【作者】 崔香淑；

【导师】 金元哲；

【作者基本信息】 延边大学 ， 生理学， 2008， 博士

【摘要】 1.黄芩苷对S₁₈₀荷瘤小鼠抑瘤作用机制的研究目的:探讨黄芩苷对S₁₈₀荷瘤小鼠的抑瘤作用机制。方法:取60只小鼠复制S₁₈₀瘤模型,随机分成5组:黄芩苷大、中、小剂量组,对照组,环磷酰胺组。观察黄芩苷对S₁₈₀荷瘤小鼠的抑制率、脾指数及胸腺指数,NK（natural killer,NK）细胞活性及血清肿瘤坏死因子-a（tumor necrosis facter-a,TNF-a）、白介素-2（interleukin-2,IL-2）的含量,肿瘤组织血管内皮生长因子（vascularendothelial growth factor,VEGF）的表达。结果:各种剂量的黄芩苷对小鼠S₁₈₀肉瘤均有明显的抑制效应,且呈现剂量依赖性,大、中、小剂量组小鼠抑瘤率分别为56.0%、48.0%、36.0%,与对照组比较均有显著性差异（P＜0.05）。此外,与对照组比较,黄芩苷大、中剂量组的脾指数有显著性差异（P＜0.05）;大、中、小剂量组的胸腺指数、NK细胞活性、血清TNF-a、IL-2含量均有显著性差异（P＜0.05）。Western blot结果表明,VEGF在黄芩苷大、中、小剂量组小鼠瘤组织中的表达与对照组比较,有显著性差异（P＜0.05）。结论:黄芩苷能够抑制S₁₈₀荷瘤小鼠肿瘤生长;并能明显升高S₁₈₀荷瘤小鼠脾细胞NK细胞活性及血清TNF-a、IL-2的含量,对小鼠的免疫功能有调节作用;同时黄芩苷可以下调S₁₈₀荷瘤小鼠肿瘤组织VEGF的表达。2.黄芩苷对HepG-2细胞诱导凋亡作用的研究目的:研究黄芩苷体外对HepG-2细胞抑制增殖、诱导凋亡的作用,并初步探讨其作用机制。方法:采用体外细胞培养方法,应用MTT法检测黄芩苷对HepG-2细胞的抗增殖作用及细胞毒活性;通过倒置显微镜、HE染色、扫描电镜（scannin electron microscope,SEM）观察凋亡细胞的形态学及超微结构的改变;应用流式细胞术（flow cytometry,FCM）分析细胞周期和凋亡率,观察黄芩苷对HepG-2细胞的诱导凋亡作用;通过免疫细胞化学（S-P法）检测凋亡相关基因bcl-2、bax、p53蛋白表达的改变;采用Westernblot技术检测Caspase-3蛋白表达情况,初步探讨黄芩苷在体外对HepG-2细胞诱导凋亡作用的可能机制。结果:（1）MTT比色法结果表明,黄芩苷在体外对HepG-2细胞有明显的抑制作用,且这种抑制作用呈现浓度和时间依赖性。（2）形态学观察:倒置显微镜、扫描电镜及HE染色结果表明,黄芩苷作用于肿瘤细胞后,可诱导较典型的细胞凋亡形态学变化及超微结构改变,如细胞胞体缩小,胞质浓缩,微绒毛结构减少甚至消失,胞核向外呈锐角突起,染色质高度浓缩,电子密度增高,边集于核膜下或呈新月形,有的出现核固缩,核碎裂以及凋亡小体形成等。（3）免疫细胞化学（S-P法）检测凋亡相关基因bel-2、p53蛋白表达明显减少、bax表达明显增加;Bcl-2/Bax比例明显降低。（4）流式细胞仪分析结果,50μg/ml黄芩苷作用于HepG-2细胞48 h后,出现明显的细胞凋亡峰,其凋亡率为27.01%,与对照组细胞的凋亡率5.47%相比差异有显著性（P＜0.01）。且G2/M期细胞明显减少,而S期细胞比率显著上升,多数细胞阻滞在S期。（5）Western blot结果显示,随着药物浓度的增加Caspase-3酶原蛋白条带逐渐变细,并且可见到蛋白裂解产物（Caspase-3蛋白）逐渐增多。结论:黄芩苷对HepG-2细胞具有较强的抑制增殖和诱导凋亡的作用;黄芩苷诱导HepG-2细胞凋亡的机制可能与下调p53基因表达和降低Bcl-2/Bax比例,从而促进Caspase-3的活化有关。更多还原

【Abstract】 1.Study on the inhibition effect mechanism of baicalin in S₁₈₀ miceObjectives:To study the inhibition effect mechanism of baicalin in S₁₈₀ mice.Methods:The S₁₈₀ sarcomas was transplanted in 60 KM mice,then they were divided randomly into 5 groups to receive different treatment(high,medium and low dose group of baicalin chloroform extract,cyclophosphosphamide group and control group.The inhibition ratios of the tumor,the spleen index and thymus index were calculated out,the activities of NK cell and the contents of TNF-a,IL-2 in serum were detected.And the levels of VEGF protein was assayed with Western blot.Results:Baicalin could increase the levels of the inhibition ratios of the tumor weight,the spleen index and thymus index in mice,and the effects were in a dose dependent manner.The inhibition ratios of tumor in high,medium and low dose group of baicalin were 56.0%,48.0%,36.0%,and the difference was significant compared with the control group（P＜0.05）.The research also finds that the difference of the thymus index,the activities of NK cell and the contents of TNF-a,IL-2 in serum were significant in high,medium and low dose group of baicalin comparing with the control group （P＜0.05）.Western blot disclosed that bicalin also could markly downregulate the expression of VEGF gene and compared with control group the difference was significant （P＜0.05）.Conclusions:The baicalin could suppres mouse S180 tumor growth and increase the activities of NK cell and the contents of TNF-a,IL-2 in serum significantly.It also could regulate the mice immunity and markly downregulate the expression of VEGF gene.2.Study on the apoptosis-inducing effect mechanism of baicalin on HepG-2 cell lineObjective:To study the inhibition proliferation and the apoptosis-inducing effect of Baicalin and the perhaps mechanism.Methods:The HepG-2 cell were cultured in vitro.To detect the change of the correlated index.The antiproliferation and cytotoxic efficiency of Baicalin on HepG-2 cell was assessed with MTT assay.The cell apoptosis effect was observed by inverted microscope,HE staining and scannin electron microscope（SEM）.The cell growth cycle and apoptosis ratio were measured by flow cytometry（FCM）.The expressions of caspase-3 protein was detected by Western blot.Results:（1） MTT assay disclosed that Baicalin inhibited the proliferation of HepG-2 cell and the effects were in a time-and-concentration dependent manner.（2） Inverted microscope,HE staining and SEM disclosed that Baicalin induced the typical apoptosis change in cell morphology.（3） FCM disclosed that the ratio of apoptosis was significanfly different as compared with that of control group compared with the control group 5.47%,deviation was significant（P＜0.01） in 48 hours.the tumor cells on G2/M stage decrease obviously and on S stage,the cells increase notablely.（4） Immunocytochemistry disclosed bcl-2,p53 were significantly decreased and bax increased.（5） Western blot disclosed that followed by the increase of Baicalin,caspase-3 protein expression was step up gradually.Conclusions:Baicalin could make some effect on inhibiting proliferation and apoptosis- inducing on HepG-2 cell line,perhaps related to activating of Caspase-3.更多还原