利用乳腺上皮細胞建構一可合成與分泌醫藥用重組蛋白質之類乳腺組織生物反應系統

Abstract

我們已由泌乳母猪乳腺組織中分離出一自發可持續繼代生長的乳腺上皮細胞株，此 稱為SI-PMEC（spontaneously immortalized porcine mammary epithelial cells），培養在適 當基質的SI-PMEC 細胞於泌乳激素刺激下，可分化形成類乳腺組織形態，即形成具泌 乳功能性的乳腺泡、腺管及腺側芽等結構，並大量表現、合成及分泌乳蛋白質。SI-PMEC 細胞可被用來測試將應用於基因轉殖泌乳動物，構築有乳腺專一表現調控序列之轉殖基 因（transgene）的表現功能；SI-PMEC 細胞也可被用來建構一生產外源重組蛋白質的生 物反應系統，該產物應會經過轉譯後的修飾作用，因此可保有原本蛋白質的結構與功 能。據此，本計畫主旨為利用乳腺上皮細胞建構一可合成與分泌醫藥用重組蛋白質之類 乳腺組織生物反應系統，在此三年期計畫中包括三項研究主軸，其分別為 (1) 篩選一在 膠原蛋白基質上具分化功能的乳腺上皮細胞株，(2) 由山羊 β-酪蛋白（casein）基因調 控序列構築而成的表現載體；(3) 建立一中空纖維束生物反應系統（hollow fiber reactor）。 研究策略一：馴化、篩選一遺傳穩定而生長快速的乳腺上皮細胞株，此株細胞對動物血 清與荷爾蒙的需求性降低，增殖時間不應超過24 小時。同時此細胞株在膠原蛋白 基質上仍具有可分化成能合成與分泌乳蛋白質功能的類乳腺組織結構之性質。 研究策略二：構築一系列在乳腺與乳腺上皮細胞中可被誘發高表現的載體，其表現調控 序列為山羊 β-casein 基因。在這些表現載體的基因選殖位置的5’端是一約5 ∼ 6 kb DNA 片段，它包括 β-casein 基因起動子、exon 1、intron 1 及部分exon 2 等序列； 3’端是一約3 ∼ 4 kb DNA 片段，它為 β-casein 基因exon 7 往後延伸的序列。綠色 螢光蛋白（green fluorescent protein）和螢光素酶（luciferase）報導基因將被構築於 上述表現載體中，以用於測試SI-PMEC 細胞和生物反應器的功能。利用這些表現 載體，我們也將構築可表現人類抗菌胜肽cathelicidin LL-37 與猪cathelicidin PR-39 之transgenes，此將用以轉染SI-PMEC 細胞以建立可高表現LL-37 和PR-39 的重組 SI-PMEC 細胞株（recombinant SI-PMEC; rSI-PMEC）。 研究策略三：rSI-PMEC 細胞將被植入中空纖維束生物反應器之柱狀管內的中空纖維束 外腔（extracapillary space; ECS），並供以生長培養基，細胞以貼附方式生長直到高 緻密度（confluence）時，置換成分化培養基，此時細胞應在ECS 中形成類乳腺組 織狀的網絡三維結構，並持續合成與分泌重組蛋白質，這些蛋白質產物將被濾過、 釋放入反應器中空纖維束內（intracapillary space; ICS），並於ICS 循環流動的化學 培養液中被逐漸濃縮。 本研究的目標是可長期（至少42 天）將rSI-PMEC 細胞培養於中空纖維束生物反應 系統中，並能持續高效率生產抗菌胜肽cathelicidin LL-37 and PR-39。此外，本計畫所建 立的SI-PMEC、表現載體及中空纖維束生物反應器模式皆可被利用於生產其他生技藥物 製品。

We have established a spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) from mammary gland of lactating sow. The SI-PMEC cells can differentiate into mammary gland-like structures (gland ducts, lateral buds, and alveoli), and strongly express the transcripts encoding milk proteins and secrete the proteins in vitro in a matrix- and lactogenic hormone-dependent manner. The cells appeared to provide an efficient and convenient system for assessing the expression of transgenes containing mammary gland-specific promoter before transgenic milk livestock generated. Moreover, the SI-PMEC can reasonably be used as a bioreactor system for producing exogenous recombinant proteins which have undergone appropriate post-translational modifications making them structurally and functionally identical to the native proteins. In this plan, we attempt to use the SI-PMEC cells to construct a bioreactor of artificial mammary gland which can be used to produce therapeutic recombinant proteins. We propose three research strategies addressing on the subject for three years planning, 1) screening a SI-PMEC which can differentiate into functional structures on collagen substratum, 2) constructing expression vectors with the regulatory sequence of goat β-casein gene, and 3) fabricating a system of hollow fiber bioreactor. In Strategy 1, a SI-PMEC cell line with genetic stability will be screened and cloned. The cells can duplicate within 24 hours in the medium containing less hormones and animal serum. Moreover, the cell line should keep the capacity of differentiation of secretory structures and functions as mammary gland-like when the cells grow on the collagen substratum. In Strategy 2, we attempt to construct the inducible highly expression vectors in mammary gland and mammary epithelial cells with the regulatory sequences of goat β-casein gene. Sequences between cloning site of the expression vectors are promoter, exon 1, intron 1, partial exon 2 of goat β-casein gene around 5 - 6 kb fragment at 5’ side and a 3 - 4 kb fragment of the β-casein gene from exon 7 to 3’-flanking sequence at 3’ side. The report genes, green fluorescent protein and luciferase, are also applied to construct the control expression vectors which are used to evaluate the functions of SI-PMEC and bioreactor. We aim to construct two transgenes which carry the sequences encoding antibacterial peptides, human cathelicidin LL-37 and porcine cathelicidin PR-39. Both transgenes will be transfected into SI-PMEC and to screen the recombinant SI-PMEC (rSI-PMEC) clones. In Strategy 3, adhered cultures of rSI-PMEC will be performed in a hollow fiber bioreactor. The cells are implanted into the extracapillary space (ECS) of cartridge in a hollow fiber bioreactor and grow to be confluence supplied with growth medium. Then differentiation medium supplied, the cells will differentiate to be three dimensional network structures as artificial mammary gland, and then synthesize as well as secrete recombinant proteins. The produced recombinant protein in the ECS will be released into the intracapillary space (ICS) through the hollow fibers (small tube-like filter) and concentrated in the chemical medium in the ICS circulation. The aim of this plan is that the rSI-PMEC can be cultivated and maintained in hollow fiber reactor for at least 42 days to continuously produce cathelicidin LL-37 and PR-39 at an extremely high level. In addition, the system established herein, SI-PMEC, expression vectors and hollow fiber module, can be applied to produce the others protein pharmaceuticals.