【作者】 廖轶；

【导师】 邓俊良；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2010， 硕士

【摘要】 鸡新城疫(ND)是由新城疫病毒(NDV)引起的一种急性、高度接触性传染病。该病是危害养鸡业发展的主要疫病之一,至今仍未得到有效控制。早期研究表明复方中药禽康散在体内具有明显的抗NDV作用。本课题在此基础上,研究复方中药禽康散防治鸡新城疫的细胞生物学机理,为该药临床应用及新药研发提供理论依据。为此进行一系列试验并取得如下结果：1.禽康散对鸡胚成纤维细胞(CEF)增殖影响本试验将禽康散提取液(1g／ml)用培养液稀释成2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10g／ml九个浓度,按每孔0.1ml分别加入到体外培养的CEF中,通过测定细胞OD值变化,判定不同浓度中药对细胞增殖的影响。结果表明,禽康散在浓度为2-6、2-7、2-8g／ml时具有明显促进CEF增殖的作用,在浓度为2-2、2-3、2-4g／ml时具有明显抑制CEF增殖的作用。药物最大无毒浓度为2-5g／ml。2.禽康散体外抗NDV作用本试验将2-5、2-7、2-9g／ml三个浓度中药提取液0.1ml和0.1ml NDV(200倍TCID50)以三种方式[即中药与病毒混合感作后加入(直接杀灭组)、先加入中药后接种病毒(预防组)、先接种病毒后加入中药(治疗组)]加入体外培养的CEF中,通过测定CEF OD值的变化判定各浓度中药在三个环节上的抗病毒作用。结果显示,直接杀灭组的病毒抑制率最高(0.749),表明禽康散对NDV的体外直接杀灭作用最好。预防组与治疗组的病毒抑制率分别为0.525、0.474,但两者无显著性差异。3.禽康散对体外培养鸡脾淋巴细胞(SPL)增殖的影响本试验将禽康散提取液(1g／ml)用培养液稀释成2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10g／ml九个浓度,按每孔0.1ml分别加入到体外培养的鸡脾淋巴细胞中,通过MTT法测定细胞OD值变化,判定不同浓度中药对细胞增殖的影响。结果显示,禽康散在浓度为2-4、2-5、2-6、2-7g／ml时具有明显促进体外培养SPL增殖的作用,在浓度为2-2g／ml时具有明显抑制SPL增殖的作用。4.禽康散对鸡脾淋巴细胞NO分泌的影响本试验将2-2、2-5、2-8g／ml三个浓度中药提取液0.1ml分别加入体外培养的SPL中,用试剂盒测定培养液中NO及一氧化氮合酶(NOS)含量,判定禽康散对淋巴细胞NO分泌的影响。结果显示,三个浓度中药提取液均能极显著增加NO及NOS含量,以中药浓度2-2g／ml组(接种量0.1ml,即25mg)效果最明显。因此,禽康散可以通过促进CEF、SPL的增殖,通过促进SPL分泌NO来发挥抗NDV作用；禽康散在体外培养的CEF内对NDV具有强的杀灭作用。更多还原

【Abstract】 New Castle disease is an acute and highly contagious disease caused by Newcastle disease virus, which do great harms to the poultry industry and has not been controlled effectually till today. Earlier period studies indicated that Complex Chinese crude drug Qinkangsan had good therapeutic effect. Based on these studies, the purpose of this topic is to survey the cytobiology mechanism of Qinkangsan for clinical application and advanced development.The experiment and consequence as follows:1. Effect of Qinkangsan on CEF proliferation Qinkangsan(1g/ml) was diluted into 2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10g/ml concentrations and used for CEF (0.1ml each hole).The effects of drug was judged according to OD value.The reasults showed that Qinkangsan could promote cell proliferation obviously in concentrations of 2-6、2-7、2-8 g/ml and restrain cell proliferation obviously in concentrations of 2-2、2-3、2-4g/ml.The maximal safety concentration is 2-5g/ml.2. Effect of Qinkangsan on cellular infectivity of virus Three concentrations(2-5、2-7、2-9g/ml) of Qinkangsan(0.1ml) and NDV(200 TCID50) were respectively added into CEF in three different ways [synchronously(sterilization group), before(prophylaxis group) and after(cure group) inoculation of virus].Cell proliferation were tested by The effects of Qinkangsan on infection of virus to CEF were evaluated by OD value’s changes.The result shows that virus inhibition in sterilization group, prophylaxis group and cure group was 0.794,0.525 and 0.474 respectively.3. Effect of Qinkangsan on SPL proliferation Qinkangsan(lg/ml) was diluted into 2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10g/ml concentrations and used for SPL(0.1ml each hole).The effects of drug was judged according to OD value.The reasults showed that Qinkangsan could promote cell proliferation obviously in concentrations of 2-4、2-5、2-6、2-7g/ml and restrain cell proliferation obviously in concentrations of 2-2g/ml. 4. Effect of Qinkangsan on NO secretion of SPL Qinkangsan was diluted into 2-2、2-5、2-8 g/ml concentrations and used for SPL(0.1ml each hole). Contents of NO and NOS in culture solution were determined by kits to judge effects of Qinkangsan on NO secretion. The reasults showed that Qinkangsan of all concentrations could increase contents of NO and NOS, result of 2"2g/ml group(0.1ml 25mg) was most obviously.更多还原