鎝99m(I)-三羰基標誌組胺酸耦合Annexin V作為細胞凋亡造影之研究

Study on 99mTc(I) Labeled His-Annexin V as an Apoptosis- Detecting Radioligand

陳宜璘(Yi-Lin Chen)

指導教授 ： 羅建苗 ； 李德偉

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2007年)

https://doi.org/10.6843/NTHU.2007.00088

摘要

細胞凋亡(apoptosis)，又稱細胞計畫性死亡(celled programmed cell death)，無論在正常生理或病變過程中都扮演著相當重要的角 色。細胞凋亡發生初期， 原本位於細胞膜內的磷脂絲胺酸 (phosphatidylserine, PS) 受到凋亡蛋白酶 (caspase) 的調控會轉移到細胞膜外。而人體內生性的蛋白質annexin V 和磷脂絲胺酸有很高的親和力。基於此，放射性標誌annexin V 可發展為細胞凋亡的造影藥物， 在文獻上已有許多報導， 其中藉由雙官能基螯合劑 hydrazinonicotinamide (HYNIC) 標誌99mTc 於annexin V 藥物 (99mTc(V)-HYNIC-annexin V) 已經進入人體實驗。本研究參考過去文獻所報導的方法，利用分生技術合成在N端接有6 個組胺酸 (histidine)的annexin V (簡稱his-annexin V)，進而以鎝99m(I)三羰基離子標誌his-annexin V。 在實驗上，我們建立在N 端帶有6 個組氨酸 (histidine) 的 pETBlue-1/his-annexin V 質體，接著轉型至E. coli 使大量表現 hisannexin V。利用Ca2+離子做為可逆性 (reversible) 的媒介來溶解質含體(inclusion body)，可有效的得到大量的水溶性(soluble)目標蛋白質，最後再使用親和層吸管柱Ni2+ sepharose column 予以純化。由本研究結果，我們成功地獲得高產率 (39 mg/l) 高純度 (~98%) 的his-annexin V。 在鎝99m 標誌his-annexin V 之試驗，利用鎝99m(I)三羰基離子， [99mTc(CO)3(OH2)3]+為前驅物標誌his-annexin V，藉由鎝99m(I) 三羰基離子與組胺酸配位鍵結， 可得高標誌產率之 99mTc(I)-his-annexin V，以薄層層析(TLC)、高效能液相層析 (HPLC)和分子大小排阻層析(SEC) 等測定標誌產率、放化純度和穩定度。由本實驗結果得知99mTc(I)- his-annexin V 之標誌產率可達87%以上，且在PBS 和血清中具有高度的穩定性，值得進一步發展為細胞凋亡之造影劑。

關鍵字

99mTc(I) tricarbonyl ion ； his-annexin V

並列摘要

Apoptosis, also called programmed cell death, plays an important role in normal physiology and many disease processes. The early indication of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane. Once
exposed to the extracellular environment, PS will display available sites for binding with annexin V, which is an endogenous 35-36 kDa,Ca2+-dependent, phospholipid binding protein. Due to the high affinity for PS, annexin V has proven useful for detecting the early stage of apoptosis.
Radiolabeled annexin V has been studied extensively for the
detection of apoptosis in both animals and humans; most clinical experience has been gained with a 99mTc-labeled annexin V complex modified with a hydrazinenicotinamide ligand (99mTc(V)-HYNICannexin V).
In this study, it has been attempted to develop a form of annexin V constructed with N-terminal extention containing six histidine residues. The his-tagged annexin V (abbreviated as his-annexin V) can be directly labeled with 99mTc(I) tricarbonyl ion, [99mTc(CO)3(OH2)3]+ .In experiment, a pETBlue-1-his-annexin V plasmid has been
constructed. The recombinant his-annexin V was allowed to overexpress in E. coli by the plasmid. Calcium ion was utilized as a reversible medium to solve the inclusion body. Finally, his-annexin V was purified by a single-step affinity chromatography via Ni2+ sepharose column. The
his-annexin V product could be obtained approximately 39 mg of protein/l of culture with a high purity of ~98% as judged by SDS-PAGE.For the 99mTc labeling, 99mTc-tricarbonyl ion, [99mTc(CO)3(OH2)3]+ , was
synthesized and utilized as the precursor to conjugate directly with his-annexin V. The resultant 99mTc(I)-his-annexin V was characterized by thin layer chromatography(TLC), high performance liquid chromatography (HPLC) and size exclusion chromatography (SEC). The labeled yield for 99mTc(I)-his-annexin V was ≧87%. 99mTc(I)-his-annexin
V exhibited good stability in PBS and serum. 99mTc(I)-his-annexin V is worthy to be be further investigated as a potent apoptosis imaging agent.

參考文獻

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