Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96551
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
引用: Andrade, N., V. Komnenovic, S. M. Blake, Y. Jossin, B. Howell, A. Goffinet, W. J. Schneider, and J. Nimpf. 2007. ApoER2/VLDL receptor and Dab1 in the rostral migratory stream function in postnatal neuronal migration independently of Reelin. Proc. Natl. Acad. Sci. U S A. 104:8508-8513. Barber, D. L., E. J. Sanders, R. Aebersold, and W. J. Schneider. 1991. The receptor for yolk lipoprotein deposition in the chicken oocyte. J. Biol. Chem. 266:18761-18770. Beisiegel, U., W. Weber, and G. Bengtsson-Olivecrona. 1991. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc. Natl. Acad. Sci. U S A. 88:8342-8346. Benoist, F., and T. Grand-Perret. 1996. ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools. Arterioscler Thromb. Vasc. Biol. 16:1229-1235. Berkowitz, E. A., and I. M. Evans. 1992. Functional analysis of regulatory regions upstream and in the first intron of the estrogen-responsive chicken very low density apolipoprotein II gene. J. Biol. Chem. 267:7134-7138. Binder, R., S. P. Hwang, R. Ratnasabapathy, and D. L. Williams. 1989. Degradation of apolipoprotein II mRNA occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-noncoding region. J. Biol. Chem. 264:16910-16918. Blade, A. M., M. A. Fabritius, L. Hou, R. B. Weinberg, and S. G. Shelness. 2011. Biogenesis of apolipoprotein A-V and its impact on VLDL triglyceride secretion. J. Lipid Res. 52:237-244. Blue, M. L., A. A. Protter, and D. L. Williams. 1980. Biosynthesis of apolipoprotein B in rooster kidney, intestine, and liver. J. Biol. Chem. 255:10048-10051. Borén, J., S. Rustaeus, and S. O. Olofsson. 1994. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells. J. Biol. Chem. 269:25879-25888. Bujo, H., K. L. Lindstedt, M. Hermann, L. M. Dalmau, J. Nimpf, and W. J. Schneider. 1995. Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor. J. Biol. Chem. 270:23546-23551. Bujo, H., M. Hermann, M. O. Kaderli, L. Jacobsen, S. Sugawara, J. Nimpf, T. Yamamoto, and W. J. Schneider. 1994. Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family. EMBO. J. 13:5167-5175. Bujo, H., T. Yamamoto, K. Hayashi, M. Hermann, J. Nimpf, and W. J. Schneider. 1995. Mutant oocytic low density lipoprotein receptor gene family member causes atherosclerosis and female sterility. Proc. Natl. Acad. Sci. U S A. 92:9905-9909. Bussmann, U. A., J. M. Perez Saez, L. E. Bussmann, and J. L. Baranao. 2013. Aryl hydrocarbon receptor activation leads to impairment of estrogen-driven chicken vitellogenin promoter activity in LMH cells. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 157:111-118. Cartwright, A. L. 1991. Adipose cellularity in Gallus domesticus: investigations to control body composition in growing chickens. J. Nutr. 121:1486-1497. Chan, L., R. L. Jackson, B. W. O'Malley, and A. R. Means. 1976. Synthesis of very low density lipoproteins in the cockerel. J. Clin. Invest. 58:368-379. Chen, S. E., D. W. Long, K. E. Nestor, R. L. WalzemL, V. L. Meuniot, H. Zhu, R. J. Hansen, and W. L. Bacon. 1999. Effect of divergent selection for total plasma phosphorus on plasma and yolk very low density lipoproteins and plasma concentrations of selected hormones in laying Japanese quail. Poult. Sci. 78:1241-1251. Elkin, R.G., I. MacLachlan, M. Hermann, and W. J. Schneider. 1995. Characterization of the Japanese quail oocyte receptor for very low density lipoprotein and vitellogenin. J. Nutr. 125:1258-1266. Elkin, R. G., R. Bauer, and W. J. Schneider. 2012. The restricted ovulator chicken strain: an oviparous vertebrate model of reproductive dysfunction caused by a gene defect affecting an oocyte-specific receptor. Anim. Reprod. Sci. 136:1-13. Eresheim, C., J. Plieschnig, N. E. Ivessa, W. J. Schneider, and M. Hermann. 2014. Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo. Biochimie. 101:67-74. Fayad, T., R. Lefebvre, J. Nimpf, D. W. Silversides, and J. G. Lussier. 2007. Low-density lipoprotein receptor-related protein 8 (LRP8) is upregulated in granulosa cells of bovine dominant follicle: molecular characterization and spatio-temporal expression studies. Biol. Reprod. 76:466-475. Gåfvels, M. E., G. Coukos, R. Sayegh, C. Coutifaris, D. K. Strickland, and J. F. Strauss. 1992. Regulated expression of the trophoblast alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Differentiation and cAMP modulate protein and mRNA levels. J. Biol. Chem. 267:21230-21234. George, R., D. L. Barber, and W. J. Schneider. 1987. Characterization of the chicken oocyte receptor for low and very low density lipoproteins. J. Biol. Chem. 262:16838-16847. Gibbons, G. F. 1990. Assembly and secretion of hepatic very-low-density lipoprotein. Biochem. J. 268:1-13. Gordon, D. A., H. Jamil, R. E. Gregg, Sven-Olof Olofsson, and J. Bore. 1996. Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. J. Biol. Chem. 51:33047-33053. Griffin, H., G. Grant, and M. Perry. 1982. Hydrolysis of plasma triacylglycerol-rich lipoproteins from immature and laying hens (Gallus domesticus) by lipoprotein lipase in vitro. Biochem. J. 206:647-654. Hamilton, R. L., J. S. Wong, C. M. Cham, L. B. Nielsen, and S. G. Young. 1998. Chylomicron-sized lipid particles are formed in the setting of apolipoprotein B deficienc. J. Lipid Res. 39:1543-1557. Hayashi, K., J. Nimpf, and W. J. Schneider. 1989. Chicken oocytes and fibroblasts express different apolipoproteins-B specific receptors. J. Biol. Chem. 264:3131-3139. Hermann, M., F. Seif, W. J. Schneider, and N. E. Ivessa. 1997. Estrogen dependence of synthesis and secretion of apolipoprotein B-containing lipoproteins in the chicken hepatoma cell line, LMH-2A. J. Lipid Res. 38:1308-1317. Hermier, D. 1997. Lipoprotein metabolism and fattening in poultry. J. Nutr. 127:805-808. Hermier, D., P. Forgez, and M. J. Chapman. 1985. A density gradient study of the lipoprotein and apolipoprotein distribution in the chicken, Gallus domesticus. Biochim. Biophys. Acta. 22:105-118. Hiesberger, T., M. Hermann, L. Jacobsen, S. Novak, R. A. Hodits, H. Bujo, M. Meilinger, M. Hüttinger, W. J. Schneider, and J. Nimpf. 1995. The chicken oocyte receptor for yolk precursors as a model for studying the action of receptor-associated protein and lactoferrin. J. Biol. Chem. 270:18219-18226. Holdsworth, G., R. H. Michell, and J. B. Finean. 1974. Transfer of very low density lipoprotein from hen plasma into egg yolk. FEBS. Lett. 39:275-277. Hu, S., H. Liu, Z. Pan, L. Xia, X. Dong, L. Li, F. Xu, H. He, and J. Wang. 2014. Molecular cloning, expression profile and transcriptional modulation of two splice variants of very low density lipoprotein receptor during ovarian follicle development in geese (Anser cygnoide). Anim. Reprod. Sci. 149:281-296. Hummel, S., A. Osanger, T. M. Bajari, M. Balasubramani, W. Halfter, J. Nimpf, and W. J. Schneider. 2004. Extracellular matrices of the avian ovarian follicle: molecular characterization of chicken perlecan. J. Biol. Chem. 279:23486-23494. Hummel, S., E. G. Lynn, A. Osanger, S. Hirayama, J. Nimpf and W. J. Schneider. 2003. Molecular characterization of the first avian LDL receptor: role in sterol metabolism of ovarian follicular cells. J. Lipid Res. 44:1633-1642. Hummel, S., S. Christian, A. Osanger, H. Heid, J. Nimpf and W. J. Schneider. 2007. Identification of a novel chondroitin-sulfated collagen in the membrane separating theca and granulosa cells in chicken ovarian follicles: the granulosa-theca cell interface is not a bona fide basement membrane. J. Biol. Chem. 282:8011-8018. Hussain, M. M., J. Shi, and P. Dreizen. 2002. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly. J. Lipid Res. 44:22-32. Ishibashi, S., M. S. Brown, J. L. Goldstein, R. D. Gerard, R. E. Hammer, and J. Herz. 1993. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J. Clin. Invest. 92:883-893. Ivessa, N. E., E. Rehberg, B. Kienzle, F. Seif, R. Hermann, M. Hermann, W. J. Schneider, and D. A. Gordon. 2013. Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein. Gene. 523:1-9. Jacobsen, L., M. Hermann, P. M. Vieira, W. J. Schneider, and J. Nimpf. 1995. The chicken oocyte receptor for lipoprotein deposition recognizes alpha 2-macroglobulin. J. Biol. Chem. 270:6468-6475. Johnson, A. L., and, D. C. Woods. 2009. Dynamics of avian ovarian follicle development: cellular mechanisms of granulosa cell differentiation. Gen. Comp. Endocrinol. 163:12-17. Kompiang, I. P., A. Bensadoun, and M. W. Yang. 1976. Effect of an anti-lipoprotein lipase serum on plasma triglyceride removal. J. Lipid Res. 17:498-505. Kudzma, D. J., F. St. Claire, L. DeLallo and S. J. Friedberg. 1975. Mechanism of avian estrogen-induced hypertriglyceridemia: evidence for overproduction of triglyceride. J. Lipid Res. 16:123-133. Kudzma, D. J., J. B. Swaney, and E. N. Ellis. 1979. Effects of estrogen administration on the lipoproteins and apoproteins of the chicken. Biochim. Biophys. Acta. 572:257-268. Kudzma, D. J., P. M. Hegstad, and R. E. Stall. 1973. The chick as a laboratory model for the study of estrogen-induced hyperlipidemia. Metabolism. 22:423-434. Leeb, C., C. Eresheim, and J. Nimpf. 2014. Clusterin is a ligand for apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR) and signals via the Reelin-signaling pathway. J. Biol. Chem. 289:4161-4172. Li, B., Y. Zhang, X. Chen, Q. Shi, D. Fu, Y. Yin, Z. Zhang, B. Gao, and G. Chen. 2010. Directional differentiation of chicken primordial germ cells into adipocytes, neuron-like cells, and osteoblasts. Mol. Reprod. Dev. 77:795-801. Li, J., I. H. Leghari, B. He, W. Zeng, Y. Mi, and C. Zhang. 2014. Estrogen stimulates expression of chicken hepatic vitellogenin II and very low-density apolipoprotein II through ER-alpha. Theriogenology. 82:517-524. Maclachlan, I., E. Steyrer, A. Hermetter, J. Nimpf, and W. J. Schneider. 1996. Molecular characterization of quail apolipoprotein very-low-density lipoprotein II: disulphide-bond-mediated dimerization is not essential for inhibition of lipoprotein lipase. Biochem. J. 317:599-604. Mead, J. R., S. A. Irvine, and D. P. Ramji. 2002. Lipoprotein lipase: structure, function, regulation, and role in disease. J. Mol. Med. (Berl) 80:753-769. Nimpf, J., and W. J. Schneider. 1991. Receptor-mediated lipoprotein transport in laying hens. J. Nutr. 121:1471-1474. Nimpf, J., and W. J. Schneider. 1998. The VLDL receptor: an LDL receptor relative with eight ligand binding repeats, LR8. Atherosclerosis. 141:191-202. Nimpf, J., M. J. Radosavljevic, and W. J. Schneider. 1989. Oocytes from the mutant restricted ovulator hen lack receptor for very low density lipoprotein. J. Biol. Chem. 25:1393-1398. Nimpf, J., R. George, and W. J. Schneider. 1988. Apolipoprotein specificity of the chicken oocyte receptor for low and very low density lipoproteins: lack of recognition of apolipoprotein VLDL-II. J. Lipid Res. 29:657-667. Oka, K., K. Ishimura-Oka, M. J. Chu, M. Sullivan, J. Krushkal, W. H. Li and L. Chan. 1994. Mouse very-low-density-lipoprotein receptor (VLDLR) cDNA cloning, tissue-specific expression and evolutionary relationship with the low-density-lipoprotein receptor. Eur. J. Biochem. 224:975-982. Peebles, E. D., M. R. Burnham, R. L. Walzem, S. L. Branton, and P. D. Gerard. 2004. Effects of fasting on serum lipids and lipoprotein profiles in the egg-laying hen (Gallus domesticus). Comp Biochem Physiol. A Mol. Integr. Physiol. 138:305-311. Perry, M. M., and A. B. Gilbert. 1979. Yolk transport in the ovarian follicle of the hen (Gallus domesticus): lipoprotein-like particles at the periphery of the oocyte in the rapid growth phase. J. Cell. Sci. 39:257-272. Pinchasov, Y., S. Elmaliah, and S. Bezdin. 1994. Plasma apolipoprotein VLDL-II and egg production in laying hens, establishment of an ELISA method. Reprod. Nutr. Dev. 34:361-369. Ram, P. T., C. M. Horvath, and R. Iyengar. 2000. Stat3-mediated transformation of NIH-3T3 cells by the constitutively active Q205L galphao protein. Sci. 287:142-144. Ratna, W. N., and C. Oyeamalu. 2002. The upstream stem-loop domain of the 3' untranslated region of apolipoprotein II mRNA binds the estrogen-regulated mRNA. J. Steroid. Biochem. Mol. Biol. 80:383-393. Ratna, W. N., V. D. Bhatt, K. Chaudhary, A. Bin Ariff, S. A. Bavadekar, and H. N. Ratna. 2016. Estrogen-responsive genes encoding egg yolk proteins vitellogenin and apolipoprotein II in chicken are differentially regulated by selective estrogen receptor modulators. Theriogenology. 85:376-383. Read, J., T. A. Anderson, P. J. Ritchie, B.Vanloo, J. Amey, D. Levitt, M. Rosseneu, J. Scott, and C. C. Shoulders. 2000. A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein. J. Biol. Chem. 275:30372-30377. Rustaeus, S., K. Lindberg, P. Stillemark, C. Claesson, L. Asp, T. Larsson, J. Borén, and S. O. Olofsson. 1999. Assembly of very low density lipoprotein: A two-step process of apolipoprotein B core lipidation. J. Nutr. 129:463-466. Ryoo, S. R., Y. K. Kim, M. H. Kim, and D. H. Min. 2010. Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes:proliferation, focal adhesion, and gene transfection studies. ACS. Nano 23:6587-6598. Salvante, K. G., G. Lin, R. L. Walzem, and T. D. Williams. 2007. Characterization of very-low density lipoprotein particle diameter dynamics in relation to egg production in a passerine bird. J. Exp. Biol. 210:1064-1074. Schaap, F. G., P. C. Rensen, P. J. Voshol, C. Vrins, H. N. van der Vliet, R. A. Chamuleau, L. M. Havekes, A. K. Groen, and K. W. Dijk. 2004. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis. J. Biol. Chem. 279:27941-27947. Schmidinger, B., A. M. Weijler, W. J. Schneider, and M. Hermann. 2016. Hepatosteatosis and estrogen increase apolipoprotein O production in the chicken. Biochimie. 127:37-43. Schneider, W. J. 1996. Vitellogenin receptors: oocyte-specific members of the low-density lipoprotein receptor supergene family. Int. Rev. Cytol. 166:103-137. Schneider, W. J. 2009. Receptor-mediated mechanisms in ovarian follicle and oocyte development. Gen. Comp. Endocrinol. 163:18-23. Schneider, W. J. 2016. Lipid transport to avian oocytes and to the developing embryo. J. Biomed. Res. 30:174-180. Schneider, W. J., and J. Nimpf. 1993. Lipoprotein receptors: old relatives and new arrivals. J. Biol. Chem. 4:205-209. Schneider, W. J., R. Carroll, D. J. Severson, and J. Nimpf. 1990. Apolipoprotein VLDL-II inhibits lipolysis of triglyceride-rich lipoproteins in the laying hen. J. Lipid Res. 31:507-513. Sensel, M. G., R. Binder, C. B. Lazier, and D. L. Williams. 1994. Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription. Mol. Cell Biol. 14:1733-1742. Shelness, G. S., and D. L. Williams. 1989. Apolipoprotein II messenger RNA. Transcriptional and splicing heterogeneity yields six 5'-untranslated leader sequences. J. Biol. Chem. 259:9925-9935. Spring, D. J., L. W. Chen-Liu, J. E. Chatterton, J. Elovson, and V. N. Schumaker. 1992. Lipoprotein assembly. Apolipoprotein B size determines lipoprotein core circumference. J. Biol. Chem. 267:14839-14845. Steyrer, E., D. L. Barber, and W. J. Schneider. 1990. Evolution of lipoprotein receptors. The chicken oocyte receptor for very low density lipoprotein and vitellogenin binds the mammalian ligand apolipoprotein E. J. Biol. Chem. 265:19675-19681. Stifani, S., D. L. Barber, J. Nimpf, and W. J. Schneider. 1990. A single chicken oocyte plasma membrane protein mediates uptake of very low density lipoprotein and vitellogenin. Proc. Natl. Acad. Sci. U S A. 87:1955-1959. Stifani, S., D. L. Barber, R. Aebersold, E. Steyrer, X. Shen, J. Nimpf, and W. J. Schneider. 1991. The laying hen expresses two different low density lipoprotein receptor-related proteins. J. Biol. Chem. 266:19079-19087. Sundaram M, and Yao. Z. 2010. Recent progress in understanding protein and lipid factors affecting hepatic VLDL assembly and secretion. Nutr. Metab. (Lond) 7:7-35. Tarugi, P., G. Ballarini, B. Pinotti, A. Franchini, E. Ottaviani, and S. Calandra. 1998. Secretion of apoB- and apoA-I-containing lipoproteins by chick kidney. J. Lipid Res. 39:731-741. Walzem, R. L. 1996. Lipoproteins and the laying hen: form follows function. Poult. Avian Biol. Rev. 7:31-64. Walzem, R. L., P. A. Davis, and R. J. Hansen. 1994. Overfeeding increases very low density lipoprotein diameter and causes the appearance of a unique lipoprotein particle in association with failed yolk deposition. J. Lipid Res. 35:1354-1366. Walzem, R. L., R. J. Hansen, D. L. Williams, and R. L. Hamilton. 1999. Estrogen induction of VLDLy assembly in egg-laying hens. J. Nutr. 129:467-472. Wang, C., S. J. Li, W. H. Yu, Q. W. Xin, C. Li, Y. P. Feng, X. L. Peng, and Y. Z. Gong. 2011. Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos). Genet. Sel. Evol. 43:1-9. Wang, X. J., Y. Li, Q. Q. Song, Y. Y. Guo, H. C. Jiao, Z. G. Song, and H. Lin. 2013. Corticosterone regulation of ovarian follicular development is dependent on the energy status of laying hens. J. Lipid Res. 54:1860-1876. Weinberg, R. B., J. W. Gallagher, M. A. Fabritius, and G. S. Shelness. 2012. ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich lipoproteins. J. Lipid Res. 53:736-743. Williams, D. L. 1979. Apoproteins of avian very Low density lipoprotein: demonstration of a single high molecular weight apoprotein. Biochemistry. 18:1056-1063. Wiskocil, R., P. Bensky, W. Dower, R. F. Goldberger, J. I. Gordon, and R. G. Deeley. 1980. Coordinate regulation of two estrogen-dependent genes in avian liver. Proc. Natl. Acad. Sci. U S A. 77:4474-4478. Yen, C. F., Y. N. Jiang, T. F. Shen, I. M. Wong, C. C. Chen, K. C. Chen, W. C. Chang, Y. K. Tsao, and S. T. Ding. 2005. Cloning and expression of the genes associated with lipid metabolism in Tsaiya Ducks. Poult. Sci. 84:67-74.
摘要: 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.
URI: http://hdl.handle.net/11455/96551
文章公開時間: 2019-08-14
Appears in Collections:動物科學系

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.