解毒益气方中的主要单体化合物体外抗子宫内膜癌的作用研究

发布时间：2018-03-12 11:50

本文选题：白花蛇舌草黄酮　切入点：半枝莲黄酮　出处：《北京中医药大学》2014年硕士论文　论文类型：学位论文

【摘要】：[目的] 肿瘤微环境可以使抑制肿瘤生长的巨噬细胞分化为促进肿瘤生长和转移的肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs),已知子宫内膜癌(Endometrial Carcinoma,EC)内存在一定数量的TAMS,其数量与EC的恶性程度呈正相关。名老中医郭志强先生在长达数十年的临床实践经验中总结出治疗子宫内膜癌的解毒益气方,在此方基础上加减治疗EC疗效确切。本实验拟通过观察解毒益气方中的主要单体化合物半枝莲黄酮、半枝莲多糖、白花蛇舌草黄酮、白花蛇舌草多糖对子宫内膜癌Ishikawa细胞增殖的抑制作用及对Ishikawa细胞条件培养基孵育的TAMs的抑制作用,初步探讨解毒益气方体外抗EC的作用,从而为开发抗EC中药制剂提供一定的理论和实验依据。 [方法] 1、解毒益气方中的4种单体化合物分别作用于Ishikawa细胞24h、48h,MTT方法检测各单体化合物对Ishikawa细胞增殖的抑制作用。 2、收集体外培养的Ishikawa细胞条件培养基,孵育人淋巴瘤U973细胞,建立体外诱导TAMs活化模型。 3、用流式细胞术检测[AMs表型分子CD163、CD206的表达。 4、用Elisa方法检测巨噬细胞上清液中IL-10的水平。 [结果] 1、半枝莲黄酮、白花蛇舌草黄酮(1.61ng/ml、800μg/ml、400μg/ml、200μg/ml、100μg/ml、50μg/ml、1、25μg/ml、12.5μg/ml)分别作用于Ishikawa细胞24h、48h后,MTT法检测其对Ishikawa细胞增殖的抑制作用,结果表明：1.6rμg/ml、800μg/ml、400μg/ml、200μg/ml、组的半枝莲黄酮较对照组对Ishikawa细胞增殖有明显的抑制作用,其中1.6mg/ml、800μg/ml、400μg/ml与对照组相比p0.001；200μg/ml组与对照组相比p0.05,差异均有统计学意义；浓度200μ g/ml组对Ishikawa细胞的增殖无抑制作用,与对照组相比p0.05,差异无统计学意义；1.6mg/ml、800μg/ml的白花蛇舌草黄酮较对照组对Ishikawa细胞有明显抑制作用(p0.001),差异有统计学意义；低浓度(≤400μg/ml)白花蛇舌草黄酮对Ishikawa细胞增殖无抑制作用；根据抑制率曲线计算出半枝莲黄酮、白花蛇舌草黄酮的有效浓度分别约为：300μg/ml、800μg/ml、半枝莲多糖(1.25μg/ml、0.625mg/ml、0.3125mg/ml、156.25μg/ml、78.125μg/ml、39.0625μ g/m1)白花蛇舌草多糖(2.5：mg/ml、1.25mg/ml、0.625mg/ml、0.3125ng/ml、156.25μg/ml、78.125μg/ml、)作用于Ishikawa细胞24h、48h后,对其增殖抑制作用不明显,与对照组相比p0.05,差异无统计学意义。 2、Ishikawa细胞培养液可以上调TAMS特征表型分子CD206、CD163的表达,与对照组相比p0.05,差异有统计学意义,表明Ishikawa细胞培养液可以将巨噬细胞驯化为TAMS,即体外诱导TAMS活化模型成功建立。 3、300μ g/ml组半枝莲黄酮、800μg/ml组白花蛇舌草黄酮较对照组可显著下调TAMs特征性表型分子CD163、CD206的表达(p0.001),差异有统计学意义。 4、300μg/ml、组半枝莲黄酮、800μg/ml组白花蛇舌草黄酮较对照组可明显下调巨噬细胞上清液中细胞因子IL-10的表达(p0.01),差异有统计学意义。 [结论] 1、解毒益气方中的半枝莲黄酮、白花蛇舌草黄酮能够显著抑制Ishikawa细胞增殖。 2、Ishikawa细胞培养液可以将巨噬细胞驯化为TAMs。 3、解毒益气方中的半枝莲黄酮、白花蛇舌草黄酮可以抑制TAMs的形成,从而起到抗子宫内膜癌的作用。
[Abstract]:[Objective]
The tumor microenvironment can make macrophage differentiation inhibiting tumor growth to promote tumor growth and metastasis of tumor associated macrophages (tumor-associated, macrophages, TAMs), known for endometrial cancer (Endometrial Carcinoma, EC) TAMS memory in a certain number, the number of malignant degree and EC were positively correlated. Mr. Guo Zhiqiang summed up the old Chinese medicine treatment endometrial cancer yiqi'formula on decades of experience in clinical practice, and based on this decoction is effective in treating EC. The experiment mainly monomeric compounds of Scutellaria barbata flavonoid yiqi'formula observation of Scutellaria barbata polysaccharide, Hedyotis diffusa flavonoids, inhibition of Oldenlandia polysaccharide on proliferation of endometrium cancer Ishikawa cells and incubationa549 TAMs inhibited by Ishikawa on cell culture conditions, to explore the effect of Jiedu Yiqi Decoction against EC in vitro, and It provides a certain theoretical and experimental basis for the development of anti EC traditional Chinese medicine preparation.
[method]
1, 4 kinds of monomer compounds in Jiedu Yiqi recipe acted on Ishikawa cells 24h, 48h and MTT respectively to detect the inhibitory effect of monomer compounds on Ishikawa cell proliferation.
2, the conditioned medium of Ishikawa cells cultured in vitro was collected and the lymphoma U973 cells were incubated and the activation model of TAMs was induced in vitro.
3, the expression of [AMs phenotypic molecule CD163 and CD206 was detected by flow cytometry.
4, the level of IL-10 in macrophage supernatant was detected by Elisa method.
[results]
1, Banzhilian Hedyotis diffusa flavonoids, flavonoids (1.61ng/ml, 800 g/ml, 400 g/ml, 200 g/ml, 100 g/ml, 50 g/ml, 1,25 g/ml, 12.5 g/ml) Ishikawa cells were treated with 24h, after 48h, the results showed that detection of its inhibitory effect on the proliferation of Ishikawa cells by MTT 1.6r g/ml, 800 g/ml, 400 g/ml, 200 g/ml, the inhibitory effect of Scutellaria barbata flavonoid group, obvious proliferation of Ishikawa cells compared with control 1.6mg/ml, 800 g/ml, 400 g/ml compared with the control group p0.001; 200 g/ml P0.05 group compared with the control group, the differences were statistically. Significance; no inhibition concentration of 200 g/ml group on the proliferation of Ishikawa cells compared with the control group P0.05, the difference was not statistically significant; 1.6mg/ml, 800 g/ml diffusa flavonoid group on Ishikawa cells significantly inhibited compared with the control (p0.001), the difference was statistically significant; the low concentration (less than or equal to 400 mu g/ml white) Diffusa flavonoids had no inhibitory effect on the proliferation of Ishikawa cells; according to the inhibition rate curve to calculate the concentration of flavonoids of Scutellaria barbata, Hedyotis diffusa flavonoids respectively: 300 g/ml, 800 g/ml, Scutellaria barbata polysaccharide (1.25 g/ml, 0.625mg/ml, 0.3125mg/ml, 156.25 g/ml, 78.125 g/ml, 39.0625 g/m1). Oldenlandia polysaccharide (2.5:mg/ml, 1.25mg/ml, 0.625mg/ml, 0.3125ng/ml, 156.25 g/ml, 78.125 g/ml, 24h) in Ishikawa cells, after 48h, no obvious inhibitory effect on the proliferation of P0.05, compared with the control group, the difference was not statistically significant.
2, Ishikawa cell culture medium can upregulate the expression of TAMS characteristic phenotype CD206 and CD163. Compared with the control group, P0.05 has a statistically significant difference, indicating that Ishikawa cell culture medium can be macrophaged domesticated into TAMS, namely, in vitro induced TAMS activation model is successfully established.
3300 g/ml group 800 g/ml group of Flavonoids from Scutellaria barbata, Hedyotis diffusa flavonoids than the control group significantly reduced the TAMs phenotype in molecular CD163, the expression of CD206 (p0.001), the difference was statistically significant.
4300 g/ml group, 800 g/ml group of Flavonoids from Scutellaria barbata, Hedyotis diffusa flavonoids than control group could significantly decrease the expression of cytokines in the supernatant of IL-10 macrophages (P0.01), the difference was statistically significant.
[Conclusion]
1, yiqi'formula in flavonoids of Scutellaria barbata diffusa, flavonoids can significantly inhibit the proliferation of Ishikawa cells.
2, Ishikawa cell culture can domesticate macrophages into TAMs.
3, yiqi'formula in flavonoids of Scutellaria barbata diffusa, flavonoids can inhibit the formation of TAMs, which plays the role of anti endometrial cancer.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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