發(fā)布時(shí)間：2018-03-12 11:50

  本文選題：白花蛇舌草黃酮　切入點(diǎn)：半枝蓮黃酮　出處：《北京中醫(yī)藥大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：[目的] 腫瘤微環(huán)境可以使抑制腫瘤生長(zhǎng)的巨噬細(xì)胞分化為促進(jìn)腫瘤生長(zhǎng)和轉(zhuǎn)移的腫瘤相關(guān)巨噬細(xì)胞(tumor-associated macrophages,TAMs),已知子宮內(nèi)膜癌(Endometrial Carcinoma,EC)內(nèi)存在一定數(shù)量的TAMS,其數(shù)量與EC的惡性程度呈正相關(guān)。名老中醫(yī)郭志強(qiáng)先生在長(zhǎng)達(dá)數(shù)十年的臨床實(shí)踐經(jīng)驗(yàn)中總結(jié)出治療子宮內(nèi)膜癌的解毒益氣方,在此方基礎(chǔ)上加減治療EC療效確切。本實(shí)驗(yàn)擬通過(guò)觀察解毒益氣方中的主要單體化合物半枝蓮黃酮、半枝蓮多糖、白花蛇舌草黃酮、白花蛇舌草多糖對(duì)子宮內(nèi)膜癌Ishikawa細(xì)胞增殖的抑制作用及對(duì)Ishikawa細(xì)胞條件培養(yǎng)基孵育的TAMs的抑制作用,初步探討解毒益氣方體外抗EC的作用,從而為開(kāi)發(fā)抗EC中藥制劑提供一定的理論和實(shí)驗(yàn)依據(jù)。 [方法] 1、解毒益氣方中的4種單體化合物分別作用于Ishikawa細(xì)胞24h、48h,MTT方法檢測(cè)各單體化合物對(duì)Ishikawa細(xì)胞增殖的抑制作用。 2、收集體外培養(yǎng)的Ishikawa細(xì)胞條件培養(yǎng)基,孵育人淋巴瘤U973細(xì)胞,建立體外誘導(dǎo)TAMs活化模型。 3、用流式細(xì)胞術(shù)檢測(cè)[AMs表型分子CD163、CD206的表達(dá)。 4、用Elisa方法檢測(cè)巨噬細(xì)胞上清液中IL-10的水平。 [結(jié)果] 1、半枝蓮黃酮、白花蛇舌草黃酮(1.61ng/ml、800μg/ml、400μg/ml、200μg/ml、100μg/ml、50μg/ml、1、25μg/ml、12.5μg/ml)分別作用于Ishikawa細(xì)胞24h、48h后,MTT法檢測(cè)其對(duì)Ishikawa細(xì)胞增殖的抑制作用,結(jié)果表明：1.6rμg/ml、800μg/ml、400μg/ml、200μg/ml、組的半枝蓮黃酮較對(duì)照組對(duì)Ishikawa細(xì)胞增殖有明顯的抑制作用,其中1.6mg/ml、800μg/ml、400μg/ml與對(duì)照組相比p0.001；200μg/ml組與對(duì)照組相比p0.05,差異均有統(tǒng)計(jì)學(xué)意義；濃度200μ g/ml組對(duì)Ishikawa細(xì)胞的增殖無(wú)抑制作用,與對(duì)照組相比p0.05,差異無(wú)統(tǒng)計(jì)學(xué)意義；1.6mg/ml、800μg/ml的白花蛇舌草黃酮較對(duì)照組對(duì)Ishikawa細(xì)胞有明顯抑制作用(p0.001),差異有統(tǒng)計(jì)學(xué)意義；低濃度(≤400μg/ml)白花蛇舌草黃酮對(duì)Ishikawa細(xì)胞增殖無(wú)抑制作用；根據(jù)抑制率曲線計(jì)算出半枝蓮黃酮、白花蛇舌草黃酮的有效濃度分別約為：300μg/ml、800μg/ml、半枝蓮多糖(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細(xì)胞24h、48h后,對(duì)其增殖抑制作用不明顯,與對(duì)照組相比p0.05,差異無(wú)統(tǒng)計(jì)學(xué)意義。 2、Ishikawa細(xì)胞培養(yǎng)液可以上調(diào)TAMS特征表型分子CD206、CD163的表達(dá),與對(duì)照組相比p0.05,差異有統(tǒng)計(jì)學(xué)意義,表明Ishikawa細(xì)胞培養(yǎng)液可以將巨噬細(xì)胞馴化為T(mén)AMS,即體外誘導(dǎo)TAMS活化模型成功建立。 3、300μ g/ml組半枝蓮黃酮、800μg/ml組白花蛇舌草黃酮較對(duì)照組可顯著下調(diào)TAMs特征性表型分子CD163、CD206的表達(dá)(p0.001),差異有統(tǒng)計(jì)學(xué)意義。 4、300μg/ml、組半枝蓮黃酮、800μg/ml組白花蛇舌草黃酮較對(duì)照組可明顯下調(diào)巨噬細(xì)胞上清液中細(xì)胞因子IL-10的表達(dá)(p0.01),差異有統(tǒng)計(jì)學(xué)意義。 [結(jié)論] 1、解毒益氣方中的半枝蓮黃酮、白花蛇舌草黃酮能夠顯著抑制Ishikawa細(xì)胞增殖。 2、Ishikawa細(xì)胞培養(yǎng)液可以將巨噬細(xì)胞馴化為T(mén)AMs。 3、解毒益氣方中的半枝蓮黃酮、白花蛇舌草黃酮可以抑制TAMs的形成,從而起到抗子宮內(nèi)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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33

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本文編號(hào)：1601430