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標題: 輔脂蛋白apo-VLDL-II影響極低密度脂蛋白粒徑之研究
The role of apolipoprotein VLDL-II in regulating VLDL particle size profile
作者: 鍾正玗
Cheng-Yu Chung
關鍵字: 輔脂蛋白apoVLDL-II;極低密度脂蛋白;粒徑分佈;蛋雞;雌性素;Apo VLDL-II;VLDL;Laying hens;Particle size distribution;Estradiol
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鳥類產蛋期時,因肝臟受到雌性素 (estradiol) 的作用下大量產生三酸甘油脂 (Triglyceride, TG),血漿中出現較小粒徑VLDL脂蛋白顆粒,此時VLDL以apo B-100及鳥類特有的載脂蛋白 (apolipoprotein VLDL-II, apo VLDL-II) 所組成,產生富含TG的VLDL運輸至卵巢卵母細胞中,堆積形成蛋黃以提供胚胎發育所用,故稱為VLDLy。產蛋期母雞apoVLDL-II與較小粒徑VLDLy的同時出現,因沒有直接的證據證明,使得apoVLDL-II一直被猜測是導致VLDLy較小粒徑的主因,以便利其通過濾泡內各層藩籬,最後被攝入形成蛋黃。但此推論一直缺乏明確的證據,因此本研究目的為證明apo VLDL-II是影響VLDLy粒徑變小的主因。本研究成功選殖apo VLDL-II基因,並構築apo VLDL-II基因表達載體。然後以分離小雞初代肝臟細胞 (chicken primary hepatocytes) 及3T3 fibroblates (3T3) 為實驗模式,轉染 (transfection) apo VLDL-II基因後,以細胞免疫染色分析結果顯示,apo VLDL-II蛋白在3T3及初代肝臟細胞皆會表達。Western blot分析顯示apo VLDL-II蛋白大量表現於3T3中極少分泌至培養液中,而初代肝臟細胞則相反,少量表現於細胞,大量分泌至培養液中,推測apo VLDL-II蛋白在肝臟細胞合成後能與VLDL結合而分泌至細胞外,而3T3因無法合成VLDL,故滯留於細胞內。初代肝臟細胞在>2.5 mM油酸 (oleate, OA) 培養下,會造成細胞死亡,而在<2.5 mM oleate刺激下,其分泌TG (triacylglycerol) 隨劑量遞增。外源性estradiol處理與apo VLDL-II過度表達 (overexpression) 皆會刺激肝細胞TG分泌,且會增強oleate所誘發TG分泌。培養液經超高速離心分離出VLDL,以動態雷射掃描儀 (Dynamic Laser Scattering, DLS) 分析其粒徑大小,結果顯示在以顆粒強度 (particle intensity)、體積 (volume) 和數量 (number) 為參數分析下,VLDL粒徑peak分別出現在8.7、5.6-6.5、與4.8-5.6 nm,而oleate、estradiol處理與apo VLDL-II overexpression皆會使VLDL顆粒分佈往大粒徑遞移。而在電子顯微鏡觀察下,外源性estradiol處理造成VLDL粒徑略變大,apo VLDL-II overexpression會造成VLDL粒徑變小,而oleate刺激會使VLDL粒徑分佈兩極化,而其粒徑大小皆>20 nm。此結果顯示apo VLDL-II 會影響VLDL粒徑,但可能因細胞培養條件、轉染效率或是分離肝細胞雞隻年紀的選擇不佳,以致DLS分析方法無法有力證明apo VLDL-II是造成粒徑變小的主因,但電子顯微鏡觀察下,apo VLDL-II的確會導致分泌VLDL粒徑變小。此外本結果亦證明apo VLDL-II表達可能影響雞隻肝細胞內脂質可運用性,進而影響VLDL合成與分泌,並導致產蛋期VLDL-lipid 組成的改變。

Plasma VLDL biology in laying hens differs distinctively from that of immature hens, including a smaller VLDL particle diameter, dramatic increases of actual and fractional of levels of VLDL-TG (triacylglycerol) and -PL (phospholipid) concentration, and the presence of an avian specific apolipoprotein; VLDL-II (apoVLDL-II). These estrogen-induced alterations were suggested to provide TG-rich VLDL for yolk formation for embryo development, and thereby termed VLDLy. Despite lacking direct evidences, due to the presence of apoVLDL-II in VLDLy simultaneously with smaller particle size, apoVLDL-II was postulated to mediate smaller particle diameter of VLDLy in order to penetrate the follicle layer barriers for yolk deposition. Therefore, the study aimed to prove that apoVLDL-II really mediates physical changes of VLDLy with smaller particle size than those from non-laying hens. ApoVLDL-II gene was successfully cloned, constructed into a expression vector,.and then transfected into to chick primary hepatocytes and 3T3 cells. Western blot analysis showed that apo VLDL-II protein was expressed in 3T3 cells and primary hepatocytes. A dramatically greater abundance of apo VLDL-II protein was found within the 3T3 cells with a merely detectable level in the culture medium, whereas a dramatic level of apo VLDL-II protein was found in the culture medium but very minor abundance was found in the primary hepatocytes. These results suggest that hepatocytes can synthesize apoVLDL-II to associate with VLDL for secretion, but apoVLDL-II is retained within 3T3 cells due to the incapability for VLDL synthesis and secretion. Treatment of chick hepatocytes with oleate (OA) at levels >2.5 mM resulted in significant cell death, whereas oleate (OA) at levels < 2.5 mM promoted TG (triacylglycerol) secretion in a dose-dependent manner. VLDL isolated from the collected medium through ultracentrifugation was used for particle size measurement through dynamic laser scanning (Dynamic Laser Scattering, DLS) method. Results showed that VLDL particle size peaked at 8.7, 5.6-6.5, and 4.8-5.6 nm under intessity, volume and number parameter as a function, respectively. Treatment of oleate amd estradiol, as well as apo VLDL-II overexpression shifted VLDL particle profile toward larger size. Under the examination of scanning electron microscope (SEM), however, estradiol treatment slightly increased VLDL particle size, whereas apo VLDL-II overexpression caused smaller VLDL particle size in the secretion and oleate treatment deviated the particle size distribution toward the two sides. All of the examinations by SEM showed VLDL particle size larger than 20 nm. Collectively, results in the study suggested that the presence apo VLDL-II indeed physically affects VLDL particle size, but did not prove smaller particle size mediated by apo VLDL-II, which may be attributed to unoptimal culture conditions, poor transfection efficiency, the age of chicks for primary hepatocytes isolation, and/or particle size analysis by DLS. In addition, overexpression of apoVLDL-II enhanced oleate-induced TG secretion, suggesting that apoVLDL-II expression is responsive to intracellular lipid availability to facilitate VLDL assembly and secretion.
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