转化生长因子α对LoVo细胞增殖及其它生物学功能影响的研究
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摘要
目的:转化生长因子α(TGF-α)是由50个氨基酸残基构成的一类小分子多肽,其广泛存在于人体多种器官和体液中,研究表明TGF-α对人体胃肠道功能起着重要的调节作用,能够刺激人体胃肠道细胞的生长和分化,对胃肠道粘膜损伤具有修复和保护的功能,并可促进新生儿胃肠道的生长发育。
本课题旨在研究人初乳、常乳及婴儿配方乳源性及重组人转化生长因子α(TGF-α)对人结肠上皮细胞LoVo促增殖作用,以及不同浓度外源性TGF-α对LoVo细胞RNA和蛋白质含量及细胞迁移能力的影响。
方法:以人结肠细胞LoVo为研究模型,采用四甲基偶氮唑盐比色法(MTT法)检测人初乳、常乳、婴儿配方乳粉和重组人TGF-α(浓度分别为0.1、1、10和100ng/mL)作用人结肠上皮细胞模型24h后细胞增殖率的变化,以及各成分添加TGF-α抗体作用24h后人结肠上皮细胞模型增殖率的变化。
分别添加重组人TGF-α0.1、1(接近人乳TGF-α含量最低值)、10(接近人乳TGF-α含量最高值)、100ng/mL后,观察LoVo细胞增殖能力、细胞总RNA、总蛋白质含量以及细胞迁移能力的变化。
结果:各种成分乳以及不同浓度重组人转化生长因子α均能够显著提高人结肠上皮细胞增殖率。并且随着TGF-α浓度的升高细胞增殖程度也相应得到提高。而TGF-α抗体的中和作用使得重组人TGF-α对肠上皮细胞促增殖作用在四个浓度组中分别降低了11±3.2%(P＜0.05)、12±0.3%(P＜0.05)、3±2.1%(P＞0.05)和4±3.3%(P＞0.05),同时也使得人初乳、常乳以及婴儿配方乳粉对肠上皮细胞促增殖作用分别降低了56±13%(P＜0.01)、35±3%(P＜0.05)和9±3%(P＞0.05)。
添加TGF-α24小时后,细胞较对照组有明显的增殖现象,且细胞增殖率随细胞浓度增加而呈上升趋势,并在10ng/mL时达到最大(P＜0.01)。添加TGF-α48小时后,细胞增殖率依然随TGF-α浓度增大而依次升高,且在100ng/mL时达到最大(P＜0.01)。而48h与24h相比细胞增殖率有所提高,说明增殖率与TGF-α成一定的时间依赖关系。
细胞总RNA含量随TGF-α浓度增加而上升为控制组的1.67倍(P＜0.05)至4.20倍(P＜0.01)。而100ng/mL组细胞总蛋白质含量明显高于控制组和其它各剂量组。为对照组的1.28倍(P＜0.05)。另外,添加重组人TGF-α与对照组相比可明显提高LoVo细胞迁移能力。
结论:TGF-α抗体对含有人初乳、常乳和基于牛乳成分的婴儿配方乳粉以及重组人TGF-α培养基的中和作用使得它们对人结肠上皮细胞的促增殖作用显著降低,证明不同源性的TGF-α都能够对人体外肠道上皮细胞起到明显的促增殖作用。
外源性TGF-α同样能够促进人结肠上皮细胞RNA含量的增加和蛋白质的合成。并且提高LoVo细胞的迁移能力。
Objective:transforming growth factor-alpha(TGF-α) is a small 50 amino acid residue long mitogenic protein,It generally exist in many human organs and body fluids.Studies confirm that TGF-αcan accommodate gastrointestinal tract physiologic function,suscitate proliferation and differentiation of gastrointestinal tract cells.It also can repair and protect the membrana mucosa of human gastrointestinal tract,and stimulate the growth and development in gastrointestinal tract of infant.
In our study,we try to explore the proliferation effects of transforming growth factor-alpha from human colostrum,natural human milk,infant formula milk powder and recombinant transforming growth factor-alpha(TGF-α) on human colon epidermal cell model(LoVo) cells.And to investigate the effect of transforming growth factorα(TGF-α) on growth,total cellular RNA and protein also the ransference capability of human colon(LoVo) cells.
Methods:Using MTT to assay the effects of human colostrums,natural human milk,infant formula milk powder and recombinant TGF-α(0.1,1,10 and 100ng/mL) on human colon epidermal LoVo cell proliferation in the presence or absence of TGF-αantibody.
And the effects of proliferation,total cellular RNA and protein also the immigration capability of LoVo cells' were examined after the addition of TGF-αat 0.1,1(close to TGF-αmin concentration in human milk),10(close to TGF-αmax concentration inhuman milk) or 100ng/mL.
Results:Whether human colostrum,natural human milk,infant formula milk powder or recombinant TGF-αcan significant stimulate the proliferation of human colon epidermal cell.And there was a dose-dependent effect of TGF-αon cell proliferation.The stimulatory effect of TGF-αwas decreased in the presence of TGF-αantibody 11±3.2%(P＜0.05),12±0.3%(P＜0.05),3±2.1% (P＞0.05) and 4±3.3%(P＞0.05).In the presence of TGF-αantibody there was a 56±13%decline in colostrum stimulated growth(P＜0.01),and a 35±3%decline in natural human milk stimulated growth(P＜0.05),and a 9±3%decline in infant formula milk powder stimulated growth(P＞0.05).
After incubation with TGF-αfor 24 h,there was a significant proliferation of LoVo cells by concentration of TGF-αwere increased.The proliferation came to the top when concentration of TGF-αwere at 10ng/mL(P＜0.01).And after incubation with TGF-αfor 48 h,there was also a significant proliferation increased of LoVo cells by concentration of TGF-α.,and the proliferation came to the top when concentration of TGF-αwere at 100ng/mL(P＜0.01).And the 48h proliferation was bigger than 24h proliferation of LoVo cells.So the proliferation can increased by the time.
And the total cellular RNA in the TGF-αtreatment groups was elevated from 1.67(P＜0.05) to 4.20(P＜0.01) fold of the control group as the concentration of TGF-αwere in creased.The 100ng/ml TGF-αtreatment group had significantly higher cellular protein as compared to the control and other TGF-αtreatment groups.It was 1.28 fold(P＜0.01) of the control group.And the immigration capability was notable increased by add rhTGF-αcompare with the control group.
Conclusion:Neutralization of recombinant TGF-αand that present in human colostrums, natural human milk or infant formula milk powder which made mostly by bovine milk medium by TGF-αantibody led to a decrease in in vitro human colon epidermal cell proliferation.These results support that TGF-αderived from different sources has a measurable trophic effect on in vitro human gut epithelial cells.
Also exogenous TGF-αcan make LoVo cells total cellular RNA and protein increased.It also can make the transference capability increased of LoVo cells
本课题旨在研究人初乳、常乳及婴儿配方乳源性及重组人转化生长因子α(TGF-α)对人结肠上皮细胞LoVo促增殖作用,以及不同浓度外源性TGF-α对LoVo细胞RNA和蛋白质含量及细胞迁移能力的影响。
方法:以人结肠细胞LoVo为研究模型,采用四甲基偶氮唑盐比色法(MTT法)检测人初乳、常乳、婴儿配方乳粉和重组人TGF-α(浓度分别为0.1、1、10和100ng/mL)作用人结肠上皮细胞模型24h后细胞增殖率的变化,以及各成分添加TGF-α抗体作用24h后人结肠上皮细胞模型增殖率的变化。
分别添加重组人TGF-α0.1、1(接近人乳TGF-α含量最低值)、10(接近人乳TGF-α含量最高值)、100ng/mL后,观察LoVo细胞增殖能力、细胞总RNA、总蛋白质含量以及细胞迁移能力的变化。
结果:各种成分乳以及不同浓度重组人转化生长因子α均能够显著提高人结肠上皮细胞增殖率。并且随着TGF-α浓度的升高细胞增殖程度也相应得到提高。而TGF-α抗体的中和作用使得重组人TGF-α对肠上皮细胞促增殖作用在四个浓度组中分别降低了11±3.2%(P＜0.05)、12±0.3%(P＜0.05)、3±2.1%(P＞0.05)和4±3.3%(P＞0.05),同时也使得人初乳、常乳以及婴儿配方乳粉对肠上皮细胞促增殖作用分别降低了56±13%(P＜0.01)、35±3%(P＜0.05)和9±3%(P＞0.05)。
添加TGF-α24小时后,细胞较对照组有明显的增殖现象,且细胞增殖率随细胞浓度增加而呈上升趋势,并在10ng/mL时达到最大(P＜0.01)。添加TGF-α48小时后,细胞增殖率依然随TGF-α浓度增大而依次升高,且在100ng/mL时达到最大(P＜0.01)。而48h与24h相比细胞增殖率有所提高,说明增殖率与TGF-α成一定的时间依赖关系。
细胞总RNA含量随TGF-α浓度增加而上升为控制组的1.67倍(P＜0.05)至4.20倍(P＜0.01)。而100ng/mL组细胞总蛋白质含量明显高于控制组和其它各剂量组。为对照组的1.28倍(P＜0.05)。另外,添加重组人TGF-α与对照组相比可明显提高LoVo细胞迁移能力。
结论:TGF-α抗体对含有人初乳、常乳和基于牛乳成分的婴儿配方乳粉以及重组人TGF-α培养基的中和作用使得它们对人结肠上皮细胞的促增殖作用显著降低,证明不同源性的TGF-α都能够对人体外肠道上皮细胞起到明显的促增殖作用。
外源性TGF-α同样能够促进人结肠上皮细胞RNA含量的增加和蛋白质的合成。并且提高LoVo细胞的迁移能力。
Objective:transforming growth factor-alpha(TGF-α) is a small 50 amino acid residue long mitogenic protein,It generally exist in many human organs and body fluids.Studies confirm that TGF-αcan accommodate gastrointestinal tract physiologic function,suscitate proliferation and differentiation of gastrointestinal tract cells.It also can repair and protect the membrana mucosa of human gastrointestinal tract,and stimulate the growth and development in gastrointestinal tract of infant.
In our study,we try to explore the proliferation effects of transforming growth factor-alpha from human colostrum,natural human milk,infant formula milk powder and recombinant transforming growth factor-alpha(TGF-α) on human colon epidermal cell model(LoVo) cells.And to investigate the effect of transforming growth factorα(TGF-α) on growth,total cellular RNA and protein also the ransference capability of human colon(LoVo) cells.
Methods:Using MTT to assay the effects of human colostrums,natural human milk,infant formula milk powder and recombinant TGF-α(0.1,1,10 and 100ng/mL) on human colon epidermal LoVo cell proliferation in the presence or absence of TGF-αantibody.
And the effects of proliferation,total cellular RNA and protein also the immigration capability of LoVo cells' were examined after the addition of TGF-αat 0.1,1(close to TGF-αmin concentration in human milk),10(close to TGF-αmax concentration inhuman milk) or 100ng/mL.
Results:Whether human colostrum,natural human milk,infant formula milk powder or recombinant TGF-αcan significant stimulate the proliferation of human colon epidermal cell.And there was a dose-dependent effect of TGF-αon cell proliferation.The stimulatory effect of TGF-αwas decreased in the presence of TGF-αantibody 11±3.2%(P＜0.05),12±0.3%(P＜0.05),3±2.1% (P＞0.05) and 4±3.3%(P＞0.05).In the presence of TGF-αantibody there was a 56±13%decline in colostrum stimulated growth(P＜0.01),and a 35±3%decline in natural human milk stimulated growth(P＜0.05),and a 9±3%decline in infant formula milk powder stimulated growth(P＞0.05).
After incubation with TGF-αfor 24 h,there was a significant proliferation of LoVo cells by concentration of TGF-αwere increased.The proliferation came to the top when concentration of TGF-αwere at 10ng/mL(P＜0.01).And after incubation with TGF-αfor 48 h,there was also a significant proliferation increased of LoVo cells by concentration of TGF-α.,and the proliferation came to the top when concentration of TGF-αwere at 100ng/mL(P＜0.01).And the 48h proliferation was bigger than 24h proliferation of LoVo cells.So the proliferation can increased by the time.
And the total cellular RNA in the TGF-αtreatment groups was elevated from 1.67(P＜0.05) to 4.20(P＜0.01) fold of the control group as the concentration of TGF-αwere in creased.The 100ng/ml TGF-αtreatment group had significantly higher cellular protein as compared to the control and other TGF-αtreatment groups.It was 1.28 fold(P＜0.01) of the control group.And the immigration capability was notable increased by add rhTGF-αcompare with the control group.
Conclusion:Neutralization of recombinant TGF-αand that present in human colostrums, natural human milk or infant formula milk powder which made mostly by bovine milk medium by TGF-αantibody led to a decrease in in vitro human colon epidermal cell proliferation.These results support that TGF-αderived from different sources has a measurable trophic effect on in vitro human gut epithelial cells.
Also exogenous TGF-αcan make LoVo cells total cellular RNA and protein increased.It also can make the transference capability increased of LoVo cells
引文
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Maiden LT, Novak U, Burgess AW. 1989. Expression of transforming growth factor alpha messenger RNA in the normal and neoplastic gastrointestinal tract. Int J Cancer. 43:380-384.
Malo C, Menard D. 1982. Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology. 83: 28—35.
Markowitz, S.D.Molkentin K et al. 1990. Growth stimulation by coexpression of transforming growth factor-a and epidermal growth factor-receptor in normal and adenomatous human colon epithelium. J.Clin Invest. 86:356—362
Marti U, Burwen SJ, Jones AL. 2004. Biological effects of epidermal growthfactor, with emphasis on the gastrointestinal tract and liver: an update. Hepatology. (9): 126—133
Masaki Okada, Eiji Ohmura, Yoshinobu Kamiya, Hitomi Murakami et al. 1991 Transforming Growth Factor (TGF)-a in Human milk. Life Sciences. 48:115—1156
Mashimo H, Wu DC, Podolsky DK, Fishman MC.1996. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science. 27(4):262—265
Massague J. 1987. The TGF-b family of growth and differentiation factors. Cell. 4(9):437—440
McCleary MJ. 1991. Epidermal growth factor: An important constituent of human milk. J Hum Lact .7(3): 123—128.
Miettinen PJ, Berger JE, Meneses J, et al.2005. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature.37(6):337—339
Nightingale JM, Kamm MA, van der Sijp JR, Ghatei MA, Bloom SR, Lennard-Jones JE. 1996.Gastrointestinal hormones in short bowel syndrome. Peptide YY may be the 'colonic brake to gastric emptying. Gut. 3(9):267-269。
Odle J, Zijlstra RT, Donovan SM. 1996. Intestinal effects of milkborne growth factors in neonates of agricultural importance. J Animal Sci. 7(4):2509-2512
Peptrides PE, Hosang M, Shooter EM, Bohlen P. 1984. Transforming growth factors in human milk: Isolation and partial chemical and biological characterization. Proc 7th Int Congr Endocrinol.p1139.
Plaut AG. 1997. Trefoil peptides in the defense of the gastrointestinal tract. N Engl J Med 33(6):506-509
Playford RJ, Marchbank T, Goodlad RA, Chinery RA, Poulsom R, Hanby AM. 1996.
Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage. Proc Nat Acad Sci USA. 9(3): 2137—2139
Playford RJ, Wright NA. 1006. Why is epidermal growth factor present in the gut lumen Gut 3(8):303—306
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R. Pakkanen, J. Aalto. 1997. Review paper growth factors and antimicrobial factors of bovine colostrums. Dairy Journal. (7):285—297
Rao RK. 1991. Biologically active peptides in the gastrointestinal lumen. Life Sci. 4(8):1685-1688
Raymond J Playford, Christopher E Macdonald, and Wendy S Johnson. 2000. Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders. Am J Clin Nutr 72:5—14.
Sandgren EP, Luetteke NC, Plamiter RD, Brinster RL, Lee DC. 1990. Overexpression of TGF α in transgenic mice: induction of epithelialhyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 6(1):1121-1125
Sands BE, Podolsky DK. 2006. The trefoil peptide family. Ann Rev Physiol.. 5 (8): 253—255
Seare NJ, Playford RJ. 1998. Growth factor and gut function. Proc Nutr Soc. 57: 403—408
Seydel AS, Miller JH, Sarac TP, Ryan CK, Sax HC.1996. Octreotide diminishes luminal nutrient transport activity, which is reversed by epidermal growth factor. Am J Surg.l7(2):267-270
Sibilia M, Wagner EF.2002. Strain dependent epithelial defects mice lacking the EGF receptor. Science. 26(9):234-235
Stig Purup, Mogens Vestergaard, Lone O Pedersen and Kris Sejrsen. 2007. Biological activity of bovine milk on proliferation of human intestinal cells. Journal of Dairy Research. 74: 58—65
Strain AJ.1993. Hepatocyte growth factor: another ubiquitous cytokine. J Endocrinol. 13 (7): 1-8
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Uribe JM, Barrett KE.1997. Nonmitogenic actions of growth factors: an integrated view of their role in intestinal physiology and pathophysiology. Gastroenterology .11(2):255—258
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Malo C, Menard D. 1982. Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology. 83: 28—35.
Markowitz, S.D.Molkentin K et al. 1990. Growth stimulation by coexpression of transforming growth factor-a and epidermal growth factor-receptor in normal and adenomatous human colon epithelium. J.Clin Invest. 86:356—362
Marti U, Burwen SJ, Jones AL. 2004. Biological effects of epidermal growthfactor, with emphasis on the gastrointestinal tract and liver: an update. Hepatology. (9): 126—133
Masaki Okada, Eiji Ohmura, Yoshinobu Kamiya, Hitomi Murakami et al. 1991 Transforming Growth Factor (TGF)-a in Human milk. Life Sciences. 48:115—1156
Mashimo H, Wu DC, Podolsky DK, Fishman MC.1996. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science. 27(4):262—265
Massague J. 1987. The TGF-b family of growth and differentiation factors. Cell. 4(9):437—440
McCleary MJ. 1991. Epidermal growth factor: An important constituent of human milk. J Hum Lact .7(3): 123—128.
Miettinen PJ, Berger JE, Meneses J, et al.2005. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature.37(6):337—339
Nightingale JM, Kamm MA, van der Sijp JR, Ghatei MA, Bloom SR, Lennard-Jones JE. 1996.Gastrointestinal hormones in short bowel syndrome. Peptide YY may be the 'colonic brake to gastric emptying. Gut. 3(9):267-269。
Odle J, Zijlstra RT, Donovan SM. 1996. Intestinal effects of milkborne growth factors in neonates of agricultural importance. J Animal Sci. 7(4):2509-2512
Peptrides PE, Hosang M, Shooter EM, Bohlen P. 1984. Transforming growth factors in human milk: Isolation and partial chemical and biological characterization. Proc 7th Int Congr Endocrinol.p1139.
Plaut AG. 1997. Trefoil peptides in the defense of the gastrointestinal tract. N Engl J Med 33(6):506-509
Playford RJ, Marchbank T, Goodlad RA, Chinery RA, Poulsom R, Hanby AM. 1996.
Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage. Proc Nat Acad Sci USA. 9(3): 2137—2139
Playford RJ, Wright NA. 1006. Why is epidermal growth factor present in the gut lumen Gut 3(8):303—306
Playford RJ.1005. Peptides and gastrointestinal mucosal integrity. Gut. 37:595-598
Polk DH: 1992. Do breast milk-derived hormones play a role in neonatal development. Early Hum Dev. 29:329-331.
R. Pakkanen, J. Aalto. 1997. Review paper growth factors and antimicrobial factors of bovine colostrums. Dairy Journal. (7):285—297
Rao RK. 1991. Biologically active peptides in the gastrointestinal lumen. Life Sci. 4(8):1685-1688
Raymond J Playford, Christopher E Macdonald, and Wendy S Johnson. 2000. Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders. Am J Clin Nutr 72:5—14.
Sandgren EP, Luetteke NC, Plamiter RD, Brinster RL, Lee DC. 1990. Overexpression of TGF α in transgenic mice: induction of epithelialhyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 6(1):1121-1125
Sands BE, Podolsky DK. 2006. The trefoil peptide family. Ann Rev Physiol.. 5 (8): 253—255
Seare NJ, Playford RJ. 1998. Growth factor and gut function. Proc Nutr Soc. 57: 403—408
Seydel AS, Miller JH, Sarac TP, Ryan CK, Sax HC.1996. Octreotide diminishes luminal nutrient transport activity, which is reversed by epidermal growth factor. Am J Surg.l7(2):267-270
Sibilia M, Wagner EF.2002. Strain dependent epithelial defects mice lacking the EGF receptor. Science. 26(9):234-235
Stig Purup, Mogens Vestergaard, Lone O Pedersen and Kris Sejrsen. 2007. Biological activity of bovine milk on proliferation of human intestinal cells. Journal of Dairy Research. 74: 58—65
Strain AJ.1993. Hepatocyte growth factor: another ubiquitous cytokine. J Endocrinol. 13 (7): 1-8
suckling. JNutr. 119:15431—551.
Taylor RG, Fuller PJ. Humoral regulation of intestinal adaptation. 1994. Baillieres Clin Endocrinol Metab 8:165—169
Thornberg W, Matrisian L, Magun B, Koldovsky O. 1984. Gastrointestinal absorption of epidermal growth factor in suckling rats. Am J Physiol. 246:G80—G85.
Threadgill DW, Dlugosz AA, Hansen LA, et al. 1995. Targeted disruption of the mouse EGF receptor: effect of genetic background on mutant phenotype. Science.26(9):230—232
Uehara Y, Minowa O, Mori C, et al. 1995. Placental defect and embryoniclethality in mice lacking hepatocyte growth factor/scatter factor. Nature. 37(3):702—704
Uribe JM, Barrett KE.1997. Nonmitogenic actions of growth factors: an integrated view of their role in intestinal physiology and pathophysiology. Gastroenterology .11(2):255—258
Wagner CL, Anderson DM, Pittard WB. 2004. Special properties of human milk. Clin Pediatr.35:283-293.
Wagner CL, Forsythe DM, 1995. Pittard WB III: Variation in the biochemical forms of transforming growth factor-alpha present in human milk and secreted by human milk macrophages.Biol Neonate. 68:325—333.
Watson H.-S. Hsieh. Jane C.-J. Chao et al. 1997. Effects of epidermal growth factor on total cellular RNA, protein and leucine aminopeptidase in human colon adenocarcinoma (Caco-2) cells.Nutritional Sciences Journal. 22:445—456
Weaver LT, Laker MF, Nelson R. 1984. Intestinal permeability in the newborn. Arch Dis Child. 59:236-241.
Whitman M, Melton DA. 1989. Growth factors in early embryogenesis. AnnRev Cell Biol .5:93—96
Wright NA, Pike C, Elia G. 1990. Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature. 343:82—85
Yasui W, Ji Z, Kuniyasu K, Ayhan A, Yokozaki H, Ito H, Tahara E.2002. Expression of transforming growth factor alpha in human tissues: Immunohistochemical study and Northern blot analysis. VirchowsArch A Pathol Anat .421:513—519.