發(fā)布時間：2018-06-16 13:09

  本文選題：家蠶 + 糖尿病誘發(fā)劑��； 參考：《山東農(nóng)業(yè)大學(xué)》2014年碩士論文

【摘要】：以家蠶作為藥物篩選、病理學(xué)、毒理學(xué)等試驗的模式動物具有諸多優(yōu)勢，是一個很有意義的研究領(lǐng)域，但是國內(nèi)外尚未有以家蠶作為糖尿病模型和化療藥物篩選模型的研究報道。本文利用生物化學(xué)、分子生物學(xué)、細胞學(xué)、藥理學(xué)等研究手段，探討了糖尿病誘發(fā)劑四氧嘧啶和鏈脲菌素以及化療模藥環(huán)磷酰胺對家蠶的生理效應(yīng)；采用原核表達系統(tǒng)和改良的純化技術(shù)獲得了家蠶素Ⅱ（bombyxin-Ⅱ，BBXⅡ）的純品，試驗比較了BBXⅡ和胰島素對家蠶和小鼠的降血糖作用和生物效應(yīng)。本研究旨在為建立家蠶糖尿病模型和化療藥物篩選模型、開發(fā)BBXⅡ降血糖藥物提供理論幫助。主要試驗結(jié)果如下： 1.給予家蠶幼蟲注射適宜劑量的四氧嘧啶和鏈脲菌素，均會在一定時間內(nèi)引起血液中海藻糖含量顯著升高，同時海藻糖酶活性顯著降低，與其對高等動物的作用相似，證明利用糖尿病誘發(fā)劑能建立高血糖模型家蠶。四氧嘧啶和鏈脲菌素還引起家蠶脫水、提前入眠、三眠蠶、眠蠶脫肛（蛻皮異常）、產(chǎn)生不滯育卵等現(xiàn)象，，同時也表現(xiàn)出不同程度的中毒癥狀，說明糖尿病誘發(fā)劑對調(diào)控家蠶代謝和發(fā)育的內(nèi)分泌系統(tǒng)的功能具有廣泛影響，進而影響蠶的水分代謝、眠性和化性變化。 2.四氧嘧啶和鏈脲菌素對家蠶BBXⅡ基因和PTTH基因的表達具有顯著影響，其中兩種藥劑對PTTH基因表達及四氧嘧啶對BBXⅡ基因表達的影響因發(fā)育時期、藥劑種類和劑量不同而異，但鏈脲菌素對BBXⅡ基因的表達在一定時間內(nèi)均表現(xiàn)出顯著的抑制作用。四氧嘧啶和鏈脲菌素是通過特異性損傷BBXⅡ分泌細胞而引起家蠶高血糖，還是由于其他原因引起血糖升高及生長發(fā)育、眠性和化性變化的，還有待于進一步研究。 3.從家蠶頭部mRNA中經(jīng)過反轉(zhuǎn)錄獲得BBXⅡ基因的cDNA，并構(gòu)建了原核表達載體pET28a-BBXⅡ，經(jīng)過IPTG誘導(dǎo)，使BBXⅡ獲得了大量表達，進一步用HisTrapHP親和層析，成功得到大量純化的BBXⅡ。為進一步開展BBX的分泌機制和作用機理研究奠定了基礎(chǔ)。 4.利用純化的BBXⅡ制備了BBXⅡ的兔多克隆抗體。對家蠶戊3-己2期胚胎及家蠶幼蟲腦-咽側(cè)體聯(lián)合體進行了BBXⅡ抗體染色，但均未獲得預(yù)期的對BBX分泌細胞的特異性染色結(jié)果，不過發(fā)現(xiàn)該抗體能夠?qū)ε咛テ诘耐懫は俸徒z腺進行特異性染色，其原因有待于進一步研究。BBXⅡ抗體可以作為胚胎期特異性識別蛻皮腺和絲腺的有效試劑。 5.研究比較了BBXⅡ和人胰島素的降血糖作用。結(jié)果證明，無論對正常飼養(yǎng)的家蠶幼蟲、饑餓幼蟲或注射糖尿病誘發(fā)劑的幼蟲，注射BBXⅡ后均在一定時間內(nèi)引起血液中海藻糖含量顯著降低，海藻糖酶活性升高。對BBXⅡ和胰島素的降血糖作用比較發(fā)現(xiàn)，無論對正常飼養(yǎng)的小鼠，還是利用鏈脲菌素誘導(dǎo)的糖尿病小鼠，注射BBXⅡ的降血糖效果均與胰島素相近，并表現(xiàn)出一定的劑量依賴性。另外還發(fā)現(xiàn)，給家蠶注射人的胰島素，也能極顯著地降低家蠶的血糖，并顯著提高血液中海藻糖酶的活性。說明家蠶的BBXⅡ與人的胰島素?zé)o論對家蠶還是對哺乳動物均具有相似的調(diào)節(jié)血糖的生理功能，它們不僅在結(jié)構(gòu)上相似，而且在生理作用上的進化也是相似和保守的。本試驗不僅證明了家蠶是一種理想的胰島素類藥物的試驗動物，為今后開展胰島素類藥理學(xué)和糖尿病病理學(xué)研究提供了一條更加簡單的途徑，而且為開發(fā)BBX新型降血糖藥物奠定了重要基礎(chǔ)。 6.研究了化療模藥環(huán)磷酰胺對家蠶的生理效應(yīng)，發(fā)現(xiàn)該藥物在低劑量時對家蠶血球密度影響不大，甚至有增加血球密度的趨勢，但達到一定劑量后能有效抑制家蠶血球的增殖，降低血球密度，而且還能改變不同類型血球的比例。另外還發(fā)現(xiàn)，環(huán)磷酰胺能夠顯著抑制家蠶精母細胞的減數(shù)分裂和精子變形成熟過程，說明其能抑制家蠶的有絲分裂和減數(shù)分裂過程。本研究證明了環(huán)磷酰胺對低等動物家蠶的細胞分裂和增殖的抑制作用與在高等動物中的作用相同或相似，因此，家蠶有望成為環(huán)磷酰胺類似化療藥物、免疫抑制藥物和不育模型藥物的試驗動物。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R965.1

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