槲皮素对宫颈癌Hela细胞放疗增敏及抑制转移的作用研究

发布时间：2018-06-28 23:31

本文选题：槲皮素 + Hela细胞　；参考：《湖北中医药大学》2014年硕士论文

【摘要】：目的：探讨槲皮素对宫颈癌Hela细胞的辐射增敏效应及抑制侵袭、转移的作用，探讨辐射增敏作用与给药时序、药物浓度的影响，，探讨槲皮素放射增敏及抑制转移可能的机制。方法：（1）MTT法检测槲皮素单独作用以及12Gy的X射线与槲皮素联合作用时对Hela细胞增殖的抑制作用,绘制出细胞抑制率曲线，计算槲皮素对Hela细胞作用的IC50值。（2）克隆形成实验观察不同给药时序、不同药物浓度的槲皮素对宫颈癌Hela细胞的放疗增敏作用，比较单独放射以及药物与辐射联合后细胞的成瘤能力，确定最佳给药时序并观察药物浓度对放射增敏的影响。（3）流式细胞法检测槲皮素、放射线单独以及联合作用后Hela细胞的周期分布及凋亡率情况。（4）光镜及DAPI染色分别观察不同处理后Hela细胞的凋亡变化。（5）划痕实验和Transwell体外侵袭实验分别观察射线及槲皮素对Hela细胞的迁移和侵袭能力的影响。（6）Western-blot法检测槲皮素、放射线单独以及联合应用后Hela细胞中Bcl-2、Bax、VEGF、MMP-9蛋白的表达。结果：（1）不同浓度的槲皮素对Hela细胞均呈量效性和时效性的生长抑制作用；不同浓度槲皮素联合12Gy剂量射线共同作用后，Hela细胞的增殖进一步受到不同程度的抑制，并具有浓度和时间依赖性。计算出24小时槲皮素对Hela细胞的IC50值约为120μmoL/L。考虑临床常规放疗剂量的实用性及参考文献资料，后续试验中选取的放疗剂量为4Gy，槲皮素选取高、低两个浓度进行比较（20%的IC50值和IC50值）。（2）在先药后放及先放后药两种不同时序下，单独放射与射线联合槲皮素作用，均能抑制Hela细胞集落形成，联合组抑制作用更加明显，先放后药比先药后放时序的抑制效果更加具有优势，因此，在后续试验中采用先放后药作用时序。（3）槲皮素与放射线均能使细胞周期阻滞于放射敏感的G2/M期，放射线与槲皮素联合作用比单独作用效果更加显著。（4）光镜下观察射线单独作用后，细胞的形态无明显的变化，槲皮素单独作用后，细胞间隙变宽，细胞形态不规整，呈不同程度的皱缩，槲皮素联合射线处理后，细胞形态变化更加明显，间隙进一步增宽，细胞变圆、皱缩，且高浓度槲皮素作用的细胞形态改变更加显著；DAPI染色后，射线及槲皮素均可引起细胞核不同程度的皱缩，二者合用后，大量细胞核皱缩、碎裂，高浓度药物作用细胞核变化更加显著。（5）射线照射24h后细胞迁移与侵袭能力增加，槲皮素对HeLa细胞的迁移与侵袭有抑制作用，槲皮素与射线共同处理细胞后，与空白组及单放组比较仍具有明显的抑制迁移作用。（6）Western blot法检测各处理组Bax、Bcl-2、VEGF、MMP-9蛋白表达情况，结果表明槲皮素单药，以及联合放射线均能升高Bax蛋白的表达，降低Bcl-2蛋白的表达；射线单独作用后VEGF、MMP-9蛋白表达在一定时间内略有增加，槲皮素处理后VEGF、MMP-9蛋白表达降低，二者共同处理后VEGF、MMP-9蛋白表达较空白组与单放组降低。结论：（1）槲皮素及X射线均能抑制Hela细胞增殖。（2）槲皮素对Hela细胞具有放射增敏作用，并有浓度、时序的依从性。（3）射线在一定时间内可能促进Hela细胞的迁徙转移，槲皮素则可抑制Hela细胞放疗后的迁移与侵袭。（4）槲皮素对Hela细胞的放疗增敏作用可能与细胞凋亡的发生、G2/M期阻滞、下调Bcl-2蛋白及上调Bax蛋白表达有关。（5）槲皮素抑制Hela细胞放疗后的迁移与侵袭可能与下调VEGF、MMP-9蛋白表达有关。
[Abstract]:Objective: To investigate the effect of quercetin on radiation sensitization of cervical cancer Hela cells and the effect of inhibition of invasion and metastasis. The effect of radiation sensitization and drug delivery time, drug concentration, and the possible mechanism of quercetin radiosensitization and inhibition of metastasis were explored.
Methods: (1) MTT method was used to detect the effect of quercetin alone and the inhibitory effect of X ray and quercetin on the proliferation of Hela cells in combination with quercetin. The cell inhibition rate was plotted and the IC50 value of quercetin on Hela cells was calculated. (2) the clone formation experiment was used to observe the time sequence of different drug delivery, and the Hela fine of cervical cancer with different concentration of quercetin on cervical cancer. Radioactivity sensitization of cell, compare the tumor formation ability of single radiation and combination of drug and radiation, determine the best time sequence and observe the effect of drug concentration on radiosensitivity. (3) flow cytometry was used to detect quercetin, the cycle distribution and apoptosis rate of Hela cells after radiation alone and after combined action. (4) light and DAPI staining The changes in the apoptosis of Hela cells after different treatments were observed. (5) the effects of ray and quercetin on the migration and invasion of Hela cells were observed in the scratch test and Transwell in vitro invasion test. (6) the Western-blot method was used to detect quercetin, radiation alone and the expression of Bcl-2, Bax, VEGF, and MMP-9 protein in Hela cells after combined application. Results: (1) different concentrations of quercetin had a dose-response and aging inhibitory effect on Hela cells. The proliferation of Hela cells was further inhibited by different concentrations of quercetin combined with 12Gy dose radiation. The IC50 value of quercetin to Hela cells was calculated. The IC50 value of quercetin for 24 hours was calculated. About 120 mu moL/L., considering the practical and reference data of the conventional radiotherapy dose, the dose of radiotherapy was 4Gy, quercetin was selected, and the low two concentrations were compared (20% IC50 and IC50). (2) the effect of radiation and radiation combined with quercetin alone was performed under the first post release and first post release drugs. It can inhibit the formation of Hela cell colonies, and the inhibition effect of the combined group is more obvious. The inhibition effect of the drug is more advantageous than that of the first post release sequence. Therefore, the sequence of pre release drug action is adopted in the follow-up test. (3) the cell cycle of Quercetin and the radiant line can block the cell cycle in the G2/M period sensitive to radiation, and the combination of radioactivity and quercetin is combined. (4) there is no obvious change in the morphology of cells after the observation of ray alone under the light microscope. After quercetin alone, the cell gap widens and the cell morphology is irregular, and the cell morphology changes to a different degree. After the quercetin combined with radiation, the cell morphology changes more clearly, the gap is further widened, the cells become round and wrinkled. The cell morphology changed more significantly with the effect of high concentration of quercetin; after DAPI staining, the radiation and quercetin could cause different degrees of crinkling of the nucleus. After the combination of the two, a large number of nuclei crinkled, fractured, and high concentration of the drug acted more significantly. (5) the cell migration and invasion ability increased after radiation of 24h, and the quercetin The effect of quercetin on the migration and invasion of HeLa cells was inhibited. After quercetin and ray treated the cells, they still had obvious inhibitory effect on the migration. (6) the expression of Bax, Bcl-2, VEGF, MMP-9 protein in each treatment group was detected by Western blot method. The results showed that quercetin single drug and combined radiation could increase Bax eggs. The expression of white protein decreased the expression of Bcl-2 protein; the expression of VEGF and MMP-9 protein was slightly increased in a certain time after the irradiation alone. The expression of VEGF and MMP-9 protein were reduced after quercetin treatment. The expression of VEGF and MMP-9 protein were lower than that in the blank group and the single release group after the two cases were treated together.
Conclusions: (1) quercetin and X rays can inhibit the proliferation of Hela cells. (2) quercetin has radiosensitizing effect on Hela cells, and has concentration and timing compliance. (3) radiation may promote migration and metastasis of Hela cells in a certain time, quercetin can inhibit the migration and invasion of Hela after cell radiotherapy. (4) quercetin's release to Hela cells The effect of sensitization may be related to the occurrence of apoptosis, G2/M phase block, down regulation of Bcl-2 protein and up regulation of the expression of Bax protein. (5) the inhibition of migration and invasion of Hela cells by quercetin may be related to the downregulation of VEGF and the expression of MMP-9 protein.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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