丹玄口康对槟榔提取物刺激下口腔粘膜成纤维细胞增殖活性的影响
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摘要
目的:运用血清药理学的方法,观察丹玄口康含药血清对槟榔提取物(ANE)刺激下的口腔粘膜成纤维细胞(FB)增殖活性及相关增殖因子表达的影响。
方法:1.将40只SD大鼠随机分成5组,分别给予生理盐水,雷公藤多甙片,丹玄口康片(小,中,大剂量)灌胃制备正常血清和含药血清。2.运用组织块法进行体外口腔粘膜成纤维细胞的常规培养,取第三或第四代细胞用于实验。(1)免疫细胞化学染色SP法鉴定口腔粘膜成纤维细胞。(2)将上述细胞接种后分为六组,加入不同浓度的ANE(0、5、50、100、150、200ug/ml)作用48小时后,MTT法检测细胞增殖,寻找ANE促进成纤维细胞增殖的最佳浓度。(3)将细胞接种后分为六组:正常组,ANE刺激组,西药对照组,丹玄口康组小、中、大剂量组,分别加入正常血清,含药血清和100ug/ml的ANE,作用48小时后。MTT法检测细胞增殖,记录细胞的吸光度值(OD);免疫细胞化学染色法检测细胞PCNA的表达,并对检测结果进行图像分析,记录平均光密度值(AO)。
结果:1.口腔粘膜成纤维细胞经SP法染色可见波形蛋白表达阳性,而细胞角蛋白表达阴性。2.槟榔提取物干预48小时后,50ug/ml,100ug/ml组OD值明显高于0 ug/ml组(P＜0.01)。3.西药对照组,丹玄口康组OD值均低于ANE刺激组(P＜0.05或P＜0.01),丹玄口康中、大剂量组OD值和西药对照组相比差异具有显著性意义(P＜0.01),丹玄口康小剂量组OD值与西药对照组相比无显著性差异(P＞0.05)。4.正常组,西药对照组,丹玄口康中、大剂量组AO值与ANE刺激组相比具有显著性差异(P＜0.05或P＜0.01),丹玄口康中、大剂量组与西药对照组相比具有显著性差异(P＜0.05或P＜0.01)。
结论:1.在50ug/ml～100ug/ml的范围内槟榔提取物促进口腔粘膜成纤维细胞增殖。2.雷公藤多甙含药血清抑制槟榔提取物作用下的口腔粘膜FB增殖及PCNA的表达。3.丹玄口康含药血清呈剂量依赖性的抑制槟榔提取物刺激下的口腔粘膜成纤维细胞增殖和PCNA的表达,能有效拮抗槟榔提取物对口腔粘膜成纤维细胞的促增殖作用,其作用优于雷公藤多甙片含药血清。
Objective:To observe effect of serum containing-DXKK on human oral mucosal fibroblasts stimulated by areca nut extract proliferation and related cytokines by serum pharmacological method in vitro.
Method:1) 40 SD rats were randomly divided into five groups, intragastric administration by normal saline,Lei Gong Teng Duo Dai(LGTDD) and DXKK(low、medium、large dose) separately to make blank serum and containing medicine serum.2) Cell culture experiments were performed using cutted human oral mucosa tissues.The cultured third or forth passage of human mucosa fibroblasts were used in the next steps.1)human mucosa fibroblasts were determined by the method of SP.2)These above-mentioned cells were divided into following six groups,were added areca nut extract(ANE)to different concentration(0、5、50、100、150、200ug~*ml~(-1)),after 48 hours,cells proliferation was detected by MTT,looking for the best concentration of ANE that promote cell proliferation.3) Those above-mentioned cells were divided into following six groups:normal groups,ANE stimulation groups,western medicine groups(LGTDD),DXKK groups(low、medium、large dose),added blank serum and containing medicine serum and 100ug*ml~(-1)ANE,after 48 hours, cells proliferation was detected by MTT,noted the optical density (OD);meantimes the expression of Proliferating cell nuclear nuclear antigen(PCNA) in each groups were detected through immunocytochemical stain technique,and the result were analyzed by videodensitomet,noted the average optical(AO).
Result:1)In our cultured fibroblasts,vimentin showing positive stain while keratin showing negative stain.2)After ANE treated FB 48 hours,50ug/ml and 100ug/ml groups OD was significant higher than 0 ug/ml groups(P＜0.01).3)DXKK and LGTDD groups OD was significantly lower than ANE stimulation groups(P＜0.05 or P＜0.01),MDXKK and LDXKK groups OD was significantly lower than LGTDD groups(P＜0.01).4)Normal groups,LGTDD groups,MDXKK、LDXKK groups AO was significantly lower than ANE stimulation groups(P＜0.05 or P＜0.01),and MDXKK,LDXKK groups compared with LGTDD groups,the difference was significant(P＜0.05 or P＜0.01).
Conclusion:1) ANE could promoted proliferation of oral mucosal fibroblasts at the concentration between 50ug/ml and 100ug/ml.2)Serum containing-LGTDD could inhibit expression of PCNA and proliferation of oral mucosal fibroblasts treated with ANE.3) Serum containing-DXKK could with relativity dose-dependent inhibit expression of PCNA and proliferation of oral mucosal fibroblasts treated with ANE,and this effection is better than Serum containing-LGTDD.
方法:1.将40只SD大鼠随机分成5组,分别给予生理盐水,雷公藤多甙片,丹玄口康片(小,中,大剂量)灌胃制备正常血清和含药血清。2.运用组织块法进行体外口腔粘膜成纤维细胞的常规培养,取第三或第四代细胞用于实验。(1)免疫细胞化学染色SP法鉴定口腔粘膜成纤维细胞。(2)将上述细胞接种后分为六组,加入不同浓度的ANE(0、5、50、100、150、200ug/ml)作用48小时后,MTT法检测细胞增殖,寻找ANE促进成纤维细胞增殖的最佳浓度。(3)将细胞接种后分为六组:正常组,ANE刺激组,西药对照组,丹玄口康组小、中、大剂量组,分别加入正常血清,含药血清和100ug/ml的ANE,作用48小时后。MTT法检测细胞增殖,记录细胞的吸光度值(OD);免疫细胞化学染色法检测细胞PCNA的表达,并对检测结果进行图像分析,记录平均光密度值(AO)。
结果:1.口腔粘膜成纤维细胞经SP法染色可见波形蛋白表达阳性,而细胞角蛋白表达阴性。2.槟榔提取物干预48小时后,50ug/ml,100ug/ml组OD值明显高于0 ug/ml组(P＜0.01)。3.西药对照组,丹玄口康组OD值均低于ANE刺激组(P＜0.05或P＜0.01),丹玄口康中、大剂量组OD值和西药对照组相比差异具有显著性意义(P＜0.01),丹玄口康小剂量组OD值与西药对照组相比无显著性差异(P＞0.05)。4.正常组,西药对照组,丹玄口康中、大剂量组AO值与ANE刺激组相比具有显著性差异(P＜0.05或P＜0.01),丹玄口康中、大剂量组与西药对照组相比具有显著性差异(P＜0.05或P＜0.01)。
结论:1.在50ug/ml～100ug/ml的范围内槟榔提取物促进口腔粘膜成纤维细胞增殖。2.雷公藤多甙含药血清抑制槟榔提取物作用下的口腔粘膜FB增殖及PCNA的表达。3.丹玄口康含药血清呈剂量依赖性的抑制槟榔提取物刺激下的口腔粘膜成纤维细胞增殖和PCNA的表达,能有效拮抗槟榔提取物对口腔粘膜成纤维细胞的促增殖作用,其作用优于雷公藤多甙片含药血清。
Objective:To observe effect of serum containing-DXKK on human oral mucosal fibroblasts stimulated by areca nut extract proliferation and related cytokines by serum pharmacological method in vitro.
Method:1) 40 SD rats were randomly divided into five groups, intragastric administration by normal saline,Lei Gong Teng Duo Dai(LGTDD) and DXKK(low、medium、large dose) separately to make blank serum and containing medicine serum.2) Cell culture experiments were performed using cutted human oral mucosa tissues.The cultured third or forth passage of human mucosa fibroblasts were used in the next steps.1)human mucosa fibroblasts were determined by the method of SP.2)These above-mentioned cells were divided into following six groups,were added areca nut extract(ANE)to different concentration(0、5、50、100、150、200ug~*ml~(-1)),after 48 hours,cells proliferation was detected by MTT,looking for the best concentration of ANE that promote cell proliferation.3) Those above-mentioned cells were divided into following six groups:normal groups,ANE stimulation groups,western medicine groups(LGTDD),DXKK groups(low、medium、large dose),added blank serum and containing medicine serum and 100ug*ml~(-1)ANE,after 48 hours, cells proliferation was detected by MTT,noted the optical density (OD);meantimes the expression of Proliferating cell nuclear nuclear antigen(PCNA) in each groups were detected through immunocytochemical stain technique,and the result were analyzed by videodensitomet,noted the average optical(AO).
Result:1)In our cultured fibroblasts,vimentin showing positive stain while keratin showing negative stain.2)After ANE treated FB 48 hours,50ug/ml and 100ug/ml groups OD was significant higher than 0 ug/ml groups(P＜0.01).3)DXKK and LGTDD groups OD was significantly lower than ANE stimulation groups(P＜0.05 or P＜0.01),MDXKK and LDXKK groups OD was significantly lower than LGTDD groups(P＜0.01).4)Normal groups,LGTDD groups,MDXKK、LDXKK groups AO was significantly lower than ANE stimulation groups(P＜0.05 or P＜0.01),and MDXKK,LDXKK groups compared with LGTDD groups,the difference was significant(P＜0.05 or P＜0.01).
Conclusion:1) ANE could promoted proliferation of oral mucosal fibroblasts at the concentration between 50ug/ml and 100ug/ml.2)Serum containing-LGTDD could inhibit expression of PCNA and proliferation of oral mucosal fibroblasts treated with ANE.3) Serum containing-DXKK could with relativity dose-dependent inhibit expression of PCNA and proliferation of oral mucosal fibroblasts treated with ANE,and this effection is better than Serum containing-LGTDD.
引文
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[11]蔡锐等.桃红四物汤加丹参对肝星状细胞增殖和凋亡的影响.湖 南中医学院学报,2005,25(6):22-24
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[2]方文贤,宋崇顺,周立孝.医用中药药理学,北京,人民卫生出版社,1998:899-910
[3]谭劲,陈安.活血化纤方治疗口腔粘膜下纤维性变的临床观察.中国中医药信息杂志,2001,8(1):71
[4]胡淳.疏肝活血冲剂治疗口腔粘膜下纤维性变20例.湖南中医学院学报,1999,19(1):46
[5]李元聪.中西医结合治疗口腔粘膜下纤维化22例[J].中国中医药科技,2002,9(5):308
[6]李元聪.丹玄口康治疗口腔粘膜下纤维化22例临床疗效观察.中国中医药科技,2002,9(2):308
[7]陈可冀,史载祥.实用血瘀证学.北京:人民卫生出版社,1999:120-207
[8]中国中西医结合学会活血化瘀研究学会.活血化瘀研究.北京:中国医学药科技出版社,1995;23-28
[9]孙红军,孙立群,阎秀欣等.丹参及细胞因子广对成纤维细胞增殖和细胞凋亡的影响.中国实验临床免疫学杂志,1997;9(3):25-27
[10]王益民等.丹参对体外培养成纤维细胞自分泌因子的影响.中国修复重建外科杂志,2002,16(6):408
[11]蔡锐等.桃红四物汤加丹参对肝星状细胞增殖和凋亡的影响.湖 南中医学院学报,2005,25(6):22-24
[12]张炜等.生地对特发性肺间质纤维化基质重建调控作用的实验研究.中国中医基础医学杂志,2001,7(6):34-35
[13]李自成等.当归提取液对培养的兔主动脉平滑肌细胞增殖的影响.中药药理与临床,1998,14(1):28-30[14]Ma RH,Tsaicc,Shieh TY.Increased lysyl oxides activity fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Tin Wan.J oral patholmed,1999;24(9):407-412
[15]成令忠主编.组织学和胚胎学.第四版.北京:人民卫生出版社,1995.30-32.
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