【作者】 冯莉芳；

【导师】 赖仁胜；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2009， 硕士

【摘要】 目的1对狭叶番泻叶蒽酮生物合成代谢途径关键酶基因苯丙氨酸解氨酶(phenylalanineammonia-lyase,PAL)基因和查尔酮合酶(chalcone synthase,CHS)基因的部分序列进行测序,寻找基因标志生物信息。2分析研究对象服用狭叶番泻叶后的肠道临床表现,泻下及副作用,肠液中番泻苷A、B含量变化,肠粘膜病理形态,探讨PAL和CHS基因标志与临床表现的生物关联性。方法1设计PAL和CHS基因引物,对狭叶番泻叶DNA进行双向测序,BLAST程序比对测序结果,并利用DNAStar Megalign软件与模式植物PAL和CHS基因序列配对分析相似性指数。2观察HPLC试验组60例与LC-MS/MS试验组5例的临床表现并评分分组,统计分析差异性。两组肠液样本分别进行番泻苷A、B的ttPLC分析和番泻苷B的LC-MS/MS分析,并与临床表现关联分析。探讨基因标志与各组肠粘膜病理形态6项指标的关联性。所用统计软件为SPSS16.0。结果1从狭叶番泻叶获得三条番泻苷基因片段,733bp、573bp和262bp,与NCBI的plant结构树比对呈短相似序列,未发现NCBI已收录番泻PAL和CHS基因相似序列。三条未鉴定基因片段与拟南芥和大豆的PAL和CHS基因相似性指数在29.7～37.1之间。2 HPLC试验组60例分组,无泻下组1例,轻度泻下组共25例,中度泻下组共27例,重度泻下组共7例。经t检验比较,副作用总评分在轻度泻下组与中度泻下组有显著性差异(P=0.008,P＜0.05)。HPLC分析狭叶番泻叶样品和番泻苷A、B对照品保留时间一致,肠液样本中未测出番泻苷。LC-MS/MS试验组5例分别存在于为轻、中、重度泻下组,各副作用总评分≥4,由于例数太少,与临床泻下相关性不明确。LC-MS/MS分析肠液样本,7/16管肠液样本出现番泻苷B吸收峰,都系中、重度泻下组。3依据该PAL和CHS基因标志使用本品番泻叶,肠粘膜病理形态在临床泻下各组间总体间无显著性差异(P＞0.05),但无充血出血和轻度充血出血程度在轻、中度泻下组间有显著性差异(P=0.041,P＜0.05)。大多研究对象表现为中度炎症、轻度充血出血、无水肿(P＞0.05)。结论1对狭叶番泻叶检出PAL和CHS基因三条未鉴定序列,其与拟南芥的PAL和CHS基因的同源性高于与大豆的相同基因的同源性。发现该三条序列的微卫星重复特征及生物信息学特征等基因标志,据此为进一步进行基因与药效关联分析做了基础研究。2狭叶番泻轻、中度泻下组的人数多于其它组,中度泻下组的副作用高于轻度泻下组。清肠状况良好。HPLC分析番泻叶及番泻苷A、B对照品中番泻苷A、B的保留时间在以误差为1.5%的范围内一致,HPLC对照组的肠液专属特异性好,HPLC试验组肠液中番泻苷A、B未检出。LC-MS/MS分析,多次腹泻者肠液中番泻苷的含量较高,随着时间的推移番泻苷的含量逐渐增大,提示LC-MS/MS法比较可靠的得到试验数据,番泻苷的代谢受肠道环境和菌群分解作用的影响,由于例数太少,番泻苷含量与番泻叶副作用的相关性不明确。3依据该狭叶番泻叶PAL和CHS基因标志背景,肠粘膜的病理形态在临床泻下各组间比较总体间无显著性差异,即安全性好,但轻、中度泻下组的充血出血程度差异显著(P＜0.05),提示在临床有明显泻下情况时,进行肠镜检查或用药时要注意充血出血倾向。更多还原

【Abstract】 Objective1 The analysis on Gene sequencing and bioinformatics of partial nucleotide sequences of phenylalanine ammonia-lyase(PAL) gene and chalcone synthase(CHS) gene,which is the key enzyme gene of biosynthesis metabolic pathway of anthrone in Cassia angustifolia Vahi.2 Research on the clinical condition of intestine,clicnical purgation and side effects,the content of sennoside A,B in intestine fluid,compared with pathologic morphology of intestinal mucosa in subjects took Cassia angustifolia Vahi,the biological correlation were investigated between clinical performance and PAL,CHS gene marker.Methods1 DNA was extracted from Cassia angustifolia Vahi and primers were designed for PCR and bidirectional sequencing.The sequencing results were searched by NCBI BLAST program,as well as were compared with PAL and CHS gene sequences to model plants with pairwise alignment to get Similarity Index under DNAStar Megalign software.2 Observed and scored on clinical performance of 60 cases in HPLC test group and 5 cases in LC-MS/MS test group.All cases were divided into four groups and made with statistical analysis of significant difference.Sennoside A,sennoside B with HPLC,and sennoside B with LC-MS/MS in intestinal fluid samples were analyzed from the two groups,respectively. Moreover,it was statistically analyzed the correlation of the results and clicnical performance and carried on research to the relationship of gene marker and six pathologic morphology indexes of intestinal mucosa of each group.The statistical software is SPSS16.0 is available. Results1 Three sennoside gene sequence fragments were obtained,733bp,573bp and 262bp by size. The similary sequences searched were short and no similary sequences of PAL gene or CHS gene of senna leaf in NCBI were found of NCBI Map Viewer plant structure trees.The Similarity Index of the three gene sequences compared with PAL and CHS gene sequences of Arabidopsis thaliana and Glycine max were 29.7～37.1.2 The 60 cases in HPLC test group were divided into four groups:1 case of group without diarrhea,25 cases of light group,27 cases of moderate group and 7 cases of severe group.Total scores of side effects were analyzed with group t test,the significant difference was:light group compared with moderate group(P=0.008,P＜0.05).The Retention Time of sennoside A,B detected in senna leaf and sennoside A,B reference substances was uniform with HPLC analysis, but could not be detectable in intestinal fluid samples of the 60 cases.The 5 cases in LC-MS/MS test group belong to three groups:light,moderate and severe group,total scores of side effects of all groups were above 4,because of the number of cases,correlation of the side effects and clinical effect was unclear yet.Absorption peaks of sennoside B appeared in 7/16 tubes of intestinal fluid samples of moderate and severe group by LC-MS/MS analysis.3 According to gene marker of this research to use this product,there were no significant difference between each group of population rates with pathologic morphology,but significant difference was found between moderate and light group of not and light congestive and hemorrhage phenomenon(P=0.041,P＜0.05).Most cases were moderate inflammation,light congestive and hemorrhage,without edema(P＞0.05).Conclusion1 Three unidentified sequences of Cassia angustifolia Vahi were found with resemblance to PAL and CHS gene sequences of Arabidopsis thaliana than that of Glycine max.Gene markers of this research were found for the initial basic tendency in correlation analysis of the further pharmacodynamical effect and gene.2 Cases in light and moderate group outnumbered cases in other groups.The total scores of side effects of moderate group were higher than that of light group.The intestine cleared was fine. The Retention Time of sennoside A,B in senna leaf and reference substances analyzed with HPLC is uniform in the range of 1.5%error,but could not be detected in samples of test group. Intestinal fluid of control group have noninterference to HPLC analysis of sennoside A,B.The more diarrheas in subjects,the more content of sennoside in intestine fluid analyzed with LC-MS/MS.Furthermore,the content of sermoside increased gradually with the passage of time, which suggested that LC-MS/MS method could get experiment data reliably.The metabolism of sennoside need intestinal environment and discomposition of intestinal microflora,because of the number of cases,correlation of the content of sennoside and the side effects was unclear yet.3 There were no significant differences between each group of pdpulation rates about pathologic morphology based on the PAL and CHS gene marker of Cassia angustifolia Vahi,which suggested high safety.The extent of congestive and hemorrhage of light group was lighter than that of moderate group,implied that the severe diarrhea compared with the possibility of congestive and hemorrhage should be cared when performed enteroscopy and taking senna leaf.更多还原