本文選題：宮頸腺癌　切入點(diǎn)：Hela細(xì)胞　出處：《蘭州大學(xué)》2014年碩士論文

【摘要】：目的：探討槐定堿聯(lián)合順鉑對宮頸腺癌Hela細(xì)胞增殖的影響,以期對宮頸腺癌的臨床治療提供新的思路和實(shí)驗(yàn)室理論依據(jù)。 方法：體外培養(yǎng)宮頸腺癌細(xì)胞系Hela細(xì)胞：采用細(xì)胞增殖與毒性試驗(yàn)法即MTT法檢測不同濃度的槐定堿及其聯(lián)合順鉑對Hela細(xì)胞的增殖抑制率的影響,并計(jì)算半數(shù)抑制率IC50,且用金正均Q值法判斷兩藥的聯(lián)合效應(yīng),倒置相差顯微鏡下觀察Hela細(xì)胞的形態(tài)學(xué)改變：Hoechst33342染色法觀察不同濃度的槐定堿聯(lián)合順鉑作用24h后Hela細(xì)胞的形態(tài)學(xué)改變及凋亡情況；應(yīng)用流式細(xì)胞術(shù)測定不同濃度的槐定堿聯(lián)合順鉑作用24h后對Hela細(xì)胞周期分布及凋亡率的影響；應(yīng)用Western Blot法檢測不同濃度的槐定堿聯(lián)合順鉑作用24h后Hela細(xì)胞凋亡相關(guān)蛋白Bcl-2、Bax、P53、Caspase-3的表達(dá)情況。 結(jié)果：(1)MTT結(jié)果表明：槐定堿能夠明顯抑制宮頸腺癌Hela細(xì)胞的體外增殖(P0.05),其作用呈明顯的劑量-時(shí)間依賴性,計(jì)算24h、48h和72h的半數(shù)抑制率IC50分別為5.5mg/ml、4.2mg/ml及2.1mg/ml,應(yīng)用金正均Q值法判斷兩藥的聯(lián)合效應(yīng)為：槐定堿聯(lián)合順鉑作用于Hela細(xì)胞24h后,槐定堿濃度1.0mg/ml,兩藥為拮抗作用(Q0.85)；槐定堿濃度≥1.0mg/ml兩藥為協(xié)同作用(Q0.85)；槐定堿聯(lián)合順鉑作用于Hela細(xì)胞48h及72h后,不同濃度的槐定堿聯(lián)合順鉑組均表現(xiàn)出協(xié)同作用(Q0.85),各組兩兩比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)；槐定堿及其聯(lián)合順鉑處理24h后,Hela細(xì)胞的形態(tài)發(fā)生顯著變化,且隨著槐定堿濃度的增加形態(tài)學(xué)變化愈加明顯。 (2)Hoechst33342染色證實(shí)槐定堿聯(lián)合順鉑可以誘導(dǎo)宮頸腺癌Hela細(xì)胞凋亡,并可見典型的凋亡小體； (3)流式細(xì)胞術(shù)檢測細(xì)胞周期結(jié)果表明：槐定堿聯(lián)合順鉑可影響宮頸腺癌Hela細(xì)胞周期分布,隨著槐定堿濃度的增加,G0/G1期細(xì)胞的百分比逐漸降低而S期細(xì)胞的百分比逐漸增高,呈現(xiàn)S期阻滯,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；與順鉑組相比較,聯(lián)合用藥組①的G0/G1期細(xì)胞的百分比高于順鉑組,S期細(xì)胞的百分比低于順鉑組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；聯(lián)合用藥組②③④的G0/G1期細(xì)胞的百分比低于順鉑組,S期細(xì)胞的百分比高于順鉑組,與順鉑組及各組間比較均具有統(tǒng)計(jì)學(xué)差異(P0.05)。 (4)流式細(xì)胞術(shù)檢測細(xì)胞凋亡率結(jié)果表明：槐定堿聯(lián)合順鉑能夠誘導(dǎo)宮頸腺癌Hela細(xì)胞發(fā)生凋亡,與陰性對照組相比,順鉑組及聯(lián)合用藥組的凋亡率升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；與順鉑組相比較,聯(lián)合用藥組①組的凋亡率低于順鉑組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；聯(lián)合用藥組②③④的凋亡率高于順鉑組,協(xié)同順鉑促凋亡效應(yīng),與順鉑組及各組間比較均具有統(tǒng)計(jì)學(xué)差異(P0.05)。 (5) Western Blot結(jié)果證實(shí)：槐定堿聯(lián)合順鉑作用于宮頸腺癌Hela細(xì)胞24h后,與陰性對照組相比較,順鉑組及槐定堿聯(lián)合順鉑組均可使Bcl-2蛋白表達(dá)下調(diào),P53蛋白、Bax蛋白及Caspase-3蛋白表達(dá)上調(diào),差異有統(tǒng)計(jì)學(xué)意義(P0.05)；與順鉑組相比較,聯(lián)合用藥組①組的Bcl-2蛋白表達(dá)率高于順鉑組,Bax蛋白、P53蛋白及Caspase-3蛋白表達(dá)率低于順鉑組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),聯(lián)合用藥組②③④隨著槐定堿濃度的增加,Bcl-2蛋白表達(dá)逐漸降低,Bax、P53及Caspase-3蛋白表達(dá)逐漸增加,與順鉑組及各組間相比較均具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：(1)槐定堿可以時(shí)間-劑量依賴性的抑制宮頸腺癌Hela細(xì)胞的增殖�；倍▔A與順鉑聯(lián)合應(yīng)用具有協(xié)同抗腫瘤作用。 (2)槐定堿聯(lián)合順鉑抑制宮頸腺癌Hela細(xì)胞的增殖作用的機(jī)制可能與誘導(dǎo)Hela細(xì)胞凋亡,S期阻滯,下調(diào)Bcl-2蛋白的表達(dá),上調(diào)Bax、P53、Caspase-3蛋白的表達(dá)及下調(diào)Bcl-2/Bax的比值有關(guān),因此槐定堿聯(lián)合順鉑引起Hela細(xì)胞的凋亡可能是通過P53依賴的凋亡途徑完成的。
[Abstract]:Objective: To investigate the effects of sophoridine combined with cisplatin on the proliferation of cervical carcinoma cell line Hela, to provide ideas and laboratory new theoretical basis for the clinical treatment of cervical adenocarcinoma.
Methods: Hela cells of cervical adenocarcinoma cell lines cultured in vitro: the effects of proliferation and cytotoxicity test method of detecting Huai MTT of different concentration by proliferation inhibition rate of alkali and cisplatin on Hela cells, and calculate the half inhibition rate of IC50, and Jin Zhengjun Q value method to determine the combined effect of the two drugs, inverted to observe the morphological changes of Hela cells under the microscope: Observation of different concentrations of Hoechst33342 staining, sophoridine morphological changes and apoptosis of Hela cells after 24h alkali combined cisplatin; different concentrations were determined by flow cytometry of sophoridine combined with cisplatin 24h effect on cell cycle distribution and apoptosis rate of Hela detection; different Huai the application of Western Blot concentration Hela apoptosis related protein Bcl-2, 24h Bax after alkali combined with cisplatin, P53, Caspase-3 expression.
Results: the results showed that: (1) MTT sophoridine could significantly inhibit the proliferation of cervical carcinoma cell line Hela in vitro (P0.05), the effect was dose and time-dependent, calculation of 24h, half 48h and 72h inhibition rate of IC50 were 5.5mg/ml, 4.2mg/ml and 2.1mg/ml, the application of Nintaus Q value judgment method the combined effects of two drugs: sophoridine combined with cisplatin in Hela cells after 24h, sophoridine concentration 1.0mg/ml, the two drugs for antagonism (Q0.85); sophoridine concentration higher than two 1.0mg/ml drug synergy (Q0.85); sophoridine combined with cisplatin on Hela cell 48h and 72h, different the concentration of sophoridine combined with cisplatin group showed synergistic effect (Q0.85), the 22 groups was statistically significant difference (P0.05); sophoridine and cisplatin after 24h treatment, the morphology change of Hela cells, and with the increase of the concentration of sophoridine more morphological changes Obviously.
(2) Hoechst33342 staining confirmed that sophoridine combined with cisplatin can induce apoptosis of cervical carcinoma cell line Hela, and the typical apoptotic bodies;
(3) flow cytometry results showed that sophoridine combined with cisplatin can affect the cervical gland cancer Hela cell cycle distribution, with the increase of sophoridine concentration, the percentage of cells in G0/G1 phase decreased and the percentage of S phase cells increased gradually, showed S arrest, the difference was statistically significant (P0.05); compared with cisplatin group, combination group the percentage of G0/G1 cells was higher than that of cisplatin group, the percentage of cells in S phase was lower than that of cisplatin group, the difference was statistically significant (P0.05); the combination group the G0/G1 phase cells percentage lower than that of cisplatin group, the percentage of cells in S phase was higher than that of cisplatin group and cisplatin group and between groups were statistically different (P0.05).
(4) flow cytometry results showed that the apoptosis rate of sophoridine combined with cisplatin can induce cervical adenocarcinoma Hela cell apoptosis, compared with the negative control group, the apoptosis of cisplatin group and the combination group increased, the difference was statistically significant (P0.05); compared with cisplatin group, combination group, apoptosis the group was lower than that of cisplatin group, the difference was statistically significant (P0.05); apoptosis in combination group was higher than that of the 4 cisplatin group and cisplatin induced apoptosis, and cisplatin group and between groups were statistically different (P0.05).
(5) Western Blot results confirmed that sophoridine combined with cisplatin on cervical carcinoma cell line Hela after 24h, compared with the negative control group, cisplatin group and sophoridine combined with cisplatin group can make the down-regulation of Bcl-2 protein expression of P53 protein, up-regulated expression of Bax protein and Caspase-3 protein, the difference was statistically significant (P0.05); compared with cisplatin group, combined treatment group 1 group the expression rate of Bcl-2 protein was higher than that of cisplatin group, Bax protein, P53 protein expression and Caspase-3 protein was lower than that of cisplatin group, the difference was statistically significant (P0.05), combination group and with the increase of the concentration of sophoridine, Bcl-2 protein expression decreased gradually the expression of Bax, P53 and Caspase-3 protein gradually increased, compared with DDP group and between groups were statistically significant (P0.05).
Conclusion: (1) sophoridine can time dose dependent inhibition of cervical carcinoma cell line Hela proliferation. Sophoridine has anti-tumor effect co application of alkali with cisplatin.
(2) the proliferation mechanism of sophoridine alkali combined with cisplatin inhibiting cervical adenocarcinoma Hela cells may be associated with the induction of Hela cell apoptosis, S phase arrest, down-regulation of Bcl-2 protein expression, up regulation of Bax, P53, Caspase-3 protein expression and reducing the ratio of Bcl-2/Bax, so the sophoridine combined with cisplatin induced apoptosis in Hela cells may is the completion of apoptosis via a P53 dependent pathway.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.33

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