雷公藤甲素对人口腔鳞状癌细胞增殖及PTEN基因表达的影响

发布时间：2018-05-19 17:22

本文选题：口腔鳞状细胞癌 + 雷公藤属　；参考：《华北理工大学》2017年硕士论文

【摘要】：目的通过研究雷公藤甲素(triptolide,TP)对人口腔鳞癌细胞株的增殖和10号染色体同源丢失性磷酸酶张力蛋白(phosphataseand tensin homolog,PTEN)基因表达的影响,探讨TP抗口腔鳞状癌的作用机制,以期为口腔鳞状细胞癌的治疗提供实验依据。方法以口腔鳞状癌细胞作为研究对象,将口腔鳞状癌细胞分为对照组(A)和四个实验组(B、C、D、E),分别给予浓度为0、1.25、2.5、5.0和10μg/L的TP进行处理。1通过甲基噻唑基四唑(methyl thiazolyl tetrazolium method,MTT)染色法检测24、48和72h后TP对口腔鳞状癌细胞增殖变化的影响;2流式细胞检测技术检测48h后TP对口腔鳞状癌细胞周期的影响;3实时荧光定量(Real-time Quantitative polymerase chain reaction,RT-PCR)检测24、48和72h后TP对PTEN的信使核糖核酸(Messenger RNA m RNA)表达的影响;4蛋白印迹法(Western-blot)检测72h后TP对PTEN蛋白表达的影响。结果1 MTT结果:24h后B、C、D、E组的细胞抑制率分别为(12.48±0.86)%、(23.61±0.10)%、(35.37±0.10)%、(46.58±0.91)%,与对照组相比,差异具有统计学意义(P0.05);72h后四个实验组的细胞抑制率分别为(26.92±0.14)%、(38.67±0.11)%、(72.62±0.89)%、(90.42±0.28)%,与对照组相比,差异有统计学意义(P0.05);48h后,细胞抑制率随浓度增加而增加,统计分析结果显示各组间两两比较均有统计学意义(P0.05);说明TP可抑制口腔鳞状癌细胞增殖。2.流式细胞检测技术结果显示,TP作用口腔鳞状癌细胞48后,A、B、C、D、E各组G1期细胞比例分别为(58.78±0.98)%、(70.85±0.41)%、(74.90±0.28)%、(79.14±0.45)%、(84.13±0.47)%,S期细胞比例分别为(25.40±0.42)%、(23.14±0.30)%、(20.90±0.25)%、(16.65±0.89)%、(9.41±0.73)%;统计结果显示各组之间两两比较均有统计学差异(P0.05);说明TP可影响口腔鳞状癌细胞的周期并有浓度依赖性。3.TRPCR结果显示,24h后B、C、D、E组PTEN基因m RNA的表达率分别为对照组的1.67、2.22、2.86、3.5倍,72h后四组PTEN基因m RNA的表达率分别为对照组的3.95、5.27、7.18、9.16倍,各时间点的对照组与实验组以及各实验组组间两两比较均有统计学意义(P0.05);说明TP可上调PTEN中m RNA的表达,并呈浓度依赖性。4.Western-blot结果显示,TP作用口腔鳞状癌细胞72h后,PTEN蛋白表达均上调,分别为对照组的3.8倍、6.8倍、10.8倍、17.5倍,对照组和实验组以及各实验组间两两比较均有统计学差异(P0.05),说明了TP显著上调了PTEN中蛋白的表达,随着TP浓度的增加,PTEN蛋白表达也呈现规律性增高。结论1在本实验条件下,TP可以有效抑制口腔鳞状癌细胞的增殖;2 TP可将口腔鳞状癌细胞抑制在G1期,减少了G1期向S期的转变;3 TP可以提高口腔鳞状癌细胞中PTEN的m RNA和蛋白的表达。
[Abstract]:Objective to investigate the effects of triptolide (TPTP) on the proliferation of human oral squamous cell carcinoma cell lines and the expression of homologous lost phosphatase (PTENs) on chromosome 10, and to explore the mechanism of TP in the treatment of oral squamous cell carcinoma (OSCC). To provide experimental evidence for the treatment of oral squamous cell carcinoma. Methods Oral squamous carcinoma cells were used as research objects. Oral squamous carcinoma cells were divided into two groups: control group (A) and four experimental groups. The cells were treated with TP of 0 1.25 渭 g / L of 2.55.0 and 10 渭 g / L respectively. 1. The increase of TP in oral squamous carcinoma cells was detected by methylthiazolyl tetrazolium methyl thiazolyl tetrazolium method MTTstaining after 24 h and 72 h. Effect of colonization changes on the effect of TP on the Cell cycle of Oral squamous carcinoma cells after 48 h Detection by flow Cytometry the effect of TP on the expression of Messenger RNA m RNA) in PTEN was detected by Real-Time Quantitative polymerase chain reactionation RT-PCR after 24 h and 72 h after Detection The effect of TP on the expression of PTEN protein was detected by Western-blot after 72 hours. Results (1) the cell inhibitory rates of MTT group were 12.48 卤0.86 and 35.37 卤0.105.37 卤0.1010, respectively. The cell inhibition rates of the four experimental groups were 26.92 卤0.14, 38.67 卤0.112.62 卤0.89, 90.42 卤0.280.42 卤0.280.42 卤0.284h, respectively, compared with those of the control group, which were significantly higher than that of the control group (P 0.05H, P 0.05h), the cell inhibition rates of the four experimental groups were 26.92 卤0.147.67 卤0.112.62 卤0.86.42 卤0.281.Compared with the control group, the cell inhibition rates of the four experimental groups were significantly higher than those of the control group. The results showed that the cell inhibition rates of the four experimental groups were 26.92 卤0.14, 38.67 卤0.112.62 卤0.89 and 90.42 卤0.280.42 卤0.28, respectively, compared with the control group. The cell inhibition rate increased with the increase of concentration, and the statistical analysis showed that there was a significant difference between the two groups, indicating that TP could inhibit the proliferation of oral squamous carcinoma cells. 2. The results of flow cytometry showed that the percentage of G 1 phase cells in the group treated with TP was 58.78 卤0.98, 74.90 卤0.281 卤0.41 and 79.14 卤0.455.The percentage of cells in S phase was 25.40 卤0.42 and 20.90 卤0.2565 卤0.899.41 卤0.899.41 卤0.73.The statistical results showed that there was a statistical difference between the two groups, and the statistical results showed that there was a statistical difference between the two groups, indicating that there was a significant difference between the two groups in the percentage of cells in the G _ 1 phase, and the statistical results showed that there was a statistical difference between the two groups, indicating that there were significant differences between the two groups in the percentage of cells in the G _ 1 phase, and the statistical results showed that there was a significant difference between the two groups in the percentage of cells in the S phase, which was 25.40 卤0.42 and 20.90 卤0.259.65 卤0.899.41 卤0.73.The statistical results showed that there was a statistical difference between the two groups. TP could affect the cell cycle of oral squamous carcinoma cells in a concentration-dependent manner. The results of TRPCR showed that the expression rate of m RNA of PTEN gene in group E was 3.955.277.187.189.16 times higher than that in control group (1.67 2.222.863.5-fold) after 72 hours, and the expression rate of PTEN gene m RNA in the four groups was 3.955.277.181.16 times higher than that in the control group. The comparison between the control group and the experimental group at each time point had statistical significance (P 0.05), which indicated that TP could up-regulate the expression of m RNA in PTEN. In a concentration dependent manner, Western-blot showed that the expression of PTEN protein was up-regulated in oral squamous carcinoma cells 72 hours after treatment with TP, which was 6.8 times, 10.8 times and 17.5 times as much as that of the control group, respectively. There was a significant difference between the control group and the experimental group (P 0.05), which indicated that TP upregulated the expression of protein in PTEN, and the expression of PTEN increased regularly with the increase of TP concentration. Conclusion (1) under this condition, TP can effectively inhibit the proliferation of oral squamous carcinoma cells and can inhibit the proliferation of oral squamous carcinoma cells in G1 phase. The expression of m RNA and protein of PTEN in oral squamous carcinoma cells was increased by decreasing the transformation from G1 phase to S phase.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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