中华猕猴桃根总三萜抗肿瘤细胞增殖和肠癌细胞侵袭及转移研究

发布时间：2018-03-04 10:10

本文选题：中华猕猴桃根总三萜　切入点：提取　出处：《桂林医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:优化大孔吸附树脂对野生中华猕猴桃根总三萜(tt-RAC)的提取工艺,并进一步探究总三萜对多种肿瘤细胞的增殖抑制作用和肠癌细胞LOVO的侵袭和转移抑制作用。方法:用75%乙醇浸泡样品粉末,分别以40%,50%,60%,70%,80%及95%乙醇为有机溶剂,以中华猕猴桃根总三萜百分含量为指标,确定AB-8型大孔吸附树脂对总三萜的提取工艺条件;选取中华猕猴桃根总三萜百分含量最高组作为后续实验样品,采用细胞增殖与活性检测试验(CCK-8法)检测不同浓度(25,50,100,200,400 ug/ml)野生中华猕猴桃根总三萜(tt-RAC)对人肠癌SW480细胞、LOVO细胞,人乳腺癌MCF-7细胞及人肝癌SMMC-7721细胞的增殖作用,并计算tt-RAC作用于不同细胞的半数抑制浓度(IC50);采用流式细胞术(FCM)分析其对肠癌SW480细胞周期分布的影响;采用划痕实验观察tt-RAC对LOVO细胞迁移影响,以划痕两侧细胞向中心迁移的距离为指标;并进一步用transwell小室试验观察tt-RAC对LOVO细胞穿透能力的影响,以判断对肿瘤细胞侵袭运动的作用;最后,运用Western Blotting(WB)实验检测上皮-间质转化(epithelial-to-mesenchymal transition,EMT)相关基因E-cadherin、N-cadherin和vimentin在蛋白水平上的表达差异。结果:AB-8大孔吸附树脂提取中华猕猴桃根总三萜的最佳工艺条件为:中华猕猴桃根上样液浓度为2.8mg/ml,PH值为7,上样流速为1BV/h;洗脱体积为4倍树脂柱床体积(4BV),洗脱流速为2BV/h。经40%乙醇洗脱提取的中华猕猴桃根总三萜的百分含量最高为75.89%。因此,选取40%乙醇提取的中华猕猴桃根总三萜(tt-RAC)做后续实验,其对四种肿瘤细胞增殖均有抑制作用并呈浓度和时间依赖性;其中对SW480细胞的抑制作用最强,其机制可能是通过诱导SW480细胞发生S和G2期阻滞引起的。在划痕试验中,将药物浓度低于IC50的tt-RAC作用于LOVO细胞结果显示,低浓度tt-RAC可抑制划痕两侧LOVO细胞向划痕中心迁移。Transwell实验同样显示低浓度tt-RAC抑制上室细胞向下室侵袭,随浓度增高侵入下室细胞数量逐渐减少,呈浓度依赖性。WB实验结果显示tt-RAC抑制LOVO细胞侵袭和转移作用可能是通过上调E-cadherin蛋白表达水平及下调N-cadherin和vimentin蛋白表达水平实现的。结论:以乙醇作为有机溶剂,使用AB-8大孔吸附树脂对中华猕猴桃根总三萜进行提取,该工艺具有成本低、安全稳定、操作简单和取得率高等优势;且所得tt-RAC对人肠癌SW480细胞、LOVO细胞,人乳腺癌MCF-7细胞及人肝癌SMMC-7721细胞都具有细胞毒作用,其机制可能是通过诱导细胞周期发生S和G2期阻滞;tt-RAC亦可抑制肿瘤细胞侵袭和迁移,抑制上皮细胞向间质细胞转化,可能的机制是与E-cadherin蛋白表达水平上调及N-cadherin和vimentin蛋白的表达水平下调相关。
[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.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3

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