低強度間歇性超音波刺激對於骨細胞cGMP增加機轉之探討

Abstract

低強度間歇性超音波（low-intensity pulsed ultrasound, LIPUS）提供非侵入式、非熱傳導的機械訊息傳遞，可以加速骨癒合速度、增加骨骼形成。人體中，一氧化氮（nitric oxide, NO）和C-type natriuretic peptide（CNP）均和骨骼系統代謝有關。 一氧化氮由一氧化氮合成酶（nitric oxide synthase, NOS）產生，其中誘導一氧化氮酶（inducible nitric oxide synthase, iNOS）會因為外界刺激而產生，當大量iNOS產生後，可以產生多量的NO。而CNP藉由它的受體- B型鳥糞環化酵素（guanylyl cyclase type B receptor, GC-B）結合進行生理作用。兩者路徑在造骨細胞中，皆會活化環鳥嘌呤核甘單磷酸（cyclic guanosine monphosphate, cGMP）促進骨骼生長。 本論文中，利用低強度間歇性超音波（frequency =1.5MHz, intensity =30m/cm2, duration =20 minutes, pulse at 1:4）給予初級造骨細胞刺激進行iNOS/NO/cGMP和CNP/GC-B/cGMP路徑之研究。另外，利用蛇毒蛋白Rhodostomin阻滯造骨細胞膜上的integrins作用後，觀察超音波是否仍可以將機械訊息傳遞至細胞內。 結果顯示，超音波可以刺激造骨細胞的一氧化氮產生、誘導一氧化氮酶和GC-B在蛋白質與基因表現增加，同時促進細胞內環鳥嘌呤核甘單磷酸濃度增加，強化CNP之感受性。在原子力顯微鏡實驗中，也發現經過超音波刺激後，骨細胞表面的細胞小泡增加、肌動蛋白絲重組、細胞膜韌度增加之現象。 綜合言之，低強度間歇性超音波能刺激iNOS/NO/cGMP和CNP/GC-B/cGMP路徑，並且活化骨細胞。

Low-intensity pulsed ultrasound（LIPUS）is regarded as a non-invasive and non-thermal mechanotransduction which accelerates fracture healing and promotes bone formation. Nitric oxide（NO）and C-type natriuretic peptide（CNP）are relevant to bone metabolism in human beings. NO is generated by nitric oxide synthase enzyme（NOS）in human beings. Inducible nitric oxide synthase enzyme（iNOS）is one of the NOS, which is responsible to external stimulation and be capable of generating a larger quantity of NO. CNP can bind with its specific receptor, guanylyl cyclase type B receptor（GC-B）, and modulates physiological effects. Both of above two pathways transduce extracellualr signals to activate cyclic guanosine monphosphate（cGMP）to regulate bone development. In this study, we explored the effects of LIPUS（frequency =1.5MHz, intensity =30mW/cm2, duration =20 minutes, pulse at 1:4）on the formation of primary osteoblasts by iNOS/NO/cGMP and CNP/GC-B/cGMP pathways. Moreover, we examined the antagonism of Rhodostomin（Rn）on LIPUS-stimulated effects in osteoblasts. The results show that LIPUS increased nitrite concentration in culture medium, both mRNA and protein levels of iNOS and GC-B. LIPUS also enhanced intracellular cGMP content and the sensitivity to exogenous CNP in cultured osteoblasts. Treatment of disintegrin rhodostomin attenuated the US-induced NO、iNOS and GC-B expression. The cell morphological change in response to LIPUS was examined by atomic force microscope（AFM）and the results show that vacuoles formed in the cell surface and the actin reorganized. The stiffness of the osteoblastic membrane was also increased by LIPUS exposure. In summary, we demonstrated that LIPUS not only enhanced iNOS/NO/cGMP and CNP/GC-B/cGMP pathways but also changed cellular activity by the influence on the cytoskeleton and membrane structures.