 48h,and after 24h,take five pictures per well,and count migrated cells by IPP software to observe change of cellular migration ability.4.3 Effect on proliferation of IEC-6 cells after RNA interferenceSet blank group,group siRNA1,group siRNA2,group siRNA3.Count cell number after interference 24h,48h,72h by cell counting.Set blank group,transfection control group,group siRNA1,group siRNA2,group siRNA3. Transfection conditions are same to 3.1,As 96 well plate for an example,plant 2.5×10~4cells every well,on the second day,transfect following this condition:whole transfection volume is 0.15mL、final concentration of siRNA is 160nM,the volume of Lipofectamine2000 is 4.2μL.Then detect the proliferation efficiency of cells after transfection 24h,48h,72h by MTT colorimetric assay.4.4 Effect on apoptosis of IEC-6 cells after RNA interference 48h Set blank group,transfection control group,negative control group,group siRNA1,group siRNA2,group siRNA3.Transfection conditions are same to 2,observe effect on apoptosis of IEC-6 cells after RNA interference 48h by Annexin V-FITC.Results1.Observation to growth conditions of IEC-6 cellsIEC-6 cells had typically morphological characteristic of normal epithelial crypt cell,high viability;the third day after planted,IEC-6 cells got into exponential phase of growth, period from 7th day to 10th day is platform,then IEC-6 cells grew slowly.2.Optimization of transfect conditions of IEC-6 cells in the process of RNA interference Detected transfection efficiency by fluorescence microscope and scored to go on rank sum test;we found,the score was 0 when there was only transfection agent or FAM-siRNA;the groups with transfection agent and FAM-siRNA,had strong or weak fluorescence intensity. Detected transfection efficiency by flow cytometry,the combination of Lipofectamine2000 1.0μL+ FAM-siRNA 160nM,had the highest transfection efficiency,but blank group was only 1%.3.Screen the best efficient siRNA to RPS203.1 Detect the expression of RPS20 mRNA by RT-PCRComparison to blank group,the expression of RPS20 mRNA in group siRNA1 decreased significantly(P＜0.05)after interference 24h,however,group siRNA2 and group siRNA3 didn’t suppress expression of RPS20 mRNA significantly;After interference 48h,72h, group siRNA1,group siRNA2 and group siRNA3 didn’t suppress the expression of RPS20 mRNA significantly.3.2 Detect the expression of RPS20 mRNA by fluorescence quantitative RT-PCRComparison to blank group,the expression of RPS20 mRNA in group siRNA1,siRNA2, siRNA3 was interfered successfully,interference efficiency was about 80%-90%.3.3 Detect the expression of RPS20 protein by western-blotComparison to blank group,after interference 24h,the expression of RPS20 protein in group siRNA1,siRNA2 decreased significantly(P＜0.028,P＜0.049),the expression of RPS20 protein in group siRNA3 trended to low,but the variance wasn’t significantly(P＜0.065);Comparison to blank group,after interference 48h,the expression of RPS20 protein in group siRNA2,siRNA3 decreased significantly(P＜0.028,P＜0.006),the expression of RPS20 protein in group siRNA1 trended to low,but the variance wasn’t significantly(P＜0.619);Comparison to blank group,after interference 72h,the expression of RPS20 protein in group siRNA2,siRNA3 decreased significantly(P＜0.019,P＜0.015),the expression of RPS20 protein in group siRNA1 trended to low,but the variance wasn’t significantly(P＜0.056).4.Change of cell morphology and function after RNA interference4.1 Change of cells morphology after RNA interferenceUnder the detection of phase contrast microscope,comparison to blank group,the cell number of group siRNA1、siRNA2、siRNA3 decreased,but there was no change on cell morphology.Under the detection of transmission electron microscope after interference 48h, cellular membrane and nucleus were complete in blank group,there were abundant cell organelles;transfection control group and negative control group had integrated nucleus, and a few of vacuoles and some plasmid;group siRNA1,siRNA2,siRNA3 had no integrated membrane,and there were lots of vacuoles,cytolysosome number increased, vacuoles in group siRNA2,siRNA3 maybe comprise high electron density content.Cell organelle decreased,and some of them were obscure.4.2 Effect on migration of IEC-6 cells after RNA interferenceComparison to blank group,after interference 48h,group siRNA1,siRNA2,siRNA3 suppressed cell migration significantly(P＜0.01,P＜0.01,P＜0.01).4.3 Effect on proliferation of IEC-6 cells after RNA interferenceComparison to blank group,after interference 24h,48h,72h,the growth cell number of group siRNA1,siRNA2,siRNA3 was suppressed significantly(P＜0.01)by cell counting. To detect proliferation of IEC-6 cells by MTT,comparison to blank group,after interference 24h,group siRNA2,siRNA3 suppressed cell proliferation significantly(P＜0.05),after interference 48h,group siRNA2 could suppress cell proliferation significantly(P＜0.05),after interference 72h,group siRNA2,siRNA3 could suppress significantly cell proliferation(P＜0.01,P＜0.05).4.4 Effect on apoptosis of IEC-6 cells after RNA interference 48hComparison to blank group,viable cells of group siRNA1 decreased,the difference was significantly(P＜0.05),and early apoptotic cells increased,but the difference was not significantly.Comparison to blank group,cells dead normally and late apoptotic cells of group siRNA1,siRNA2,siRNA3,had no significantly difference.Conclusion:1.After planted IEC-6 cells 10 days,growth curve changed from platform to the period increased slowly.Due to begin to present differentiation,IEC-6 cells grew slowly.2.This study design and compose three pairs siRNAs to RPS20 of rats,and they interference the expression of RPS20 on the aspect of mRNA and protein successfully,and prove their validity.3.After interference,IEC-6 cells cytoplasm present vacuolization,and its migration,and proliferation were suppressed,but there was no apoptosis to happen.These results indicate that interfering expression of RPS20 may affect the function of digestion and absorption and gastrointestinal mucosa healing,which is in accordance with condition of low level on digestive and absorptive function and gastrointestinal mucosa healing.This result support characteristic from clinical spectrum of gene expressed differently,support the hypothesis that essence of pi-deficiency syndrome maybe relate with RPS20 lowly expression.更多还原