【摘要】：目的:本實(shí)驗(yàn)通過觀察海兔素對(duì)人肝癌Hep G2細(xì)胞株增殖以及對(duì)小鼠H22肝癌移植瘤的抑制效應(yīng),以此探討海兔素對(duì)肝癌的抑制作用及可能的分子機(jī)制。方法:1.人肝癌Hep G2細(xì)胞株常規(guī)培養(yǎng)于含10%胎牛血清的DMEM/HIGH GLUCOSE完全培養(yǎng)基,置于37℃、體積分?jǐn)?shù)為0.05 CO2的細(xì)胞恒溫培養(yǎng)箱中培養(yǎng)。取對(duì)數(shù)生長期細(xì)胞,分別加入終末濃度為20、40、60、80、100、120mg/L的海兔素溶解液對(duì)其進(jìn)行干預(yù),Cell Counting Kit-8(CCK-8)法檢測海兔素作用24h、48h后對(duì)肝癌Hep G2細(xì)胞株增殖的影響;熒光顯微鏡Annexin V-FITC/PI雙染實(shí)驗(yàn)檢測海兔素對(duì)Hep G2肝癌細(xì)胞株凋亡的誘導(dǎo)作用。2.昆明種雄性小鼠40只,將其按體重隨機(jī)分為模型組和海兔素低、中、高劑量組,每組10只。將以生理鹽水稀釋的H22瘤細(xì)胞懸液接種于各組小鼠左前肢腋下皮下組織,建立小鼠H22皮下移植瘤模型。次日,海兔素低、中、高劑量組分別給予海兔素25、50、100mg·kg-1·d-1 0.5m L灌胃;模型組動(dòng)物則予以等體積大豆色拉油灌胃,實(shí)驗(yàn)進(jìn)行2周。小鼠末次灌胃24h后,稱取體質(zhì)量,摘眼球法取全血,頸椎脫臼法處死動(dòng)物。完整剝離新鮮移植瘤組織,稱重,計(jì)算抑瘤率;免疫組化法檢測腫瘤組織中基質(zhì)金屬蛋白酶-9(MMP-9)、血管內(nèi)皮生長因子(VEGF)及增殖細(xì)胞核抗原(PCNA)的表達(dá)情況;酶聯(lián)免疫吸附試驗(yàn)(ELISA法)測定小鼠血清中白介素6(IL-6)和腫瘤壞死因子α(TNF-α)的含量。結(jié)果:1.海兔素對(duì)人肝癌Hep G2細(xì)胞株抑制作用的體外試驗(yàn)效果評(píng)價(jià)CCK-8實(shí)驗(yàn)結(jié)果表明,Hep G2肝癌細(xì)胞經(jīng)海兔素干預(yù)后,生長增殖能力顯著受到抑制,且隨藥物劑量的增加和干預(yù)時(shí)間的延長,抑制作用增強(qiáng)(P0.05),其24h IC25值和IC50值分別為65.36mg/L和103.77mg/L。熒光顯微鏡Annexin V-FITC/I雙染實(shí)驗(yàn)檢測發(fā)現(xiàn),經(jīng)65mg/L和105mg/L海兔素干預(yù)24h后,Hep G2肝癌細(xì)胞凋亡數(shù)目較對(duì)照組明顯增加,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.海兔素對(duì)H22荷瘤小鼠體內(nèi)抑瘤試驗(yàn)效果評(píng)價(jià)H22荷瘤小鼠經(jīng)低、中、高劑量海兔素處理后,腫瘤的生長水平顯著受到抑制,與模型組相比,瘤塊質(zhì)量呈劑量依賴性顯著減輕(P0.05),其抑瘤率分別為28.31%、33.84%、42.96%。海兔素各劑量干預(yù)組小鼠移植瘤組織中MMP-9的陽性表達(dá)率與模型組相比,均有不同程度地降低,尤其是50mg/kg、100mg/kg劑量組降低更為明顯,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);各海兔素組與模型動(dòng)物組相比,小鼠腫瘤組織中VEGF和PCNA的陽性表達(dá)率逐漸降低,并呈一定的劑量依賴關(guān)系(P0.05)。經(jīng)50mg/kg、100mg/kg海兔素處理后,小鼠血清中IL-6及TNF-α表達(dá)水平均顯著高于模型組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.通過體外細(xì)胞培養(yǎng)的方法,運(yùn)用CCK-8法及熒光顯微鏡Annexin V-FITC/PI雙染實(shí)驗(yàn)法,初步證實(shí)了海兔素能夠有效抑制肝癌細(xì)胞的增殖作用,同時(shí)顯著誘導(dǎo)肝癌細(xì)胞發(fā)生凋亡。2.海兔素能夠在體內(nèi)抑制小鼠H22肝癌移植瘤的生長,其作用機(jī)制主要表現(xiàn)為以下幾個(gè)方面:①抑制腫瘤組織周圍細(xì)胞外基質(zhì)降解過程②阻礙新血管形成過程③提高機(jī)體免疫功能。3.體內(nèi)、外研究結(jié)果顯示,海兔素對(duì)肝癌具有明顯的抑制作用,有望成為一種新型的天然抗癌藥物。然而海兔素能否成為有效逆轉(zhuǎn)和治療肝癌的藥物,尚需要進(jìn)一步的實(shí)驗(yàn)研究進(jìn)行佐證。
[Abstract]:OBJECTIVE: To investigate the inhibitory effect of pirarin on the proliferation of human hepatocellular carcinoma Hep G2 cell line and H22 transplanted tumor in mice, and to explore the possible molecular mechanism of pirarin on hepatocellular carcinoma. Methods: 1. The human hepatocellular carcinoma Hep G2 cell line was cultured in DMEM / HIGH GLUCOSE complete medium containing 10% fetal bovine serum and placed in 37% medium. Cells in logarithmic growth phase were cultured in a constant temperature incubator with 0.05 CO2 volume fraction and 20,40,60,80,100,120 mg/L Kainin solution respectively. Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of Kainin on proliferation of Hep G2 cell line after 24 hours and 48 hours. V-FITC/PI double staining assay was used to detect the induction of apoptosis of Hep G2 hepatoma cell line. 2. Forty Kunming male mice were randomly divided into two groups according to their body weight: model group and low, medium and high dosage groups, 10 in each group. The next day, the mice were given 25,50,100 mg kg 1 D 10.5 ml of kaempferol by gastric lavage respectively, and the mice in the model group were given soybean salad oil by gastric lavage for 2 weeks. After 24 hours of the last gastric lavage, the mice were weighed, eyeballs were taken out, and the animals were killed by cervical vertebra dislocation. The expression of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in tumor tissues were detected by immunohistochemistry, and the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in serum of mice were determined by enzyme linked immunosorbent assay (ELISA). The results of CCK-8 assay showed that the growth and proliferation of Hep G2 hepatoma cells were significantly inhibited after the intervention of kaempferol, and the inhibition increased with the increase of drug dosage and the prolongation of intervention time (P 0.05). The 24-hour IC25 and IC50 values of Hep G2 hepatoma cells were 65.36 m, respectively. G/L and 103.77 mg/L. Fluorescence microscope Annexin V-FITC/I double staining assay found that after 65 mg/L and 105 mg/L kaempferol intervention for 24 hours, the number of apoptosis of Hep G2 hepatoma cells was significantly increased compared with the control group, the difference was statistically significant (P 0.05). 2. Compared with the model group, the tumor growth level was significantly inhibited, and the tumor mass was significantly reduced in a dose-dependent manner (P 0.05). The tumor inhibition rates were 28.31%, 33.84% and 42.96% respectively. The positive expression rates of VEGF and PCNA in tumor tissues of mice were gradually decreased and showed a dose-dependent relationship (P 0.05). After treatment with 50 mg/kg and 100 mg/kg Haituansu, the expression of IL-6 and TNF-alpha in serum of mice was significantly decreased (P 0.05). CONCLUSION: 1. The CCK-8 method and fluorescence microscope Annexin V-FITC/PI double staining method were used in vitro cell culture, and the preliminary results showed that pikacin could effectively inhibit the proliferation of hepatocellular carcinoma cells and induce apoptosis of hepatocellular carcinoma cells. It can inhibit the growth of H22 liver cancer xenograft tumor in vivo. The main mechanisms are as follows: 1. Inhibiting the degradation of extracellular matrix around tumor tissue; 2. Inhibiting the formation of new blood vessels; 3. In vivo and in vitro studies show that hippocampin has a significant inhibitory effect on liver cancer. It is expected to become a new natural anticancer drug. However, further experimental studies are needed to confirm whether kaempferol can be an effective drug for reversing and treating liver cancer.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7

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