單步驟製備含細胞明膠微泡細胞支架模擬肺泡構造作為肺癌藥物篩選之用途探討

Abstract

前言：癌症是全球死亡的主要原因。在各種癌症類型中，不論男女，肺癌都是最惡性的癌症。但是，從體外研究到體內臨床前試驗，腫瘤學藥物開發的失敗率極高，這表明現今評估體外藥物療效的方法並不可靠。傳統的二維細胞培養已被證明不足以模擬體內的生理條件。三維細胞培養方法，包含細胞球體培養法和細胞支架培養法，並不能充分表現肺泡的微妙結構。因此，本篇研究中，吾人開發具有均一類肺泡孔徑的含肺癌細胞生物支架製備方法。 方法：為模擬肺泡結構，將含有界面活性劑的明膠溶液與微生物轉谷氨酰胺酶(microbial transglutaminase)混合的A549細胞懸浮液在四通道流動聚焦微流體晶片中混合，以產生含細胞之酵素交聯明膠微泡支架。以WST-1分析和活/死染色評估支架內細胞之存活。在支架中測試肺癌細胞對抗癌藥物吉西他濱(gemcitabine)的敏感性，並與二維培養進行比較。使用IVIS冷光成像系統、組織學染色、西方墨點法和即時定量聚合酶連鎖反應評估吉西他濱治療後的細胞反應機轉。 結果：該明膠微泡支架具有約100微米的均勻孔徑，其結構和機械性質似於肺泡，具有良好的生物相容性，並提供了不同於二維培養的微環境。誘導微泡支架中細胞凋亡所需的吉西他濱有效濃度與藥物處理時間皆顯著高於二維培養。通過蛋白和基因表現證實細胞死亡機制是吉西他濱誘導的細胞凋亡。此外，組織學分析與TUNEL assay顯示在藥物處理的支架中確實造成細胞萎縮並有核酸斷裂損傷現象。 結論：吾人成功製備模仿肺泡構造和細胞藥物反應的含細胞微泡支架，並透過諸多實驗證實藥物處理結果。吉西他濱治療後的細胞反應類似於治療肺癌的臨床方案。因此，微泡支架有望透過提供更準確的臨床前預測促進抗癌藥物發現。

Cancer is the leading cause of mortality worldwide, and lung cancer is the most malignant. However, the high failure rate in oncology drug development from in vitro studies to in vivo preclinical models indicates that the modern methods of evaluating drug efficacies in vitro are not reliable. Traditional 2-dimensional (2D) cell culture has been proved inadequate to mimic real physiological conditions. Current 3-dimensional (3D) cell culture methods do not represent the delicate structure of lung alveoli. To mimic lung alveoli structure, a cell containing enzyme-cross-linked gelatin microbubble scaffold was produced by mixing surfactant-containing gelatin solution with microbial transglutaminase (mTGase)-mixed A549 cell suspension in a four-channel flow focusing microfluidic device. With uniform pore size of about 100 μm in diameter, this gelatin microbubble scaffold resembled the lung alveoli in structure and in mechanical properties with good biocompatibility. Effective gemcitabine concentration required to induce cell death in microbubble scaffolds was significantly higher than in 2D culture together with a longer treatment time. Cell death mechanisms were confirmed to be gemcitabine-induced cell apoptosis through Western blotting and real-time PCR. H E staining and TUNEL assay showed rounded cells with DNA damage in drug-treated scaffolds. Taken together, the cell-containing microbubble scaffolds successfully mimicked lung alveoli in structure and cellular responses after gemcitabine treatment were similar to clinical regimen of treating lung carcinoma. The microbubble scaffold is promising to facilitate anticancer drug discovery by providing more accurate preclinical predictions.