中華獼猴桃根總?cè)瓶鼓[瘤細(xì)胞增殖和腸癌細(xì)胞侵襲及轉(zhuǎn)移研究
發(fā)布時(shí)間：2018-03-04 10:10
本文選題：中華獼猴桃根總?cè)?/strong> 切入點(diǎn)：提取 出處：《桂林醫(yī)學(xué)院》2017年碩士論文 論文類型：學(xué)位論文
【摘要】：目的:優(yōu)化大孔吸附樹(shù)脂對(duì)野生中華獼猴桃根總?cè)?tt-RAC)的提取工藝,并進(jìn)一步探究總?cè)茖?duì)多種腫瘤細(xì)胞的增殖抑制作用和腸癌細(xì)胞LOVO的侵襲和轉(zhuǎn)移抑制作用。方法:用75%乙醇浸泡樣品粉末,分別以40%,50%,60%,70%,80%及95%乙醇為有機(jī)溶劑,以中華獼猴桃根總?cè)瓢俜趾繛橹笜?biāo),確定AB-8型大孔吸附樹(shù)脂對(duì)總?cè)频奶崛」に嚄l件;選取中華獼猴桃根總?cè)瓢俜趾孔罡呓M作為后續(xù)實(shí)驗(yàn)樣品,采用細(xì)胞增殖與活性檢測(cè)試驗(yàn)(CCK-8法)檢測(cè)不同濃度(25,50,100,200,400 ug/ml)野生中華獼猴桃根總?cè)?tt-RAC)對(duì)人腸癌SW480細(xì)胞、LOVO細(xì)胞,人乳腺癌MCF-7細(xì)胞及人肝癌SMMC-7721細(xì)胞的增殖作用,并計(jì)算tt-RAC作用于不同細(xì)胞的半數(shù)抑制濃度(IC50);采用流式細(xì)胞術(shù)(FCM)分析其對(duì)腸癌SW480細(xì)胞周期分布的影響;采用劃痕實(shí)驗(yàn)觀察tt-RAC對(duì)LOVO細(xì)胞遷移影響,以劃痕兩側(cè)細(xì)胞向中心遷移的距離為指標(biāo);并進(jìn)一步用transwell小室試驗(yàn)觀察tt-RAC對(duì)LOVO細(xì)胞穿透能力的影響,以判斷對(duì)腫瘤細(xì)胞侵襲運(yùn)動(dòng)的作用;最后,運(yùn)用Western Blotting(WB)實(shí)驗(yàn)檢測(cè)上皮-間質(zhì)轉(zhuǎn)化(epithelial-to-mesenchymal transition,EMT)相關(guān)基因E-cadherin、N-cadherin和vimentin在蛋白水平上的表達(dá)差異。結(jié)果:AB-8大孔吸附樹(shù)脂提取中華獼猴桃根總?cè)频淖罴压に嚄l件為:中華獼猴桃根上樣液濃度為2.8mg/ml,PH值為7,上樣流速為1BV/h;洗脫體積為4倍樹(shù)脂柱床體積(4BV),洗脫流速為2BV/h。經(jīng)40%乙醇洗脫提取的中華獼猴桃根總?cè)频陌俜趾孔罡邽?5.89%。因此,選取40%乙醇提取的中華獼猴桃根總?cè)?tt-RAC)做后續(xù)實(shí)驗(yàn),其對(duì)四種腫瘤細(xì)胞增殖均有抑制作用并呈濃度和時(shí)間依賴性;其中對(duì)SW480細(xì)胞的抑制作用最強(qiáng),其機(jī)制可能是通過(guò)誘導(dǎo)SW480細(xì)胞發(fā)生S和G2期阻滯引起的。在劃痕試驗(yàn)中,將藥物濃度低于IC50的tt-RAC作用于LOVO細(xì)胞結(jié)果顯示,低濃度tt-RAC可抑制劃痕兩側(cè)LOVO細(xì)胞向劃痕中心遷移。Transwell實(shí)驗(yàn)同樣顯示低濃度tt-RAC抑制上室細(xì)胞向下室侵襲,隨濃度增高侵入下室細(xì)胞數(shù)量逐漸減少,呈濃度依賴性。WB實(shí)驗(yàn)結(jié)果顯示tt-RAC抑制LOVO細(xì)胞侵襲和轉(zhuǎn)移作用可能是通過(guò)上調(diào)E-cadherin蛋白表達(dá)水平及下調(diào)N-cadherin和vimentin蛋白表達(dá)水平實(shí)現(xiàn)的。結(jié)論:以乙醇作為有機(jī)溶劑,使用AB-8大孔吸附樹(shù)脂對(duì)中華獼猴桃根總?cè)七M(jìn)行提取,該工藝具有成本低、安全穩(wěn)定、操作簡(jiǎn)單和取得率高等優(yōu)勢(shì);且所得tt-RAC對(duì)人腸癌SW480細(xì)胞、LOVO細(xì)胞,人乳腺癌MCF-7細(xì)胞及人肝癌SMMC-7721細(xì)胞都具有細(xì)胞毒作用,其機(jī)制可能是通過(guò)誘導(dǎo)細(xì)胞周期發(fā)生S和G2期阻滯;tt-RAC亦可抑制腫瘤細(xì)胞侵襲和遷移,抑制上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)化,可能的機(jī)制是與E-cadherin蛋白表達(dá)水平上調(diào)及N-cadherin和vimentin蛋白的表達(dá)水平下調(diào)相關(guān)。
[Abstract]:Objective: to optimize the extraction process of total triterpenoids from wild kiwifruit roots by macroporous adsorption resin. The inhibitory effects of total triterpene on the proliferation of various tumor cells and the invasion and metastasis of intestinal cancer cell LOVO were further investigated. Methods: the sample powder was soaked with 75% ethanol, and 80% and 95% ethanol were used as organic solvents, respectively. Taking the total triterpene content of Actinidia chinensis root as an index, the extraction conditions of total triterpene from AB-8 macroporous adsorption resin were determined, and the group with the highest total triterpene content in the root of Actinidia chinensis was selected as the following experimental sample. Cell proliferation and activity test (CCK-8) was used to detect the proliferation of human colorectal cancer SW480 cells, human breast cancer MCF-7 cells and human liver cancer SMMC-7721 cells. The effects of tt-RAC on the cell cycle distribution of SW480 cells were analyzed by flow cytometry, and the effects of tt-RAC on the migration of LOVO cells were observed by scratch test. The distance of migration to the center of the cells on both sides of the scratches was taken as the index, and the influence of tt-RAC on the penetration of LOVO cells was further observed by transwell chamber test to determine the effect on the invasion and movement of tumor cells. The expression difference of E-cadherin and vimentin related genes of epithelial-to-mesenchymal transition of epithelial-mesenchymal transition was detected by Western blottingsWB.Results the best technological conditions for extracting total triterpenoids from root of Actinidia chinensis were: Actinidia chinensis root total triterpene extracted by macroporous adsorption resin: Actinidia chinensis. The pH value of the sample solution was 2.8 mg / ml 路h, the flow rate was 1 BV / h, the elution volume was 4 times the volume of resin column bed, the elution rate was 2 BV / h. The highest content of total triterpenoid was 75.89% in the root of Actinidia chinensis, which was eluted by 40% ethanol. Total triterpenoids of Actinidia chinensis root extracted from 40% ethanol were selected as follow-up experiments. The results showed that the inhibitory effects on the proliferation of four kinds of tumor cells were concentration-dependent and time-dependent, and the inhibitory effect on SW480 cells was the strongest. The mechanism may be caused by inducing S and G 2 arrest in SW480 cells. In scratch test, the effect of tt-RAC with concentration lower than IC50 on LOVO cells showed that, Low concentration of tt-RAC inhibited the migration of LOVO cells from both sides of scratch to the center of scratch. Transwell experiments also showed that low concentration of tt-RAC inhibited the invasion of upper chamber cells down the ventricle, and decreased gradually with increasing concentration of tt-RAC. The results of concentration dependent. WB assay showed that the inhibitory effect of tt-RAC on invasion and metastasis of LOVO cells might be achieved by up-regulating the expression of E-cadherin protein and down-regulating the expression of N-cadherin and vimentin proteins. Conclusion: ethanol is used as an organic solvent to inhibit the invasion and metastasis of LOVO cells. The extraction of total triterpenoids from Actinidia chinensis root by AB-8 macroporous adsorption resin has the advantages of low cost, safety and stability, simple operation and high yield, and the obtained tt-RAC can be used in the extraction of human intestinal cancer SW480 cells. Both human breast cancer MCF-7 cells and human liver cancer SMMC-7721 cells have cytotoxic effects, which may be due to the inhibition of invasion and migration of tumor cells and the transformation of epithelial cells to interstitial cells by inducing cell cycle arrest in S and G2 phases. The mechanism may be related to the up-regulation of E-cadherin protein expression and the down-regulation of N-cadherin and vimentin protein expression.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.3
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