【作者】 杨文超；

【导师】 缪晓青；

【作者基本信息】 福建农林大学 ， 农产品加工与贮藏工程， 2010， 博士

【摘要】 本文考察了采用层析法分离蜂毒得到的纯蜂毒肽和神蜂精(内含蜂毒肽0.05 mg/ml的中药外搽剂)的抗HSV-1病毒作用,同时对其抗HSV-1病毒的机理进行了探索,并研究了HSV-1感染后小鼠脑细胞中p53和p21 WAF1/CIP1蛋白表达的变化。结果如下:1、QF-1取毒器蜂箱内取毒法采集意大利蜜蜂的蜂毒,过滤提纯蜂毒后经葡聚糖凝胶G-25,G-50,G-75层析和溶血试验分离纯化后得到蜂毒肽。经SDS-PAGE电泳Marker及蜂毒肽标准品对分离所得的组分进行鉴定,证实其为电泳级纯度的蜂毒肽,利用分离所得的蜂毒肽进行后续试验。2、CPE法测定并经过Reed- Meuench法计算得出HSV-1病毒的半数感染量TCID50为10-5.2 / 100uL,MTT法测定和Reed- Meuench法计算得出蜂毒肽和神蜂精对Vero细胞的半数中毒剂量CC50分别为10-2.1 mg/100uL和10-2.4mg /100uL。体外实验结果表明1、3、6、9倍CC50的蜂毒肽对HSV-1病毒的抑制率分别为55.12%,55.12%,59.30%,66.51%,药物对照组(阿昔洛韦试验组)的抑制率为74.88%。四个梯度浓度的神蜂精对HSV-1病毒的抑制率分别为69.50%,71.52%,62.11%,45.74%,阿昔洛韦试验组的抑制率为75.34%。3、体内试验结果表明蜂毒肽和神蜂精均有提高小鼠存活率和延长小鼠存活时间的作用。蜂毒肽在低、中和高剂量(1×10-3mg/mL,3×10-3mg/mL,6×10-3mg/mL)时小鼠的存活率分别为40%,40%和50%。神蜂精试验组的小鼠存活率分别为40%,50%和60%,均高于病毒对照组的20%。蜂毒肽试验组小鼠的平均存活时间分别为11.4d,12d,12.7d,中、高剂量蜂毒肽试验组的平均存活时间明显高于病毒对照组(p<0.05)。神蜂精试验组小鼠的平均存活时间分别为11.6d,11.8d,12.8d,明显高于病毒对照组(p<0.05)。高剂量组的蜂毒肽和神蜂精的小鼠平均存活时间均高于阳性对照组(阿昔洛韦试验组)。4、蜂毒肽和神蜂精的抗HSV-1病毒机理的研究结果表明,蜂毒肽和神蜂精没有直接杀死HSV-1病毒的作用,但是对HSV-1病毒的吸附、合成和释放均有明显的抑制作用。1CC50,3CC50,6CC50,9CC50的蜂毒肽对HSV-1病毒的吸附均有抑制作用,抑制率分别为31.25%,32.92%,32.92%,32.08%。四个梯度浓度1CC50,3CC50,6CC50,9CC50的神蜂精对HSV-1病毒吸附的抑制率分别为29.17%,31.25%,32.08%,32.08%。四个梯度浓度的蜂毒肽对HSV-1病毒合成的抑制率分别为29.05%,29.91%,31.69%,33.06%。四个梯度浓度的神蜂精对HSV-1病毒合成的抑制率分别为23.12%,23.49%,24.25%,23.72%。四个浓度的蜂毒肽对HSV-1病毒释放的抑制率分别为29.31%,18.37%,13.28%,10.70%。四个梯度浓度的神蜂精对HSV-1病毒释放的抑制率分别为42.89%,33.77%,31.32%,30.67%。5、小鼠在注射0.05 mL 100LD50 HSV-1病毒液感染后,连续两天进行蜂毒肽和神蜂精腹腔注射给药,小鼠感染病毒后第4天进行免疫组化试验,结果显示:正常试验组,病毒对照组,低、中、高剂量蜂毒肽组,低、中、高剂量神蜂精组小鼠的脑细胞p21 WAF1/CIP1蛋白阳性细胞率分别为100%,2.1%±0.6, 14.2%±0.4,28.5%±0.6,33.8%±0.8, 13.7%±0.5,22.3%±0.4,29.4%±0.6。各试验组小鼠脑细胞的p53蛋白的阳性细胞率分别为3.5%±0.2,40.2%±0.5,31.2%±0.5,22.4%±0.4,20.5%±0.3,27.3%±0.6,20.9%±0.3,14.1%±0.2。经层析分离蜂毒得到的电泳级纯度的蜂毒肽和蜂毒制剂神蜂精通过抑制病毒的吸附、合成和释放,从而达到体内外抗HSV-1病毒的作用,进而调节小鼠脑细胞p21 WAF1/CIP1和p53蛋白的表达,调节细胞周期,保护细胞DNA免受HSV-1的损伤。神蜂精中蜂毒肽的含量约为0.05 mg/mL,100uL 1CC50神蜂精所含蜂毒肽约为0.0002 mg,100uL 1CC50蜂毒肽溶液含蜂毒肽0.00799mg。神蜂精中的蜂毒肽含量只有相应试验浓度的蜂毒肽溶液的1/40。比较试验组神蜂精和蜂毒肽的体内外抗HSV-1病毒的能力,抑制病毒吸附、合成和释放的能力,对蛋白p53和p21的阳性表达的影响,二者的差别却并不大。这可能有以下几个原因:一方面,神蜂精中的中药成分可能也参与了病毒释放的抑制;另一方面,中药成分改变了蜂毒肽的构象,使得蜂毒肽的抗病毒释放能力大大增强,或者兼而有之。此类增效作用的研究结果为蜂毒肽的开发利用和蜂毒制剂神蜂精的抗HSV-1病毒等的药效作用机理提供新的理论依据。更多还原

【Abstract】 In this paper, the antiviral effects as well as the mechanism of melittin purified from bee venom by chromatography and Shen Fengjing on HSV-1 virus were investigated. At the same time, the differences of the expression of p53 and p21 WAF1/CIP1 proteins in the brain cells of mice infected by HSV-1 were explored. The results were as follow:1. The melittin was isolated and purified through polydextran gel G-25,G-50,G-75 combined with hemolysis test from venom of honeybee (Apis mellifera lijustica) workers which was collected by QF-1 Bee Venom Collector in the top of hives. The separated component was used in next experiments after confirmed by comparing to melittin standard and protein marker in SDS-PAGE.2. The TCID50 of HSV-1 virus on Vero cell was 10-5.2 /100uL, which was tested by CPE method and calculated by the Reed-Meuench method. And the CC50 of melittin and Shen Fengjing, which were tested by MTT method, were 10-2.1mg/100uL and 10-2.4mg/100uL. In vitro test results showed that suppression rates of 1,3,6,9 times CC50 of melittin on the virus HSV-1 were 55.12%, 55.12%, 59.30% and 66.51% respectively, and the inhibition rate of drug control (Acyclovir) was 74.88%. Those of Shen Fengjing were 69.50%, 71.52%, 62.11% and 45.74% respectively, and the control was 75.34%.3. In vivo results showed that melittin and Shen Fengjing could improve the survival rate and the survival time of mice. The mice’survival rates subjected to melittin at the low, medium and high dose(1×10-3mg/mL,3×10-3mg/mL,6×10-3mg/mL) were 40%, 40% and 50% respectively, and those to Shen Fengjing were 40%, 50% and 60%, 20% higher than that of virus control group. The average mice’survival duration subjected to melittin were 11.4d, 12d, 12.7d, respectively, and those of median and high doses of melittin were significantly higher than that of the virus control group (p <0.05). Those of Shen Fengjing were 11.6d, 11.8d, and 12.8d respectively, significantly higher than that of virus control group (p <0.05). The survival time of high dose of melittin and Shen Fengjing were higher than the positive control group(ACV).4. The antiviral mechanism research of melittin and Shen Fengjing showed that HSV-1 virus couldn’t directly be killed, but the adsorption, synthesis and release of HSV-1 virus were inhibited. The suppression rates of 1, 3, 6 and 9 times CC50 of melittin against adsorption of HSV-1 were 31.25%, 32.92%, 32.92% and 32.08% respectively. The inhibitive percentages caused by Shen Fengjing against virus adsorption were 29.17%, 31.25%, 32.08% and 32.08% respectively. The inhibition rates of melittin against viral synthesis were 29.05%, 29.91%, 31.69% and 33.06% respectively. Those of Shen Fengjing against synthesis were 23.12%, 23.49%, 24.25% and 23.72% respectively. The suppression rates of the four concentrations of melittin against the release of virus were 29.31%, 18.37%, 13.28% and 10.70% respectively, and as for Shen Fengjing groups they were 42.89%, 33.77%, 31.32% and 30.67% respectively.5. Mice were injected with 0.05 mL 100LD50 HSV-1 virus, Melittin and Shen Fengjing was injected for the following two days, then immunohistochemical tests were conducted 4 days after virus injection. The results showed that the protein p21 WAF1/CIP1 positive rates of the normal test group, the virus control group, low , median and high doses of melittin group, low, medium and high doses of Shen Fengjing group were 100%, 2.1%±0.6%, 14.2%±0.4%, 28.5%±0.6%, 33.8%±0.8%, 13.7%±0.5%, 22.3%±0.4% and 29.4%±0.6% respectively. The protein p53 positive cells rates were 3.5%±0.2, 40.2%±0.5%, 31.2%±0.5%, 22.4%±0.4%, 20.5%±0.3%, 27.3%±0.6%, 20.9%±0.3% and 14.1%±0.2% respectively.Melittin purified by chromatogram methods and Shen Fengjing had the antiviral effects on HSV-1 virus in vivo and in vitro by inhibiting the virus adsorption, synthesis and release. And the expression of p21 WAF1/CIP1 and p53 proteins and the cell cycle were adjusted and DNA damage by HSV-1 was protected.The content of bee venom in Shen Fengjing is about 0.1 mg / mL, and the content of melittin is about 0.05 mg / mL. The melittin in 100uL 1CC50 Shen Fengjing and melittin solution are about 0.0002 mg and 0.0079mg. The concentration of melittin tested is 40 times higher than that of Shen Fengjing. There were no significant differences between Shen Fengjing and melittin in the ability of in vivo and in vitro viral resistance to HSV-1, the inhibition against viral adsorption, synthesis and release, and the effects on the positive expression rates of protein p53 and p21. The reasons maybe as followings: The Chinese medicine in Shen Fengjing inhibited the virus releasing too, they might change the conformation of melittin so the antiviral effects were improved. This enhancement effects provided new theoretic basis on pharmacodynamic mechanism for the development and utilization of melittin and Shen Fengjing.更多还原