医学平肺口服液抑制肺成纤维细胞生长和诱导凋亡专题.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,平肺口服液抑制肺成纤维细胞生长和诱导凋亡,研究背景,放射性肺炎及肺纤维化,是胸部放疗中部分正常肺组织因受到一定剂量射线照射而发生的放射损伤,肺成纤维细胞,是射线作用的效应细胞之一。它在射线作用下发生活化和增殖，持续合成并分泌细胞因子，从而导致胶原代谢调控失衡和肺组织纤维化,Imbalanced wound-healing response*,*Wilson MS,Wynn TA.Pulmonary fibrosis:pathogenesis,etiology and regulation.Mucosal Immunol.2009 Mar;2(2):103-21.,2,平肺口服液是中日友好医院药学部中药复方制剂，该方源自中西医结合肿瘤专家李佩文教授经验方，能用于放射性肺炎的辩证论治,1,。治疗原则：养阴清肺，解毒散结,实验研究表明，平肺口服液能有效预防大鼠放射性肺损伤,2,1 程志强，张嘉，刘轩等.平肺口服液在非小细胞肺癌放疗中的应用.中日友好医院学报,2009,23(6):334-336,2 刘轩，程志强，徐波等.平肺口服液对放射性肺炎大鼠肺组织细胞因子的调节作用.中国免疫学杂志,2009,25(9):808-812,3,A,B,C,D,E,HE staining in lung tissue of rats after a single whole thoracic irradiation with a dose of 20Gy,A:Normal control;B:Model group,4 weeks after irradiation;C:Model group,8 weeks after irradiation;D:Pingfei group,4 weeks after irradiation;E:Pingfei group,8 weeks after irradiation.,4,*,#,*,#,*,#,mRNA of IL-6,IL-10 and IFN-expression in the rat lung tissue 4 weeks after a single whole thoracic irradiation.,Note:Total RNA from the lung tissue was harvested at the 4th week after a dose of 20Gy irradiation in rat whole thoraces.mRNA of IL-6,IL-10 and IFN-expression was assayed by RT-PCR.Products of RT-PCR were electrophoresed in 2%agarose gel in the presence of ethidium bromide（80V,20min）,with the same samples beta-actin as internal control.The ratios of target gene vs internal control were calculated,and the results were expressed as meanstandard deviation（meansd）.The intensities of the specific bands were analysed using the software Quantity One 4.4.0(Bio-Rad)to determine whether the differences were statistically significant.,Compared with Control group,*.,P,0.01;Compared with Model group,#.,P,0.01;Compared with Model group,#.,P,0.05.,5,研究目的,采用血清药理学方法，研究平肺口服液对人胚肺成纤维细胞增殖及诱导细胞凋亡的作用。,6,材料与方法,平肺口服液10,g/kg,或20,g/kg,灌服,SD,大鼠,每日一次，连续7,d,第81,0d，,每日两次，两次灌服间隔12,h,第10,d,晚上灌服后禁食不禁水,第11,d,上午末次灌服后2,h,无菌腹主动脉取血,4,C,静置6,h，,离心，取上清，分装，-20,C,冻存,平肺口服液药物组成：百合15,g，,麦冬15,g，,五味子10,g，,白及10,g，,瓜蒌10,g，,鱼腥草30,g，,白花蛇舌草30,g，,桑白皮30,g，,浙贝母10,g。,生药含量 2,g/mL（,中日友好医院药学部，生产批号20070308）。,7,实验分组与检测方法,人胚肺成纤维细胞,CCC-HPF-1（,第14代）购自中国医学科学院基础所细胞库,普通培养组：完全培养基（含10%胎牛血清的高糖,DMEM）,血清对照组：完全培养基,+10%,正常大鼠血清,生药对照组：完全培养基,+,平肺口服液终浓度60,mg/mL,含药血清,A,组：完全培养基,+10g/kg,平肺口服液灌服所得血清,含药血清,B,组：完全培养基,+20g/kg,平肺口服液灌服所得血清,噻唑蓝（,MTT）,法检测细胞的增殖情况,流式细胞术检测细胞周期和凋亡,8,结 果,图,1,正常大鼠血清对,CCC-HPF-1,生长的影响,(meansd，,n,5),于完全培养基中加入10%正常大鼠血清，培养120,h,之内大鼠血清不会对细胞生长产生明显影响，因此以该实验条件研究平肺口服液含药血清对,CCC-HPF-1,生长的影响(图1)。,9,表,1,平肺口服液含药血清对,CCC-HPF-1,生长的抑制作用(,meansd,n,5),组别,含药血清或,药物剂量,OD,550nm,24,h,48,h,72,h,96,h,120,h,普通培养组,完全培养基(高糖,DMEM,中含10%,FBS),0.230.01,0.340.01,0.450.006,0.670.03,1.180.14,血清对照组,完全培养基10%正常大鼠血清,0.220.01,0.330.02,0.450.03,0.770.10,1.030.10,生药对照组,完全培养基平肺口服液60,mg/mL,0.230.02,0.370.02,0.420.05,0.400.03,*,0.510.07,*,含药血清,A,组,完全培养基10%平肺,A,组大鼠血清,0.200.003,0.310.03,0.420.04,0.600.06,1.080.10,含药血清,B,组,完全培养基10%平肺,B,组大鼠血清,0.230.002,0.340.02,0.450.03,0.460.08,0.530.06,注：与普通培养组相比，*,P,0.01,；与血清对照组相比，,P,0.05,；与血清对照组相比，,P,0.01。,应用,MTT,法检测,CCC-HPF-1,的增殖显示，各含药血清组在体外作用72,h,内对细胞生长均无明显影响。平肺口服液20,g/kg,灌服所获大鼠血清(含药血清,B,)在体外作用96,h,和120,h,可分别抑制细胞生长,(,表1，图2,)。,10,图,2,平肺口服液含药血清作用96,h,的,CCC-HPF-1,注：,a：,普通培养组；,b：,血清对照组；,c：,生药对照组；,d：,含药血清,A,组；,e：,含药血清,B,组,a,C,b,d,e,11,b,d,a,c,图,3,平肺口服液含药血清作用,96 h,对,CCC-HPF-1,细胞凋亡率的影响,注：,a.,普通培养组；,b.,血清对照组；,c.,生药对照组；,d.,含药血清,B,组,12,表,2,平肺口服液含药血清作用96,h,对,CCC-HPF-1,细胞周期、细胞凋亡率的影响,(meansd,n,3),组别,细胞周期/%,细胞,凋亡率/%,G1/G0,G2/M,S,普通培养组,68,2.59,17.8,1.7,14.23,1.1,0.58,0.16,血清对照组,51.4,2.65,25.1,2.44,23.43,3.36,4.19,0.96,生药对照组,53.4,5.40,26.93,2.87,19.7,2.56,1.01,0.44,含药血清,B,组,68.17,1.64,*,17.87,1.25,13.93,2.22,15.9,4.97,*,注：与血清对照组相比，*,P,0.05，*,P,0.01；,与普通培养组相比，,P,0.05，,P,0.01。,应用流式细胞术检测显示，平肺口服液,20 g/kg,灌服所获大鼠血清(含药血清,B),可使,CCC-HPF-1,细胞周期阻滞于,G1/G0,期，细胞凋亡增加(图3，表2)。,13,讨 论,中药血清药理学研究方法是目前联系体内与体外研究、揭示中药作用机理的重要桥梁。,建立平肺口服液血清药理学研究方法依据：,1.平肺口服液20,g/kg,灌服,(,造模前一周开始灌服，每日一次，每周5,d，,共5周,),可有效预防大鼠放射性肺损伤,2.血清药理学通法给药和采血原则，即将待测药每天给药两次，连续给药3,d，,末次给药后1,h2 h,采血,14,结 论,平肺口服液,20 g/kg,灌服所获含药血清作用,96 h,能抑制人胚肺成纤维细胞的生长，诱导细胞凋亡，以及使细胞周期发生,G1/G0,期阻滞。结合体内研究结果，认为该药有可能从抑制肺成纤维细胞增殖和诱导凋亡方面预防放射性肺损伤的发生。,谢谢！,15,