• 1.廣西醫(yī)科大學(xué)第一附屬醫(yī)院胃腸外科（南寧 530021);;
  • 2.廣西醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院組織胚胎學(xué)教研室（南寧 530021);

目的 　探討survivin反義寡核苷酸(antisense oligonucleotide，ASODN)對(duì)人胰腺癌細(xì)胞PANC-1增殖的影響。
方法 　設(shè)計(jì)并合成特異性靶向ASODN及正義寡核苷酸(sense oligodeoxynucleotides，SODN)。ASODN經(jīng)FAM標(biāo)記,以陽(yáng)離子脂質(zhì)體為載體轉(zhuǎn)染至體外培養(yǎng)的PANC-1細(xì)胞中（ASODN組），另設(shè)空白對(duì)照組（正常培養(yǎng)的細(xì)胞）、脂質(zhì)體空轉(zhuǎn)染組（脂質(zhì)體轉(zhuǎn)染的為正常的培養(yǎng)基）及SODN組作為對(duì)照組。采用流式細(xì)胞儀(flow cytometry，F(xiàn)CM)測(cè)轉(zhuǎn)染率,依次篩選最佳細(xì)胞接種數(shù)量、ASODN濃度和脂質(zhì)體的量； 熒光顯微鏡觀察轉(zhuǎn)染后的細(xì)胞； MTT法測(cè)定轉(zhuǎn)染后細(xì)胞的抑制率； 透射電鏡觀察轉(zhuǎn)染后細(xì)胞的超微結(jié)構(gòu)變化； 吖叮橙(AO)與溴化乙啶(EB)染色觀察轉(zhuǎn)染后細(xì)胞凋亡的形態(tài)學(xué)改變； DNA凝膠電泳觀察轉(zhuǎn)染后細(xì)胞凋亡情況； FCM測(cè)轉(zhuǎn)染后細(xì)胞凋亡率及細(xì)胞周期； 免疫組織化學(xué)技術(shù)檢測(cè)轉(zhuǎn)染后survivin蛋白的表達(dá)。
結(jié)果 　①ASODN濃度為50、100、150、200、250、400、600及800 nmol/L時(shí),轉(zhuǎn)染率分別為31.9%、37.4%、41.4%、52.6%、24.2%、11.4%、16.1%和　15.5%； 接種細(xì)胞數(shù)量為2×104 、2×105及2×106個(gè)/ 孔時(shí)，轉(zhuǎn)染率分別為12.0%、50.8%和11.2%； 轉(zhuǎn)染所用的脂質(zhì)體量為2、3及4 μl時(shí),轉(zhuǎn)染率分別為58.8%、34.0%和23.6%。②轉(zhuǎn)染ASODN后熒光顯微鏡發(fā)現(xiàn)轉(zhuǎn)染后細(xì)胞之間空隙加大，形態(tài)變圓，貼壁細(xì)胞生長(zhǎng)較少，部分漂浮于培養(yǎng)液中。③ASODN組24、36及48 h后細(xì)胞抑制率明顯高于各對(duì)照組（P lt;0.05），隨著時(shí)間延長(zhǎng)，細(xì)胞抑制率增高(P lt;0.05)。④ASODN組電泳圖上凋亡細(xì)胞呈明顯的“梯狀”條帶。⑤AO與EB染色觀察ASODN組細(xì)胞出現(xiàn)凋亡的形態(tài)學(xué)改變，其核染色質(zhì)均高度聚集或核染色呈新月形聚集于核膜一邊，核仁消失。⑥ASODN組細(xì)胞凋亡率為（38.10±3.4）%，明顯高于SODN組的(4.16±1.7)% (P lt;0.05)。⑦ASODN組細(xì)胞周期阻滯于G2／M期。⑧轉(zhuǎn)染后ASODN組survivin蛋白表達(dá)明顯低于各對(duì)照組（P lt;0.05）。
結(jié)論 　survivin ASODN轉(zhuǎn)染至胰腺癌細(xì)胞最佳轉(zhuǎn)染條件為接種細(xì)胞數(shù)量為2×105個(gè)/孔，ASODN的濃度為200 nmol/L，脂質(zhì)體的量為2 μl； survivin ASODN能明顯下調(diào)survivin蛋白的表達(dá)，抑制胰腺癌PANC-1細(xì)胞的增殖，并誘導(dǎo)細(xì)胞凋亡。

引用本文： 劉海威,程樂,吳非,厲成杰,陳維平,劉志明. 脂質(zhì)體轉(zhuǎn)染survivin反義寡核苷酸對(duì)人胰腺癌細(xì)胞凋亡的影響. 中國(guó)普外基礎(chǔ)與臨床雜志, 2010, 17(10): 1056-1061. doi: 復(fù)制

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7. 　Kawasaki H, Toyoda M, Shinohara H, et al. Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis [J]. Cancer, 2001; 91(11): 2026-2032.
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10. 　文亞淵, 劉寶華, 洪勝龍, 等. 凋亡相關(guān)基因survivin、caspase-3及cyclin-B1在胃癌發(fā)生、發(fā)展中的作用 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006; 13(1): 34-37.
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14. 　陳曦海， 張豈凡， 馬榮. 反義survivin誘導(dǎo)胃癌SGC7901細(xì)胞凋亡及逆轉(zhuǎn)耐藥的研究 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2007; 14(1): 63-67.
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  1. 1. 　Guo X, Cui Z. Current diagnosis and treatment of pancreatic cancer in China [J]. Pancreas, 2005; 31(1): 13-22.
  2. 2. 　王麗， 楊功煥， 李輝， 等. 1991-2000年中國(guó)胰腺癌病死率的變遷 [J]. 中華內(nèi)科雜志， 2005； 44(7)： 509-513.
  3. 3. 　Rodriguez JA, Li M, Yao Q, et al. Gene overexpression in pancreatic adenocarcinoma: diagnostic and therapeutic implications [J]. World J Surg, 2005; 29(3): 297-305.
  4. 4. 　Devesa SS, Blot WJ, Stone BJ, et al. Recent cancer trends in the United States [J]. J Natl Cancer Inst, 1995; 87(3): 175-182.
  5. 5. 　Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma [J]. Nat Med, 1997; 3(8): 917-921.
  6. 6. 　Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs [J]. Cancer Res, 1998; 58(23): 5315-5320.
  7. 7. 　Kawasaki H, Toyoda M, Shinohara H, et al. Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis [J]. Cancer, 2001; 91(11): 2026-2032.
  8. 8. 　Satoh K, Kaneko K, Hirota M, et al. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors [J]. Cancer, 2001; 92(2): 271-278.
  9. 9. 　謝貽祥, 孟剛. suvivin在乳腺癌中的表達(dá)及其意義 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2007; 14(3): 260-264.
  10. 10. 　文亞淵, 劉寶華, 洪勝龍, 等. 凋亡相關(guān)基因survivin、caspase-3及cyclin-B1在胃癌發(fā)生、發(fā)展中的作用 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006; 13(1): 34-37.
  11. 11. 　Reed JC. The Survivin saga goes in vivo [J]. J Clin Invest, 2001; 108(7): 965-969.
  12. 12. 　Li FZ, Ackermann EJ, Bennett CF, et al. Pleiotropic cell-division defects and apoptosis induced by interference with survivin function [J]. Nat Cell Biol, 1999; 1: 461-466.
  13. 13. 　Carter BZ, Wang RY, Schober WD, et al. Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells [J]. Cell Cycle, 2003; 2(5): 488-493.
  14. 14. 　陳曦海， 張豈凡， 馬榮. 反義survivin誘導(dǎo)胃癌SGC7901細(xì)胞凋亡及逆轉(zhuǎn)耐藥的研究 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2007; 14(1): 63-67.
  15. 15. 　Xia C, Xu Z, Yuan X, et al. Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides [J]. Mol Cancer Ther, 2002; 1(9): 687-694.