利用基因傳送的技術生成抗傷害性感受的(antinociceptive)分子用以治療疼痛為目前熱門之研究。在內生性的鴉片類(opioids)中，衍生自前-腦啡-黑細胞促素-皮促素(pro-opiomelanocortin, POMC)的腦內啡(b-endorphin, b-EP)是最具效力者。除b-endorphin，POMC也是許多神經胜&#32957;諸如腎上腺皮促素(ACTH)、黑細胞促素（MSH）…等之前驅物。為了使POMC在臨床上成功地應用，適當地施予POMC基因是首要條件；基因傳送為可以有效地持續表現b-endorphin以緩和疼痛之處理方式。由於重組腺病毒(recombinant adenovirus)具有高效價、應用宿主範圍大及傳送效率高之優點，故將其應用在b-endorphin、ACTH及α-MSH之基因傳送系統。目前，已證實腺病毒載體在細胞培養及脊髓中，能有效地傳送基因，蛋白質產物的表現效率很高。在本實驗中，利用PCR的技術構築出重組的腺病毒載體Ad-POMC，以西方點墨法加以確認；並建立放射免疫分析法及化學冷光免疫分析法之ACTH、α-MSH及b-endorphin分析系統；利用傳送Ad-POMC之GH3老鼠腦下垂體細胞，其細胞培養液中之ACTH、b-endorphin及α-MSH濃度均明顯高於對照組，顯示Ad-POMC之傳送成功。利用帶有綠螢光蛋白之腺病毒(Ad-GFP)作為指標，找出腺病毒感染不同型細胞之最適感染條件，分別找出50、 500、 50、 500、 500 及 200 MOI為腺病毒轉染神經細胞GH3 cells、星狀細胞C6 cells、肝臟細胞Hep3B cells、平滑肌細胞G8 cells、纖維母細胞CCD-966SK cells及內皮細胞EA.hy926 cells之最適MOI。以RIA 測定分泌型ACTH及b-endorphin之濃度，進而評估基因傳送後不同型細胞處理POMC的效能。結果顯示週邊細胞雖只有低濃度的表現，仍具有如中樞神經系統神經細胞之能力，能在傳送POMC基因後切割並釋放鴉片類胜&#32957;。建立足爪縮回時間延遲及福馬林試驗之動物疼痛模式，研究以POMC基因傳送治療疼痛之效果；結果椎管內POMC基因傳送相較於施打生理食鹽水或Ad-GFP之對照組，明顯降低福馬林引起的退縮反應第一期(P < 0.05)及第二期(P < 0.001)之幅度。綜合以上之研究結果顯示椎管內POMC基因傳送具有緩和發炎性疼痛之效果。目前已有各種基因傳送研究確證POMC用於止痛之潛力，而α-MSH未來尚有治療肥胖之展望有待吾人繼續鑽研！

The use of gene-based techniques to produce antinociceptive molecules has been actively investigated for treatment of neuropathic pain and trauma of central nervous system. Among the endogenous opioids, b-endorphin (b-EP) is the most potent one, which is derived from pro-opiomelanocortin (POMC). In addition to b-endorphin, POMC is also the precursor of many neuropeptides such as adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (a-MSH), …etc. Appropriate administration of POMC gene is essential for the success of its clinical application. Thus, gene transfer approach seems to be suitable for continuous supply of b-endorphin to alleviate intractable pain. Recombinant adenovirus was used as gene delivery system for POMC because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant adenovirus encoding POMC (Ad-POMC) by PCR and western blot analysis, and detect the presence of opioid peptides including ACTH, a-MSH and b-EP by RIA and chemilluminiscent assay. GH3 cells infected with Ad-POMC showed significantly higher levels of ACTH, b-endorphin, and a–MSH comparing with cells of control groups. By using Ad-GFP, the optimal MOI for adenovirus vector to infect neuronal GH3 cells, glial C6 cells, hepatoma Hep3B cells, smooth muscle G8 cells, fibroblast CCD-965K cells, and endothelial EA.hy926 cells was determined at 50, 500, 50, 500, 500, and 200, respectively. The results of determining the efficiency of POMC processing in different types of cells after in vitro cell cultures gene delivery indicated that peripheral cells, though at a lower extent, are capable of cleaving POMC and releasing opioid peptides after POMC gene delivery like neuronal cells of central nervous system. In formalin test, the intrathecal POMC gene delivery significantly decreased the magnitude of the formalin-evoked flinching response phase 1 (P < 0.05) and phase 2 (P < 0.001) when compared with rats receiving saline or Ad-GFP. In conclusion, the intrathecal POMC gene delivery can produce effectively attenuation on the inflammatory pain response. So far, there have been various gene delivery studies confirming the potential role of POMC in antinociception. In the future, more experiments will be needed to characterize the effects of POMC expression on cellular lipid metabolism. This will enable us to evaluate the therapeutic potential of POMC on treatment of obesity.

目錄 頁次
摘要 4
英文摘要 6
縮寫 7
前言 8
前-腦啡-黑細胞促素-皮促素 8
腎上腺皮促素和皮質醇 8
黑細胞促素 9
腦內啡和疼痛 10
腺病毒基因傳送 11
材料和方法 13
細胞培養 13
量產及純化腺病毒載體 13
檢測和定量綠螢光蛋白 14
分離RNA及以RT-PCR選殖人類POMC的cDNA 15
生產POMC基因重組腺病毒 15
足爪縮回時間延遲足底試驗及福馬林試驗 16
分析b-endorphin和a-MSH 17
分析ACTH 17
萃取腳掌組織之鴉片類胜&#32957; 17
免疫組織化學染色 18
統計分析 18
結果 19
構築並確認POMC重組腺病毒 19
測定腺病毒感染不同型細胞之最適感染條件 19
各種不同型細胞處理及切割POMC基因之效能 20
椎管內POMC基因傳送減緩疼痛反應之福馬林試驗 20
討論 21
參考文獻 27

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