【作者】 李伟；

【导师】 曾小平； 闵卫平；

【作者基本信息】 南昌大学 ， 免疫学， 2015， 硕士

【摘要】 目的:观察瑞香素联合树突状细胞(DC)疫苗对小鼠H22肝癌移植瘤抗肿瘤免疫效应及其对荷瘤小鼠肿瘤细胞中抑凋亡基因Bcl-2及促凋亡基因Bax mRNA表达的影响,初步探讨瑞香素联合DC疫苗抗肿瘤的可能机制。方法:1、采用MTT法检测不同浓度瑞香素对人肝癌HepG2细胞和小鼠肝癌H22体外增殖的抑制作用;2、体外培养DC并在倒置显微镜下观察细胞形态,应用反复冻融法制备的肿瘤细胞全细胞抗原冲击DC制备DC肿瘤疫苗;3、瑞香素联合DC疫苗的体内抑瘤作用:建立小鼠H22肝癌荷瘤模型,将实验动物随机分为荷瘤对照组、5-Fu治疗组、瑞香素组、DC疫苗组、瑞香素联合DC疫苗组,另设健康对照组,每组8只,健康对照组和荷瘤对照组每鼠每天腹腔注射生理盐水0.2mL,共14天;5-Fu组按20mg/kg·d剂量每鼠每天腹腔注射0.2mL,共14天;瑞香素组按4mg/kg·d剂量每鼠每天腹腔注射瑞香素0.2mL,共14天;DC疫苗组于接种肿瘤细胞后的第1d、7d在接种部位瘤周皮下注射DC疫苗(5×106/只);瑞香素联合DC疫苗组:瑞香素按4mg/kg·d剂量每鼠每天腹腔注射瑞香素0.2mL,同时于接种肿瘤细胞后的第1d、7d在接种部位瘤周皮下注射DC疫苗(5×106/只)。第15天处死治疗小鼠,计算抑瘤率、脾脏指数、胸腺指数;4、检测小鼠NK细胞活性以及ConA诱导的脾T淋巴细胞增殖指数(MTT法);5、HE染色,光镜下观察H22肝癌组织病理学改变;6、ELISA法检测各组小鼠血清中细胞因子IFN-γ、ΙL-12、ΙL-4的水平;7、RT-qPCR法检测各组小鼠肿瘤组织Bcl-2、Bax mRNA的表达。结果:1、①瑞香素浓度(μg/mL)为250、125、62.5、31.25、15.62、7.81、3.90、1.95、0.97、0.48时,作用hepg2细胞48h,其抑制率(%)分别为:63.71±2.20、62.69±0.76、61.68±1.80、59.09±1.08、56.40±1.33、47.92±1.65和28.02±2.23、20.05±1.60、16.60±1.53、10.10±1.71,ic50值(μg/ml)为21.89±0.70。②瑞香素浓度(μg/ml)为250、125、62.5、31.25、15.62、7.81、3.90、1.95、0.97、0.48时,作用于h22细胞48h,其抑制率(%)分别为:85.52±5.15、65.04±4.72、61.04±5.94、50.77±7.40、47.26±8.49、45.21±6.37、36.52±3.53、33.55±8.26、23.62±8.67、19.79±5.47,ic50值(μg/ml)为16.89±9.64。2、各用药组对小鼠移植瘤生长均有抑制作用,以5-fu组最为明显。荷瘤对照组、5-fu组、瑞香素组、dc疫苗组、瑞香素联合dc疫苗组瘤质量(g)分别为1.817±0.388、0.424±0.309、0.833±0.314、1.206±0.523和0.493±0.306,均低于荷瘤对照组(p<0.05)。三治疗组间两两相比,差异有统计学意义(p<0.05),联合治疗组与5-fu相比,差异无统计学意义(p>0.05)。3、①瑞香素组、dc疫苗组、瑞香素联合dc疫苗组脾脏指数分别是8.504±0.508、10.878±3.916和12.902±2.927,均高于荷瘤对照组7.075±0.379(p<0.05)。联合治疗组高于与瑞香素组、dc疫苗组(p<0.05),但瑞香素组与dc疫苗组间无差异(p>0.05)。5-fu组脾脏指数为5.638±1.567,低于荷瘤对照组(p<0.05)。②三治疗组胸腺指数虽略高于荷瘤对照组,但差异无统计学意义(p>0.05)。而5-fu组胸腺指数低于荷瘤对照组和健康对照组,差异有统计学意义(p<0.05)。4、瑞香素组、瑞香素联合dc疫苗组nk细胞活性高于荷瘤对照组和5-fu组(p<0.05),其中瑞香素联合dc疫苗组的最高为(64.40±9.99)%,其次为瑞香素组(59.50±10.90)%。5-fu组则大大降低了脾淋巴细胞杀瘤活性,低于健康对照组和荷瘤对照组(p<0.05)。5、瑞香素组、瑞香素联合dc疫苗组cona诱导的t淋巴细胞增殖刺激指数高于荷瘤对照组和5-fu组(p<0.05);5-fu组cona诱导的t淋巴细胞增殖刺激指数,低于荷瘤对照组和健康对照组(p<0.05)。6、各组肿瘤组织的病理学变化:he染色显示,瑞香素联合dc疫苗组可见大面积坏死区,较多肿瘤细胞呈细胞核碎裂,核固缩等凋亡和坏死的形态学改变;荷瘤对照组坏死区较少,且坏死面积较小,肿瘤细胞生长旺盛,坏死及凋亡细胞较少;瑞香素组和dc疫苗组亦有明显坏死,但坏死程度较瑞香素联合dc疫苗组明显减轻。7、治疗组小鼠外周血细胞因子检测:①三治疗组血清IL-12含量均较荷瘤对照组高(P<0.05),其中,瑞香素联合DC疫苗组>瑞香素组>DC疫苗组(P<0.05)。②三治疗组血清IFN-γ含量均高于荷瘤对照组和5-Fu组,其中瑞香素联合DC疫苗组高于瑞香素组和DC疫苗组(P<0.05),但瑞香素组和DC疫苗组相比差异无显著性(P>0.05),瑞香素联合DC疫苗组也高于健康对照组(P<0.05)。③三治疗组血清IL-4含量均低于荷瘤对照组(P<0.05),其中瑞香素联合DC疫苗组又低于瑞香素组和DC疫苗组(P<0.05),瑞香素组和DC疫苗组相比,差异无统计学意义(P>0.05)。8、RT-qPCR实验结果显示,瑞香素组、DC疫苗组、瑞香素联合DC疫苗组均能下调瘤组织中Bcl-2 mRNA的表达,同时能上调瘤组织中Bax mRNA的表达,与荷瘤对照组相比,差异有统计学意义(P<0.05);其中瑞香素联合DC疫苗组瘤组织Bcl-2 mRNA与Bax mRNA比值最小。结论:1、瑞香素对HepG2细胞生长有不同程度的抑制作用,在0.48~250μg/mL浓度范围内呈现良好的剂量依赖性;瑞香素对H22细胞生长也有抑制作用,在0.48~250μg/mL浓度范围内亦呈现良好的剂量依赖性;2、瑞香素联合DC疫苗能够改善荷瘤小鼠的免疫功能状态,对小鼠H22移植瘤具有抑瘤作用,且效果好于单独使用瑞香素或DC疫苗。更多还原

【Abstract】 Objective:To observe the therapeutic effect of daphnetin combined dendritic cells(DCs) vaccine on H22-bearing mice and preliminarily explore its possible mechanism of anti-tumor. Methods:1、Use MTT methods to exam the surpress effect of inhibition daphnetin of different concentrations to human hepatoma Hep G2 cells and mice hepatoma proliferation of H22;2 、 Culture DC in vitro and observe cell morphology under an inverted microscope, use repeated freezing and thawing prepared tumor cells whole cell antigen pulse DC to prepare DC tumor vaccine;3、The anti-tumor effect of daphnetin combined with DC vaccines in vivo: establish a mouse hepatoma H22 tumor-bearing model, random Ly divide the tumor-bearing animals into a control group, 5-Fu group, daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group, set another healthy control groups, each group with eight mice. Inject saline 0.2m L intraperitoneally to both control group and tumor-bearing mice every day for 14 days;As for 5-Fu group, inject 0.2m L 5-Fu to each mouse intraperitoneally by 20 mg / kg·d dose everyday for 14 days; For daphnetin group, inject 0.2m L Daphnetin by 4mg / kg·d dose per mouse intraperitoneally everyday for 14 days; for DC vaccine group, subcutaneously inject DC vaccine around the inoculation peritumoral area after the first 1d and 7d of tumor cell inoculation(5×106/mouse). For daphnetin combined with DC vaccine group: inject 0.2m L daphnetin by 4mg/kg·d dose per mouse intraperitoneally everyday for 14 days; At the same time, subcutaneously inject DC vaccine around the inoculation peritumoral area after the first 1d and 7d of tumor cell inoculation(5×106/mouse). Sacrifice all the Mice under treatment after 15 days, count the tumor inhibition rate, spleen index and thymus index;4、Use MTT method to assay the activity of mice spleen natural killer(NK) cell and the proliferation index of Con A-induced T lymphocytes;5、HE staining, observe the pathological changes of liver cancer H22 under light microscope;6、Use ELISA to detect the levels of IFN-gamma、IL-12、IL-4 levels of cytokines in serum;7、Use real-time quantitative PCR method to detect the expression of Bcl-2、Bax m RNA in the tumor tissue of mice in each group. Results:1、①Inhibition rate(%) were from 63.71±2.20 to 10.10±1.71,when Hep G2 cells were treated by the daphnetin with the concentration from 250 to 0.48(μg/m L) lasts for 48 hours.IC50 value(μg/m L) was 21.89±0.70;②Inhibition rate(%) were from 85.52±5.15 to 19.79±5.47,when H22 cells were treated by the daphnetin with the concentration from 250 to 0.48(μg/m L) lasts for 48 hours. IC50 value(μg/m L) was 16.89±9.64.2、Treated groups have inhibition effects on the xenografts growth of mice of which the 5-Fu group is the most obvious. The tumor weight of tumor-bearing control group, 5-Fu group, daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group respeictively(1.817±0.388) g,(0.424±0.309) g,(0.833±0.314) g,(1.206±0.523) g,(0.493±0.306) g, the tumor weight of each group is lower than that of the tumor-bearing control group(P<0.05).The inhibition rate of daphnetin combined with DC vaccine group is the highest, the significance level of differences when compared to the daphnetin group was less than 0.5, and the significance level of differences when compared to the DC vaccine group was less than 0.5; The significance level of differences when DC vaccine group compared to the daphnetin group was less than 0.05.3、①The spleen index of daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group separately was 8.504±0.508,10.878±3.916 and 12.902±2.927, higher than the 7.075±0.379 of tumor-bearing control group(P<0.05). The significance level of the differences when combination therapy group compared to the daphnetin group and DC vaccine group was less than 0.5, but the difference between daphnetin group and DC vaccine group showing no statistically significant(P>0.05).The spleen index of 5-Fu group is 5.638±1.567, lower than that of the tumor-bearing control group(P<0.05). ② The thymus index of daphnetin group,daphnetin combined with DC vaccine group is higher than that of tumor-bearing control group, indicating that it can promote the thymus development of mice, but the difference is no statistically significant(P>0.05). But the thymus index of 5-Fu group is lower than that of tumor-bearing control group and the healthy control group, the significance level of differences was less than 0.5.4、Tumoricidal rate of NK cells of Daphnetin group, daphnetin combined with DC vaccine group is higher than that of tumor-bearing control group and 5-Fu group(P<0.05), the tumoricidal rate of daphnetin combined with DC vaccine group is up to(64.40±9.99)%, followed by daphnetin group of(59.50 ±10.90)%,while the 5-Fu group greatly reduces the NK cell killing activity, lower than the control group and tumor-bearing control group(P<0.05).5、T lymphocyte proliferation stimulation index induced by Con A of daphnetin group, daphnetin combined with DC vaccine is higher than tumor-bearing control group and 5-Fu group(P<0.05); T lymphocyte proliferation stimulation index induced by Con A of 5-Fu group is lower than that of the tumor-bearing control group and the healthy control group(P<0.05).6、Pathological changes of tumor tissues for each group:HE staining showes that daphnetin combined with DC vaccine group presenting large areaof necrosis,more tumor cells showing nuclear fragmentation,morphological changes of apoptosis and necrosis like nuclear condensation and so on; less necrotic area presents in tumor-bearing control group, and the necrotic area is less,vigorous growth of tumor cells, necrosis and apoptosis cells are less; daphnetin group and the DC vaccine group also present significant necrosis, but the degree of necrosis significantly reduces compared to daphnetin combined with DC vaccine group.7、Peripheral cytokines of the mice of the treated groups: ①The content of serum IL-12 of three treatment groups is higher than that of tumor-bearing control group(P<0.05), among which, daphnetin combined with DC vaccine group> daphnetin group>DC vaccine group(P<0.05). ②the content of serum IFN-gamma of three treatment groups is higher than that of the tumor-bearing control group and 5-Fu group, among which,the daphnetin combined with DC vaccine group is higher than that of daphnetin group and DC vaccine group(P<0.05), but there is no significant difference when daphnetin group compared to DC vaccine group(P>0.05), daphnetin combined with DC vaccine group is also higher than that of the healthy control group(P<0.05). ③the content of serum IL-4 of three treatment groups is lower than that of the tumor-bearing control group(P<0.05),among which the daphnetin combined with DC vaccine group is lower than that of daphnetin group and DC vaccine group(P<0.05), there is no statistical significance when daphnetin group compared to DC vaccine group(P>0.05).8 、 The RT-q PCR results show that Daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group can reduce the expression of Bcl-2 m RNA in tumor tissue and increase the expression of Bax m RNA in tumor tissues, the significance level of the diffierences when compared to the tumor-bearing control group was less than 0.5; wherein the Bcl-2 m RNA and Bax m RNA ratio of the daphnetin combined with DC tumor vaccine group is the smallest. Conclusion:1、Daphnetin has inhibition effect of different degrees on Hep G2 cell growth, showing a good dose-dependent in the concentration range of 0.48~250μg/m L; Daphnetin also has inhibition effect on H22 cell growth in the concentration range of 0.48~250μg/m L, also showing a good dose-dependent;2、Daphnetin combined with DC vaccine can improve the immune function in tumor-bearing mice,has inhibitory effect on mouse H22 transplanted tumor, and the effect is better than single use of daphnetin or DC vaccine.更多还原