新穎微脂體 Lipid calcium phosphate組合siRNA下調HIF-1α與EGFR癌化基因治療之口腔癌研究

Novel liposomal Lipid calcium phosphate combined with siRNA for knockdown of HIF-1α and EGFR genes in oral cancer treatment

吳俊賢(Jyun-Sian Wu)

指導教授 ： 陳信銘

共同指導教授 ： 許毅芝

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2019年)

https://doi.org/10.6342/NTU202000476

摘要

癌症是台灣人排行第一的死亡原因，占總死亡人數27.7%，平均每天約有130人死於癌症。其中台灣口腔癌的發生率是世界第一，主要原因與飲食文化中有嚼食檳榔的行為有關。本研究使用新型微脂體磷酸鈣技術Lipid calcium phosphate (LCP)，組合Hypoxia-inducible factors-1α (HIF-1α) 與Epidermal growth factor receptor (EGFR) 的small interfering RNA (siRNA) 利用DOPA (1,2-dioleoyl-sn-glycero-3-phosphate, sodium salt)、DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt)、DSPE–PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], ammonium salt) 等化合物包覆成為奈米大小之微脂體粒子。LCP HIF-1α & EGFR siRNA微脂體的平均粒徑約在38.6 ± 7.4 nm，膜電荷為50.2 ± 0.8 mV。以體外細胞實驗驗證siRNA序列可以有效抑制60% HIF-1α蛋白質表現和抑制81% EGFR蛋白質表現。

體內動物實驗使用異種移植SAS口腔癌細胞之裸鼠的動物模型，用BALB/cAnN.Cg-Foxn1nu/CrlNarl品系的裸鼠在其皮下移植SAS口腔癌細胞，並等待其腫瘤成長是180-201 mm3的口腔癌腫瘤隨機分配5組實驗，分別為正向控制組 (PBS)、負向控制組 (LCP Control siRNA)、HIF-1α siRNA對照組 (LCP HIF-1α siRNA)、EGFR siRNA對照組 (LCP EGFR siRNA) 和HIF-1α & EGFR siRNA組合治療組 (LCP HIF-1α & EGFR siRNA)，在第 0、1、2、5、6 和7天注射LCP藥物，其間隔為每24小時一次。每天持續觀察生理狀況，並每日量測腫瘤體積大小和體重，在第13天結束實驗犧牲動物。第13天的結果，PBS組腫瘤體積為500 mm3；LCP Control siRNA組腫瘤體積為542 mm3；LCP HIF-1α siRNA組腫瘤體積為526 mm3；LCP EGFR siRNA組腫瘤體積為532 mm3；LCP HIF-1α & EGFR siRNA組腫瘤體積為379 mm3。受到LCP藥物治療後，LCP HIF-1α & EGFR siRNA組比較PBS組減少121 mm3的腫瘤體積 (P<0.05)。LCP HIF-1α & EGFR siRNA組比較LCP Control siRNA組、LCP HIF-1α siRNA組與LCP EGFR siRNA組的腫瘤體積都有達到顯著的抑制 (P<0.01)。

蘇木精-伊紅染色 (hematoxylin and eosin stain, H&E stain) 觀察動物肝、腎和腫瘤切片，LCP藥物對動物的肝腎組織是沒有毒性，腫瘤切片觀察LCP HIF-1α & EGFR siRNA組有較多凋亡細胞的產生，用Mitotic Figure方法分析確認LCP HIF-1α & EGFR siRNA治療可以減少細胞有絲分裂的發生。LCP HIF-1α & EGFR siRNA在免疫組織染色中與PBS組相比Ki-67細胞增生因子和CD31血管新生因子的表現都有顯著減少，Caspase 3細胞凋亡因子的表現也比PBS組高出71%，代表LCP HIF-1α & EGFR siRNA可以抑制細胞增生，使癌細胞凋亡，並阻止腫瘤繼續生長。免疫組織染色HIF-1α和EGFR的切片染色分析證實LCP包覆HIF-1α與EGFR siRNA可以抑制HIF-1α和EGFR蛋白質表現的效果 (P<0.001)。血液生化分析分析15項與肝、腎和心肌有關的生化因子，數據顯示使用LCP HIF-1α & EGFR siRNA治療的動物，沒有出現與肝、腎和心肌功能相關的毒性產生。總體而言，LCP組合HIF-1α siRNA和EGFR siRNA可以比單獨使用HIF-1α siRNA或EGFR siRNA能更有效的抑制SAS口腔癌腫瘤的生長，並能抑制腫瘤中蛋白質的表現，且不會對動物有毒性產生副作用發生。

關鍵字

口腔癌 ； 脂質磷酸鈣 ； DOPA ； 缺氧誘導因子-1α ； 上皮生長因子受體 ； 微小干擾RNA

並列摘要

Cancer is most of the people cause of death in Taiwanese, accounting for 27.7% of deaths. About one day have 130 people die from cancer. The incidence of oral cancer in Taiwan is the highest in the world. The main reason is related to the behavior of chewing areca. Lipid calcium phosphate (LCP) compose of DOPA (1,2-dioleoyl-sn-glycero-3-phosphate, sodium salt)、DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt)、DSPE–PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], ammonium salt). The LCP technology encapsulated of hypoxia-inducible factors-1α (HIF-1α) and epidermal growth factor receptor (EGFR) small interfering RNA (siRNA). LCP HIF-1α & EGFR siRNA liposome average size was 38.6 ± 7.4 nm, zeta potential was 50.2 ± 0.8 mV. In vitro cell experiments confirmed that siRNA sequences can effectively inhibit the expression of 60% HIF-1α protein and inhibit the expression of 81% EGFR protein.

In vivo animal study used xenograft SAS oral cancer cell animal model. The animal used BALB/cAnN.Cg-Foxn1nu/CrlNarl mice. Inject the SAS oral cancer cell under the mice back skin, and waited tumor growth to 180-201 mm3. The animal group had PBS group, LCP Control siRNA group, LCP HIF-1α siRNA group, LCP EGFR siRNA group and LCP HIF-1α & EGFR siRNA. LCP drugs were injected on days 0, 1, 2, 5, 6 and 7 at intervals of 24 hours. The physiological condition was continuously observed every day, and the tumor volume and body weight were measured daily, and the animals were sacrificed on the 13th day. The day 13th result, PBS group tumor volume was 500 mm3；LCP Control siRNA group tumor volume was 542 mm3；LCP HIF-1α siRNA group tumor volume was 526 mm3；LCP EGFR siRNA group tumor volume was 532 mm3；LCP HIF-1α & EGFR siRNA group tumor volume was 379 mm3。After being treated with LCP drug, LCP HIF-1α & EGFR siRNA group tumor volume was smaller than PBS group 121 mm3 (P<0.05). LCP HIF-1α & EGFR siRNA group tumor volume compares to LCP Control siRNA group, LCP HIF-1α siRNA group and LCP EGFR siRNA group tumor volume had been inhibited (P<0.01).

H&E stain (hematoxylin and eosin stain) slice about the animal liver and kidney was showing no toxicity of LCP drug. The tumor H&E stain slice LCP HIF-1α & EGFR siRNA group had apoptosis more than other groups. Mitotic figure data show LCP HIF-1α & EGFR siRNA can reduce mitosis. The expression of Ki-67 and CD31 was significantly decreased in LCP HIF-1α & EGFR siRNA groups compared with PBS group. The LCP HIF-1α & EGFR siRNA group expression of caspase 3 was higher than the PBS groups. The LCP HIF-1α & EGFR siRNA can inhibit cell proliferation, causes apoptosis, and prevents tumor growth. IHC staining of HIF-1α and EGFR protein analysis confirmed that LCP encapsulated HIF-1α and EGFR siRNA could inhibit the expression of HIF-1α and EGFR proteins (P<0.001). Biochemical analysis analyzed 15 biochemical factors related to liver, kidney and heart muscle. The data showed that animals treated with LCP HIF-1α & EGFR siRNA did not develop toxicity associated with liver, kidney and heart.

Overall, LCP combined with HIF-1α siRNA and EGFR siRNA can inhibit the growth of SAS oral cancer tumors more effectively than HIF-1α siRNA or EGFR siRNA alone, and can inhibit the expression of proteins in tumors, and not to animals. LCP will not cause side effects.

並列關鍵字

Oral cancer ； Lipid calcium phosphate ； DOPA ； Hypoxia-inducible factor-1α ； Epidermal growth factor receptor ； Small interference RAN

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