三維培養之髓核細胞退化模型探討

Abstract

椎間盤退化目前臨床上並沒有有效的治療方法。椎間盤退化主要的原因之一是髓核細胞老化後的增殖緩慢。為了瞭解髓核細胞老化與再生，髓核細胞的體外培養與研究有其必要性。傳統2D培養中髓核細胞會因為離開其原生環境而導致細胞型態產生變化。我們為了能了解椎間盤退化機制，與髓核細胞在其中所扮演的角色，我們分別使用了Type I collagen以及Hydrogel來建立3D細胞培養系統，為髓核細胞的生長提供一個更接近真實環境的研究平台。我們將髓核細胞培養於3D環境當中，並觀察其形態上的變化，之後進行超音波刺激，確認細胞是否會發生平面培養中無法觀察到的現象，同時我們對3D細胞模型進行破壞，來模擬真實椎間盤的損傷模型，並進行細胞內信號傳導抑制實驗來確認刺激反應路徑。 我們成功的將髓核細胞種植到3D細胞模型當中，發現在3D環境時，細胞會呈現圓球狀而非平面培養的扁平狀，在活體上，髓核細胞同樣也是圓球狀的，因此我們認為髓核細胞以3D的模式培養能夠保持其原有的細胞結構。我們在此模型中觀察到髓核細胞的p-PKCz會進入到初級纖毛當中，而這個現象可以透過超音波刺激來提升，但需一定的反應時間。在破壞實驗當中，我們觀察到髓核細胞在缺口的周圍會產生拉長的現象，且此反應可能是經由ASIC3通道來進行調控。另外同樣在破壞實驗當中，我們發現如果給予超音波刺激時，缺口週遭的細胞排列會有所改變，並且是透過TRP通道進行調控。這些實驗結果證明髓核細胞體外3D與損傷模型的有效性，可作為將來的髓核細胞再生研究之用。

Degeneration of intervertebral disc (IVD) and especially that of nucleus pulposus (NP) has been incurable at present. NP cell proliferates slowly and can not regenerate after being damaged. To better understand potential mechanisms of the degeneration, particularly the NP cell's role, we used TYPE II collagen and Hydrogel, respectively, to build up 3D cell cultures that provide a more in vivo-like platform for disc cells to grow on. In this study, the NP cells were cultured in 3D cell culture. We expected to observe phenotypic changes in NP cells . Subsequently, we used the customized chamber to ultrasonically stimulate the NP cell 3D cell cultures, hoping to confirm whether the cells in the 3D culture have special response to ultrasound is not found in the past 2D culture. Lastly, we simulated real disc damage models by creating damages in the 3D cultures. We successfully planted the NP cell into a 3D cell model. At the same time, we also found that the NP cell is spherical in the 3D structure, rather than flat in the 2D structure. In histology, the shape of NP cell is also spherical, so we believe that when NP cell grows in a 3D structure, it can maintain its original cellularity. After immunofluorescence , We found that p-PKC  will enter cilia(k40), and the percentage will increase under ultrasound stimulation, but it will not be significantly different until 24hr after stimulation. In damage experiments, we have observed that NP cells will undergo morphological changes, which is regulated by the ASIC3 channel. In addition, we also found that ultrasound can disarrange cell orientations around the damage through TRP channel. These experimental results indicate that we have developed an in vitro injury model for further study.