人體關節老化與發炎反應研究 (I)：人體關節軟骨細胞之不朽化與定性

關節老化及發炎有普遍化及年輕化之趨勢，其最大之原因是關節軟骨細胞的病變；但目前尚無保持原良好特性之不朽化軟骨細胞株，可以用來進行關節老化及發炎的分子基因的研究。本研究成功地利用 retrovirus infection system 將 HPV-16 E6/E7 DNA 感染且不朽化人類關節軟骨細胞（ immortalized human articular chondrocytes ），並對此不朽化軟骨細胞株 (immortalized cell line) 進行實驗分析及細胞定性，利用 RT-PCR 、 Western blotting 、 Alcian Blue staining 、三度立體空間培養(3 dimensional cultures) 以及 Immunohistology 等方法，來觀察證明此不朽化軟骨細胞株與初代軟骨細胞之間的差異性，並將此細胞株並命名為 hPi cells 。經由各項實驗分析證明，此不朽化軟骨細胞株（ hPi cells ）已可被成功地培養超過六十代，而且定性分析結果顯示，軟骨細胞最主要的分化 marker : type II collagen ，不論是在 mRNA level 或是 Protein level ，都可以在此 hPi cells 明顯偵測出和初代軟骨細胞有相同的表現，而且將 hPi cells 植入 type I collagen 為scaffold 的三度空間立體培養中，在 hPi cells 的周圍亦有明顯的 lacunae 產生，經由對 GAGs 的專一性Alcian Blue 染色結果，不論是初代軟骨細胞或是 hPi cells ，都可明顯被染色。由於此細胞株是經由 HPV-16 E6/E7 DNA 之轉感染，亦作了 hPi cells 在 NOD/SCID mice 身上是否會生成腫瘤（ tumorigenic ）之測試，經過長達三個月的培養， hPi cells 在 mice 身上並無任何腫瘤生成反應，亦證明了此細胞株與正常細胞相同性質。本實驗室所建立之軟骨細胞株 hPi cells 不需經過 re-differentiation 的過程，即可在體外培養到接近五十代，仍然有明顯的軟骨細胞分化 markers 表現，諸如 type II collagen 、 Aggrecan ，甚至是在 3-dimension 的培養環境下，都可看出此 hPi 細胞株和初代軟骨細胞具有相同之分化能力及特性，是其他報告文獻中所欠缺的。希望藉由此 hPi 細胞株之成功建立，除了作為人體軟骨再生研究外，更重要是進而拓寬進行關節老化及發炎分子基因的研究領域！

Articular aging and inflammation has became a tendency of being popular and young. Pathological changes of articular cartilage chondrocytes are the main reason of these diseases. There is, however, still no well-established immortalized articular chondrocyte system to be used to study the molecular mechanism of articular aging and inflammation.In this experiment, we have successfully established an immortalized human articular chondrocyte transfected with HPV 16 E6/E7 DNA by using retrovirus infection system and this cell line, named hPi, was analyzed and well characterized. RT-PCR, Western blotting, Alcian Blue staining, 3 dimensional cultures and immunohistology were used to demonstrate the characteristic similarity between human articular immortalized and primary chondrocytes.The results showed that the major differentiation marker of articular chondrocytes, type II collagen in mRNA level or protein level in hPi cells were apparently identical with that in primary chondrocytes over 60 passages culture. Lacunae were formed between cells and collagen matrices obviously in hPi cells cultured in type I collagen scaffold in 3 dimension. The GAGs-specific Alcian Blue staining also demonstrated that both primary chondrocytes and hPi cells were stained in blueness. Tumor was not formed on NOD/ SCID mice injected with hPi cells for 3 months. This result indicated that the hPi cell line, chondrocytes immortalized by HPV-16 E6/E7, is non-tumorigenic and could still express cartilage-specific differentiation markers, such as type II collagen and aggrecan through 50 passages in vitro cultures without any redifferentiation strategies. The immortalized human articular chondrocytes will be used to investigate human articular regeneration, cartilage aging and inflammation molecular mechanisms !

Human Joint Aging and Inflammatory Studies (I) :Human Articular Chondrocytes Immortalization and Characterization