【作者】 邢晓静；

【导师】 蔡玉文；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2008， 博士

【摘要】 目的:肝癌是原发于肝细胞或肝内胆管上皮细胞的恶性肿瘤之一。在肿瘤的发病中,原发性肝癌在我国男性居第三位,女性居第四位.中国每年约有12万人死于肝癌。由于其恶性度极高,现有的治疗方法均达不到满意的效果,寻找有效的治疗药物和方法是目前肝癌治疗的一项重要课题。越来越多的证据表明,中医药在防治肝癌的发病及改善患者的免疫状态、提高患者生存率方面有其独特疗效。许多中药具有诱导细胞凋亡(APO)、影响DNA、RNA合成及保持相关酶活性的作用。健脾理气方是临床常用验方,探讨健脾理气方对肝癌作用的机制,为中药治疗肝癌提供可靠的依据已成为当务之急。方法:以中药血清学方法测定健脾理气方药物血清对人肝癌HepG2细胞株作用的最佳量效、时效点;以倒置相差显微镜、光镜、透射电镜观察经健脾理气方药物血清处理后的人肝癌HepG2细胞株的形态学变化,以流式细胞化学方法研究人肝癌HepG2细胞株在健脾理气方药物血清的影响下细胞形态学变化及联合DNA琼脂凝胶电泳法检测细胞凋亡典型的梯状带分析;以Western blot法测定经药物血清处理后的人肝癌HepG2细胞株P53、bcl-2基因的蛋白表达水平;以酶细胞化学染色方法观察健脾理气方药物血清对人肝癌HepG2细胞株Mg2+-ATP酶、G-6-P酶活性的影响。结果:以不同时相药物血清作用人肝癌HepG2细胞株后,除2t-1h组药物血清未见明显增殖抑制作用,其余各组均表现明显抑制作用,以3t-2h组药物血清抑制作用最好,抑制率达54.81%,以不同浓度药物血清作用24h、48h后,除中药中小剂量药物血清作用24h组外,其余各组均表现明显的抑制作用,以中药大剂量药物血清作用48h组抑制作用最佳,抑制率达53.96%;倒置相差显微镜下,经药物血清处理后的HepG2细胞彼此失去连结,收缩变圆,贴壁能力减弱,易从培养瓶上脱落下来,光镜下观察药物血清作用后,细胞收缩变圆,部分细胞出现固缩,细胞核染色质聚集,断裂成块状、颗粒状或月形小体,细胞形态呈凋亡特征性改变,电镜下,经药物血清处理的HepG2细胞,表面微绒毛减少甚至消失,核染色质浓集,胞质内有空泡形成,细胞膜起皱呈出芽状,可见凋亡小体形成,以DNA琼脂凝胶电泳法检测,健脾理气方药物血清能诱导人肝癌HepG2细胞形成凝胶电泳特异梯状带,以流式细胞化学方法定量分析健脾理气方药物血清作用24、48h,在G1峰前出现的明显凋亡峰(Ap峰),测得凋亡细胞占整个细胞群分别为1.8%和8.2%;人肝癌HepG2细胞株经药物血清处理前后蛋白表达Western-Blot结果显示,P53蛋白表达水平增高、bcl-2蛋白表达水平降低;经健脾理气方药物血清处理后,光镜下见用药组人肝癌HepG2细胞株Mg2+-ATPase和G-6-Pase酶反应颗粒较正常变小,数量减少,密度减低,酶活性下降。结论:1.健脾理气方药物血清对人肝癌HepG2细胞株体外具有抑制肿瘤细胞增殖的作用,作用的最佳量效、时效点为大剂量,3t-2h;2.健脾理气方药物血清对人肝癌HepG2细胞株具有诱导细胞凋亡的作用;3.健脾理气方药物血清可上调人肝癌HepG2细胞株P53基因的蛋白表达水平,下调bcl-2基因的蛋白表达水平;4.健脾理气方药物血清具有降低人肝癌HepG2细胞株Mg2+-ATP酶和G-6-P酶的活性的作用。更多还原

【Abstract】 Purpose: Liver cancer is one of the malignant tumors, which is due to liver cells or intrahepatic bile duct epithelial cells. In all kinds of tumour, the incidence of PHC is the third of the male in China, the fourth of female. There are over 120 thousand persons dying of liver cancer in China every year. Due to the high malignancy, current therapy methods are not of satisfied effect. To search active drugs and methods is an important program to cure liver cancer. More and more truths prove that the traditional Chinese medicine has especially curative effect on preventing from the invasions of liver cancer, improving sufferers’immune and raising the survival rate. Lots of herbs can induce APO, influence the synthesis of DNA and RNA and hold the activity of correlated enzyme. Jianpi Liqi herbs are commonly applied in clinic. It is necessary to research the mechanism of the herbs and supply reliable evidence to treat liver cancer.Method: With the serological method, to determine the best quant potency and time limitation of effecting human liver cancer HepG2 cell strain by drug serum of Jianpi Liqi herbs. With inverted phase contrast microscope, light microscope and transmission electron microscope, to observe the liver cancer HepG2 cell strain’s morphological change after dealing by drug serum; to determine the liver cancer HepG2 cell strain gene P53 and bc1-2’s express level; to observe the activity of Enzyme Mg2+-ATP and G-6-P through enzyme cytochemical staining method.Result: With different phase drug serum, inhibitory action was not obviously seen in the liver cancer HepG2 cell strain except Group 2t-1h. Other groups were opposite. Group 3t-2h was the best, inhibition ratio approaching to 54.81%. In different concentration drug serum, within 24 hours and 48 hours, the effect of each group was obvious but in low concentration within 24h. In all the groups, the high concentration within 48h was the best, inhibition ratio 53.96%. Under the inverted phase contrast microscope, the HepG2 blanked and shrink to round. Their adherent abitity was attenuated and the cells easily ablated from the culture flasks. Under the light microscope, the cells shrink to round and part cells pyknosised. The nuclear chromatins aggregated, collapsing to massive, granulo- or selene corpuscles. The cells were to die. Under the electron microscope , the surface microvilli of HepG2 decreased even disappeared. The nuclear chromatins enriched , intracytoplasm cavitating. The cell membranes corrugated as buds, apoptotic body forming could be seen clearly. With the DNA abstraction of cells and agarose gel electrophoresis,there was a“ladder strip”appearance. Collected the cells treated with drug serum about 24h,48h,there is a apoptotic peak obviously. inhibition ratio approaching to 1.8% and 8.2%.Westerm-blot result displayed that the increase of P53 express level and the decrease of bc1-2. Under the light microscope, after Mg2+-ATPase and G-6-Pase reacted, it was seen that the grains were shrinking and decreasing comparing to normal, and the density was degraded.Conclusion: The effect of drug serum of Jianpi Liqi herbs on human liver cancer HepG2 cell strain was obvious. The best effective dose and time limitation is 3t-2h, in high concentration. There was manifest generation inhibitory action of the drug serum, which could induce the apoptosis, which also up-regulate the proternum express level of human liver cancer HepG2 cell strain Gene P53 but down-regulate bc1-2, and restrain the activity of HepG2 cell strain Mg2+-ATPase、G-6-Pase.更多还原