苦參素對結(jié)腸癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化影響的實(shí)驗(yàn)研究
發(fā)布時間：2018-08-02 08:53
【摘要】：背景和目的苦參素是傳統(tǒng)中藥苦參提取的一種生物堿活性成分,近來研究表明它具有廣泛的抗腫瘤作用,然而其在結(jié)腸癌具體的作用機(jī)制不清。本研究的目的是探討苦參素在結(jié)腸癌細(xì)胞的作用,闡明其對結(jié)腸癌上皮間質(zhì)轉(zhuǎn)化的影響及其可能涉及的機(jī)制,為中藥單體治療結(jié)腸癌提供實(shí)驗(yàn)依據(jù)。方法1.體外培養(yǎng)結(jié)腸癌細(xì)胞,觀察細(xì)胞的生長狀態(tài),取處于對數(shù)生長期的細(xì)胞用于實(shí)驗(yàn)。試驗(yàn)分為:(1)實(shí)驗(yàn)組:用不同濃度的苦參素(mg/ml)(0.25,0.5,0.75,1,1.25,1.5,1.75,2)處理結(jié)腸癌細(xì)胞。(2)對照組:未經(jīng)藥物處理,僅加入細(xì)胞懸液。(3)空白對照組:未加入細(xì)胞,僅加入細(xì)胞培養(yǎng)液。運(yùn)用MTT法使用不同濃度的苦參素(mg/ml)(0.25,0.5,0.75,1,1.25,1.5,1.75,2)作用于SW480,RKO,HCT116等結(jié)腸癌細(xì)胞24h后檢測細(xì)胞的增殖活性;不同濃度的苦參素(mg/ml)(0,0.25,0.5,0.75)干預(yù)結(jié)腸癌細(xì)胞RKO 24小時后,應(yīng)用細(xì)胞劃痕實(shí)驗(yàn)和Transwell小室模型分別進(jìn)行細(xì)胞遷移、細(xì)胞侵襲實(shí)驗(yàn)檢測細(xì)胞遷移、侵襲能力;苦參素干預(yù)之前和干預(yù)之后的結(jié)腸癌細(xì)胞形態(tài)學(xué)上的改變由應(yīng)用倒置相差顯微鏡觀察。將結(jié)腸癌細(xì)胞RKO分為四組,即苦參堿素干預(yù)的終濃度分別為0、0.25、0.5、0.75(mg/ml),應(yīng)用免疫印跡法(Western Blotting)檢測各組細(xì)胞E-cadherin、N-cadherin、Snail、P65蛋白水平的表達(dá)情況。2.利用基因干擾技術(shù)設(shè)計(jì)并合成干擾效率較高的P65sh RNA。實(shí)驗(yàn)分為四組,分別是對照組、苦參素組(終濃度0.25mg/ml)、P65sh RNA組、苦參素(終濃度0.25mg/ml)+P65sh RNA組,應(yīng)用免疫印跡法(Western Blotting)檢測各組細(xì)胞E-cadherin、N-cadherin、Snail的蛋白水平表達(dá)情況,應(yīng)用倒置相差顯微鏡觀察各組細(xì)胞的形態(tài)改變,應(yīng)用細(xì)胞劃痕實(shí)驗(yàn)和Transwell小室模型分別進(jìn)行細(xì)胞遷移、細(xì)胞侵襲實(shí)驗(yàn)檢測細(xì)胞遷移、侵襲能力。結(jié)果1.MTT實(shí)驗(yàn)表明苦參素能夠抑制結(jié)腸癌細(xì)胞的增殖,當(dāng)苦參素的干預(yù)濃度在0~2mg/ml范圍時,隨著苦參素干預(yù)濃度的增大,其對結(jié)腸癌細(xì)胞的增殖抑制越明顯。不同濃度的苦參素(mg/ml)(0、0.25、0.5、0.75)作用于結(jié)腸癌RKO細(xì)胞24小時后,各組的遷移距離分別為263.00±25.51,170.25±46.38、103.87±33.90、75.46±20.19。Transwell小室侵襲實(shí)驗(yàn)中,0mg/ml組、0.25mg/ml組、0.5mg/ml組、0.75mg/ml組中穿過上室的細(xì)胞數(shù)分別是:166±8.89、130.65±10.23、93.60±9.21、62.28±7.07。倒置相差顯微鏡下可見苦參素處理前細(xì)胞呈間質(zhì)樣的細(xì)長梭形,細(xì)胞連接松散;苦參素處理后細(xì)胞呈上皮樣的形態(tài),細(xì)胞間連接緊密,幾乎看不到偽足。Western Blotting法結(jié)果顯示,0mg/ml組、0.25mg/ml組、0.5mg/ml組、0.75mg/ml組中所對應(yīng)的E-cadherin的蛋白表達(dá)結(jié)果分別為:0.30±0.08、0.48±0.19、0.72±0.13、1.06±0.44;N-cadherin的蛋白表達(dá)結(jié)果分別為:0.50±0.10、0.33±0.09、0.25±0.08、0.18±0.05;Snail的蛋白表達(dá)結(jié)果分別為:0.88±0.04、0.43±0.11、0.33±0.11、0.20±0.07。P65的蛋白水平結(jié)果分別為:0.75±0.03、0.40±0.08、0.34±0.08、0.20±0.06。綜上,隨著苦參素干預(yù)濃度的增大,N-cadherin、Snail、P65的蛋白表達(dá)減少,而E-cadherin的蛋白表達(dá)增加。2.成功設(shè)計(jì)、合成出一條干擾效率較高的P65sh RNA。對照組、苦參素組、P65sh RNA組、苦參素+P65sh RNA組中所對應(yīng)的E-cadherin蛋白表達(dá)結(jié)果分別為:0.16±0.01、0.23±0.02、0.33±0.03、0.5±0.06;N-cadherin的蛋白表達(dá)結(jié)果分別為:0.60±0.04、0.46±0.07、0.32±0.06、0.20±0.05;Snail的蛋白表達(dá)結(jié)果分別為:0.93±0.04、0.71±0.12、0.62±0.10、0.35±0.07。綜上,N-cadherin、Snail的蛋白表達(dá)水平,P65sh RNA組的低于苦參堿組,苦參素+P65sh RNA均低于苦參素組及P65sh RNA組,E-cadherin蛋白表達(dá)水平恰好相反。倒置相差顯微鏡下可見對照組細(xì)胞呈間質(zhì)樣的細(xì)長梭形,大多數(shù)細(xì)胞出現(xiàn)細(xì)長的絲狀偽足,細(xì)胞連接松散,苦參素組或P65sh RNA組較對照組細(xì)胞連接緊密。相對于苦參素組或P65sh RNA,苦參素+P65sh RNA組細(xì)胞進(jìn)一步呈明顯的上皮樣形態(tài),細(xì)胞間連接緊密,幾乎看不到偽足,細(xì)胞呈緊密連接的鵝卵石樣改變�？瞻讓φ战M,苦參素組、P65sh RNA、苦參素+P65sh RNA組相對應(yīng)的遷移距離分別為297.31±65.91、180.00±50.24、116.84±36.89、59.09±15.36。Transwell小室侵襲實(shí)驗(yàn)中,對照組,苦參素組、P65sh RNA組、苦參素+P65sh RNA組相對應(yīng)的穿過上室的細(xì)胞數(shù)分別是143.67±10.97、119.33±20.23、97.67±18.77、50.33±8.50。結(jié)論1.苦參素在體外具有抗結(jié)腸癌細(xì)胞的作用,具體表現(xiàn)在其能夠抑制結(jié)腸癌細(xì)胞的增殖、遷移和侵襲。2.苦參素能影響結(jié)腸癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化相關(guān)蛋白,其能抑制結(jié)腸癌細(xì)胞的遷移和侵襲能力,與抑制結(jié)腸癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化有關(guān)。3.苦參素能影響NF-?B信號通路相關(guān)蛋白,其抑制結(jié)腸癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化的能力,可能是通過抑制NF-?B通路來實(shí)現(xiàn)。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.35
本文編號：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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.35
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 劉武;徐燕;王剛;;復(fù)方苦參注射液聯(lián)合同步放化療治療局部晚期胃癌近期臨床觀察[J];中國中醫(yī)藥信息雜志;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 劉寶玉;黃杰安;劉詩權(quán);李素艷;徐春燕;梁夢紫;譚林;覃蒙斌;;NF-κB對人結(jié)腸癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化及侵襲轉(zhuǎn)移的影響[J];世界華人消化雜志;2014年23期
4 劉武;程曉磊;董凡;王剛;顧華勇;;復(fù)方苦參注射液聯(lián)合同步放化療治療局部晚期直腸癌的臨床研究[J];天津藥學(xué);2014年03期
5 阮新建;劉暢;韋麗麗;王飛;;康艾注射液聯(lián)合XELOX方案治療晚期大腸癌療效觀察[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2014年18期
6 張俊;唐安洲;;苦參堿及其衍生物對人鼻咽癌細(xì)胞的耐藥逆轉(zhuǎn)作用[J];中藥材;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 李圓;周雪華;劉亭彥;崔彩霞;;苦參堿對喉鱗癌細(xì)胞株Hep-2侵襲轉(zhuǎn)移的影響[J];中國中西醫(yī)結(jié)合外科雜志;2012年03期
9 屈飛;崔艷茹;徐鏡;;苦參堿對小鼠H22細(xì)胞抗腫瘤作用及其機(jī)制研究[J];腫瘤藥學(xué);2011年04期
10 陸彬彬;王朝霞;王娟;李娟;王繼榮;王科明;;復(fù)方苦參注射液對術(shù)后胃癌DFC方案輔助治療毒副反應(yīng)的影響[J];中國醫(yī)藥科學(xué);2011年16期
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