【作者】 马玲娣；

【导师】 蒋纪恺； 康格非；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2005， 博士

【摘要】 研 究 目 的 苦参碱是从我国传统中药苦参中提取出的一种生物碱活性成分,具有广泛的药理学作用。我们多年来的研究证明,苦参碱在体外可明显抑制人白血病细胞 K562 的增殖,诱导其向正常形态分化。近年来,苦参碱对于其他肿瘤细胞作用的研究报道日见增多,其体内外的抗肿瘤活性受到极大关注。目前,对祖国传统医学宝库中的传统中草药进行大力开发,对其药理学作用给予新的理解和诠释,致力于研究开发出新的抗癌药物,大力弘扬祖国传统医学已成为当前中医药研究的发展趋势,在这样的大背景下,深入探讨苦参碱抑制肿瘤尤其是抑制实体瘤方面的作用,揭示其体内外抗癌作用的分子机制,无疑是一项很有前景和研究价值的课题,是非常有意义的。 本着这样的目的,我们通过体外细胞培养,体内建立小鼠荷瘤模型,全面系统地研究了苦参碱对小鼠 H22 肝癌细胞体内外的抗肿瘤作用,并进一步探讨了苦参碱对机体免疫功能的影响,试图从免疫学角度分析和阐述苦参碱的抑瘤作用机制。由于荷瘤机体普遍存在有免疫功能低下的状态,我们将小鼠的一种新的共刺激分子 TIM2 基因转入 H22 细胞内,经过单克隆稳定筛选,得到了小鼠 TIM2 基因修饰的 H22 肝癌细胞瘤苗,以期提高肿瘤细胞的免疫原性,用此瘤苗再次在小鼠体内建立移植瘤模型,进一步了解苦参碱在机体免疫功能改善情况下,对荷瘤小鼠的体内抑瘤作用,为苦参碱将来应用于肿瘤临床治疗提供有价值的理论依据。更多还原

【Abstract】 Objective Matrine is one of the active ingredients—an alkaloid from the extract of traditional Chinese medical herb sophora flavesscens Ait. root. Matrine had been reported to have various pharmacological functions. Our long-term research had proved that matrine could significantly inhibit the proliferation of human leukemic cell lines K562 and induce K562 cells differentiate into normal morphological cell in vitro. Recently, there are increasing study on the matrine’s inhibitory effects on tumor cell lines other than K562 cells, and more attention has been paid to its anti-tumor effects both in vitro and vivo. Nowadays, exploiting our Chinese medicine treasury, excavating common traditional medical herbs and endowing them up-to-date pharmcological explication has been thought to be the general trend of Chinese medicine development. Now, many people are devoting to develop new anti-cancer pharmaceuticals from Chinese herbs medicine, which is necessary to carry forward our precious traditional Chinese medicine. Under this background, it is undoubtedly a promising and singnificant work to investigate thorough the inhibitory effects of matrine on tumor, especial on solid-tumor, and explain its mechanism of anti-tumor effects in vitro and in vivo. Ginven that, we investigated the inhibitory effects of matrine in vitro and in vivo with cell cultivating method, H22 xenograft tumor models estabilishing in BALB/C, and explored the probable mechanisms of matrine in tumor inhibition. We also observed the effects of matrine on the BALB/C’s immunofunction in vivo, trying to analyze the mechanisms of anti-tumor effects of matrine and bring them to the light from immunological perspective. Owing to the immunological response lower in tumor-bearing body, we constructed the eukaryotic expression vector pIRES-EGFP-TIM2 with a murine co-stimulation molecule gene—TIM2 gene inserted and transfected it into H22 hepatocarcinoma cells by Lipofectamin 2000. After electing by G418 pressure and limited dilution method in turn, we obtained the monoclone of the positive H22 cells stably espressing TIM2 gene mRNA and EGFP protein in vitro named as H22-TIM2. We regarded it as the murine H22 hepatocarcinoma whole cell vaccination applied to perform the following experiment in BALB/C. The H22-TIM2 cells were subcutaneously inoculated into BALB/C, expecting improving the immunogenecity of H22 cells, and established the tumor-bearing mice models again. Then we researched the inhibitory efficacy of matrine under the immunofunction improved in this model mice. Our data may provide important clues to clinic application of matrine in tumor treatment in the future. Methods 1. Taking the murine hepatocarcinoma cell line H22 as our experimental object, we observed the H22 cell’s proliferation inhibition treated with matrine and discussed the probable mechanisms of anti-tumor effects of matrine in vitro from the following aspects: the morphological changes of H22 cells under the optical microscope was observed; changes of cell number、the inhibitory rate of cell proliferation and cell growth curve were detected using MTT assay and cell counting method, respectively; cell necrosis was manifested with trypan blue staining method;the effection of matrine on the cell cycle of H22 cells was examined with fluorescein activated cell sortor (FACS) and the cell apoptosis induced by matrine at the early stage was detected with Annexin Ⅴ-FITC/PI double marked assay; the expression of Bcl-2 and Bax protein in H22 cells were detected using immunohistochemistry assay. 2. H22 xenograft tumor models were established in BALB/C. The BALB/C mice in experimental groups were administed matrine with 50mg/kg weight and 100mg/kg weight dosage through abdominal cavity injection, respectively; while those in the control groups were administed 20mg/kg weight CTX and N.S solution, respectively. Matrine were injected into BALB/C mice 7 times q.o.d. The tumor suppression effects of matrine was studied in vivo from the following aspects: the xenograft tumor were excis更多还原