Oral Plasmids Delivery Using Polymeric Micelles for SCI Treatment

1. 利用聚合載體口服投與專一性髓鞘基質蛋白質質體脊髓傳遞 • 近年來，脊髓損傷（spinal cord injury，SCI）的人數日益增多，使之成為社會的經濟負擔，因此越來越多的研究學者朝著改善脊髓損傷後的症狀，甚至希望治癒。脊髓損傷後，會使寡樹突膠質細胞（oligodendrocyte）產生細胞凋亡（apoptosis），使去髓鞘化（demyelination）產生，造成神經傳導失調（neurologic dysfunction）；而Bcl-2家族蛋白質是近幾年來發現具有調控細胞凋亡機制的功能，其中Bcl-xL蛋白質是屬於抗凋亡（anti-apoptotic）的蛋白質，可以抑制凋亡（pro-apoptotic）蛋白質Bid以及Bax，使細胞存活（survival）。 因此本實驗利用oligodendrocyte上的特異蛋白質基因：髓鞘基質蛋白質（myelin basic protein；MBP）基因的啟動子（promoter），形成兩種質體（plasmid，（1）pMBP-LacZ，（2）pMBP-Bcl-xL-EGFP），以口服聚合微膠體（polymeric micelles；PM）投與到動物體內，觀察在脊髓中基因表現的情形。初步結果發現，質體pMBP-LacZ 是可以在口服六個劑量下，48小時後有其表現，並直接從裸鼠脊髓組織中以定量β-Galactosidase活性（193.3± 25.5 mU/g；p<0.05）或脊髓組織切片免疫染色，觀察LacZ的基因表現，同時利用即時聚合?連鎖反應法（Real Time-PCR）絕對定量方式，LacZ mRNA表現為82.25± 56.48 copies/μg total RNA；而在胃、十二指腸和肝臟則均無明顯LacZ的基因表現。而在利用脊髓損傷之老鼠模式下，口服投與質體 pMBP-Bcl-xL-EGFP（P）和PM後，利用即時聚合?連鎖反應法（Real Time-PCR），定量Bcl-xL mRNA，在投與P/PM與MP的脊髓中， Bcl-xL 基因的表現量與受傷後脊髓比較，有上升的趨勢；並且在P、P/PM也可見EGFP的表現。而西方點墨法（Western Blotting）也發現到，投與P、P/PM、Methylprednisolone（MP）與MP/PM的脊髓， Bcl-xL 基因的表現量與受傷後脊髓比較，也有上升的趨勢；同時也利用脊髓組織切片免疫染色法，觀察到EGFP與專一髓鞘基質蛋白質有表現在相同的位置上。

2. Oral Specific Myelin Basic Protein (MBP) Promoter Plasmids Delivery by Polymeric Micelles in Mouse Spinal Cord • The population of spinal cord injury (SCI) was increased, resently. Reseachers hope that they could improve symptome after SCI. After SCI, oligodendrocytes of spinal cord area would undergo apoptosis phenonemia and following demyelination, that could result in neurologic dysfunction. The pivotal anti-apoptotic protein Bcl-xL gene can be one of the candidate genes for treatment of spinal cord injury (SCI). We formulated a nano-sized particle of polymeric micelles (PM) with two plasmids：（1）pMBP-LacZ encoding reporter gene LacZ, （2）pMBP-Bcl-xL-EGFP encoding Bcl-xL fused with enhanced green fluoresce protein whose expression are driven by specific, myelin basic protein（MBP）gene promoter into oligodendrocytes. Our results show that after six doses of oral delivery of pMBP-LacZ plasmid with PM, gene expression is observed at 48 h in mouse spinal cord. Using CPRG substrate to evaluate β-Galactosidase enzymatic activity, the results show that administration of pMBP-LacZ significant increases（193.3± 25.5 mU/g）compared with normal control mouse. LacZ expression was found to co-localize with MBP in the spinal cord by double immunohistological staining. Real time-PCR analysis also found that administration of pMBP-LacZ plasmid with PM increases the levels of LacZ mRNA（82.25± 56.48 copies/μg total RNA）.Furthermore, after six doses of oral delivery of pMBP-Bcl-xL-EGFP with PM （P/PM）, gene expression was observed at 30 h in injuried spinal cord area. Real time-PCR analysis showed that both of administration of P/PM and methylprednisolone（MP）delivery increase Bcl-xL mRNA. Western blotting analysis also showed that administration of P, P/PM, MP and MP/PM increase Bcl-xL protein in injured spinal cord. In Western blotting analysis, it also showed EGFP protein expression at P and P/PM administration. EGFP protein expression is co-localized with MBP protein in the spinal cord by double immunohistological staining.