糖尿病诱发剂与化疗药物对家蚕的生理效应及BBXⅡ降血糖作用研究

发布时间：2018-06-16 13:09

本文选题：家蚕 + 糖尿病诱发剂　；参考：《山东农业大学》2014年硕士论文

【摘要】：以家蚕作为药物筛选、病理学、毒理学等试验的模式动物具有诸多优势，是一个很有意义的研究领域，但是国内外尚未有以家蚕作为糖尿病模型和化疗药物筛选模型的研究报道。本文利用生物化学、分子生物学、细胞学、药理学等研究手段，探讨了糖尿病诱发剂四氧嘧啶和链脲菌素以及化疗模药环磷酰胺对家蚕的生理效应；采用原核表达系统和改良的纯化技术获得了家蚕素Ⅱ（bombyxin-Ⅱ，BBXⅡ）的纯品，试验比较了BBXⅡ和胰岛素对家蚕和小鼠的降血糖作用和生物效应。本研究旨在为建立家蚕糖尿病模型和化疗药物筛选模型、开发BBXⅡ降血糖药物提供理论帮助。主要试验结果如下： 1.给予家蚕幼虫注射适宜剂量的四氧嘧啶和链脲菌素，均会在一定时间内引起血液中海藻糖含量显著升高，同时海藻糖酶活性显著降低，与其对高等动物的作用相似，证明利用糖尿病诱发剂能建立高血糖模型家蚕。四氧嘧啶和链脲菌素还引起家蚕脱水、提前入眠、三眠蚕、眠蚕脱肛（蜕皮异常）、产生不滞育卵等现象，，同时也表现出不同程度的中毒症状，说明糖尿病诱发剂对调控家蚕代谢和发育的内分泌系统的功能具有广泛影响，进而影响蚕的水分代谢、眠性和化性变化。 2.四氧嘧啶和链脲菌素对家蚕BBXⅡ基因和PTTH基因的表达具有显著影响，其中两种药剂对PTTH基因表达及四氧嘧啶对BBXⅡ基因表达的影响因发育时期、药剂种类和剂量不同而异，但链脲菌素对BBXⅡ基因的表达在一定时间内均表现出显著的抑制作用。四氧嘧啶和链脲菌素是通过特异性损伤BBXⅡ分泌细胞而引起家蚕高血糖，还是由于其他原因引起血糖升高及生长发育、眠性和化性变化的，还有待于进一步研究。 3.从家蚕头部mRNA中经过反转录获得BBXⅡ基因的cDNA，并构建了原核表达载体pET28a-BBXⅡ，经过IPTG诱导，使BBXⅡ获得了大量表达，进一步用HisTrapHP亲和层析，成功得到大量纯化的BBXⅡ。为进一步开展BBX的分泌机制和作用机理研究奠定了基础。 4.利用纯化的BBXⅡ制备了BBXⅡ的兔多克隆抗体。对家蚕戊3-己2期胚胎及家蚕幼虫脑-咽侧体联合体进行了BBXⅡ抗体染色，但均未获得预期的对BBX分泌细胞的特异性染色结果，不过发现该抗体能够对胚胎期的蜕皮腺和丝腺进行特异性染色，其原因有待于进一步研究。BBXⅡ抗体可以作为胚胎期特异性识别蜕皮腺和丝腺的有效试剂。 5.研究比较了BBXⅡ和人胰岛素的降血糖作用。结果证明，无论对正常饲养的家蚕幼虫、饥饿幼虫或注射糖尿病诱发剂的幼虫，注射BBXⅡ后均在一定时间内引起血液中海藻糖含量显著降低，海藻糖酶活性升高。对BBXⅡ和胰岛素的降血糖作用比较发现，无论对正常饲养的小鼠，还是利用链脲菌素诱导的糖尿病小鼠，注射BBXⅡ的降血糖效果均与胰岛素相近，并表现出一定的剂量依赖性。另外还发现，给家蚕注射人的胰岛素，也能极显著地降低家蚕的血糖，并显著提高血液中海藻糖酶的活性。说明家蚕的BBXⅡ与人的胰岛素无论对家蚕还是对哺乳动物均具有相似的调节血糖的生理功能，它们不仅在结构上相似，而且在生理作用上的进化也是相似和保守的。本试验不仅证明了家蚕是一种理想的胰岛素类药物的试验动物，为今后开展胰岛素类药理学和糖尿病病理学研究提供了一条更加简单的途径，而且为开发BBX新型降血糖药物奠定了重要基础。 6.研究了化疗模药环磷酰胺对家蚕的生理效应，发现该药物在低剂量时对家蚕血球密度影响不大，甚至有增加血球密度的趋势，但达到一定剂量后能有效抑制家蚕血球的增殖，降低血球密度，而且还能改变不同类型血球的比例。另外还发现，环磷酰胺能够显著抑制家蚕精母细胞的减数分裂和精子变形成熟过程，说明其能抑制家蚕的有丝分裂和减数分裂过程。本研究证明了环磷酰胺对低等动物家蚕的细胞分裂和增殖的抑制作用与在高等动物中的作用相同或相似，因此，家蚕有望成为环磷酰胺类似化疗药物、免疫抑制药物和不育模型药物的试验动物。
[Abstract]:The model animals that use silkworm as drug screening, pathology, toxicology and other experiments have many advantages, and it is a very meaningful research field. However, there has not been a research report on the model of silkworm as a diabetes model and the screening model of chemotherapeutic drugs at home and abroad. This paper uses the research means of biology, molecular biology, cytology and pharmacology. The physiological effects of diabetes inducer, four oxouracil and streptozotocin and cyclophosphamide, on the silkworm, were investigated. The pure products of bombyxin- II (BBX II) were obtained by using the prokaryotic expression system and improved purification technology. The effects of BBX II and insulin on the hypoglycemic and biological effects of insulin on silkworm and mice were compared. The purpose of this study is to provide theoretical support for establishing a silkworm diabetes model and a screening model for chemotherapeutic drugs and developing BBX II hypoglycemic drugs.
1. injecting the suitable dose of four pyrimidine and streptozocin into the silkworm larvae could cause a significant increase in the content of trehalose in the blood for a certain time, while the activity of the alginate enzyme decreased significantly, which was similar to that of the higher animals. It was proved that the diabetic model could be used to build the hyperglycemic model silkworm, four oxacil and streptozotocin. It also causes the dehydration of silkworm, sleeping in advance, three silkworms, DIUS anus (abnormal molt) and undiapause eggs. At the same time, it also shows the symptoms of different degrees of poisoning. It shows that the diabetic agents have a wide influence on the function of the endocrine system that regulate the metabolism and development of silkworm, and then affect the water metabolism, sleeping and chemical changes of the silkworm.
2. four oxouracil and streptozotocin have significant influence on the expression of BBX II gene and PTTH gene in silkworm. Among them, the expression of PTTH gene and the expression of BBX II gene on the gene expression of PTTH and four of pyrimidine are different in the developmental period, the dosage of the agent and the dosage, but the expression of the streptozotocin to the BBX II gene is significant in a certain period of time. Four oxacil and streptozotocin can cause hyperglycemia in silkworm by specific damage to BBX II secreting cells, or the rise and growth of blood sugar and growth, sleep and chemical changes due to other causes, and further study.
3. the cDNA of BBX II gene was obtained by reverse transcription from the mRNA of the head of the silkworm, and the prokaryotic expression vector pET28a-BBX II was constructed. After IPTG induction, a large number of BBX II was expressed and a large number of purified BBX II was successfully obtained by HisTrapHP affinity chromatography, which laid the foundation for further research on the mechanism of BBX secretion and the mechanism of action.
4. the rabbit polyclonal antibody of BBX II was prepared by purified BBX II. The BBX II antibody staining was carried out on the 2 stage of the silkworm and the brain pharyngeal side body of the silkworm larvae and the silkworm larvae, but the specific staining results of the BBX secretory cells were not obtained, but it was found that the antibody could specifically dye the molting gland and the silk gland at the embryonic stage. The reason for further study is that.BBX II antibody can be used as an effective reagent for the identification of molting glands and silk glands in embryonic stage.
5. the study compared the hypoglycemic effect of BBX II and human insulin. The results showed that both the larvae of the silkworm, the larvae of the silkworm, or the larvae of the injectable diabetic inducer, after the injection of BBX II, caused a significant decrease in trehalose content in the blood, the activity of alginate enzyme in the blood and the hypoglycemic effect on BBX II and insulin. It was found that the hypoglycemic effect of injecting BBX II was similar to that of insulin in both normal and streptozotocin induced diabetic mice, and showed a certain dose dependence. In addition, it was found that injecting human insulin to the silkworm could significantly reduce the blood sugar of the silkworm, and significantly increase the blood algae in the blood. The activity of the glycase. It shows that the BBX II of the silkworm and human insulin have similar physiological functions to regulate blood sugar both in the silkworm and in the mammals. They are not only similar in structure but also in the physiological function of similar and conservative. This test not only proved that the silkworm is an ideal test of insulin drugs. Animal testing provides a more simple way for the development of insulin pharmacology and diabetes pathology in the future, and has laid an important foundation for the development of a new BBX hypoglycemic drug.
6. the physiological effects of cyclophosphamide on the silkworm were studied. It was found that the drug had little influence on the density of the silkworm in the low dose and even increased the density of blood cells, but it could effectively inhibit the proliferation of silkworm blood cells and reduce the density of blood cells after a certain dose, and also could change the proportion of different types of blood cells. Now, cyclophosphamide can significantly inhibit meiosis and sperm deformability of silkworm spermatocytes, indicating that it can inhibit the mitosis and meiosis process of silkworm. This study proved that the inhibitory effect of cyclophosphamide on the cell division and proliferation of silkworms in lower animals is the same or similar to that in higher animals. Silkworm is expected to become a test animal for cyclophosphamide analogous chemotherapeutic drugs, immunosuppressive drugs and sterile model drugs.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965.1

【参考文献】