本文選題：溪黃草水溶性總黃酮 + HepG2細(xì)胞　； 參考：《廣州中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：肝細(xì)胞癌(Hepatocellular carcinoma,HCC)是一種全球范圍內(nèi)發(fā)病率和死亡率都很高的疾病,目前已成為威脅人類生命健康安全的惡性腫瘤,肝細(xì)胞癌的主要特點(diǎn)為疾病發(fā)展速度快、術(shù)后復(fù)發(fā)率高、癌細(xì)胞轉(zhuǎn)移能力強(qiáng),這給肝細(xì)胞癌的有效防治帶來了巨大的困難,與化學(xué)合成單體藥物相比較,傳統(tǒng)植物藥具有眾多的優(yōu)勢,越來越多的植物藥被發(fā)現(xiàn)具有抗腫瘤活性。因此,對植物藥的研究是探索天然的抗腫瘤藥物的重要途徑。狹基線紋香茶菜Isodon lophanthoides var gerardianus(Benth)Hara 為唇形科多年生草本植物,其地上部分是廣東傳統(tǒng)中藥溪黃草品種之一,為當(dāng)前溪黃草的主流種植品種,具有利膽退黃、清熱祛濕作用,為防治肝膽系統(tǒng)疾病的常用藥,實(shí)驗(yàn)表明狹基線紋香茶菜的水提液具有良好的抗炎、抗腫瘤、保肝活性,其提取物的抗腫瘤活性研究較多,但其藥效物質(zhì)基礎(chǔ)不清楚,我們研究團(tuán)隊(duì)前期篩選狹基線紋香茶菜水溶性成分的抗腫瘤活性,發(fā)現(xiàn)水溶性總黃酮部位能顯著地抑制HepG2細(xì)胞活性,預(yù)實(shí)驗(yàn)結(jié)論也顯示具有誘導(dǎo)細(xì)胞凋亡作用,有可能成為抗腫瘤候選藥物,但藥理研究基礎(chǔ)薄弱,其機(jī)制也沒有涉及。因此,研究其抑制HepG2細(xì)胞體內(nèi)外活性及作用機(jī)制,對于深入開發(fā)應(yīng)用溪黃草具有重要意義。本研究所用溪黃草藥材為狹基線紋香茶菜。目的:本論文對溪黃草水溶性總黃酮的富集純化方法進(jìn)行了改良,研究溪黃草水溶性總黃酮對人肝癌細(xì)胞HepG2生長抑制和誘導(dǎo)凋亡的作用及機(jī)制,通過建立荷瘤裸鼠動(dòng)物模型,探討研究溪黃草水溶性總黃酮對人肝癌裸鼠移植瘤抑制作用及相關(guān)機(jī)制。方法:一、溪黃草水溶性總黃酮對人肝癌細(xì)胞HepG2生長抑制作用采用細(xì)胞增殖抑制實(shí)驗(yàn)(MTT法)檢測溪黃草水溶性總黃酮對人肝癌細(xì)胞HepG2的生長抑制率。通過平皿集落形成試驗(yàn)觀察溪黃草水溶性總黃酮對HepG2細(xì)胞貼壁生長并形成克隆的能力。流式細(xì)胞術(shù)檢測水溶性總黃酮對HepG2細(xì)胞周期分布的影響。二、溪黃草水溶性總黃酮對人肝癌細(xì)胞HepG2凋亡的影響通過檢測細(xì)胞線粒體膜電位,細(xì)胞內(nèi)ROS水平和流式細(xì)胞術(shù)分析水溶性總黃酮誘導(dǎo)HepG2細(xì)胞凋亡情況。三、溪黃草水溶性總黃酮誘導(dǎo)人肝癌細(xì)胞HepG2凋亡機(jī)制研究電鏡檢測細(xì)胞內(nèi)自噬體情況,采用RT-PCR法檢測溪黃草水溶性總黃酮對各組細(xì)胞Bcl-2mRNA、Bax mRNA、Survivin mRNA的基因表達(dá)變化、運(yùn)用Western blot法檢測各組的線粒體和胞質(zhì) cytochrome c,cytosolic AIF、Bcl-2、Bax、Survivin 蛋白表達(dá),caspase-3蛋白表達(dá)。四、溪黃草水溶性總黃酮對人肝癌裸鼠移植瘤抑制作用研究建立荷瘤裸鼠動(dòng)物模型,給藥后觀察并測量裸鼠瘤體,計(jì)算體積大小,并繪出生長曲線。分離瘤體后,計(jì)算抑制率,病理組織學(xué)檢查,檢測外周血甲胎球蛋白變化,TUNEL染色檢測各組移植瘤細(xì)胞凋亡。五、溪黃草水溶性總黃酮對人肝癌裸鼠移植瘤抑制作用機(jī)制研究透射電鏡觀察瘤組織細(xì)胞的自噬小體和細(xì)胞超微結(jié)構(gòu),RT-PCR法檢測基因Beclinl、Atg5、CyclinD1、Caspase-3、bax、bcl-2、Survivin、nm23-H1 和 MMP-9 mRNA的表達(dá),Western blot 法檢測指標(biāo):Beclinl、Atg5、CyclinD1、Caspase-3、bax、bcl-2、Survivin、nm23-H1 和 MMP-9 蛋白表達(dá)。結(jié)果:一、溪黃草水溶性總黃酮對人肝癌細(xì)胞HepG2生長抑制作用MTT法結(jié)果顯示,水溶性總黃酮對HepG2細(xì)胞增殖有明顯的抑制作用,呈現(xiàn)濃度依賴性和一定的時(shí)間依賴關(guān)系。給藥后顯微鏡下觀察到細(xì)胞出現(xiàn)各期凋亡細(xì)胞形態(tài),產(chǎn)生凋亡小體,提示可能與促進(jìn)HepG2細(xì)胞凋亡有關(guān)。細(xì)胞平皿集落形成實(shí)驗(yàn)顯示,水溶性總黃酮處理組的細(xì)胞集落形成數(shù)明顯少于溶媒對照組。提示可能與抑制細(xì)胞錨定依賴性生長作用有關(guān)。細(xì)胞周期實(shí)驗(yàn)結(jié)果顯示,總黃酮各濃度組G0/G1期細(xì)胞比例均明顯高于對照組,而S期細(xì)胞比例均明顯低于對照組,這說明水溶性總黃酮能有效地將HepG2細(xì)胞阻滯于G0/G1期,進(jìn)而抑制細(xì)胞增殖和生長,呈現(xiàn)劑量依賴性。二、溪黃草水溶性總黃酮對人肝癌細(xì)胞HepG2凋亡的影響流式細(xì)胞術(shù)分析結(jié)果顯示,水溶性總黃酮各濃度組和各時(shí)間點(diǎn)的凋亡率均明顯高于對照組,呈劑量和時(shí)間依賴關(guān)系。細(xì)胞線粒體膜電位實(shí)驗(yàn)顯示,水溶性總黃酮和5-FU均能誘導(dǎo)HepG2細(xì)胞線粒體膜電位去極化。檢測細(xì)胞內(nèi)ROS水平,結(jié)果顯示,水溶性總黃酮能誘導(dǎo)HepG2細(xì)胞內(nèi)ROS升高。這些提示水溶性總黃酮能通過干擾細(xì)胞線粒體膜電位,升高細(xì)胞內(nèi)ROS水平,誘導(dǎo)HepG2細(xì)胞凋亡。三、溪黃草水溶性總黃酮誘導(dǎo)人肝癌細(xì)胞HepG2凋亡機(jī)制研究透射電鏡下觀察,水溶性總黃酮具有抑制細(xì)胞自噬作用;RT-PCR法檢測結(jié)果顯示,抗凋亡蛋白基因bcl-2 mRNA和Survivin mRNA隨著水溶性總黃酮濃度升高而減少,呈濃度依賴關(guān)系,相反,促凋亡蛋白基因BaxmRNA和Caspase-3mRNA隨著藥物濃度升高而增加,也呈濃度依賴關(guān)系。Western blot法分析結(jié)果也進(jìn)一步證實(shí)水溶性總黃酮是通過下調(diào)抗凋亡蛋白(bcl-2 and mcl-1)和survivin和上調(diào)促凋亡蛋白(Bax)的表達(dá)水平誘導(dǎo)HepG2細(xì)胞凋亡。WB分析還發(fā)現(xiàn),水溶性總黃酮還增高線粒體膜通透性,向胞質(zhì)內(nèi)釋放促凋亡因子Cytochrome c和Cytosolic AIF,引起細(xì)胞凋亡。四、溪黃草水溶性總黃酮對人肝癌裸鼠移植瘤抑制作用研究各組連續(xù)給藥21天,繪制腫瘤生長曲線,計(jì)算抑瘤率,與模型組相比,水溶性總黃酮高、中劑量藥物組和5-FU組均能抑制腫瘤的生長,呈劑量相關(guān)性,其中以藥物高劑量組、5-FU組和水溶性總黃酮高劑量+5-FU組效果最佳。各給藥組與模型組比較,均能顯著地降低AFP。而各給藥組的腫瘤/體重比結(jié)果顯示,水溶性總黃酮高劑量+5-FU組動(dòng)物的基本情況明顯好轉(zhuǎn),說明水溶性總黃酮和5-FU聯(lián)合應(yīng)用既抑制了腫瘤生長,又克服了化藥的副作用,改善了生活質(zhì)量。病理組織學(xué)檢查發(fā)現(xiàn),水溶性總黃酮各組的瘤體中腫瘤細(xì)胞發(fā)生凋亡,有中度壞死灶,這表明水溶性總黃酮對癌細(xì)胞具有促進(jìn)凋亡、抑制分裂增殖的作用。荷瘤裸鼠胸腹腔未見積液,心、肝、腎、脾、肺、腦等臟器未見癌細(xì)胞轉(zhuǎn)移結(jié)節(jié)及其他異常。Tunel染色發(fā)現(xiàn),水溶性總黃酮組隨著濃度的增大凋亡細(xì)胞數(shù)量增加,顯示出一定的量效關(guān)系。五、溪黃草水溶性總黃酮對人肝癌裸鼠移植瘤抑制作用機(jī)制研究電鏡觀察水溶性總黃酮抑制肝癌細(xì)胞形成自噬體,這與細(xì)胞實(shí)驗(yàn)中的結(jié)論相一致。細(xì)胞超微結(jié)構(gòu)也出現(xiàn)了明顯的形態(tài)學(xué)改變和大量的凋亡小體。RT-PCR法檢測表明水溶性總黃酮是通過下調(diào)抗凋亡蛋白基因bcl-2 mRNA和survivinmRNA和上調(diào)促凋亡蛋白Bax mRNA和Caspase-3 mRNA的表達(dá)水平誘導(dǎo)移植瘤HepG2細(xì)胞凋亡。水溶性總黃酮能抑制自噬基因Beclin1mRNA、Atg5mRNA的表達(dá),能抑制CyclinD1 mRNA、MMP-9mRNA基因的表達(dá),且呈現(xiàn)濃度越高越明顯的濃度依賴關(guān)系,相反,水溶性總黃酮能促進(jìn)nm23-H1基因的表達(dá),濃度越高越明顯。Western blot法分析結(jié)果也進(jìn)一步證實(shí)水溶性總黃酮是通過下調(diào)抗凋亡蛋白基因bcl-2和Survivin和上調(diào)促凋亡蛋白Bax和Caspase-3的表達(dá)水平誘導(dǎo)移植瘤HepG2細(xì)胞凋亡。水溶性總黃酮還能抑制Beclin1、Atg5、CyclinD1、MMP-9等蛋白的表達(dá),相反,水溶性總黃酮能促進(jìn)nm23-H1蛋白的表達(dá),結(jié)論:本研究以溪黃草水溶性總黃酮為研究對象,應(yīng)用了多種方法研究了其體內(nèi)外抑制HepG2細(xì)胞的活性及其機(jī)制。在體外,水溶性總黃酮有效地將HepG2細(xì)胞阻滯于G0/G1期,干擾細(xì)胞線粒體膜電位,升高細(xì)胞內(nèi)ROS水平,誘導(dǎo)HepG2細(xì)胞凋亡。其機(jī)制可能是通過下調(diào)抗凋亡蛋白(bcl-2 and mcl-1)和survivin和上調(diào)促凋亡蛋白(Bax)的表達(dá)水平,促使細(xì)胞色素C向胞質(zhì)內(nèi)釋放,誘導(dǎo)HepG2細(xì)胞凋亡,抑制細(xì)胞增殖。在體內(nèi),水溶性總黃酮能抑制裸鼠移植瘤的生長。其可能機(jī)制一方面通過抑制自噬,阻止腫瘤細(xì)胞從周圍的基質(zhì)細(xì)胞中獲取能量和營養(yǎng)物質(zhì),從而抑制腫瘤細(xì)胞生長,另一方面,通過下調(diào)抗凋亡蛋白基因bcl-2和Survivin和上調(diào)促凋亡蛋白Bax和Caspase-3的表達(dá)水平誘導(dǎo)移植瘤HepG2細(xì)胞凋亡,并通過有效地抑制HepG2細(xì)胞體內(nèi)侵襲和轉(zhuǎn)移能力抑制腫瘤生長。這些研究為溪黃草臨床應(yīng)用及進(jìn)一步開發(fā)應(yīng)用提供了可靠的理論依據(jù)。
[Abstract]:Hepatocellular carcinoma (HCC) is a disease with high morbidity and mortality in the world. It has become a malignant tumor that threatens the health and safety of human life. The main characteristics of hepatocellular carcinoma are rapid development of disease, high recurrence rate after operation and strong metastasis of cancer cells. This is an effective prevention and control zone for hepatocellular carcinoma. Compared with chemical synthetic monomers, traditional plant drugs have many advantages, and more and more plant drugs have been found to have anti-tumor activity. Therefore, the study of plant drugs is an important way to explore natural antitumor drugs. The narrow baselines Isodon lophanthoides var gerardianus (Benth) Hara As one of the perennial herbaceous plants of the family lip family, the upper part of the family is one of the traditional Chinese herbs of Guangdong, one of the main varieties of Creek yellow grass. It is the main main plant of the current Herba yellowgrass. It is a common medicine for the prevention and treatment of liver and gallbladder diseases. The experiment shows that the water extracts of the narrow baselines have good anti-inflammatory, anti-tumor and liver preservation activities. The antitumor activity of the extract is not clear, but our research team screened the antitumor activity of the water-soluble components of the narrow baselines, and found that the water soluble total flavonoids could significantly inhibit the activity of HepG2 cells. The pre experimental conclusion also showed that it has the effect of inducing cell apoptosis. Anti tumor drugs, but the pharmacological research foundation is weak and its mechanism is not involved. Therefore, it is of great significance to study its inhibitory activity and mechanism of HepG2 cells in vivo and in depth for the development and application of Creek grass. The purification method was improved to study the effect and mechanism of the water soluble total flavonoids of Creek grasses on the growth inhibition and induction of apoptosis of human hepatoma cell HepG2. By establishing a tumor bearing nude mouse model, the inhibitory effects and related mechanisms of water soluble total flavonoids of Creek grass on human hepatoma in nude mice were investigated and studied. The inhibitory effect of ketone on the growth of human hepatoma cell HepG2 using cell proliferation inhibition test (MTT method) to detect the growth inhibition rate of water soluble total flavonoids of Creek grasses on human hepatoma cell HepG2. The ability of water soluble total flavonoids of Creek grass to observe the growth and clone of HepG2 cells was observed by the plate colony formation test. Flow cytometry was used to detect water solubility. The effect of total flavonoids on the cycle distribution of HepG2 cells. Two, the effect of water soluble total flavonoids on HepG2 apoptosis in human hepatoma cells by detecting cell mitochondrial membrane potential, intracellular ROS level and flow cytometry analysis of water soluble total flavonoids to induce apoptosis in HepG2 cells. Three, the water soluble total flavonoids of Creek hay induced human hepatoma cells Hep The mechanism of G2 apoptosis was studied by electron microscopy, and RT-PCR method was used to detect the changes in the expression of Bcl-2mRNA, Bax mRNA, Survivin mRNA, and cytochrome c of each group by Western blot method. Protein expression. Four, study on the inhibitory effect of water soluble total flavonoids on human hepatoma xenografts in nude mice, to establish a nude mouse model of nude mice, to observe and measure the tumor in nude mice after administration, to calculate the size, and to draw the growth curve. After the separation of the tumor, the inhibition rate, pathological examination, detection of the changes of peripheral blood alpha fetoglobulin and TUNEL staining were detected. Detection of the apoptosis of transplanted tumor cells in each group. Five, the mechanism of the inhibitory effect of water soluble total flavonoids on human hepatoma xenografts in nude mice, the autophagic corpuscles and ultrastructure of the tumor tissue cells were observed by transmission electron microscopy, and the expression of Beclinl, Atg5, CyclinD1, Caspase-3, Bax, Bcl-2, Survivin, nm23-H1 and MMP-9 mRNA were detected by the transmission electron microscope. N blot assay: Beclinl, Atg5, CyclinD1, Caspase-3, Bax, Bcl-2, Survivin, nm23-H1 and MMP-9 protein expression. Results: 1. The inhibitory effect of water soluble total flavonoids on the growth of human hepatocellular carcinoma cells showed that the water-soluble total flavonoids had a significant inhibitory effect on the proliferation of cells, showing a concentration dependence and certain degree of inhibition. Time dependence. The cell morphology of cell apoptotic cells appeared at various stages and apoptotic bodies were observed under microscopically, suggesting that it may be related to the promotion of HepG2 cell apoptosis. Cell colony formation experiment showed that the number of colony formation in the water soluble total flavonoids treatment group was less than that of the medium control group. The cell cycle test showed that the proportion of G0/G1 phase cells in the total flavonoids was significantly higher than that of the control group, while the proportion of S cells was significantly lower than that of the control group. This indicated that the water-soluble total flavonoids could effectively block the HepG2 cells in the G0/G1 phase, and then inhibit the proliferation and growth of the cells, and showed a dose dependence. Two The effects of water soluble total flavonoids on the apoptosis of human hepatoma cell HepG2 were analyzed by flow cytometry. The results showed that the apoptosis rate of water soluble total flavonoids and each time point were significantly higher than those of the control group, showing a dose and time dependence. The cell mitochondrial membrane potential experiment showed that the water-soluble total flavonoids and 5-FU could induce HepG2 cells. Mitochondrial membrane potential depolarization. Detection of intracellular ROS level, the results show that water-soluble total flavonoids can induce the increase of ROS in HepG2 cells. These suggest that water soluble total flavonoids can induce apoptosis of HepG2 cells by interfering with cell mitochondrial membrane potential and increasing intracellular ROS level. Three, the water soluble total flavonoids of Creek hay induced the apoptosis of human hepatoma cells HepG2 The results of RT-PCR assay showed that the anti apoptotic protein gene Bcl-2 mRNA and Survivin mRNA decreased with the increase of the concentration of water soluble total flavonoids, which showed a concentration dependence. On the contrary, the apoptotic protein gene BaxmRNA and Caspase-3mRNA were increased with the concentration of drugs. The results of.Western blot analysis showed that water soluble total flavonoids also induced.WB analysis by down regulating the expression level of anti apoptotic protein (Bcl-2 and Mcl-1) and Survivin and up regulation of apoptotic protein (Bax), and found that the water soluble total flavonoids also increased the membrane permeability of mitochondrial membrane. Apoptotic factor Cytochrome C and Cytosolic AIF were released in the cytoplasm. Four, the inhibitory effect of water soluble total flavonoids on human hepatoma xenografts in nude mice was administered for 21 days. The tumor growth curve was plotted and the tumor suppressor rate was calculated. Compared with the model group, the total water-soluble flavonoids were high, the medium dose drug group and the 5-FU group were all inhibited. The growth of the tumor was dose-dependent. In the high dose group, the 5-FU group and the water soluble total Huang Tonggao dose +5-FU group had the best effect. Compared with the model group, the drug group and the model group could significantly reduce the AFP. and the results of the tumor / body weight ratio of each group. The basic situation of the high dose of water soluble total flavonoids in the high dose +5-FU group was obviously improved. The combination of water soluble total flavonoids and 5-FU not only inhibited the growth of the tumor, but also overcame the side effects of the chemicals, and improved the quality of life. Histopathological examination found that the tumor cells in the tumor bodies of the total water soluble total flavonoids were apoptotic and had moderate necrosis, which showed that the water-soluble total flavonoids could promote apoptosis and inhibit the division of the cancer cells. No effusion was found in the breast and abdominal cavity of nude mice. No metastasis nodules of cancer cells and other abnormal.Tunel staining were found in the heart, liver, kidney, spleen, lung and brain. The number of apoptotic cells increased with the increase of concentration and showed a certain dose effect relationship. Five, the water soluble total flavonoids of Creek grasses inhibit the transplantation tumor of human liver cancer in nude mice. The production mechanism study electron microscope to observe the water soluble total flavonoids inhibit the formation of autophago in hepatoma cells, which is in accordance with the conclusion in the cell experiment. The ultrastructure of the cell also has obvious morphological changes and a large number of apoptotic bodies.RT-PCR assay showed that the water soluble total flavonoids were down regulated by the anti apoptotic protein gene Bcl-2 mRNA and survivi The expression of nmRNA and Bax mRNA and Caspase-3 mRNA induced the apoptosis of HepG2 cells in transplanted tumor. Water soluble total flavonoids can inhibit the expression of autophagic gene Beclin1mRNA, Atg5mRNA, and inhibit the expression of CyclinD1 mRNA, MMP-9mRNA gene, and the concentration dependence of concentration is higher and more obvious. On the contrary, water soluble total flavonoids can be found. The higher the expression of nm23-H1 gene, the higher the concentration of the.Western blot method, the results also further confirmed that the water soluble total flavonoids can induce the apoptosis of the transplanted tumor HepG2 cells by down regulation of the anti apoptotic protein gene Bcl-2 and Survivin and up regulation of the expression level of the apoptotic protein Bax and Caspase-3. The water soluble total flavonoids can also inhibit Beclin1, Atg5, Cyc. The expression of linD1, MMP-9 and other proteins, in contrast, water soluble total flavonoids can promote the expression of nm23-H1 protein. Conclusion: in this study, a variety of methods were used to study the inhibitory activity and mechanism of HepG2 cells in vitro and in vitro. In vitro, the water soluble total flavonoids effectively block the HepG2 cells in the G0/G1 phase. Interfering with the mitochondrial membrane potential and increasing the intracellular ROS level and inducing apoptosis of HepG2 cells, the mechanism may be to induce the release of cytochrome C into the cytoplasm by down regulation of the anti apoptotic protein (Bcl-2 and Mcl-1) and Survivin and up regulation of apoptotic protein (Bax), inducing the apoptosis of HepG2 cells and inhibiting the proliferation of cells. In vivo, water solubility Total flavonoids can inhibit the growth of xenografts in nude mice. Its possible mechanism, by inhibiting autophagy, prevents tumor cells from obtaining energy and nutrients from the surrounding matrix cells, thus inhibiting the growth of tumor cells. On the other hand, down regulation of anti apoptotic protein gene Bcl-2 and Survivin and up regulation of apoptotic protein Bax and Caspase-3 Levels of HepG2 cells can induce apoptosis of transplanted tumor cells and inhibit the growth of tumor by effectively inhibiting the invasion and metastasis of HepG2 cells. These studies provide a reliable theoretical basis for the clinical application and further development and application of Creek grasses.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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