【作者】 王亚军；

【导师】 盛军； 付学奇；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2010， 博士

【摘要】 新型降糖活性物质的研究:治疗糖尿病包括以下两种方式:1,使用胰岛素;2,使用其他降糖药物。对应这两种方式,本文构建和筛选了两种降糖活性物质,并分别对这两种物质的降糖效果进行了研究。对于CTB-人胰岛素融合蛋白,构建并筛选到含CTB-BA的阳性细胞并采用了Balb/c小鼠动物试验检测其降血糖活性,表明阳性细胞具有较强的降血糖活性。对于普洱茶,首先对各种茶在体外对PTP-1B的抑制作用进行了考察,并得到对PTP-1B抑制作用最强的普洱茶。对普洱茶在db/db小鼠上的降糖效果进行研究。普洱茶水饮用组表现出很高的降血糖活性,而采用灌胃给药组亦表现出一定的降糖活性。对普洱茶的减肥作用进行研究,饮用普洱茶具有减肥作用。对普洱茶对糖尿病患者和健康受试者的降血糖作用进行了研究,发现对于糖尿病受试者组,普洱茶显著地降低了他们的空腹血糖、餐后2h血糖和HbA1c（%）,健康受试者组则没有明显的变化。对试验前后血Ca²⁺,K⁺浓度进行检测,实验前后血Ca²⁺,K⁺的浓度均在正常范围内,证明喝茶不会造成Ca²⁺,K⁺流失。新型脂质体疫苗的研究:以脂质体作为流感裂解疫苗佐剂,采用冻干重建法,制备了流感脂质体疫苗。实验表明,相同剂量下,脂质体流感疫苗组与相应非脂质体流感疫苗组相比,可激发2-4倍的抗体,抗体高峰持续时间更长,在脾细胞增值比率,CD4+水平及CD4+/CD8+比例,以及细胞因子（IL-4、IFNγ）水平上均有显著的增强作用。脂质体流感疫苗不但能够刺激机体产生较无佐剂疫苗更高效持久的体液免疫,而且能够激发机体产生细胞免疫。更多还原

【Abstract】 The study of new substances with hypoglycemic activity:Diabetes is a kind of chronic disease. Diabetes mellitus and its sequelae are a major and growing public health problem. Diabetes not only cause suffering to patient but also bring burden to the service of national public health. So, the study of diabetes and its treatment may have very important sense. The treatments of diabetes may compose of two ways as follow: 1. Insulin medicating; 2, some other drugs that with hypoglycemic activity, to increase the sensitive of the insulin receptor. This study constructed and chosen two substances with hypoglycemic activity correspond to the two treatment ways:1, the CTB-human insulin（BA） fusion protein expressed in Gynostemma Pentapyhllum Makino callus cells;2,Puer tea, and their effects of glucose lowering were studied respectively.For the CTB-human insulin fusion protein, the CTB and human insulin gene without C peptide （BA） were fused using gene fusion method first to construct the cloned vector pMD18-T/CTB-BA and the GP hinge was inlet between the CTB and BA genes. The cloned vector was digested with BamHI and SacI, and the aimed CTB-BA gene was linked to the expression vector pBI121 to construct the plant expression vector pBI121/（CTB-BA）,the pBI121/（CTB-BA） was identified by BamHI and SacI. Agrobacterium tumerfaciens strain LBA4404 was transformed with previously constructed recombinant plasmid pBI121/（CTB-BA） using the freeze thawing method,then G Pentapyhllum Makino callus cells were integrated with CTB-BA gene by co-culturing the cells with the transformed LBA4404 strain in the 67V-AS medium.The transformed G Pentapyhllum Makino callus cells were identified by DNA sequence assey and RT-PCR. The expressed product was identified by western-blot and its amount was tested by ELISA kit and its blood sugar decreasing effect was tested in mice.The sequences of synthetic CTB and human insulin genes （BA） were completely identical to those designed. Restriction map proved that the length of gene fragment inserted into expression vector pBI121 was consistent with that expected. The sequence of genomic DNA of expressed product was completely identical to that designed.The result of RT-PCR was consistent with that expected. The expressed product showed a specific band with relative molecular mass of 17 000Da by western-blot. The human insulin expression amount was 6.03μIU/ml according to the ELISA result.The animal test showed that only the G Pentapyhllum Makino callus cells itself also showed activity in decreasing the blood sugar of mice, however the activity of the transformed G Pentapyhllum Makino callus cells was much higher.For the puer tea, in our present study, the puer tea and related compounds were assayed for their inhibitory activity against PTP-1B and its isozymes. The results suggested that puer tea showed higher inhibitory activity than green tea , black tea, acted as a true PTP-1B inhibitor. We studied the inhibitory activity of pure tea against TC-PTP, which was the analogue of PTP-1B, and it showed much lower activity than PTP-1B. But, it showed much higher activity with PTP-SHP1, another PTP which had negative correlation with diabetes. Because the PTP-1B and PTP-SHP1 were all negative regulating factor of diabetes, the high inhibitory activity might be favorable. We also studied the affects of temperature and autoclaving on the puer tea and we found that all the treatments had no effect on the inhibitory activity of puer tea. So the inhibitory component in puer tea had high stability. The result might give support to its commercialization production.We investigated the in vivo glucose lowering efficacy of puer tea with three similar animal tests. Using the second animal experiment as an example,the puer tea was given to db/db mice, a strain widely used as an animal model for type 2 diabetes, for 6 weeks with a twice a day dosing regimen, for the solution-drinking groups drinking puer tea all day. The puer tea drinking group with a regimen using puer tea water-solution instead of water showed higher serum glucose lowering activity compared to control. At the 6th week, puer tea reduced serum glucose level by about 42% compared to the control. The high dose puer tea group, midst dose puer tea group and low dose puer tea group with intragastric administration also have some serum glucose lowering activity compared to control at 4th, 6th week and at the 4th week with a reducing level of about 38%,28%,21% compared to the control, but their effects of lowering serum glucose were less obviously than the puer tea drinking group. It is better for the puer tea to deal with diabetes mellitus as health care drink than as curing drug. The principal composition of tea: The tea brown Amoxicillin and the tea polysaccharide had few serum glucose lowering activity in vivo indicated that the serum glucose lowering activity of the tea maybe have a new action mechanism that had not been illuminated yet.We all know Type 2 diabetes is frequently associated with obesity because obesity may cause insulin resistance and lead to hyperglycemia. Our db/db mice are the typical example of type2 diabetes with obesity. The fitness weight-lowering is beneficial for the curing of type 2 diabetes. In our study, we found that as the administration time went, the solution-drinking groups showed an obvious tendency of weight-lowering with a reducing level by about 12.2% compared to the control group. But the positive group （rosiglitazone ） had opposite effect with a increasing level by about 21.1%. This phenomenon might be a proof that, to achieve a same curative effect, the puer tea might be a better choice than the rosiglitazone in the therapy of type 2 diabetes.At last, we had studied the glucose lowering efficacy of puer tea in the diabetes and healthy people. Subjects were divided into two groups: the diabetics group （n = 60） and the healthy subjects group （n = 20）. The washout period was set for 4 weeks prior to treatment, and no tea was permitted for drinking throughout this period. The next 13 weeks was the treatment period. The fasting plasma glucose（FPG）, postprandial 2 hours plasma glucose（P2HPG） and glycosylated hemoglobin （HbA1c） in diabetics group decreased significantly （P < 0.001, respectively） after drinking puer tea but did not change for the healthy subjects group. The adverse rates were reducing sharply （from 59.3% to 3.5%） as the administration duration extended.We added HbA1c measurement, which was the king-indicator of serum glucose concentration and a obvious indicator of the diabete-complication. So the reducing of the HbA1c in our study also indicated the reducing of the risk of the diabete-complication. The concentrations of blood Ca²⁺ and K⁺ were tested before and after the test to prove whether the blood Ca²⁺ and K⁺ loss with tea drinking. The results showed that their concentrations both in the normal range both in diabetic group and healthy subjects group, proving that the blood Ca²⁺ and K⁺ did not lose with tea drinking.Above all, both the CTB-human insulin fusion protein and the puer tea all had hypoglycemic activity. But the plant expression system had very low expression ability. It was very difficult to product massively. For the diabetics, the insulin taking would bring stronger insulin resistance. On the other hand, we suggested that we could relief the diabetes or even cure it through pure tea drinking. On the whole, tea drinking is a traditional way of resting therapy that is very acceptable for people. It had been proved that the pure tea had hypoglycemic activity, could lower the risk of diabetes neopathy. And the blood Ca²⁺ and K⁺ did not lose with tea drinking. We could relief the diabetes or even cure it through pure tea drinking. This was a kind of food therapy, which would lower the painful of diabetics.The study of a new liposome vaccine:At present, most of the virus vaccines have low immunity effect. Their effects can been strengthen by using liposome as adjuvant. Take the influenza vaccine as an example. The most effective method of prevention and cure influenza was influenza vaccine vaccination. This study aimed to produce liposome-encapsulated influenza split vaccine and analyze its humoral and cellular immunological enhancement in mice.In this study, we used liposome as an adjuvant for influenza split vaccine in order to make freeze-dried liposome influenza vaccine, we explore its properties, observe the prepared vaccine for morphology under electron microscope, and determine for envelope rate. Analyze the particle distribution and size of the vaccine by laser diffraction particle size analyzer. The prepared influenza liposome vaccine under electron microscope were in forms of round or elliptic particles distributed evenly, at a mean size of 15μm. The envelope rate of the vaccine was 91%.For the humoral immunity testing of the liposome influenza vaccine, doses of 2ug/mouse and 4ug/mouse were administered i.p. to BALB/c mice （5-6 per group） with nonliposome or liposome vaccines. Serum antibodies were assayed on weeks 2-13 by the haemagglutination-inhibition（HI） test and ELISA. Following immunization with 2μg and 4μg liposome-encapsulated influenza split vaccine, there was a higher, up to 4fold stronger HI titer and ELISA titer than that obtained with non-liposome HN. For the cellular immunity testing of the liposome influenza vaccine, a single dose of 4μg-antigen/mice were administered i.p. to BALB/c mice （5-6 per group） with nonliposome or liposome vaccines. The mice were put to death after 4 weeks administration. The spleen cell proliferation was measured using MTT in vitro; flow cytometry was used to determine the levels of CD4 and CD8 T-cell and the CD4/CD8; the levels of IL-4 and IFNγin lung homogenates were tested by ELISA kit. We obtained that the combined liposome vaccine consisting of encapsulated antigen, but not the nonliposome vaccine, elicited a higher titer of he spleen cell proliferation and cell subsets like CD4+ and CD4+/CD8+ and the liposome-encapsulated influenza split vaccine triggered cytokine （IL-4 and IFNγ） production.Above all, the liposome influenza vaccine could not only stimulate much higher and longer lasting humoral immunity than non-liposome vaccine, but also could stimulate the body`s cellular immunity. The liposome influenza vaccine had a large superiority in the apply dosage reduction, production cost depression and susceptible population protection.更多还原