超音波與壓電刺激對人類骨髓間質幹細胞遷移，與軟骨細胞分佈的影響

Abstract

在軟骨損傷及退化性關節炎中，軟骨再生的速度緩慢，不易修復。在治療方面，骨髓間質幹細胞再生以及超音波刺激對於骨癒合的幫助擁有巨大的潛力，許多研究也嘗試加速幹細胞及軟骨細胞的分化、修復速率。本研究亦是針對骨髓間質幹細胞之遷移，以及軟骨細胞分佈及蛋白訊息傳遞來進行研究。 因骨骼為一種壓電材料，故超音波與壓電效應在人體內的作用難以分別討論，對於骨癒合的幫助是純超音波提供或是壓電效應造成的是一個值得探討的課題。本實驗中，使用實驗室開發的超音波刺激載台 (LIC與mini-LIC)，對細胞提供純超音波刺激及壓電刺激。為了模擬體內骨骼系統之壓電環境，本實驗使用石英片來提供細胞壓電效應的電場刺激。 骨髓間質幹細胞之遷移以傷口復原實驗來觀察遷移效率及受刺激後影響程度。在傷口復原效率中壓電刺激較未刺激組提升1.4倍，超音波刺激則較未刺激組提升1.2倍。分析細胞初級纖毛觀察細胞極性，超音波刺激與壓電刺激皆對細胞極性有所影響。 在軟骨細胞實驗中，壓電刺激造成軟骨細胞有明顯聚合現象，使用Alcian blue染色可確定其確實有軟骨特性。壓電刺激亦誘導軟骨細胞重新排列以及細胞極性改變，而純超音波刺激則沒有對軟骨細胞造成上述的明顯改變。在使用Src、PKCζ與ASIC3抑制劑抑制其蛋白作用後，前二者皆阻止了壓電刺激造成的細胞重排與極性改變，ASIC3抑制劑則是造成部分抑制，並沒有前兩者的抑制效果明顯，而三者皆沒有逆轉壓電刺激誘導的細胞大小減小。使用Western blot測定Src、ASIC3被抑制後PKCζ磷酸化程度，兩者皆阻止了壓電刺激所造成的PKCζ磷酸化，故PKCζ極可能受Src、ASIC3所調控。

Cartilage repair is an intractable issue due to difficult repair and slow regeneration in Cartilage damage and Osteoarthritis. In terms of treatment, bone marrow mesenchymal stem cell and ultrasonic stimulation both showing great potential for bone healing. Various approaches also willing to find the ways to accelerate healing process. In this study, the migration of bone marrow mesenchymal stem cells, and distribution and protein signaling of chondrocytes were the research project. Ultrasound and Piezoelectric field is difficult to separate in vivo because of the Piezoelectric property in bone. We wondering pure ultrasound or piezoelectric stimulate is the aid for bone healing. Ultrasound stimulation (US) or Piezoelectric field stimulation (PE) were provided by Ultrasound stimulation device (LIC or mini-LIC). To simulate bone skeletal system in vivo, We used quartz to provide Piezoelectric field. In bone marrow mesenchymal stem cells wound healing experiment, the wound healing (migration) efficiency in PE was 1.4 times higher than that in the unstimulated (Control) group, more significant than the US group (1.2 times to Control). Analyzing the direction of primary cilia to evaluate cell polarity, both US and PE had effects. Chondrocyte rearrangement and aggregation were significant under Piezoelectric field stimulation. Using Alcian blue staining to make sure that Chondrocyte in Piezoelectric field stimulation maintain the cartilage properties. The rearrangement of chondrocytes and the orientation of primary cilia were significantly different from Control and US groups, while pure ultrasound stimulation did not cause significant changes. After inhibiting Src, PKCζ and ASIC3, the head two prevented cell rearrangement that induced by piezo stimulation, and ASIC3 inhibitor cause partial inhibition, but none of them reversed the reduction of cell size that induced by Piezoelectric field stimulation. We determined the degree of PKCζ phosphorylation by using Western blot after adding Src and ASIC3 inhibitors. The result shows that both of them inhibited the phosphorylation of PKCζ that induced by piezoelectric stimulation. Depends on the result, PKCζ may be regulated by Src and ASIC3.