【作者】 刘鹏；

【导师】 赵志壮； 付学奇；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2013， 博士

【摘要】 第一篇：秀丽新杆线虫（Caenorhabditis elegans）是生活在泥土中的一类非寄生类线虫，它也是第一个被阐明全部基因组序列的多细胞真核生物。它具有生命周期短暂、全身透明、易培养和观察等特点使得秀丽新杆线虫成为生命科学研究领域中的一种优秀的模式生物。近年来许多的实验小组都将线虫应用于长寿模型的建立和药理学的研究中，这是由于线虫在培养的过程中会不断地摄入添加在培养基中的物质，始终保持其体内的药物浓度，而且线虫从发育到死亡过程中组织结构及行为表现的变化也可在显微镜下进行观察，因而可以对其生物学表型进行系统的研究，所以线虫就成为了我们研究中药活性成份生物学功能的优秀模式生物。本文第一部分将石斛，“螃蟹脚”（扁枝槲寄生），三七总皂苷，甜菜碱，人参多糖，瑞香素分别提取或者溶解后，配制成含有一定浓度的中药活性成份的培养基。我们利用给药组及对照组的培养基喂养线虫，观察和比较生活在两种培养基中的线虫寿命，在观察寿命的同时记录其他表型。通过观察和比较寿命得出：浓度为100μg/ml和300μg/ml的甜菜碱（从卵给药），40μg/ml和200μg/ml人参多糖，折合成生药量为0.67g/ml和0.067g/ml石斛水溶性成分都具有延长线虫寿命的功能；三七皂苷，螃蟹脚（扁枝槲寄生）对于线虫寿命没有任何作用，浓度为300μg/ml和500μg/ml的瑞香素对于线虫有缩短寿命和导致发育异常的作用，为以后这些中药活性成份的开发和利用提供理论依据。本文的第二部分研究了瑞香素对于线虫的生物学功能的影响。瑞香素对于线虫生物学功能的影响包括两个方面：缩短寿命和异常发育。从实验结果我们可以看出瑞香素对于线虫寿命的缩短是具有类似毒性累积和遗传作用的，且这种寿命的缩短现象是随着浓度的升高而增大，是浓度依赖型的；此外，线虫的异常发育现象可能与毒性累积和遗传作用无关，因为F2代线虫异常发育的比率小于F1代。这为瑞香素类药物的开发和利用提供了理论基础和依据。本文的第三部分利用建立的紫外损伤模型，对松籽油微囊粉抗紫外辐射作用进行了研究。研究结果表明适当浓度的松籽油微囊粉（50μg/ml，100μg/ml）对于低剂量的紫外辐射（100J/m2）具有保护作用，对于大剂量（800J/m2）的紫外辐射不具有保护作用；适当浓度的松籽油微囊粉（50μg/ml，100μg/ml）对于线虫的寿命具有延长作用，在一定浓度范围内是浓度依赖的。这就为松籽油微囊粉的抗紫外辐射产品的开发和利用提供了理论依据。许多中药活性成份的药理作用已经在人体及多种动物模型中得到了证明，但是对于线虫生命活动的影响尚没有系统的报道。而线虫作为高等的真核多细胞生物，其短暂的生命周期，十分利于我们进行寿命研究的。同时其生理特征也保证了我们进行药理学研究的可能。因此，已经有越来越多的实验组通过线虫来研究中药活性成份的生物学功能。在本研究中，我们利用线虫模型，系统的分析了几种中药活性成份的生物学功能，为这些中药活性成份的研究与开发提供新的理论依据。第二篇：糖尿病（DM）是一类公认的代谢紊乱综合征，以高血糖和血脂异常为主要特征的慢性疾病。糖尿病主要分为三种类型。1型糖尿病的特征是胰岛素生产的缺乏，2型糖尿病源于人体无法有效利用胰岛素，即：代谢靶组织的胰岛素抵抗，和妊娠期糖尿病。其中，2型糖尿病（T2DM）是最常见的形式，占全世界所有糖尿病患者的90％左右。胰岛素受体（IR）具有酪氨酸激酶活性区及自身磷酸化位点，受体与胰岛素结合后，催化受体的酪氨酸磷酸化而激活，启动级联反应。这种自身磷酸化能够提高IR的激酶活性，导致胰岛素受体底物（IRS）的磷酸化，随后激活磷脂酰肌醇3-激酶（PI3-K）和下游的蛋白激酶AKT，最终招募葡萄糖转运GLUT4，使其向细胞膜上易位，从而调节葡萄糖摄取。因此，提高靶组织对胰岛素的敏感性，将成为改善糖代谢异常的重要目标。蛋白酪氨酸磷酸酶（PTPs）是一类庞大的、结构不同的细胞信号调节酶家族，调节细胞内蛋白酪氨酸磷酸化水平，并在细胞信号转导和代谢过程中发挥重要作用。SHP-1(SH2-containing tyrosine phosphatase1)，又称为HCP、SHPTP1或PTP1C，是含有SH2结构域的具有高度保守序列的蛋白质酪氨酸磷酸酶的亚家族中的重要成员，它是一种胞浆蛋白，其N端有2个SH2结构域，C端有1个催化功能结构域。研究表明，SHP-1的活力增高可导致免疫缺陷综合症和白血病。最近的一些研究发现，SHP-1与2型糖尿病的发生有关，认为SHP-1的活力增高是导致高血糖的原因之一，说明内源性SHP-1在调节血糖代谢方面发挥着重要作用，这也意味着通过抑制体内SHP-1的活性，有可能降低糖尿病患者的血糖。我们应用离子交换方法，通过Q-Sepharose Fast Flow，SP-Sephadex两种离子交换柱，分离纯化得到目的蛋白SHP-1。透析冻干后，经SDS-PAGE、HPLC分析鉴定，其纯度大于90%。酶反应动力学实验显示在前20分钟内该酶显示了良好的线性关系，我们筛选出中药紫苏梗对SHP-1具有明显的抑制效果，其IC50值为4ug/ml，双倒数作图法进一步得出紫苏梗是SHP-1的非竞争性抑制剂。我们研究中药紫苏梗的降糖机制。我们发现，不同浓度的紫苏梗水提液可以显著提高HepG2细胞中蛋白质的磷酸化水平，以及胰岛素信号通路中关键蛋白胰岛素受体IRβ和细胞外信号调节激酶ERK的磷酸化水平。与对照组相比，1mg/ml和0.75mg/ml紫苏梗水提液组对HepG2细胞中蛋白质、IRβ和ERK磷酸化水平显著提高。我们推测，紫苏梗很可能是通过抑制SHP-1的酶活性，进而激活胰岛素信号通路而实现的，这为紫苏梗对2型糖尿病的治疗提供理论依据。我们采用STZ对小鼠进行腹腔注射的方式建立2型糖尿病小鼠模型，用血糖测定试剂盒每周对小鼠检测一次血糖，结果显示，建模第14天后，小鼠平均血糖明显升高，血糖浓度平均值为28.3mmol/L，大于11.1mmol/L，说明建模成功。随机选取小鼠进行分组：正常对照组，紫苏梗灌胃给药组，模型对照组。对正常对照组的健康小鼠和模型对照组的2型糖尿病小鼠按照饮用水25ml/kg体重进行灌胃，对紫苏梗灌胃给药组的2型糖尿病小鼠按照生药量10g/kg体重进行灌胃给药，每日一次，共持续25天。结果显示,中药紫苏梗具有明显的降血糖作用，使小鼠血糖平均降低了19.4mmol/L。实验表明紫苏梗这种中药很可能是通过抑制SHP-1的活性，从而降低了2型糖尿病模型小鼠的血糖。这为紫苏梗在治疗2型糖尿病中的应用提供了理论依据。更多还原

【Abstract】 PART1：Caenorhabditis elegans (referring as the C.elegans in the followingpresentation) is a category of non-parasitic C.elegans living in the soil, which isused to be the first multicellular eukaryotes to clarify the sequence of the entiregenome. Due to the short lifespan, the transparent body, and the easiness to cultivateand observe, the C.elegans becomes an excellent model organism in the field of lifescience research. In recent years, many experimental groups applied the C.elegans toestablish the longevity model and conducted the pharmacological research, becausein the process of cultivation, the C.elegans will continue to consume material addedin the medium, maintain the concentration of drug in its body, and the changes in theorganizational structure and behavior expression of the C.elegan from developmentto death can be observed under the microscope, making it possible to conduct asystematic research on the biological phenotype. Therefore, the C.elegans becomesthe appropriate model organism in the rasearching of the biological function of theactive constituent in traditional Chinese herb.In the first part of this article, we prepare a medium contained certainconcentration of the active constituent in traditional Chinese herbs, then feed themwith the different medium in the administration group and the control group, observeand compare the life span in each situation and record other biological phenotype.We conclude that all the Betaine with the concentration of100μg/ml and300μg/ml,the Ginseng polysaccharide with the concentration of40μg/ml and200μg/ml, andthe conversation of Dendrobium water-soluble components into crude drug with theconcentration of0.67g/ml and0.067g/ml can post an effect of extending the lifespan of the C.elegans, while the Notoginsenoside and “Crab legs” post none.Daphnetin with the concentration of300μg/ml and500μg/ml will shorten the lifespan and leading to dysplasia. All these provide the theoretical basis for the development and utilization of the related the active constituent in traditionalChinese herbs.The second part of the paper reveals the effect of the daphnetin on thebiological function of the C.elegans. There are two aspects that the daphnetin canaffect the the biological function of the C.elegans: shorten the life span and developewith dysplasia. From the experimental results, we conclude that the shorten effect oflife span brought by the daphnetin on the C.elegans is similar to thecumulative toxic and genomics effect, and this life shortening phenomenon isincreasing with the rising of concentration. In addition, the relations between theabnormal development of the C.elegans and the cumulative toxic and genomicseffect may be very weak, because the proportion of dysplasia of the F2is lower thanthe F1. These results provide a toxicological basis and foundation for thedevelopment and utilization of the daphnetin drugs.In the third part of this article, we conduct a research on the resistance effect toUV radiation of the pine nut oil microencapsulated powder through theestablishment of a model of UV damage. The pine nut oil microencapsulated powderwith the concentration of50μg/ml and100μg/ml presents the resistance effect on thelow level of UV radiation with100J/m2, while this protection disappears on the levelof the UV radiation rising to800J/m2. The life span of the C.elegans can beextended on the appropriate concentration of pine nut oil microencapsulated powder(50μg/ml,100μg/ml), which provides a theoretical basis for the development anduse of the products of the pine nut oil microencapsulated powder UV radiation.Although a lot of pharmacological effects of the active constituent in thetraditional Chinese Herb have been proven in the human body and a variety ofanimal models, none of the effects on the C.elegans life activities have been reportedsystematically. As a higher eukaryotic multicellular organism, the C.elegans is veryconducive to our life research due to its short life span, and of great value to conductthe pharmacology research due to its physiological characteristics. Therefore, moreand more experimental groups use the C.elegans to research the biological functionof the traditional Chinese Herb active constituent. In this research, we takeadvantage of the C.elegans model, conduct a systematic analysis of the biologicalfunction of the active constituent of Chinese herbal medicine, that provide a new theoretical basis for the research and development of the active constituent of theseherbs. PART2：Diabetic mellitus (DM) is recognized as a group of metabolic disordersand characterized by hyperglycemia and dyslipidaemia. Type1diabetes ischaracterized by a lack of insulin production and type2diabetes results from thebody’s ineffective use of insulin, Insulin resistance in its metabolic target tissues.Type3diabetes is gestational diabetes. Type2diabetes mellitus (T2DM) is the mostcommon form and accounts for around90%of all diabetes worldwide. Insulinbinding evokes a cascade of phosphorylation events, beginning with theautophosphorylation of the IR on multiple tyrosyl residues. Autophosphorylationenhances IR kinase activity and leads to recruitment of insulin receptor substrate(IRS) proteins, followed by activation of phosphatidylinositol3-kinase (PI3K) anddownstream protein kinase AKT, subsequent translocation of the glucose transporterGLUT4and regulation of glucose uptake. Therefore, Improving tissue sensitivity toinsulin is a major clinical goal to help ameliorate abnormal metabolism.Protein tyrosine phosphatases (PTPs) constitute a large and structurally diversefamily of signaling enzymes that control the cellular levels of protein tyrosinephosphorylation and play an important role in the intracellular signal transductionprocess and metabolism. SHP-1(SH2-containing tyrosine phosphatase1), also calledHCP, SHPTP1and PTP1C, is an important member of protein tyrosine phosphotasesubfamily with highly conserved sequence. SHP-1is a kind of cytosolic proteincontaining two SH2domains at the N-terminal and one catalytic domain at theC-terminal. It was reported that increased SHP-1activity led to immuno-deficientsyndrome andn leukemia. The most recent studies have shown that SHP-1is relatedto type II diabetes and high level of blood sugar is due to elevated SHP-1activitywhich means that endogenous SHP-1plays a critical role in regulating blood sugarmetabolism and SHP-1may be a potential target for reducing the blood sugar of diabetes patients by inhibiting its activity.Two steps of ion-exchange chromatography, Q-Sepharose Fast Flow andSP-Sephadex, were used to isolate and purify SHP-1protein obtained from E.coliDE3strain. The purity of target protein was more than90%determined bySDS-PAGE and HPLC analysis. The enzyme showed a linear relationship during thefirst20min. The catalytic domain of SHP-1protein tyrosine phosphatase, is moresensitive towards the Perilla stem extract than other PTPs and its IC50was4μg/ml.Based on the above experimental phenomenon, we investigated thehypoglycemic mechanism of Perilla stem. The tyrosine phosphorylation levels ofthe IR β-subunit and ERK1/2significantly increased in the HepG2cells by theconcentration of1mg/ml and0.75mg/ml of Perilla stem compared to the controlgroup.We speculate that the hypoglycemic mechanisms of Perilla stem may becaused by inhibition of the SHP-1activity, consequently, activating the signalingpathway, which also provide us a theoretical basis for Perilla stem on type2diabetictherapy.40mice were selected for establishing type2diabete animal model which wereinjected with STZ/kg per day. After the treatment, each mouse was weighed everyday and the blood sugar was detected every week using Blood Glucose Kit. Themouse with more than11.1mmol/L blood sugar was considered as established model.The mice were randomly grouped into control group, herb-treated group, and modelgroup. Both the control group (healthy mice) and the negative control group (type IIdiabetic mice) were administrated with25ml/kg water according to body weights,while the herb-treated group (type II diabetic mice) was administrated with10g/kgherbs according to body weights for25days. Our animal experiments providedstrong evidence that perilla stem significantly reduced the blood sugar of type2diabetes animal model with19.4mmol/L.The Perilla stem has hypoglycemic effectthrough inhibiting the protein phosphorylation of SHP-1, which may lead to thesecondary development of Chinese traditianal herbs.更多还原