苦参素对结肠癌细胞上皮间质转化影响的实验研究
发布时间：2018-08-02 08:53
【摘要】：背景和目的苦参素是传统中药苦参提取的一种生物碱活性成分,近来研究表明它具有广泛的抗肿瘤作用,然而其在结肠癌具体的作用机制不清。本研究的目的是探讨苦参素在结肠癌细胞的作用,阐明其对结肠癌上皮间质转化的影响及其可能涉及的机制,为中药单体治疗结肠癌提供实验依据。方法1.体外培养结肠癌细胞,观察细胞的生长状态,取处于对数生长期的细胞用于实验。试验分为:(1)实验组:用不同浓度的苦参素(mg/ml)(0.25,0.5,0.75,1,1.25,1.5,1.75,2)处理结肠癌细胞。(2)对照组:未经药物处理,仅加入细胞悬液。(3)空白对照组:未加入细胞,仅加入细胞培养液。运用MTT法使用不同浓度的苦参素(mg/ml)(0.25,0.5,0.75,1,1.25,1.5,1.75,2)作用于SW480,RKO,HCT116等结肠癌细胞24h后检测细胞的增殖活性;不同浓度的苦参素(mg/ml)(0,0.25,0.5,0.75)干预结肠癌细胞RKO 24小时后,应用细胞划痕实验和Transwell小室模型分别进行细胞迁移、细胞侵袭实验检测细胞迁移、侵袭能力;苦参素干预之前和干预之后的结肠癌细胞形态学上的改变由应用倒置相差显微镜观察。将结肠癌细胞RKO分为四组,即苦参碱素干预的终浓度分别为0、0.25、0.5、0.75(mg/ml),应用免疫印迹法(Western Blotting)检测各组细胞E-cadherin、N-cadherin、Snail、P65蛋白水平的表达情况。2.利用基因干扰技术设计并合成干扰效率较高的P65sh RNA。实验分为四组,分别是对照组、苦参素组(终浓度0.25mg/ml)、P65sh RNA组、苦参素(终浓度0.25mg/ml)+P65sh RNA组,应用免疫印迹法(Western Blotting)检测各组细胞E-cadherin、N-cadherin、Snail的蛋白水平表达情况,应用倒置相差显微镜观察各组细胞的形态改变,应用细胞划痕实验和Transwell小室模型分别进行细胞迁移、细胞侵袭实验检测细胞迁移、侵袭能力。结果1.MTT实验表明苦参素能够抑制结肠癌细胞的增殖,当苦参素的干预浓度在0~2mg/ml范围时,随着苦参素干预浓度的增大,其对结肠癌细胞的增殖抑制越明显。不同浓度的苦参素(mg/ml)(0、0.25、0.5、0.75)作用于结肠癌RKO细胞24小时后,各组的迁移距离分别为263.00±25.51,170.25±46.38、103.87±33.90、75.46±20.19。Transwell小室侵袭实验中,0mg/ml组、0.25mg/ml组、0.5mg/ml组、0.75mg/ml组中穿过上室的细胞数分别是:166±8.89、130.65±10.23、93.60±9.21、62.28±7.07。倒置相差显微镜下可见苦参素处理前细胞呈间质样的细长梭形,细胞连接松散;苦参素处理后细胞呈上皮样的形态,细胞间连接紧密,几乎看不到伪足。Western Blotting法结果显示,0mg/ml组、0.25mg/ml组、0.5mg/ml组、0.75mg/ml组中所对应的E-cadherin的蛋白表达结果分别为:0.30±0.08、0.48±0.19、0.72±0.13、1.06±0.44;N-cadherin的蛋白表达结果分别为:0.50±0.10、0.33±0.09、0.25±0.08、0.18±0.05;Snail的蛋白表达结果分别为:0.88±0.04、0.43±0.11、0.33±0.11、0.20±0.07。P65的蛋白水平结果分别为:0.75±0.03、0.40±0.08、0.34±0.08、0.20±0.06。综上,随着苦参素干预浓度的增大,N-cadherin、Snail、P65的蛋白表达减少,而E-cadherin的蛋白表达增加。2.成功设计、合成出一条干扰效率较高的P65sh RNA。对照组、苦参素组、P65sh RNA组、苦参素+P65sh RNA组中所对应的E-cadherin蛋白表达结果分别为:0.16±0.01、0.23±0.02、0.33±0.03、0.5±0.06;N-cadherin的蛋白表达结果分别为:0.60±0.04、0.46±0.07、0.32±0.06、0.20±0.05;Snail的蛋白表达结果分别为:0.93±0.04、0.71±0.12、0.62±0.10、0.35±0.07。综上,N-cadherin、Snail的蛋白表达水平,P65sh RNA组的低于苦参碱组,苦参素+P65sh RNA均低于苦参素组及P65sh RNA组,E-cadherin蛋白表达水平恰好相反。倒置相差显微镜下可见对照组细胞呈间质样的细长梭形,大多数细胞出现细长的丝状伪足,细胞连接松散,苦参素组或P65sh RNA组较对照组细胞连接紧密。相对于苦参素组或P65sh RNA,苦参素+P65sh RNA组细胞进一步呈明显的上皮样形态,细胞间连接紧密,几乎看不到伪足,细胞呈紧密连接的鹅卵石样改变。空白对照组,苦参素组、P65sh RNA、苦参素+P65sh RNA组相对应的迁移距离分别为297.31±65.91、180.00±50.24、116.84±36.89、59.09±15.36。Transwell小室侵袭实验中,对照组,苦参素组、P65sh RNA组、苦参素+P65sh RNA组相对应的穿过上室的细胞数分别是143.67±10.97、119.33±20.23、97.67±18.77、50.33±8.50。结论1.苦参素在体外具有抗结肠癌细胞的作用,具体表现在其能够抑制结肠癌细胞的增殖、迁移和侵袭。2.苦参素能影响结肠癌细胞上皮间质转化相关蛋白,其能抑制结肠癌细胞的迁移和侵袭能力,与抑制结肠癌细胞上皮间质转化有关。3.苦参素能影响NF-?B信号通路相关蛋白,其抑制结肠癌细胞上皮间质转化的能力,可能是通过抑制NF-?B通路来实现。
[Abstract]:Background and objective matrine is a kind of alkaloid active component extracted from Radix Sophorae Radix Sophora flavescens. Recent studies have shown that it has extensive anti-tumor effect. However, its specific mechanism of action in colon cancer is not clear. The purpose of this study is to explore the effect of matrine in colon cancer cells and to elucidate its effect on the transformation of colon epithelial mesenchymal transition. It may be involved in the mechanism to provide experimental basis for the treatment of colon cancer by traditional Chinese medicine. Method 1. in vitro culture of colon cancer cells, observe the growth state of the cells, take the cells in the logarithmic growth stage for the experiment. The experiment is divided into: (1) the experimental group: treatment of colon cancer cells with different concentrations of mg/ml (0.25,0.5,0.75,1,1.25,1.5,1.75,2). (2) the control group: the cell suspension was only added without the drug treatment. (3) the blank control group was not added to the cell, only the cell culture solution was added. The proliferation activity of the colon cancer cells such as SW480, RKO, HCT116 and other colon cancer cells were detected with different concentrations of matrine (0.25,0.5,0.75,1,1.25,1.5,1.75,2) using the MTT method, and the different concentrations of matrine (m). G/ml) (0,0.25,0.5,0.75) after the intervention of colon cancer cell RKO for 24 hours, cell migration and invasion ability were detected by cell scratch test and Transwell compartment model respectively. The morphological changes of colon cancer cells before and after the intervention of matrine were observed by inverted phase contrast microscope. Colon cancer cell RKO was divided into four groups, that is, the final concentration of matrine intervention was 0,0.25,0.5,0.75 (mg/ml). The expression of E-cadherin, N-cadherin, Snail, P65 protein in each group was detected by Western Blotting (Western Blotting).2. using gene interference technique to design and synthesize the P65sh RNA. experiment with high interference efficiency was divided into four The group, the control group, the Matrine group (final concentration 0.25mg/ml), the P65sh RNA group and the +P65sh RNA group of matrine (final concentration 0.25mg/ml), the expression of E-cadherin, N-cadherin, Snail protein in each group was detected by immunoblotting (Western Blotting). The morphological changes of the cells in each group should be observed by inverted phase contrast microscope, and the cells should be applied to the cells. Cell migration and invasion ability were detected by the scratch test and the Transwell compartment model. Results the 1.MTT experiment showed that matrine could inhibit the proliferation of colon cancer cells. When the concentration of matrine was in the 0~2mg/ml range, the proliferation inhibition of colon cancer cells with the increase of the preconcentration of matrine. The different concentrations of matrine (mg/ml) (0,0.25,0.5,0.75) acted on the RKO cells of colon cancer for 24 hours, and the migration distance was 263 + 25.51170.25 + 46.38103.87 + 33.90,75.46 + 20.19.Transwell, respectively. The number of cells passing through the upper chamber in group 0mg/ml, 0.25mg/ml, 0.5mg/ml, 0.75mg/ml group were respectively 166 + 8.89130.65 + 10.23,93.60 + 9.21,62.28 + 7.07. inverted phase contrast microscope showed that the cells were thin and slender spindle shaped and the cells were loosely connected before the treatment of Sophora flavescens. After the treatment of matrine, the cells were epithelioid, and the cells were closely connected. The result showed that the.Western Blotting method of pseudo foot was not found, 0mg/ml group, 0.25mg/ml group, 0.5m. The protein expression results of E-cadherin in group g/ml and group 0.75mg/ml were 0.30 + 0.08,0.48 + 0.19,0.72 + 0.13,1.06 + 0.44, and the protein expression results of N-cadherin were 0.50 + 0.10,0.33 + 0.09,0.25 + 0.08,0.18 + 0.05, and the protein expression of Snail was 0.88 +. The results were as follows: 0.75 + 0.03,0.40 + 0.08,0.34 + 0.08,0.20 + 0.06., with the increase of the concentration of Sophora flavescens, the protein expression of N-cadherin, Snail and P65 decreased, while the protein expression of E-cadherin increased successfully, and a P65sh RNA. control group with high interference efficiency was synthesized. The expression of E-cadherin protein in RNA group was 0.16 + 0.01,0.23 + 0.02,0.33 + 0.03,0.5 + 0.06, and the protein expression results of N-cadherin were 0.60 + 0.04,0.46 + 0.07,0.32 + 0.06,0.20 + 0.05 respectively. The protein expression results of Snail were respectively: 0.93 + 0.04,0.71 + 0.12,0.62. The level of white expression was lower in the P65sh RNA group than in the Matrine group. The +P65sh RNA of the Matrine was lower than that of the Matrine group and the P65sh RNA group. The expression level of the E-cadherin protein was the opposite. The cells of the control group showed a thin and elongated spindle shaped in the control group under the inverted phase contrast microscope. Most cells appeared slender filograph, loose cell connection, matrine group or P6 The 5sh RNA group was more closely connected than the control group. Compared with the Matrine group or P65sh RNA, the cells of the Matrine +P65sh RNA group further showed a distinct epithelioid form. The cells were closely connected, almost invisible, and the cells were closely connected cobblestone. The blank control group, the Matrine group, P65sh RNA, and the sophorin +P65sh RNA group corresponded. The migration distance is 297.31 + 65.91180.00 + 50.24116.84 + 36.89,59.09 + 15.36.Transwell, the control group, the Matrine group, the P65sh RNA group and the Matrine +P65sh RNA group, the number of cells passing through the upper chamber is 143.67 + 10.97119.33 + 20.23,97.67 + 18.77,50.33 + 8.50. conclusion 1. matrine in vitro The effect of anti colon cancer cells is manifested in its ability to inhibit the proliferation of colon cancer cells, migration and invasion of.2. Oxymatrine can affect the epithelial mesenchymal transformation related proteins in colon cancer cells, which can inhibit the migration and invasion of colon cancer cells, and the inhibitory effect of.3. oxymatrine on the transformation of epithelial mesenchymal transition of colon cancer cells can affect the NF-? B signal Pathway related proteins, which inhibit the epithelial mesenchymal transition of colon cancer cells, may be achieved by inhibiting the NF-? B pathway.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.35
本文编号：2158896
[Abstract]:Background and objective matrine is a kind of alkaloid active component extracted from Radix Sophorae Radix Sophora flavescens. Recent studies have shown that it has extensive anti-tumor effect. However, its specific mechanism of action in colon cancer is not clear. The purpose of this study is to explore the effect of matrine in colon cancer cells and to elucidate its effect on the transformation of colon epithelial mesenchymal transition. It may be involved in the mechanism to provide experimental basis for the treatment of colon cancer by traditional Chinese medicine. Method 1. in vitro culture of colon cancer cells, observe the growth state of the cells, take the cells in the logarithmic growth stage for the experiment. The experiment is divided into: (1) the experimental group: treatment of colon cancer cells with different concentrations of mg/ml (0.25,0.5,0.75,1,1.25,1.5,1.75,2). (2) the control group: the cell suspension was only added without the drug treatment. (3) the blank control group was not added to the cell, only the cell culture solution was added. The proliferation activity of the colon cancer cells such as SW480, RKO, HCT116 and other colon cancer cells were detected with different concentrations of matrine (0.25,0.5,0.75,1,1.25,1.5,1.75,2) using the MTT method, and the different concentrations of matrine (m). G/ml) (0,0.25,0.5,0.75) after the intervention of colon cancer cell RKO for 24 hours, cell migration and invasion ability were detected by cell scratch test and Transwell compartment model respectively. The morphological changes of colon cancer cells before and after the intervention of matrine were observed by inverted phase contrast microscope. Colon cancer cell RKO was divided into four groups, that is, the final concentration of matrine intervention was 0,0.25,0.5,0.75 (mg/ml). The expression of E-cadherin, N-cadherin, Snail, P65 protein in each group was detected by Western Blotting (Western Blotting).2. using gene interference technique to design and synthesize the P65sh RNA. experiment with high interference efficiency was divided into four The group, the control group, the Matrine group (final concentration 0.25mg/ml), the P65sh RNA group and the +P65sh RNA group of matrine (final concentration 0.25mg/ml), the expression of E-cadherin, N-cadherin, Snail protein in each group was detected by immunoblotting (Western Blotting). The morphological changes of the cells in each group should be observed by inverted phase contrast microscope, and the cells should be applied to the cells. Cell migration and invasion ability were detected by the scratch test and the Transwell compartment model. Results the 1.MTT experiment showed that matrine could inhibit the proliferation of colon cancer cells. When the concentration of matrine was in the 0~2mg/ml range, the proliferation inhibition of colon cancer cells with the increase of the preconcentration of matrine. The different concentrations of matrine (mg/ml) (0,0.25,0.5,0.75) acted on the RKO cells of colon cancer for 24 hours, and the migration distance was 263 + 25.51170.25 + 46.38103.87 + 33.90,75.46 + 20.19.Transwell, respectively. The number of cells passing through the upper chamber in group 0mg/ml, 0.25mg/ml, 0.5mg/ml, 0.75mg/ml group were respectively 166 + 8.89130.65 + 10.23,93.60 + 9.21,62.28 + 7.07. inverted phase contrast microscope showed that the cells were thin and slender spindle shaped and the cells were loosely connected before the treatment of Sophora flavescens. After the treatment of matrine, the cells were epithelioid, and the cells were closely connected. The result showed that the.Western Blotting method of pseudo foot was not found, 0mg/ml group, 0.25mg/ml group, 0.5m. The protein expression results of E-cadherin in group g/ml and group 0.75mg/ml were 0.30 + 0.08,0.48 + 0.19,0.72 + 0.13,1.06 + 0.44, and the protein expression results of N-cadherin were 0.50 + 0.10,0.33 + 0.09,0.25 + 0.08,0.18 + 0.05, and the protein expression of Snail was 0.88 +. The results were as follows: 0.75 + 0.03,0.40 + 0.08,0.34 + 0.08,0.20 + 0.06., with the increase of the concentration of Sophora flavescens, the protein expression of N-cadherin, Snail and P65 decreased, while the protein expression of E-cadherin increased successfully, and a P65sh RNA. control group with high interference efficiency was synthesized. The expression of E-cadherin protein in RNA group was 0.16 + 0.01,0.23 + 0.02,0.33 + 0.03,0.5 + 0.06, and the protein expression results of N-cadherin were 0.60 + 0.04,0.46 + 0.07,0.32 + 0.06,0.20 + 0.05 respectively. The protein expression results of Snail were respectively: 0.93 + 0.04,0.71 + 0.12,0.62. The level of white expression was lower in the P65sh RNA group than in the Matrine group. The +P65sh RNA of the Matrine was lower than that of the Matrine group and the P65sh RNA group. The expression level of the E-cadherin protein was the opposite. The cells of the control group showed a thin and elongated spindle shaped in the control group under the inverted phase contrast microscope. Most cells appeared slender filograph, loose cell connection, matrine group or P6 The 5sh RNA group was more closely connected than the control group. Compared with the Matrine group or P65sh RNA, the cells of the Matrine +P65sh RNA group further showed a distinct epithelioid form. The cells were closely connected, almost invisible, and the cells were closely connected cobblestone. The blank control group, the Matrine group, P65sh RNA, and the sophorin +P65sh RNA group corresponded. The migration distance is 297.31 + 65.91180.00 + 50.24116.84 + 36.89,59.09 + 15.36.Transwell, the control group, the Matrine group, the P65sh RNA group and the Matrine +P65sh RNA group, the number of cells passing through the upper chamber is 143.67 + 10.97119.33 + 20.23,97.67 + 18.77,50.33 + 8.50. conclusion 1. matrine in vitro The effect of anti colon cancer cells is manifested in its ability to inhibit the proliferation of colon cancer cells, migration and invasion of.2. Oxymatrine can affect the epithelial mesenchymal transformation related proteins in colon cancer cells, which can inhibit the migration and invasion of colon cancer cells, and the inhibitory effect of.3. oxymatrine on the transformation of epithelial mesenchymal transition of colon cancer cells can affect the NF-? B signal Pathway related proteins, which inhibit the epithelial mesenchymal transition of colon cancer cells, may be achieved by inhibiting the NF-? B pathway.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.35
【参考文献】
相关期刊论文 前10条
1 刘武;徐燕;王刚;;复方苦参注射液联合同步放化疗治疗局部晚期胃癌近期临床观察[J];中国中医药信息杂志;2016年10期
2 Dong-Yuan Zhu;Qi-Sen Guo;Yan-Liang Li;Bin Cui;Jun Guo;Ji-Xiao Liu;Peng Li;;Twist 1 correlates with poor differentiation and progression in gastric adenocarcinoma via elevation of FGFR2 expression[J];World Journal of Gastroenterology;2014年48期
3 刘宝玉;黄杰安;刘诗权;李素艳;徐春燕;梁梦紫;谭林;覃蒙斌;;NF-κB对人结肠癌细胞上皮间质转化及侵袭转移的影响[J];世界华人消化杂志;2014年23期
4 刘武;程晓磊;董凡;王刚;顾华勇;;复方苦参注射液联合同步放化疗治疗局部晚期直肠癌的临床研究[J];天津药学;2014年03期
5 阮新建;刘畅;韦丽丽;王飞;;康艾注射液联合XELOX方案治疗晚期大肠癌疗效观察[J];现代中西医结合杂志;2014年18期
6 张俊;唐安洲;;苦参碱及其衍生物对人鼻咽癌细胞的耐药逆转作用[J];中药材;2012年12期
7 ;Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells[J];World Journal of Gastroenterology;2012年31期
8 李圆;周雪华;刘亭彦;崔彩霞;;苦参碱对喉鳞癌细胞株Hep-2侵袭转移的影响[J];中国中西医结合外科杂志;2012年03期
9 屈飞;崔艳茹;徐镜;;苦参碱对小鼠H22细胞抗肿瘤作用及其机制研究[J];肿瘤药学;2011年04期
10 陆彬彬;王朝霞;王娟;李娟;王继荣;王科明;;复方苦参注射液对术后胃癌DFC方案辅助治疗毒副反应的影响[J];中国医药科学;2011年16期
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