創新HIF1α靜默RNA基因標靶治療結合光動力治療於口腔癌的轉譯研究-人類口腔癌細胞異種移植動物模式

Oral cancer treatment using HIF1α siRNA targeted gene combines with photodynamic therapy - A translational research study of SCC xenograft mice model

陳韋華(Wei-Hua Chen)

指導教授 ： 陳瑩山 ； 許毅芝 ； 黃力夫

中原大學/理學院/奈米科技碩士學位學程/碩士(2013年)

https://doi.org/10.6840/CYCU.2013.00010

摘要

口腔癌 (oral cancer) 為目前國人十大癌症排行榜中排名第五名，在男性癌症排行為第四名，是最為常見的癌症之一。光動力療法 (photodynamic therapy、PDT) 被視為治療人類癌前病變及口腔癌中的另一種替代治療方法，因為PDT是非侵入性的，可反覆治療，沒有累積的副作用，其治療結果形成的結疤小。為了提高口腔癌的治療效果，我們結合了靜默RNA (small interfering RNA、siRNA) 技術針對人類口腔癌治療，我們利用脂質磷酸鈣 (Lipid-calcium-phosphate、LCP) 標靶有效地運送siRNA進入人類口腔癌SCC4細胞細胞質的sigma受體表達。我們探討了在人類口腔癌細胞 (squamous cell carcinoma, SCC4) 上，標靶anisamide HIF1α (Hypoxia-inducibe factor 1-alpha) 基因的siRNA序列致癌基因針對殺死細胞的效果，在動物模式則結合光動力療法在人類口腔癌SCC4細胞上，這樣的組合治療能達到殺死細胞的綜效效果。結果顯示LCP標靶運送HIF1 siRNA治療能有效地抑制HIF1α基因表現，進而顯著地抑制腫瘤癌細胞的生長。我們在人類口腔癌細胞SCC4和SAS異種移植裸鼠上，經由尾靜脈注射LCP標靶包覆HIF1α siRNA治療，siRNA成功地傳送到腫瘤組織上濃度聚積最高的腫瘤部位，使用裸鼠基因標靶治療結合光動力療法效果顯示出腫瘤體積下降了10-30％，比只抑制HIF1α表現治療或只做光動力療法效果來得佳。靜默HIF1α能夠增強抗腫瘤活性，它也能夠抗腫瘤增生和提高PDT治療效果來抑制腫瘤生長。在腫瘤切片凋亡分析 (Tunel assay) 結果上，也顯示靜默HIF1α和PDT治療能使腫瘤細胞凋亡，而組織化學染色切片結果顯示對肝腎功能毒性沒有影響。我們的結果證明，LCP標靶奈米傳輸技術來傳送靜默HIF1α在治療腫瘤上，在人類口腔癌上運用基因治療結合光動力療法，在未來是一個很有潛在性發展的臨床治療方法。

關鍵字

口腔癌 ； 缺氧誘導因子 ； 靜默RNA

並列摘要

Oral cancers (OC) rank as the fifth most prevalent cancer in both genders and account for the fourth most common cancer in males in Taiwan. Photodynamic therapy (PDT) is considered as another alternative for treating human oral precancerous and cancerous lesions because PDT is noninvasive, can be used repeatedly without cumulative side effects, and results in little scar formation. To enhance the therapeutic effect of OC, we combined small interfering RNA (siRNA) technology for OC therapy, we have developed anisamide-targeted LCP to efficiently deliver siRNA into the cytoplasm of sigma receptor-expressing human SCC4 cells. Encapsulated HIF1α siRNA sequence targeting HIF1α oncogene in SCC4 was investigated for cell-killing efficacy, then following PDT in vitro. Synergistic cell-killing effects were observed in such combined therapy in human SCC4. The results indicated that the HIF1α siRNA delivered by the targeted LCP could effectively knock down HIF1α expressions and significantly inhibit tumor cell growth. After iv injection of mice bearing human OC SCC4 or SAS xenografted tumor with HIF1α siRNA formulated in the targeted LCP, siRNA was successfully delivered to the tumors with relatively concentrated amount in the tumor tissues. Nude mouse treated with combined therapy showed better tumor regression which is reduced tumor to 10-30% more volume than that of treated with HIF1α siRNA alone or PDT alone.The results of Tunel assay, HIF1α siRNA with combined PDT therapy make tumor cell apoptosis. The results of H&E histpathological staining don’t affect funcion of the liver and kidney.The enhanced antitumor activity is due to the fact that the silencing of HIF1α and it could anti-tumor-proliferation and enhance PDT therapeutic effect to suppress tumor growth. Our results prove that the targeted LCP is a promising nanoparticle technology to deliver HIF1α siRNA into tumors as a potentially clinical therapeutic modality in the gene therapy combined with PDT in human OC.

並列關鍵字

HIF1α ； siRNA ； oral cancer ； nanoparticle

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