在正篩選免疫磁力微流平台中計數上皮細胞與內皮細胞之研究

Abstract

近來的研究顯示血液中特定的稀少細胞可用作癌症預後或其他用途的指標。循環腫瘤細胞和循環內皮細胞是這類細胞的兩個代表。血液中循環內皮細胞的量與各色疾病相關。循環內皮細胞對周邊血單核細胞的比例一般小於千分之ㄧ，使得純化這類細胞有一定程度的難度。 本論文呈現了一種以經濟的微流碟片分離與偵測稀少細胞的可能性。在循環腫瘤細胞的試驗中，一株乳癌細胞株MCF7和血癌細胞株Jurkat Clone E6-1經由適當地標定並以磁力試捉取以驗證概念的可行性。在循環內皮細胞的試驗中，使用免疫磁珠技術和多級磁鐵有效地回收表達CD146的HUVEC。周邊血單核細胞則被標以另ㄧ株螢光抗體供識別。細胞株HUVEC/周邊血單核細胞和病人檢體皆有測試。多級磁鐵為精心設計，藉此磁捉取力可被最大化並確保良好的捉取效果。 在循環腫瘤細胞的試驗中，結果顯示當少於百顆的目標細胞在多過百萬顆的細胞海中，目標的回收率維持在約80%、甚至當目標細胞僅佔總細胞量的千萬分之ㄧ亦如此。和習用回收技術autoMACS相比，這樣的結果仍然比習用技術高了至少20%。回收下來的MCF7存活率約90±20%，表示碟片回收技術對目標造成的傷害很小。在循環內皮細胞的試驗中，當數百顆HUVEC混入百萬顆周邊血單核細胞，95% HUVEC仍可被檢出、即便60顆HUVEC在萬顆周邊血單核細胞中亦如此。回收率也和流式細胞儀做了比較。在紅斑性狼瘡病人周邊血的測試中，循環內皮細胞的量在病人 [61.1(21.5)] 和健康人 [31.2 (13.3)] 有顯著的 (P<0.01)差異。 循環內皮細胞的數目是紅斑性狼瘡的恰當指標，而微流碟片系統亦應成為偵測循環內皮細胞的重要工具。微流碟片可望應用在其他稀少細胞的分離和偵測上。

Recently studies have shown that specific rare cells in the blood can serve as an indicator of cancer prognosis, or other purposes. Circulating tumor cells (CTCs) and Circulating endothelial cells (CEnCs) are two important examples. CEnCs in the blood are rare but have been shown to be associated with various diseases. With the ratio of CEnCs to peripheral blood mononuclear cells (PBMCs) less than 1 part per thousand, their separation from PBMCs and detection are challenging. This dissertation demonstrated the concept of separating and detecting rare cells from PBMC via an economical microfluidic disk with a model system. In CTC tests, MCF7, labeled with magnetic beads, was used to simulate CTC as target. Jurkat Clone E6-1 was used to simulate leukocytes or other cells abundant in human blood. In CEnC tests, CEnC-simulating HUVECs, as target cells, were stained with primary anti-CD146-phycoerythrin antibody and bound with secondary antibody on antiphycoerythrin magnetic beads. PBMCs served as nontarget cells and were labeled with anti–CD45-FITC antibody. Both model cell system [human umbilical vein endothelial cells (HUVECs) in PBMCs] and primary samples were studied. A tailored multi-stage magnet maximized the magnetic field to ensure optimal trapping efficiency. Results indicate that, in CTC tests, while fewer than hundreds of MCF7 cells mixed in greater than one million Jurkat cells, the yield of detected MCF7 was consistent at approximately 80%. The 80% yield also held for ten MCF7 in one hundred million Jurkat (rarity of 10 7). Compared to the results from autoMACS, the performance was at least 20% higher and more independent of the number of Jurkat. The viability of the enriched cells was around 90±20%, showing this method caused little damage to trapped cells. In CEnC tests, When hundreds of HUVECs were mixed in 106 PBMCs, 95% of spiked HUVECs were detected. This yield also held for 60 HUVEC in 104 PBMCs. This dissertation compared data from flow cytometry with that from the disk: CEnC counts in 50 μl blood from patients with systemic lupus erythematosus were 61.1 (21.5), significantly higher (P<0.01) than those of healthy donors, 31.2 (13.3). The count of CEnCs should be a suitable marker for symptoms of systemic lupus erythematosus. The microfluidic disk system should be a viable platform for detection of CEnCs, and should be applicable for separation and detection of various rare cells.