乳源免疫调节肽（PGPIPN）对小鼠免疫和抗肿瘤的影响
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摘要
目的探讨乳源免疫调节肽(PGPIPN)对小鼠免疫与抗肿瘤方面的影响。
方法通过四甲基偶氮唑盐比色法(MTT法)检测在不同浓度的PGPIPN作用下体内、外Balb/c鼠的脾T淋巴细胞的转化情况;利用人卵巢癌细胞株SKOV3(人卵巢浆液性乳头状囊腺癌)建立荷人裸鼠模型,接种肿瘤细胞一周后,24只BALB/c-nu/nu(BALB/c Nude)雌性裸鼠被随机分为对照组(NS)、低剂量组(PGPIPN1)、高剂量组(PGPIPN2)和5-氟尿嘧啶组(5-fu),分别用生理盐水,低剂量免疫调节肽(0.4mg/ml),高剂量免疫调节肽(0.8mg/ml),5-氟尿嘧啶(30mg/kg/d)腹腔注射。四周后处死裸鼠,测量荷瘤裸鼠的瘤重、瘤体积,计算抑瘤率,观察其疗效,并用免疫组化、TUNEL染色、Western-blot、DNA ladder、Elisa等实验手段观察乳源免疫调节肽对卵巢癌的抑制作用及其作用机制。
结果1、PGPIPN在体外与刀豆蛋白A(ConA)合用可明显促进ConA的丝裂原作用;体内试验表明各种剂量PGPIPN饲养的小鼠,其脾淋巴细胞在适当剂量的ConA的诱导下均具有明显的T淋巴细胞转化的能力。2、荷人卵巢癌裸鼠经免疫调节肽治疗后出现肿瘤体积缩小,质量减轻。3、瘤组织中提取的DNA进行琼脂糖电泳均出现特征性DNA梯状电泳条带。4、TUNEL染色实验表明高、低剂量组均具有明显大于对照组的凋亡指数。5、在Elisa实验中,与对照组相比,血清中的TNF-α在高剂量组中有明显的升高而在低剂量组中则下降。6、免疫组化和western实验均显示了Bcl-2蛋白在高、低剂量组中的表达明显低于对照组,Bax蛋白在高、低剂量组中的表达明显高于对照组,Bcl-2与Bax的比值明显下降,说明免疫调节肽抑制卵巢癌过程过程可能与凋亡相关调节基因bcl-2下调、bax上调的表达有关。
结论1、PGPIPN具有促进T淋巴细胞转化的作用。2、PGPIPN有明显的抑瘤效应并介导凋亡,其可能机制是通过调节bax与bcl-2基因的表达。
Objective To study the effects of immunomodulating peptide (PGPIPN) deriving fromβ-cacein on immunity and anti-ovarian carcinoma in mice.
Method To investigate the effect of spleen T lymphocyte transformation impacted by PGPIPN at different concentrations with MTT method in vitro and in vivo. Twenty-four Balb/c-nu/nu (Balb/c Nude) mice were inoculated SKOV3 cells. After one week , 24 mice were randomly divided into control group, low dosage group, high dosage group and 5-fluorouracil (5-fu) group, which were given saline, the immunomodulating peptide at 0.4 and 0.8 mg/ml and 5-fluorouracil at 30mg/kg/d via celiac injection respectively. After administration of four weeks, the tumor weight, the tumor volume and tumor inhibitory rate were respectively calculated. The tumor tissue was observed by TUNEL. DNA ladder was observed by agarose gel electrophosesis. The Bcl-2 and Bax protein in tumor tissue were examined by immunohistochemistry and western blot. The levels of TNF-αand IFN-γwere detected by ELISA.
Results 1. In vitro, LTs were markedly improved under together ConA at 5ug/ml with PGPIPN at different concentrations, compared with ConA group. However, PGPIPN didn’t enhance spleen T lymphocyte transformation alone. In vivo, LT significantly improved in contrast with their control group, and there was a dose-dependent manner between PGPIPN and LT level. 2. Compared with the control group,the weights and volumes of the tumors were significantly decreased in the mice treated by PGPIPN. 3. Internucleosomal DNA fragments displayed the characteristic ladder-lane on electrophoresis in PGPIPN groups. 4. Compared with the control group,the apoptosis index (AI) were significantly increased in the treated mice by TUNEL staining. 5. The level of serum TNF-αwas significantly increased in high dosage group, but decreased in low dosage group compared with control. 6. Immunohistochemistry and western blot show that Bcl-2 expression was significant decreased, Bax experession were significantly increased, compared with the control group.
Conclusion 1. PGPIPN can promote LT both in vivo and in vitro 2. PGPIPN has significant inhibitory effect on tumor growth of human ovarian cancer in nude mice, could induce apoptosis in ovarian cancer cells by regulating the expression of Bcl-2 and Bax.
方法通过四甲基偶氮唑盐比色法(MTT法)检测在不同浓度的PGPIPN作用下体内、外Balb/c鼠的脾T淋巴细胞的转化情况;利用人卵巢癌细胞株SKOV3(人卵巢浆液性乳头状囊腺癌)建立荷人裸鼠模型,接种肿瘤细胞一周后,24只BALB/c-nu/nu(BALB/c Nude)雌性裸鼠被随机分为对照组(NS)、低剂量组(PGPIPN1)、高剂量组(PGPIPN2)和5-氟尿嘧啶组(5-fu),分别用生理盐水,低剂量免疫调节肽(0.4mg/ml),高剂量免疫调节肽(0.8mg/ml),5-氟尿嘧啶(30mg/kg/d)腹腔注射。四周后处死裸鼠,测量荷瘤裸鼠的瘤重、瘤体积,计算抑瘤率,观察其疗效,并用免疫组化、TUNEL染色、Western-blot、DNA ladder、Elisa等实验手段观察乳源免疫调节肽对卵巢癌的抑制作用及其作用机制。
结果1、PGPIPN在体外与刀豆蛋白A(ConA)合用可明显促进ConA的丝裂原作用;体内试验表明各种剂量PGPIPN饲养的小鼠,其脾淋巴细胞在适当剂量的ConA的诱导下均具有明显的T淋巴细胞转化的能力。2、荷人卵巢癌裸鼠经免疫调节肽治疗后出现肿瘤体积缩小,质量减轻。3、瘤组织中提取的DNA进行琼脂糖电泳均出现特征性DNA梯状电泳条带。4、TUNEL染色实验表明高、低剂量组均具有明显大于对照组的凋亡指数。5、在Elisa实验中,与对照组相比,血清中的TNF-α在高剂量组中有明显的升高而在低剂量组中则下降。6、免疫组化和western实验均显示了Bcl-2蛋白在高、低剂量组中的表达明显低于对照组,Bax蛋白在高、低剂量组中的表达明显高于对照组,Bcl-2与Bax的比值明显下降,说明免疫调节肽抑制卵巢癌过程过程可能与凋亡相关调节基因bcl-2下调、bax上调的表达有关。
结论1、PGPIPN具有促进T淋巴细胞转化的作用。2、PGPIPN有明显的抑瘤效应并介导凋亡,其可能机制是通过调节bax与bcl-2基因的表达。
Objective To study the effects of immunomodulating peptide (PGPIPN) deriving fromβ-cacein on immunity and anti-ovarian carcinoma in mice.
Method To investigate the effect of spleen T lymphocyte transformation impacted by PGPIPN at different concentrations with MTT method in vitro and in vivo. Twenty-four Balb/c-nu/nu (Balb/c Nude) mice were inoculated SKOV3 cells. After one week , 24 mice were randomly divided into control group, low dosage group, high dosage group and 5-fluorouracil (5-fu) group, which were given saline, the immunomodulating peptide at 0.4 and 0.8 mg/ml and 5-fluorouracil at 30mg/kg/d via celiac injection respectively. After administration of four weeks, the tumor weight, the tumor volume and tumor inhibitory rate were respectively calculated. The tumor tissue was observed by TUNEL. DNA ladder was observed by agarose gel electrophosesis. The Bcl-2 and Bax protein in tumor tissue were examined by immunohistochemistry and western blot. The levels of TNF-αand IFN-γwere detected by ELISA.
Results 1. In vitro, LTs were markedly improved under together ConA at 5ug/ml with PGPIPN at different concentrations, compared with ConA group. However, PGPIPN didn’t enhance spleen T lymphocyte transformation alone. In vivo, LT significantly improved in contrast with their control group, and there was a dose-dependent manner between PGPIPN and LT level. 2. Compared with the control group,the weights and volumes of the tumors were significantly decreased in the mice treated by PGPIPN. 3. Internucleosomal DNA fragments displayed the characteristic ladder-lane on electrophoresis in PGPIPN groups. 4. Compared with the control group,the apoptosis index (AI) were significantly increased in the treated mice by TUNEL staining. 5. The level of serum TNF-αwas significantly increased in high dosage group, but decreased in low dosage group compared with control. 6. Immunohistochemistry and western blot show that Bcl-2 expression was significant decreased, Bax experession were significantly increased, compared with the control group.
Conclusion 1. PGPIPN can promote LT both in vivo and in vitro 2. PGPIPN has significant inhibitory effect on tumor growth of human ovarian cancer in nude mice, could induce apoptosis in ovarian cancer cells by regulating the expression of Bcl-2 and Bax.
引文
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6 Kayser H, Meisel H. Stimulation of human peripheral blood lymphocytes by bioactive peptides derived from bovine milk proteins[J].FEBS Lett, 1996, 383(1-2):18-20.
7 Meisel H. Biochemical properties of regulatory peptides derived from milk proteins[J].Biopolymers, 1997, 43(2):119-28.
8徐静波,张建友,孙培龙,等.乳蛋白源生物活性肽的研究现状[J].中国酿造,2007(7):8-11
9 J.萨姆布鲁克,D.W.拉塞尔,等著;黄培堂等译分子克隆实验指南(第三版)(上,下)
10 Berthou J, Migliore-Samour D, Lifchitz A, Delettre J, Floc'h F and Jolles P, Immunostimulating properties and three-dimensional structure of two tripeptides from human and cow caseins. FEBS Lett 218:55-58 (1987).
11 Parker F, Migliore-Samour D, Floc'h F, Zerial A, et al, Immunostimulatinghexapeptide from human casein: amino acid sequence, synthesis and biological properties. Eur J Biochem 145:677-682 (1984).
12赵卫红,寿好长,阎福岭.细胞凋亡[M].河南医科大学,1997;12
13张雪玲,糜漫天,李等松,等.洛伐他汀对HL-60细胞bcl-2基因表达的影响.中国公共卫生,2004;20(6):658-659
14成军.程序性细胞死亡与疾病.北京:北京医科大学中国医科大学联合出版,1997;67
15王建东,潘星华.细胞凋亡调节的分子遗传机制[J].国外医学遗传学分册,1996;19(2):116
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17 Gillardon F, Wickert H, Zimmermann M. Up-regulation of bax and down-regulation of bcl-2 is associated with kainate-induced apoptosis in mouse brain. Neurosci Lett 1995, 192: 85-88.
18 Aadms JM, Cory S. The bcl-2 protein family: arbiters of cell surival. Science 1998, 281: 1322-1326.
19 Savory J,Rao JK,Huang Y,et al.Age-related hippocampal changes in Bcl-2:Bax ratio,oxidative stress,redox-active iron and apoptosis associated with aluminum-induced neurodegeneration: increased Susceptibility with aging. Neurotoxicology1999;20:805-17
20 Shalaby MR , Aggarwal BB , Rinderknecht E , et al.Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors[J]. J Immunol, 1985;135(3):2069-2073
21 Tsimberidou AM, Estey E, Wen S,et al. The prognostic significance of cytokine levels in newly diagnosed acute myeloid leukemia and high-risk myelodysplastic syndromes[J]. Cnncer,2008;113(7):1605-1613.
22 Talar-Wojnarowska R,Gasiorowska A,Smolarz B,et al. Tumor necrosis factor alpha and interferon gamma genes polymorphisms and serum levels in pancreatic adenocarcinoma. Neoplasma. 2009;56(1):56-62.
23 Hua-qiang Ouyang, Lu-ming Liu,Zhen Chen,et al. Effects of Qingyi Huaji decoction on serum levels of interleukin-6,interlukin-8 and tumor necrosis factor-alpha in nude mice bearing pancreatic tumors[J]. Journal of Chinese Integrative Medicine. 2010;8(7):655-660.
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2 Baldi A,Ioannis P,Chiara P,et al。Biological eeffes of milk porteins and their peptides with emphasis on these related to the gastrointestinal ecosyestm[J].J Dairy Res,2005,72(S1):66-72.
3 Migliore-Smaour D , Floch F , Jolles. P.Biologically active casein peptides implicated in immunomodulation.J Dairy Res[J],1989,56(3):357-362.
4 Miglioer-Smaour D,Jolles.P.Casein,a prohomrone with an immunomodulating role for the new bom?[J].Experientia,1988,44(3):188-193.
5 Fiat AM,Migliore-Smaour D,Jolles p,et al.Biologically active peptides from milk proteins with emphasis on two examples concerning antithrombotic and immunomodulating activities [J].J Dairy sci,1993,76(l):301-310.
6 Kayser H, Meisel H. Stimulation of human peripheral blood lymphocytes by bioactive peptides derived from bovine milk proteins[J].FEBS Lett, 1996, 383(1-2):18-20.
7 Meisel H. Biochemical properties of regulatory peptides derived from milk proteins[J].Biopolymers, 1997, 43(2):119-28.
8徐静波,张建友,孙培龙,等.乳蛋白源生物活性肽的研究现状[J].中国酿造,2007(7):8-11
9 J.萨姆布鲁克,D.W.拉塞尔,等著;黄培堂等译分子克隆实验指南(第三版)(上,下)
10 Berthou J, Migliore-Samour D, Lifchitz A, Delettre J, Floc'h F and Jolles P, Immunostimulating properties and three-dimensional structure of two tripeptides from human and cow caseins. FEBS Lett 218:55-58 (1987).
11 Parker F, Migliore-Samour D, Floc'h F, Zerial A, et al, Immunostimulatinghexapeptide from human casein: amino acid sequence, synthesis and biological properties. Eur J Biochem 145:677-682 (1984).
12赵卫红,寿好长,阎福岭.细胞凋亡[M].河南医科大学,1997;12
13张雪玲,糜漫天,李等松,等.洛伐他汀对HL-60细胞bcl-2基因表达的影响.中国公共卫生,2004;20(6):658-659
14成军.程序性细胞死亡与疾病.北京:北京医科大学中国医科大学联合出版,1997;67
15王建东,潘星华.细胞凋亡调节的分子遗传机制[J].国外医学遗传学分册,1996;19(2):116
16 Ohval ZN,Mikiman CI,Korsmeyer SJ.Bcl-2 heterodimerizes in vivo with a conserved homology,bax,that accelerates programmed cell death[J].Cell,1993;74: 609 619
17 Gillardon F, Wickert H, Zimmermann M. Up-regulation of bax and down-regulation of bcl-2 is associated with kainate-induced apoptosis in mouse brain. Neurosci Lett 1995, 192: 85-88.
18 Aadms JM, Cory S. The bcl-2 protein family: arbiters of cell surival. Science 1998, 281: 1322-1326.
19 Savory J,Rao JK,Huang Y,et al.Age-related hippocampal changes in Bcl-2:Bax ratio,oxidative stress,redox-active iron and apoptosis associated with aluminum-induced neurodegeneration: increased Susceptibility with aging. Neurotoxicology1999;20:805-17
20 Shalaby MR , Aggarwal BB , Rinderknecht E , et al.Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors[J]. J Immunol, 1985;135(3):2069-2073
21 Tsimberidou AM, Estey E, Wen S,et al. The prognostic significance of cytokine levels in newly diagnosed acute myeloid leukemia and high-risk myelodysplastic syndromes[J]. Cnncer,2008;113(7):1605-1613.
22 Talar-Wojnarowska R,Gasiorowska A,Smolarz B,et al. Tumor necrosis factor alpha and interferon gamma genes polymorphisms and serum levels in pancreatic adenocarcinoma. Neoplasma. 2009;56(1):56-62.
23 Hua-qiang Ouyang, Lu-ming Liu,Zhen Chen,et al. Effects of Qingyi Huaji decoction on serum levels of interleukin-6,interlukin-8 and tumor necrosis factor-alpha in nude mice bearing pancreatic tumors[J]. Journal of Chinese Integrative Medicine. 2010;8(7):655-660.
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