季德胜蛇药通过调控miR-335抑制肝癌细胞增殖作用机制探讨

发布时间：2018-06-12 08:25

本文选题：miR-335 + HuH-7　；参考：《南京中医药大学》2016年硕士论文

【摘要】：背景：肝细胞肝癌(hepatocellular carcinoma, HCC)是习见的恶性肿瘤之一,目前HCC确诊时已多属中、晚期,又由于肿瘤细胞的侵袭转移、凋亡逃避、血管生成以及肿瘤耐药等复杂的生物学行为也为HCC的根治带来一定的困难,预后极差。因此,深入研究肿瘤在这些复杂生物学恶性表型形成中的信号传递及调节机制具有重要的理论和临床应用价值,可为HCC的诊断及治疗提供新的干预靶点。季德胜蛇药片是南通特色中成药之一,在南通乃至全中国都有良好的应用,能够清热解毒,活血化瘀止痛。它目前主要用于治疗蛇虫咬伤伤,但近年来在临床实践中运用该药治疗肝癌也起到一定疗效。因此本研究可为HCC诊断、发病机制及治疗提供新思路,也为HCC治疗提供新的干预靶点。目的：本研究旨在探讨miR-335作为标志物在HCC诊断中的临床价值并研究季德胜蛇药含药血清对肝癌细胞的作用。方法：采用实时荧光PCR检测50例HCC患者以及40例健康人群血清中miR-335相对表达量；采用实时荧光PCR检测miR-335在54例HCC患者癌与癌旁组织中的相对表达量,以及在肝癌细胞株中的表达差异；CCK-8实验检测转染miR-335入HuH-7细胞后对该细胞增殖的影响；CCK-8实验检测季德胜蛇药含药血清对肝癌细胞的影响。实时荧光PCR检测季德胜蛇药含药血清对HuH-7细胞中miR-335的调控作用。Western-blot检测miR-335潜在靶向Bcl-w对HuH-7细胞凋亡的影响。结果：HCC患者血清中miR-335相对表达量明显低于健康对照组,差异有统计学意义(P0.05)。HCC患者血清中miR-335相对表达量与性别、年龄、肿瘤大小、TNM分期、淋巴结转移以及甲胎蛋白(AFP)无相关性(P0.05),而与有无癌栓相关,差异有统计学意义(P0.01)。ROC曲线分析确定了最佳的miR-335相对表达量的临界值为1.28,灵敏度为78%,特异性为70%,曲线下面积(AUC)为0.75。AFP诊断HCC的灵敏度为76%,特异性为97%,曲线下面积(AUC)为0.83；血清中miR-335与AFP联合检测,灵敏度为84%,特异性为97%,曲线下面积(AUC)为0.93。HCC患者癌组织中miR-335相对表达量较癌旁组织中显著降低,差异有统计学意义(P0.05)。临床病理分析表明肝细胞肝癌组织中miR-335相对表达量与有无癌栓呈正相关(P0.05),与性别、年龄、肿瘤大小、TNM分期、淋巴结转移以及甲胎蛋白(AFP)无相关性(P0.05)。细胞筛选结果显示miR-335在HuH-7细胞中显著低表达；miR-335过表达后能显著抑制HuH-7细胞增殖(P0.01)。季德胜蛇药含药血清能抑制肝癌HuH-7细胞增殖且对肝癌细胞中miR-335起到一定的调控作用。miR-335过表达后通过下调靶向Bcl-w能促进细胞凋亡。结论：肝细胞肝癌患者血清中miR-335表达降低,有望成为一种新的生物标志物用于肝癌的诊断,且与AFP联合检测可提高肝癌的诊断效率。miR-335在HCC患者癌及癌旁组织中相对表达量有显著差异；miR-335过表达能显著抑制HuH-7细胞的增殖能力。季德胜蛇药含药血清能抑制肝癌HuH-7细胞增殖且对肝癌细胞中miR-335起到一定的调控作用。miR-335过表达通过下调靶向Bcl-w促进细胞凋亡。miR-335将有可能成为新的HCC治疗靶向。季德胜蛇药将有望成为治疗肝癌新的药物。
[Abstract]:Background: hepatocellular carcinoma (HCC) is one of the common malignant tumors. At present, the diagnosis of HCC is mostly in the middle and late stages, and the complicated biological behavior, such as invasion and metastasis of tumor cells, apoptosis evasion, angiogenesis and tumor resistance, also brings some difficulties and extremely poor prognosis for the radical cure of HCC. The study of the signal transduction and regulation mechanism of tumor in the formation of these complex biological malignant phenotypes has important theoretical and clinical value. It can provide new targets for the diagnosis and treatment of HCC. The serpent is one of the Chinese traditional Chinese medicine, and has good application in Nantong and in China. It can clear heat and detoxify and live. Blood stasis and pain relief. It is mainly used in the treatment of snake bite and injury, but in recent years the use of this drug in clinical practice also plays a certain effect in the treatment of liver cancer. Therefore, this study can provide new ideas for HCC diagnosis, pathogenesis and treatment, and also provide new intervention targets for the treatment of HCC. Purpose: This study aims to explore miR-335 as a marker in H The clinical value of the CC diagnosis and the effect of the sera containing the medicine of Ji Desheng on the liver cancer cells. Methods: real-time fluorescence PCR was used to detect the relative expression of miR-335 in 50 patients with HCC and 40 healthy people, and the relative expression of miR-335 in 54 cases of cancer and para cancerous tissues in HCC patients and in the liver were detected by real-time fluorescent PCR. The difference of expression in the cancer cell lines; the effect of the CCK-8 test on the proliferation of the cells after transfection of miR-335 into HuH-7 cells; the effect of the CCK-8 test on the sera containing the serpant on the serpant of the serpant. Real time fluorescence PCR detects the regulation of the sera containing the sera containing the serpant on the miR-335 in HuH-7 cells,.Western-blot detection miR-335 potential The effect of target Bcl-w on HuH-7 cell apoptosis. Results: the relative expression of miR-335 in serum of HCC patients was significantly lower than that of the healthy control group. The difference was statistically significant (P0.05) in.HCC patients, there was no correlation between the relative expression of miR-335 and sex, age, tumor size, TNM staging, lymph node metastasis and alpha fetoprotein (AFP) without correlation (P0.05). No tumor thrombus related, the difference was statistically significant (P0.01).ROC curve analysis determined that the best miR-335 relative expression of the critical value of 1.28, sensitivity 78%, specificity of 70%, AUC for 0.75.AFP diagnostic HCC sensitivity of 76%, specificity 97%, AUC is 0.83 under the curved line (AUC); serum miR-335 and AFP combined detection, spirit The sensitivity was 84% and the specificity was 97%. The relative expression of miR-335 in the cancer tissue under the curve (AUC) was significantly lower than that in the adjacent tissue (P0.05). The clinicopathological analysis showed that the relative expression of miR-335 was positively correlated with the tumor suppository (P0.05) in the hepatocellular carcinoma tissue (P0.05), and the sex, age, tumor size, T NM staging, lymph node metastasis and alpha fetoprotein (AFP) have no correlation (P0.05). Cell screening results showed that miR-335 was significantly lower in HuH-7 cells; miR-335 overexpression could significantly inhibit the proliferation of HuH-7 cells (P0.01). .miR-335 overexpression can promote cell apoptosis after overexpression of the target Bcl-w. Conclusion: the expression of miR-335 in the serum of hepatocellular carcinoma patients is reduced, and it is expected to be a new biomarker for the diagnosis of liver cancer, and the combined detection of AFP can improve the diagnostic efficiency of hepatocellular carcinoma (.MiR-335) in the relative expression of.MiR-335 in the cancer and adjacent tissues of the patients with HCC MiR-335 overexpression can significantly inhibit the proliferation of HuH-7 cells. The sera of snake-snakes can inhibit the proliferation of hepatoma HuH-7 cells and play a certain regulatory role on miR-335 in hepatoma cells..miR-335 overexpression by down regulation of target Bcl-w to promote apoptosis.MiR-335 may be a new target for HCC therapy. Ji Desheng snake medicine is expected to become a new drug for the treatment of liver cancer.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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