双歧杆菌脂磷壁酸体外诱导胃癌细胞BGC823凋亡的实验研究
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摘要
目的：探讨双歧杆菌脂磷壁酸（LTA）体外诱导胃癌细胞凋亡的作用及机制。
方法：采用MTT试验、电镜观察及流式细胞仪检测双歧杆菌LTA对体外培养的胃癌细胞BGC823的抑制作用、超微结构及细胞周期的变化，并用免疫组化技术检测LTA对凋亡相关蛋白c-fos、p53、bcl-2、c-myc表达的影响。
结果： MTT试验结果显示20μg～80μg/ml的LTA对体外培养的BGC823细胞具有明显的生长抑制作用，且LTA的浓度越大、作用时间越长，其抑制作用越强，具有浓度和时间依赖性。20μg/ml LTA作用72h后,对BGC823细胞的生长抑制率达到53%，80μg/ml LTA作用72h后，对BGC823细胞的生长抑制率达到79%。电镜观察发现未经LTA作用的对照组细胞核大，常染色质丰富，细胞浆中线粒体、内质网、高尔基体等细胞器少，微绒毛较丰富。经50μg/ml LTA作用72h后细胞微绒毛消失，细胞质电子密度加深，出现大量脱落的凋亡小体。流式细胞仪检测发现，50μg/ml LTA作用48h后能将细胞阻滞于G0/G1期, 在细胞周期G1峰的左侧出现了凋亡峰。免疫组化结果显示，LTA
能上调c-fos、p53蛋白的表达、并使bcl-2、c-myc蛋白的表达下降。
结论：双歧杆菌LTA能诱导胃癌细胞株BGC823凋亡，细胞被阻滞在G1期，同时上调BGC823细胞中c-fos、p53蛋白的表达，使bcl-2、c-myc的表达下降，这可能是双歧杆菌LTA诱导肿瘤细胞凋亡的机制之一。
Object: Explore the role and mechanism of bifidobacteria lipoteichoic acid in inducing apoptosis of gastric cancer in vitro.
Method: Through MTT, electron microscope, flow cytometer, detecting inhibitory effect of bifidobacteria lipoteichoic acid to gastric cancer cell strain BGC823, the change of cell ultrastructure and cell cycle. Meanwhile detecting changes of protein related to apoptosis c-fos, p53, bcl-2, c-myc by immunocytochemistry.
Result: Result of MTT shows LTA with concentration from 20μg/ml to 80μg/ml can suppress growth of BGC823, and that effect increase with higher concentration or longer time shows time dependence and concentration dependence. After being treated with 20μg/ml LTA for 72 hour, growth inhibitory rate is 53%. And treated with 80μg/ml LTA for 72 hour, growth inhibitory rate is 79%. In result of electron microscope, cells
of contrast group have large nuclear, plenty of euchromatin and microvillus, and organells, such as mitochondrion, Endoplasmic reticulum and Golgi apparatus are less. After being treated with 50μg/ml LTA for 72 hours, microvillus disappear, electron density of cytoplasm is high and organells is not clear, large deciduous apoptosis bodies appear. In result of flow cytometer, after dealt with 50μg /ml LTA for 48 hours, cell cycle is controlled in G0/G1, and there is a apoptosis peak left-side of G1 peak. In result of immunocytochemistry LTA induce up-regulation expression of c-fos, p53, and down-regulation expression of bcl-2, c-myc. Conclusion: Bifidobacteria lipoteichoic acid can induce apoptosis of gastric cancer BGC823 cells, cell cycle is controlled in G1. Meanwhile, LTA can induce up-regulation the expression of c-fos and p53 and down-regulation the expression of bcl-2 and c-myc，which may be the mechanism of apoptosis of gastric cancer cells by Bifidobacteria lipoteichoic acid.
方法：采用MTT试验、电镜观察及流式细胞仪检测双歧杆菌LTA对体外培养的胃癌细胞BGC823的抑制作用、超微结构及细胞周期的变化，并用免疫组化技术检测LTA对凋亡相关蛋白c-fos、p53、bcl-2、c-myc表达的影响。
结果： MTT试验结果显示20μg～80μg/ml的LTA对体外培养的BGC823细胞具有明显的生长抑制作用，且LTA的浓度越大、作用时间越长，其抑制作用越强，具有浓度和时间依赖性。20μg/ml LTA作用72h后,对BGC823细胞的生长抑制率达到53%，80μg/ml LTA作用72h后，对BGC823细胞的生长抑制率达到79%。电镜观察发现未经LTA作用的对照组细胞核大，常染色质丰富，细胞浆中线粒体、内质网、高尔基体等细胞器少，微绒毛较丰富。经50μg/ml LTA作用72h后细胞微绒毛消失，细胞质电子密度加深，出现大量脱落的凋亡小体。流式细胞仪检测发现，50μg/ml LTA作用48h后能将细胞阻滞于G0/G1期, 在细胞周期G1峰的左侧出现了凋亡峰。免疫组化结果显示，LTA
能上调c-fos、p53蛋白的表达、并使bcl-2、c-myc蛋白的表达下降。
结论：双歧杆菌LTA能诱导胃癌细胞株BGC823凋亡，细胞被阻滞在G1期，同时上调BGC823细胞中c-fos、p53蛋白的表达，使bcl-2、c-myc的表达下降，这可能是双歧杆菌LTA诱导肿瘤细胞凋亡的机制之一。
Object: Explore the role and mechanism of bifidobacteria lipoteichoic acid in inducing apoptosis of gastric cancer in vitro.
Method: Through MTT, electron microscope, flow cytometer, detecting inhibitory effect of bifidobacteria lipoteichoic acid to gastric cancer cell strain BGC823, the change of cell ultrastructure and cell cycle. Meanwhile detecting changes of protein related to apoptosis c-fos, p53, bcl-2, c-myc by immunocytochemistry.
Result: Result of MTT shows LTA with concentration from 20μg/ml to 80μg/ml can suppress growth of BGC823, and that effect increase with higher concentration or longer time shows time dependence and concentration dependence. After being treated with 20μg/ml LTA for 72 hour, growth inhibitory rate is 53%. And treated with 80μg/ml LTA for 72 hour, growth inhibitory rate is 79%. In result of electron microscope, cells
of contrast group have large nuclear, plenty of euchromatin and microvillus, and organells, such as mitochondrion, Endoplasmic reticulum and Golgi apparatus are less. After being treated with 50μg/ml LTA for 72 hours, microvillus disappear, electron density of cytoplasm is high and organells is not clear, large deciduous apoptosis bodies appear. In result of flow cytometer, after dealt with 50μg /ml LTA for 48 hours, cell cycle is controlled in G0/G1, and there is a apoptosis peak left-side of G1 peak. In result of immunocytochemistry LTA induce up-regulation expression of c-fos, p53, and down-regulation expression of bcl-2, c-myc. Conclusion: Bifidobacteria lipoteichoic acid can induce apoptosis of gastric cancer BGC823 cells, cell cycle is controlled in G1. Meanwhile, LTA can induce up-regulation the expression of c-fos and p53 and down-regulation the expression of bcl-2 and c-myc，which may be the mechanism of apoptosis of gastric cancer cells by Bifidobacteria lipoteichoic acid.
引文
戴建宜,王立生,朱惠明, 双歧杆菌的完整肽聚糖对实验性结直肠癌信号转导的影响, 中华胃肠外科杂志,2003,006(005):329-332
Biffi A, Coradini D, Larsen R, et al. Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line. Nutr Cancer, 1997, 28(1):93-99
Jung-Woo Lee, Jung-Gul Shined al. Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and bifidobacterium longum. J Vet Sci,2004,5(1):41–48
Baricault L, Denariaz G, Houri JJ, et al. Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation. Carcinogenesis.1995, 16(2):245-252
王跃,邓一平, 双歧杆菌脂磷壁酸对HL-60细胞端粒酶活性的影响, 中华微生物学和免疫学杂志,2000,020(004):316-318
蒋虹,胡宏, 双歧杆菌表面分子诱导大肠癌细胞分化的研究, 中华微生物学和免疫学杂志,2001,021(002):186-190
王立生,朱惠明,潘令嘉等, 双歧杆菌的完整肽聚糖对实验性大肠癌PCNA、caspase-3和iNOS表达的影响, 世界华人消化杂志.2001, 009(010):1196-119
Iain C. Sutcliffe, Stephen D. Hogg. Extraction of lipoteichoic acid from Streptococcus mutans with the non-ionic detergent Triton X-114. Journal of Microbiological Methods,1993,17(1993):215-225
乐军,胡宏, 非离子型去垢剂TritonX-114提取两歧双歧杆菌的脂磷壁酸, 中国微生态学杂志,1997,9(4):58-59
Into T, Kiura K, Yasuda M, et al. Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation. Cell Microbiol,2004,6(2):187- 199
Sekine K, Toida T, Saito M, et al. A new morphologically characterized cell wall preparation (whole peptidoglycan) from Bifidobacterium infantis with a higher efficacy on the regression of an established tumor in mice. Cancer Res,1985,45(3):1300-1307
Kerr JFR, Wyllie AH, Cunie AR. Apoptosis basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J CANCER, 1972; 26:239-257
Kerr JFR, Winterford CM, Harnon BV, et al. Apoptosis: its significance in cancer and cancer therapy. Cancer, 1994, 74(4):2013-2026
Martin SJ, Green D. Apoptosis as a goal of cancer therapy. Curr Oncol, 1994, 6:616
朱惠明,王立生,王玉林等, 双歧杆菌的完整肽聚糖对实验性大肠癌bak和bax基因表达的影响, 中国微生态学杂志,2002, 014(002):65-66
王立生,朱惠明,潘令嘉等, 双歧杆菌的完整肽聚糖对实验性大肠癌NF—κB和IκBα的影响, 中国微生态学杂志,2002,12,14（6）:318-319,321
Baker PE, Gillis S, Smith KA. Monoclonal cytolytic T-cell lines. J Exp Med. 1979, 149(1):273-278
Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods, 1986, 89(2):271-277
Rhee YK, Bae EA, Kim SY, et al. Antitumor activity of Bifidobacterium spp. isolated from a healthy Korean. Arch Pharm Res.2000, 23(5):482-487
Anodrew L, Kung, Anders Z, et al. Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation. Cancer research, 1999, 50:7307-7317
谢锦玉主编, 现代细胞化学技术及其在中西医药中的应用[M],第一版, 北京：中医古籍出版社,1988:205-226
Enari M, Sakahira H, Yokoyama Heat al. A Caspases-activatted DNase that degrades DNA during apoptosis, and its inhibitor ICAD. NATURE,1998,391(6662):43-50
Koshida Y,Saegusa M, Okayasu I，et al. Apoptosis, cell proliferation and expression of Bcl-2 and Bax in gastric carcinomas: Immunohisto-chemical and clinic
pathological study. British Journal of Cancer,1997,75(3):367-373
Yang E, Korsmeyer S. Molecular thanatopsis: a discourse on the Bcl-2 family and cell death. Blood,1996,88:386
Ogretmen B, Safa AR. Down-regulation of apoptosis-related bcl-2 but not bcl-xl or bax proteins in multidrug resistant MCF-7/Adr human breast cancer cells. Int J Cancer,1996,67(5):608-614
王立生,潘令嘉, 双歧杆菌的完整肽聚糖对实验性大肠癌凋亡控制基因表达的影响, 中国微生态学杂志,1999,011(004):195-198
Sionv R, Haupt Y. The cellular response to p53:decision between life and death[J]. Oncogene,1999,18(45):6145-6157
Caelles C, Heimberg A, Karin M. A new function for p53 in apoptosis. Trends cell boil,1994,4:356
Akikos S, David EF. P53 in life and death. Clin Cancer Res,1996,2(3):345
Fudazawa T, Fujiwara T, Morimoto A. Differential involvement of the CD95(Fas/Apo-1) receptor/ligand system on apoptosis induced by the wild type P53 gene transfer in human cancer cells. Oncogene,1999,18(13):2189
Tralongo V, Rodolico V, Luciani A, et al. Prognostic factors in oral squamous cell carcinoma. A review of the literature. Anticancer Res,1999,19(4C):3503-3510
孙抒,孙百研,智慧兰等, 胃癌及其癌前病变中细胞凋亡及其调控基因的表达, 中国病理生理杂志,2002,18(4):395-397
Simpson PC. Proto-oncogene and cardiac hypertrophy. Annu Rev Physiol,1989,51: 189
Miosege N, Schneider W, Gotz W, et al. The onco-proteins cerb-B2, c-fos and the tumor suppressor protein P53 in human embryos and feuses. Anat Embryol Berl,1997,195(4):345
Mils V, Piette J, Barrette C, et al. The proto-oncogene c-fos increases the sensitivity of kerationcytes to apoptosis. Onco-gene,1997,14(13):1555
Walker PR, Kwase-welfeld J, Gourdeall H, et al. Relation ship between apoptosis
and the cell cycle in lymphocytes: roles of protein Kinase C, tyrosine phosphorylation and AP1. Exp cell Res,1993,207:142
Sheerin A, Thompson KS, Goyns MH. Altered composition and DNA binding activity of the AP-1 transcription factor during the ageing of human fibroblasts. Mech Ageing Dev,2001,122(15):1813-1824
Richard J, Smeyne, Montserrat Vendrell, et al. continuous c-fos expression precedes programmed cell death in vivo. Nature,1993,363:166-169
Xu AH, Chen HS, Sun BC, et al. Therapeutic mechanism of ginkgo biloba exocarp polysaccharides on gastric cancer. World J Gastroenterol,2003,9(11): 2424-2427
Biffi A, Coradini D, Larsen R, et al. Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line. Nutr Cancer, 1997, 28(1):93-99
Jung-Woo Lee, Jung-Gul Shined al. Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and bifidobacterium longum. J Vet Sci,2004,5(1):41–48
Baricault L, Denariaz G, Houri JJ, et al. Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation. Carcinogenesis.1995, 16(2):245-252
王跃,邓一平, 双歧杆菌脂磷壁酸对HL-60细胞端粒酶活性的影响, 中华微生物学和免疫学杂志,2000,020(004):316-318
蒋虹,胡宏, 双歧杆菌表面分子诱导大肠癌细胞分化的研究, 中华微生物学和免疫学杂志,2001,021(002):186-190
王立生,朱惠明,潘令嘉等, 双歧杆菌的完整肽聚糖对实验性大肠癌PCNA、caspase-3和iNOS表达的影响, 世界华人消化杂志.2001, 009(010):1196-119
Iain C. Sutcliffe, Stephen D. Hogg. Extraction of lipoteichoic acid from Streptococcus mutans with the non-ionic detergent Triton X-114. Journal of Microbiological Methods,1993,17(1993):215-225
乐军,胡宏, 非离子型去垢剂TritonX-114提取两歧双歧杆菌的脂磷壁酸, 中国微生态学杂志,1997,9(4):58-59
Into T, Kiura K, Yasuda M, et al. Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation. Cell Microbiol,2004,6(2):187- 199
Sekine K, Toida T, Saito M, et al. A new morphologically characterized cell wall preparation (whole peptidoglycan) from Bifidobacterium infantis with a higher efficacy on the regression of an established tumor in mice. Cancer Res,1985,45(3):1300-1307
Kerr JFR, Wyllie AH, Cunie AR. Apoptosis basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J CANCER, 1972; 26:239-257
Kerr JFR, Winterford CM, Harnon BV, et al. Apoptosis: its significance in cancer and cancer therapy. Cancer, 1994, 74(4):2013-2026
Martin SJ, Green D. Apoptosis as a goal of cancer therapy. Curr Oncol, 1994, 6:616
朱惠明,王立生,王玉林等, 双歧杆菌的完整肽聚糖对实验性大肠癌bak和bax基因表达的影响, 中国微生态学杂志,2002, 014(002):65-66
王立生,朱惠明,潘令嘉等, 双歧杆菌的完整肽聚糖对实验性大肠癌NF—κB和IκBα的影响, 中国微生态学杂志,2002,12,14（6）:318-319,321
Baker PE, Gillis S, Smith KA. Monoclonal cytolytic T-cell lines. J Exp Med. 1979, 149(1):273-278
Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods, 1986, 89(2):271-277
Rhee YK, Bae EA, Kim SY, et al. Antitumor activity of Bifidobacterium spp. isolated from a healthy Korean. Arch Pharm Res.2000, 23(5):482-487
Anodrew L, Kung, Anders Z, et al. Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation. Cancer research, 1999, 50:7307-7317
谢锦玉主编, 现代细胞化学技术及其在中西医药中的应用[M],第一版, 北京：中医古籍出版社,1988:205-226
Enari M, Sakahira H, Yokoyama Heat al. A Caspases-activatted DNase that degrades DNA during apoptosis, and its inhibitor ICAD. NATURE,1998,391(6662):43-50
Koshida Y,Saegusa M, Okayasu I，et al. Apoptosis, cell proliferation and expression of Bcl-2 and Bax in gastric carcinomas: Immunohisto-chemical and clinic
pathological study. British Journal of Cancer,1997,75(3):367-373
Yang E, Korsmeyer S. Molecular thanatopsis: a discourse on the Bcl-2 family and cell death. Blood,1996,88:386
Ogretmen B, Safa AR. Down-regulation of apoptosis-related bcl-2 but not bcl-xl or bax proteins in multidrug resistant MCF-7/Adr human breast cancer cells. Int J Cancer,1996,67(5):608-614
王立生,潘令嘉, 双歧杆菌的完整肽聚糖对实验性大肠癌凋亡控制基因表达的影响, 中国微生态学杂志,1999,011(004):195-198
Sionv R, Haupt Y. The cellular response to p53:decision between life and death[J]. Oncogene,1999,18(45):6145-6157
Caelles C, Heimberg A, Karin M. A new function for p53 in apoptosis. Trends cell boil,1994,4:356
Akikos S, David EF. P53 in life and death. Clin Cancer Res,1996,2(3):345
Fudazawa T, Fujiwara T, Morimoto A. Differential involvement of the CD95(Fas/Apo-1) receptor/ligand system on apoptosis induced by the wild type P53 gene transfer in human cancer cells. Oncogene,1999,18(13):2189
Tralongo V, Rodolico V, Luciani A, et al. Prognostic factors in oral squamous cell carcinoma. A review of the literature. Anticancer Res,1999,19(4C):3503-3510
孙抒,孙百研,智慧兰等, 胃癌及其癌前病变中细胞凋亡及其调控基因的表达, 中国病理生理杂志,2002,18(4):395-397
Simpson PC. Proto-oncogene and cardiac hypertrophy. Annu Rev Physiol,1989,51: 189
Miosege N, Schneider W, Gotz W, et al. The onco-proteins cerb-B2, c-fos and the tumor suppressor protein P53 in human embryos and feuses. Anat Embryol Berl,1997,195(4):345
Mils V, Piette J, Barrette C, et al. The proto-oncogene c-fos increases the sensitivity of kerationcytes to apoptosis. Onco-gene,1997,14(13):1555
Walker PR, Kwase-welfeld J, Gourdeall H, et al. Relation ship between apoptosis
and the cell cycle in lymphocytes: roles of protein Kinase C, tyrosine phosphorylation and AP1. Exp cell Res,1993,207:142
Sheerin A, Thompson KS, Goyns MH. Altered composition and DNA binding activity of the AP-1 transcription factor during the ageing of human fibroblasts. Mech Ageing Dev,2001,122(15):1813-1824
Richard J, Smeyne, Montserrat Vendrell, et al. continuous c-fos expression precedes programmed cell death in vivo. Nature,1993,363:166-169
Xu AH, Chen HS, Sun BC, et al. Therapeutic mechanism of ginkgo biloba exocarp polysaccharides on gastric cancer. World J Gastroenterol,2003,9(11): 2424-2427
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