The Effect of Pertussis Toxin on the Growth of Porcine Vascualr Smooth Muscle Cells

• Main Authors: Wen-Ch'i Su, 蘇文祺
• Other Authors: Ming-Te Lin
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/05105865582259521918

碩士 === 國立成功大學 === 生物化學研究所 === 83 === Pertussis toxin (PTX)是一種由Bordetella pertussis所產生的蛋白質 ，它可以穿透細胞膜並對Gi/o protein的.alpha. subunit進行 ADP- ribosylation，使Gi/o protein不能將細胞外的訊息傳向細胞內。先前已 有報告 指出利用PTX對細胞生長的影響而發現Gi/o protein可能牽涉到血 管平滑肌細胞生長的調控。本篇論文便是想探討PTX-sensitive Gi/o protein是否牽涉到豬血管平滑肌細胞的增生及其可能機轉為何？利用直 接計算細胞數目來評估細胞生長情形，發現當以FBS來刺激豬血管平滑肌 細胞生長時 ，PTX可抑制細胞生長，這種效果由處理PTX30小時後即明顯 可見，並至少維持150小時；此種抑制作用亦不能藉由提昇FBS濃度來回復 。PTX也可抑制PDGF,bFGF誘發的豬血管平滑肌細胞增生。因此PTX- sensitive G protein似乎調節某些豬血管平滑肌細胞正常生長所必須的 過程。我們發現當PTX抑制細胞生長時，細胞內cAMP濃度並未增加。PTX也 可以抑制FBS所誘發的細胞中phospholipase A2及D活化，並減少細胞內 diacylglycerol的累積，若以phosphatidic acid(PA, phospholipase D 的產物)，arachidonic acid (AA, phospholipase A2的產物)或A23187( ionophore,增加[Ca++]i)和PTX一起來處理細胞，發現不能回復PTX的抑制 作用。若以PMA(PKC activator)和PTX一起處理細胞，發現可以部分回復 PTX的抑制作用。因此，Gi/o protein可能至少有部分是經由PKC 來調控 豬血管平滑肌之生長。 Pertussis toxin (PTX) is a protein produced by Bordetella pertussis. It can penetrate cell membrane and ADP-ribosylate the .alpha. subunit of Gi/o proteins. The ADP-ribosylated Gi/o proteins can't transduce extracellular signal into cells. It has been reported that Gi/o proteins may be involved the regulation of the growth of vascular smooth muscle cells (VSMCs). In this study, we are to studywhether PTX-sentitive Gi/ o proteins are involved in the proliferation of the porcine VSMCs and its possible mechanisms. Cell growth is assayed by counting cell number after 3~4 days in culture. Our results showed that PTX inhibited the proliferation of porcine VSMCs, which was induced by fetal bovine serum (FBS). This effect could be observed within 30h and persisted for at least 150h after treatment. The inhibition of PTX couldn't be overcome by increasing serum concentraction. PTX also inhibited the proliferation of porcine VSMCs induced by PDGF or bFGF. So, PTX- sensitive G proteins appear to mediate some processes required for the normal growth of these cells. The intracellular level of cAMP was not increased in the cells treated with PTX. PTX also inhibited phospholipase A2 and D activity and attenuated the intracellular diacylglycerol accumulation. The effect of PTX on the inhibition of the proliferation of porcine VSMCs could not be overcome by treating with phosphatidic acid (the product of Phospholipase D), arachidonic acid (the product ofphospholipase A2) or A23187 ( ionophore, increasing [Ca++]i). However, the addition of PMA (PKC activator) to culture could partially overcome the inhibition of PTX. This results suggest that Gi/o proteins at least partially modulate the proliferation of porcine VSMCs by PKC.