【作者】 李际君；

【导师】 刘亚娴；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2004， 硕士

【摘要】 目的：发掘研究古方，探讨启膈散抗肿瘤作用机理，扩大古方应用范围；为启膈散治疗肿瘤提供客观依据，进一步指导临床应用。方法1.采用MTT法观察启膈散水提取物对人红白血病细胞株K562、人B淋巴细胞瘤细胞株Raji、人胃腺癌细胞株BGC823、人食管癌细胞株TE13体外增殖的抑制作用。2.采用MTT法研究启膈散水提取物对小鼠脾细胞体外增殖的促进作用。3.采用ELISA法研究启膈散水提取物对人红白血病细胞株K562凋亡的影响。4.采用CTLL-2生物法观察启膈散水提取物小鼠脾T细胞分泌IL-2功能的影响。5.采用L929生物法研究启膈散水提取物对小鼠单核细胞分泌TNF-α功能的影响。6.采用MTT法观察启膈散水提取物对NK细胞对肿瘤细胞杀伤活性的影响。结果1.启膈散与对照组比较，对不同组织来源的肿瘤细胞增殖表现出很强的抑制作用，且在7.81~500mg/ml范围内增殖抑制作用有明显的量效关系。终浓度50mg（生药）/ml启膈<WP=4>散对K562、RAJI、BGC823、TE13的抑制率分别达到了82.2%、78.9%、76.7%、79%，但增殖抑制作用逊于终浓度20μg/ml顺铂。2.启膈散作用小鼠脾细胞72小时后,显著的刺激了小鼠脾细胞的增殖，并在7.81~500mg/ml范围内对增殖表现出明显的量效关系。终浓度50mg（生药）/ml启膈散对小鼠脾细胞增殖率达277.8%,增殖作用显著优于ConA、LPS。3.随作用时间的延长，两种药物促肿瘤细胞凋亡的现象越明显，各时间段结果之间比较存在显著性差异。24小时终浓度50mg（生药）/ml启膈散促凋亡作用优于终浓度20μg/mlDDP，48、72小时两种药物之间比较无差异。4.终浓度50mg（生药）/ml启膈散能显著提高小鼠脾细胞分泌IL-2的活性,作用强于启膈散的其它两个剂量组和ConA组;终浓度5mg/ml、0.5mg/ml启膈散对IL-2产生的增加作用不明显,但优于空白组。5.终浓度50mg/ml启膈散促进小鼠单核细胞分泌TNF-α的作用显著优与其它实验组和空白组。LPS作用优于空白组。6.启膈散三个剂量与小鼠脾细胞共同孵育后，收集的培养上清均可提高NK细胞对靶细胞的杀伤率，均大于对照组,杀伤率分别达到33.28%、40.8%和45.7%,与对照组相比达到极显著差异。ConA刺激NK细胞对靶细胞的杀伤率也都大于对照组,杀伤率达到29.9%,与对照组达到极显著差异。结论：通过实验结果可以发现，启膈散对肿瘤有较强的直接杀伤作用，但逊于顺铂。虽然启膈散的直接杀伤作用逊于顺铂，对肿瘤的凋亡作用，启膈散与顺铂无明显差异，提<WP=5>示启膈散在抗肿瘤作用中一些作用并不逊于化疗药。值得注意的是启膈散在对多种肿瘤细胞表现较强细胞毒活性同时，对正常免疫细胞（小鼠脾细胞）没有抑制作用，且表现出很好的促增殖作用。据此推断，启膈散在对肿瘤的直接杀伤作用并非是通过抑制DNA复制取得的，而是通过诱导肿瘤细胞凋亡发挥作用的。而对正常免疫细胞的增殖作用是通过刺激免疫细胞分泌的细胞因子发挥作用所致。对Nk细胞的作用则证实刺激免疫细胞分泌的细胞因子不仅可以促进正常免疫细胞的增殖，而且具有激活Nk细胞，提高Nk细胞杀伤肿瘤的能力。也从另一个方面证明启膈散对特异性免疫、非特异性免疫俱有促进作用。更多还原

【Abstract】 Objective:Screening of natural products with anti-tumor activity is a new subject in the field of tumor therapy. We examined the cell biology and molecular biology of carcinoma cells and spleen cells treated with the extracts of Chinese herbal medicines. Many cancer patients take these medicines, but their effects at the cellular level are largely unknown.The aim of the the present study was to investigate ancient prescription and observe the anticancer effect of Qige San extract; to enlarge the application of ancient prescription and provide experiment data;to produce the scientific evidence for developing and manufacturing new antitumor drugs.Methods1.Using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium (MTT) cell proliferation assay, we investigated the suppressive effect of Qige San extract on the proliferation of tumor cells up to 72 h such as leucocythemia cell line K562, gastric carcinoma cell line BGC823,esophagus carcinoma cell line TE13 and Burkitt’s lymphoma cell line Raji in vitro. 2.The proliferation quantity of BALB/C’s spleen cells was <WP=7>analysed using MTT assay with and without the treatment of Qige San extract in concentrations of 0.78~50mg/ml up to 72h. 3.A human leucocythemia cell line K562 was cultured in full medium with variable doses of Qige San or cisplatin for different hours. In order to assess apoptosis induction, the cells were exposed to various concentrations of test substances. Histone in the cytoplasm was monitored as an indication of apoptosis Using Enzyme-linked immunosorbent assay (ELISA) .4.The levels of interleukin-2 (IL-2) in the spleen cells at 24h after treated with and without Qige San extract were detected. The IL-2 resulting produced was measured in a bioassay using an IL-2-dependent cell line (CTLL-2).5.The macrophages were isolated from murine spleen and treated with Qige San in vitro. Tumor necrosis factor-alpha (TNF-a) production and cytotoxicity was measured by an murine L929 bioassay on cellular supernatants.6.Augmentation of natural killer (NK) killing activity was studied in vitro. In vitro NK cells from normal mice treated of cellular supernatants with and without Qige San were observed how to enhanced their cytotoxicity against YAC-1 cells.Results1.Qige San extract can markedly inhibited the proliferation of tumor cells such as leucocythemia cell line K562, gastric carcinoma cell line BGC823 and esophagoscope cell line TE13 , Burkitt’s lymphoma cell line Raji in vitro.2.When the spleen cells of BALB/c mice were specifically <WP=8>stimulated with Qige San extract, the optical densities were remarkably higher than that of the controls. The extract (50 mg/ml) significantly promote the proliferation of BALB/C’s spleen cells, the optical densitie was 0.177±0.009,three times of the control.3.Qige San can facilitate the apoptosis of tumor cells depending in a concentration-and-time-dependent manner. The extract (50mg/ml) exhibited significant inducing apoptosis of leucocythemia cell line K562,as same as DDP. The apoptotic responses induced by other extracts were also potentiated compared with the control group.4.The levels of IL-2 in the spleen cells at 24h after treated with Qige san were detected by using the methods of colorimetric analysis of MTT. it appeared that an increase in a concentration dependent manner. Conditioned medium from splenic lymphocytes treated with both ConA and the extract (50 mg/ml) further increased CTLL-2 cell proliferation indicating an addnl. increase of IL-2 secretion. 5.Qige San can significantly stimulate the exudation of TNF-αby BALB/C’s monocyte cells.The results showed that the levels of TNF were higher obviously by the drug-treated group than in the control group (P < 0.01). The macrophages treated with Qige San in vitro displayed significant cytolytic activity toward TNF-alpha-sensitive L929 cells in a dose-dependent manner.6.Pretreatment of NK cells primarily with Qige San over <WP=9>24h significantly enhanced their cytotoxicity against YAC-1 cells in vitro. Bot更多还原