γ射线对人肝癌细胞SMMC7721和HepG2灭活效果的研究

发布时间：2018-04-10 12:08

本文选题：肝癌细胞 + 自体输血　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:肝细胞肝癌在肿瘤导致死亡的疾病中排名世界第三,特别是在发展中国家,发病率还呈上升的趋势。手术治疗是根治肝癌的最有效的手段,但是仅有10%-30%的患者在诊断出肝癌时有手术指征。肝癌患者进行肝切除术或者肝移植手术时常常会伴随着大量失血,往往需要输血,异体输血存在着很多弊端,比如不良输血反应,免疫抑制以及疾病传播等,而特别是对于肿瘤患者来讲,可能还会引起肿瘤的复发,因此推荐肝癌患者进行自体输血。但是肝癌患者自体输血一直以来又存在着争议,有学者担心血中可能存在的肝癌细胞有可能会引起肿瘤的血行转移。故本实验通过研究γ射线对人肝癌细胞的增殖、迁移、侵袭、凋亡的影响,来探究γ射线对肝癌患者的自体血液中残留的肿瘤细胞的灭活效果,寻找合适的辐照剂量,为肝癌患者的自体输血的安全应用提供依据。方法:1)人肝癌细胞株SMMC7721和HepG2的体外培养。2)用不同剂量的γ射线处理HCC(Hepatoma carcinoma cell)后,用CCK8(cell counting kit-8)实验检测细胞的增殖能力,吸光度值的大小可以反应活细胞的相对数量;划痕实验检测细胞的迁移能力,细胞移动的距离可以代表迁移能力;Transwell实验以及MPP-9蛋白表达量检测来反应细胞的侵袭能力,穿过的细胞越多,MMP-9蛋白的表达越多,细胞的侵袭能力越强;annexin V/PI染色(流式细胞术)和P53凋亡蛋白的表达量测定来检测细胞的凋亡情况。3)用SPSS17.0软件统计分析实验结果,P0.05表示差异有统计学意义。结果:1)γ射线处理细胞后,细胞失去了正常形态,出现了细胞变形,漂浮,细胞膜不完整,胞质内有空泡,细胞碎裂等一系列变化;2)γ射线处理后24h,48h,72h,细胞的增殖能力都明显受到抑制,细胞活力跟培养时间和γ射线剂量成反比,随着培养时间和剂量增加,细胞活力降低,差异有统计学意义(P0.01);3)γ射线对细胞的迁移能力有抑制作用,24h划痕实验结果显示,随着剂量增加,细胞迁移的距离减小,差异有统计学意义(P0.01);4)γ射线对细胞的侵袭能力有抑制作用,随着剂量增加,穿过小室的细胞数量逐渐减小,差异具有统计学意义(P0.01);Western Blot法结果显示,辐照后,MPP-9蛋白的表达量降低(P0.01);5)γ射线会抑制细胞的克隆形成,20Gy开始,细胞没有克隆形成。6)流式凋亡检测显示,细胞早期凋亡率随着γ射线的剂量增加而增加(P0.01),Western Blot法结果显示,γ射线处理HCC后,P53凋亡蛋白的表达量增加(P0.01)。结论:γ射线可以有效地抑制肝癌细胞的增殖,迁移和侵袭,促进细胞凋亡,可以有效地灭活肝癌细胞;推荐使用40Gy的剂量应用于肝癌患者自体血液中肿瘤细胞的灭活,提高肝癌患者自体输血的安全性和有效性。
[Abstract]:Objective: hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world, especially in developing countries.Surgical treatment is the most effective method to cure liver cancer, but only 10 to 30% of the patients have surgical indications in the diagnosis of liver cancer.Liver cancer patients undergoing hepatectomy or liver transplantation are often accompanied by massive blood loss and often need blood transfusions. Allogeneic blood transfusion has many drawbacks, such as adverse transfusion reactions, immunosuppression and disease transmission.Especially for cancer patients, it may also lead to recurrence of tumor, so we recommend autologous blood transfusion for liver cancer patients.However, autologous blood transfusion in patients with liver cancer has been controversial, some scholars worry that the existence of liver cancer cells in the blood may cause tumor blood metastasis.Therefore, the effect of 纬-ray on the proliferation, migration, invasion and apoptosis of human hepatoma cells was studied in order to explore the inactivation effect of 纬-ray on the residual tumor cells in autologous blood of patients with liver cancer, and to find the appropriate irradiation dose.To provide the basis for the safe application of autologous blood transfusion in patients with liver cancer.Methods in vitro culture of human hepatoma cell lines SMMC7721 and HepG2. (2) HCC(Hepatoma carcinoma cells were treated with different doses of 纬 -rays. The proliferative ability of HCC(Hepatoma carcinoma cells was measured by CCK8(cell counting kit-8). The relative number of living cells could be reflected by the size of absorbance.The migration ability of cells was measured by scratch test. The distance of cell movement could represent the migration ability and the expression of MPP-9 protein to reflect the invasion ability of cells. The more cells passed through, the more MMP-9 protein was expressed.The stronger the invasiveness of cells was, the more annexin V/PI staining (flow cytometry) and p53 apoptotic protein expression were measured to detect the apoptosis of cells.Results after the cell was treated with 纬 -ray, the cell lost its normal shape, appeared cell deformation, floating, incomplete cell membrane, and vacuole in the cytoplasm.A series of changes, such as cell fragmentation, etc.) after 24 h ~ 48 h ~ 72 h of 纬 -ray treatment, the cell proliferation ability was obviously inhibited, and the cell viability was inversely proportional to the culture time and 纬 -ray dose, and decreased with the increase of culture time and dose.The difference was statistically significant (P 0.01) 纬 -ray could inhibit the migration ability of cells. The results of 24 h scratch test showed that the distance of cell migration decreased with the increase of dose, and the difference was statistically significant (P 0.01) 纬 -ray could inhibit the invasion ability of cells.With the increase of dose, the number of cells passing through the cell gradually decreased. The results of Western Blot analysis showed that the expression of MPP-9 protein decreased after irradiation.Flow cytometry showed that the early apoptosis rate increased with the increase of 纬 -ray dose. The results of Western Blot showed that the expression of p53 apoptosis protein increased after 纬 -ray treatment of HCC.Conclusion: 纬-ray can effectively inhibit the proliferation, migration and invasion of hepatoma cells, promote cell apoptosis, and effectively inactivate hepatoma cells. It is recommended that the dose of 40Gy be used in the inactivation of tumor cells in autologous blood of patients with liver cancer.To improve the safety and efficacy of autologous blood transfusion in patients with liver cancer.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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