本文選題：miR-335 + HuH-7��； 參考：《南京中醫(yī)藥大學》2016年碩士論文

【摘要】：背景：肝細胞肝癌(hepatocellular carcinoma, HCC)是習見的惡性腫瘤之一,目前HCC確診時已多屬中、晚期,又由于腫瘤細胞的侵襲轉移、凋亡逃避、血管生成以及腫瘤耐藥等復雜的生物學行為也為HCC的根治帶來一定的困難,預后極差。因此,深入研究腫瘤在這些復雜生物學惡性表型形成中的信號傳遞及調節(jié)機制具有重要的理論和臨床應用價值,可為HCC的診斷及治療提供新的干預靶點。季德勝蛇藥片是南通特色中成藥之一,在南通乃至全中國都有良好的應用,能夠清熱解毒,活血化瘀止痛。它目前主要用于治療蛇蟲咬傷傷,但近年來在臨床實踐中運用該藥治療肝癌也起到一定療效。因此本研究可為HCC診斷、發(fā)病機制及治療提供新思路,也為HCC治療提供新的干預靶點。目的：本研究旨在探討miR-335作為標志物在HCC診斷中的臨床價值并研究季德勝蛇藥含藥血清對肝癌細胞的作用。方法：采用實時熒光PCR檢測50例HCC患者以及40例健康人群血清中miR-335相對表達量；采用實時熒光PCR檢測miR-335在54例HCC患者癌與癌旁組織中的相對表達量,以及在肝癌細胞株中的表達差異；CCK-8實驗檢測轉染miR-335入HuH-7細胞后對該細胞增殖的影響；CCK-8實驗檢測季德勝蛇藥含藥血清對肝癌細胞的影響。實時熒光PCR檢測季德勝蛇藥含藥血清對HuH-7細胞中miR-335的調控作用。Western-blot檢測miR-335潛在靶向Bcl-w對HuH-7細胞凋亡的影響。結果：HCC患者血清中miR-335相對表達量明顯低于健康對照組,差異有統(tǒng)計學意義(P0.05)。HCC患者血清中miR-335相對表達量與性別、年齡、腫瘤大小、TNM分期、淋巴結轉移以及甲胎蛋白(AFP)無相關性(P0.05),而與有無癌栓相關,差異有統(tǒng)計學意義(P0.01)。ROC曲線分析確定了最佳的miR-335相對表達量的臨界值為1.28,靈敏度為78%,特異性為70%,曲線下面積(AUC)為0.75。AFP診斷HCC的靈敏度為76%,特異性為97%,曲線下面積(AUC)為0.83；血清中miR-335與AFP聯(lián)合檢測,靈敏度為84%,特異性為97%,曲線下面積(AUC)為0.93。HCC患者癌組織中miR-335相對表達量較癌旁組織中顯著降低,差異有統(tǒng)計學意義(P0.05)。臨床病理分析表明肝細胞肝癌組織中miR-335相對表達量與有無癌栓呈正相關(P0.05),與性別、年齡、腫瘤大小、TNM分期、淋巴結轉移以及甲胎蛋白(AFP)無相關性(P0.05)。細胞篩選結果顯示miR-335在HuH-7細胞中顯著低表達；miR-335過表達后能顯著抑制HuH-7細胞增殖(P0.01)。季德勝蛇藥含藥血清能抑制肝癌HuH-7細胞增殖且對肝癌細胞中miR-335起到一定的調控作用。miR-335過表達后通過下調靶向Bcl-w能促進細胞凋亡。結論：肝細胞肝癌患者血清中miR-335表達降低,有望成為一種新的生物標志物用于肝癌的診斷,且與AFP聯(lián)合檢測可提高肝癌的診斷效率。miR-335在HCC患者癌及癌旁組織中相對表達量有顯著差異；miR-335過表達能顯著抑制HuH-7細胞的增殖能力。季德勝蛇藥含藥血清能抑制肝癌HuH-7細胞增殖且對肝癌細胞中miR-335起到一定的調控作用。miR-335過表達通過下調靶向Bcl-w促進細胞凋亡。miR-335將有可能成為新的HCC治療靶向。季德勝蛇藥將有望成為治療肝癌新的藥物。
[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.
【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R273

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