發(fā)布時(shí)間：2018-06-28 23:31

  本文選題：槲皮素 + Hela細(xì)胞　； 參考：《湖北中醫(yī)藥大學(xué)》2014年碩士論文

【摘要】：目的：探討槲皮素對(duì)宮頸癌Hela細(xì)胞的輻射增敏效應(yīng)及抑制侵襲、轉(zhuǎn)移的作用，探討輻射增敏作用與給藥時(shí)序、藥物濃度的影響，，探討槲皮素放射增敏及抑制轉(zhuǎn)移可能的機(jī)制。 方法：（1）MTT法檢測(cè)槲皮素單獨(dú)作用以及12Gy的X射線與槲皮素聯(lián)合作用時(shí)對(duì)Hela細(xì)胞增殖的抑制作用,繪制出細(xì)胞抑制率曲線，計(jì)算槲皮素對(duì)Hela細(xì)胞作用的IC50值。（2）克隆形成實(shí)驗(yàn)觀察不同給藥時(shí)序、不同藥物濃度的槲皮素對(duì)宮頸癌Hela細(xì)胞的放療增敏作用，比較單獨(dú)放射以及藥物與輻射聯(lián)合后細(xì)胞的成瘤能力，確定最佳給藥時(shí)序并觀察藥物濃度對(duì)放射增敏的影響。（3）流式細(xì)胞法檢測(cè)槲皮素、放射線單獨(dú)以及聯(lián)合作用后Hela細(xì)胞的周期分布及凋亡率情況。（4）光鏡及DAPI染色分別觀察不同處理后Hela細(xì)胞的凋亡變化。（5）劃痕實(shí)驗(yàn)和Transwell體外侵襲實(shí)驗(yàn)分別觀察射線及槲皮素對(duì)Hela細(xì)胞的遷移和侵襲能力的影響。（6）Western-blot法檢測(cè)槲皮素、放射線單獨(dú)以及聯(lián)合應(yīng)用后Hela細(xì)胞中Bcl-2、Bax、VEGF、MMP-9蛋白的表達(dá)。結(jié)果：（1）不同濃度的槲皮素對(duì)Hela細(xì)胞均呈量效性和時(shí)效性的生長(zhǎng)抑制作用；不同濃度槲皮素聯(lián)合12Gy劑量射線共同作用后，Hela細(xì)胞的增殖進(jìn)一步受到不同程度的抑制，并具有濃度和時(shí)間依賴性。計(jì)算出24小時(shí)槲皮素對(duì)Hela細(xì)胞的IC50值約為120μmoL/L�？紤]臨床常規(guī)放療劑量的實(shí)用性及參考文獻(xiàn)資料，后續(xù)試驗(yàn)中選取的放療劑量為4Gy，槲皮素選取高、低兩個(gè)濃度進(jìn)行比較（20%的IC50值和IC50值）。（2）在先藥后放及先放后藥兩種不同時(shí)序下，單獨(dú)放射與射線聯(lián)合槲皮素作用，均能抑制Hela細(xì)胞集落形成，聯(lián)合組抑制作用更加明顯，先放后藥比先藥后放時(shí)序的抑制效果更加具有優(yōu)勢(shì)，因此，在后續(xù)試驗(yàn)中采用先放后藥作用時(shí)序。（3）槲皮素與放射線均能使細(xì)胞周期阻滯于放射敏感的G2/M期，放射線與槲皮素聯(lián)合作用比單獨(dú)作用效果更加顯著。（4）光鏡下觀察射線單獨(dú)作用后，細(xì)胞的形態(tài)無(wú)明顯的變化，槲皮素單獨(dú)作用后，細(xì)胞間隙變寬，細(xì)胞形態(tài)不規(guī)整，呈不同程度的皺縮，槲皮素聯(lián)合射線處理后，細(xì)胞形態(tài)變化更加明顯，間隙進(jìn)一步增寬，細(xì)胞變圓、皺縮，且高濃度槲皮素作用的細(xì)胞形態(tài)改變更加顯著；DAPI染色后，射線及槲皮素均可引起細(xì)胞核不同程度的皺縮，二者合用后，大量細(xì)胞核皺縮、碎裂，高濃度藥物作用細(xì)胞核變化更加顯著。（5）射線照射24h后細(xì)胞遷移與侵襲能力增加，槲皮素對(duì)HeLa細(xì)胞的遷移與侵襲有抑制作用，槲皮素與射線共同處理細(xì)胞后，與空白組及單放組比較仍具有明顯的抑制遷移作用。（6）Western blot法檢測(cè)各處理組Bax、Bcl-2、VEGF、MMP-9蛋白表達(dá)情況，結(jié)果表明槲皮素單藥，以及聯(lián)合放射線均能升高Bax蛋白的表達(dá)，降低Bcl-2蛋白的表達(dá)；射線單獨(dú)作用后VEGF、MMP-9蛋白表達(dá)在一定時(shí)間內(nèi)略有增加，槲皮素處理后VEGF、MMP-9蛋白表達(dá)降低，二者共同處理后VEGF、MMP-9蛋白表達(dá)較空白組與單放組降低。 結(jié)論：（1）槲皮素及X射線均能抑制Hela細(xì)胞增殖。（2）槲皮素對(duì)Hela細(xì)胞具有放射增敏作用，并有濃度、時(shí)序的依從性。（3）射線在一定時(shí)間內(nèi)可能促進(jìn)Hela細(xì)胞的遷徙轉(zhuǎn)移，槲皮素則可抑制Hela細(xì)胞放療后的遷移與侵襲。（4）槲皮素對(duì)Hela細(xì)胞的放療增敏作用可能與細(xì)胞凋亡的發(fā)生、G2/M期阻滯、下調(diào)Bcl-2蛋白及上調(diào)Bax蛋白表達(dá)有關(guān)。（5）槲皮素抑制Hela細(xì)胞放療后的遷移與侵襲可能與下調(diào)VEGF、MMP-9蛋白表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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