【作者】 古兆森；

【导师】 王淑玲；

【作者基本信息】 郑州大学 ， 中西医结合临床， 2012， 硕士

【摘要】 背景与目的：观察补肾疏肝方含药血清对人肺腺癌A549细胞生长的影响及补肾疏肝方对Lewis肺癌荷瘤小鼠免疫及内分泌的影响并研究其中可能存在的机制。方法：补肾疏肝方含药血清对人肺腺癌A549细胞生长的影响实验。选取健康的SD大鼠30只,随机分为五组：模型组、DDP组、补肾疏肝方低剂量组、补肾疏肝方中剂量组、补肾疏肝方高剂量组,每组6只。模型组给予生理盐水4ml/天；DDP组给予0.5mg/mlDDP液2ml,分别于第1、3、5天腹腔注射；补肾疏肝方低剂量组给予中药4ml/天灌胃(1.75g/ml共计含生药7g)；补肾疏肝方低剂量组给予中药4ml/天灌胃(3.5g/ml共计含生药14g)；补肾疏肝方低剂量组给予中药4ml/天灌胃(5.25g/ml共计含生药21g)。共干预5天。第5天干预2小时后腹主动脉取血,制备大鼠含药血清,-20℃冰箱保存。临用时配制为含血清10%的培养液培养细胞,MTT法测OD值,观察补肾疏肝方含药血清对细胞生长的抑制作用。补肾疏肝方对Lewis肺癌荷瘤小鼠免疫及内分泌的影响实验。植瘤5天后,于腋下可触及约5mm*5mm肿块。选取荷瘤成功的C57BL/6小鼠40只,随机分为四组：模型组、DDP (cisplatin)组、补肾疏肝方组、DDP+中药组。每组10只。模型组给予0.9%NaCl灌胃0.4ml/天,同时于第1、3、5天腹腔注射0.9%NaCl0.5ml。DDP组小鼠分别在实验第1、3、5天给予腹腔注射0.5mlDDP(0.2mg/m1)溶液,并给予0.9%NaCl灌胃0.4ml/天。补肾疏肝方组给予中药灌胃0.4ml/天(1.75g/ml共计含生药0.7g),同时于第1、3、5天腹腔注射0.9%NaCl0.5ml。DDP+中药组给予每日中药灌胃,同时分别于第1、3、5天腹腔注射DDPO.5ml。共干预14天。每日记录小鼠精神状态、活动状况、毛发饮食及大小便情况,每天称取小鼠体重,测肿瘤体积。于第15天摘眼球取血,剥取肿瘤称重,计算抑瘤率、肺转移抑制率等。采取酶联免疫吸附(ELISA)的方法检测血清IL-2、IL-10、E2、T含量。结果：1、体外实验中,与模型组相比,中药低剂量组48h、72h,中剂量组、高剂量组、顺铂组均可以抑制肺腺癌A549细胞生长,差异明显(p<0.01),并呈时间-剂量依赖性。高剂量组与顺铂组差异不明显(p>0.05)。2、体内实验中,与模型组相比,各用药组均能抑制肿瘤生长,差异明显(p<0.01)。与中药组相比较,中西医结合组平均瘤重显著降低,有统计学意义(p<0.05)。3、与模型组相比,其余各组均能显著抑制肿瘤的自发性肺转移(p<0.05或p<0.01)。4、实验结束后,与模型组相比,DDP组小鼠体重均明显减轻,差异显著(p<0.05)；中药组及DDP+中药组小鼠体重均明显增加,差异显著(p<0.05)。与模型组相比,各用药组小鼠肿瘤体积明显较小(p<0.05)5、与模型组相比,各组血清IL-2含量均明显增高,差异显著(p<0.01);与DDP组相比,DDP+中药组明显增高(p<0.01)。与模型组相比,各用药组IL-10显著降低(p<0.01);与中药组和顺铂组相比,中西医结合组IL-10均显著降低(p<0.01)；与顺铂组相比,中药组IL-10降低有统计学意义(p<0.01)。6、与模型组相比,各用药组E2显著降低(p<0.01)；与顺铂组和中药组相比,中西医结合组E：表达降低有统计学意义(p<0.01)。与模型组相比,各用药组T显著升高;与顺铂组和中药组相比,中西医结合组T表达升高有统计学意义(p<0.01)。与模型组相比,各用药组E2/T显著降低(p<0.01)；与顺铂组和中药组相比,中西医结合组E2/T表达降低有统计学意义(p<0.01)。结论：1、补肾疏肝方含药血清可以抑制肺腺癌A549细胞生长,并呈时间-剂量依赖关系。2、补肾疏肝方可以显著抑制小鼠Lewis市癌生长,并与顺铂联合有协同作用。3、补肾疏肝方可以调节荷瘤小鼠体内免疫及内分泌平衡,这可能是本方抑制肿瘤生长的机制之一。4、补肾疏肝方可以提高小鼠生活质量,减轻化疗引起的毒副作用。更多还原

【Abstract】 Objective:To investgate the effect of Bushen Shugan(BSSG) formula on immune function and endocrine system of lewis lung cancer mice and the effect of serum containing BSSG on the proliferation of A549cells and discuss the possible underlying mechanism.Methods:In Vitro Experiment.30healthy SD mice were randomly divided into five groups:model control group、DDP group、low-dose group、middle-dose group、 high-dose group of serum containing BSSG. Model control group was given saline4ml by intragastric administration every day; DDP group was given cisplatin solution2ml(containing DDP1mg)by the way of intraperitoneal injection on the first day、the third day and the fifth day; low-dose group was given BuShenShuGan formula4ml/d (containing crude chinese herb7g) by intragastric administration; middle-dose group was given BuShenShuGan formula4ml/d (containing crude chinese herb14g) by intragastric administration; high-dose group was given BuShenShuGan formula4ml/d (containing crude chinese herb21g) by intragastric administration; all the treaments lasted5days.At last,the mice blood were collected by puncturing the aorta abdominalis2hours after treaments on the fifth day.The serum containing BSSG was made,and store in the refrigerator of-20℃.We can observe the effect of serum containing BSSG on the proliferation of A549cells.In Vivo Experiment.40C57BL/6mice that had been successfully transplanted Lewis lung cancer model were randomly divided into four groups:model control group、DDP group、BSSG group、BSSG combined with DDP group. Model control group was given saline0.4ml by intragastric administration every day, and the same time was given saline0.5ml by the way of intraperitoneal injection on the first day、 the third day and the fifth day;DDP group was given cisplatin solution0.5ml (containing DDP0.1mg) by the way of intraperitoneal injection on the first day、the third day and the fifth day,and the same time was given saline0.4ml by intragastric administration every day; BSSG group was given BuShenShuGan formula0.4ml/d (containing crude chinese herb0.7g) by intragastric administration, and the same time was given saline0.5ml by the way of intraperitoneal injection on the first day、 the third day and the fifth day; BSSG combined with DDP group was given BuShenShuGan formula by intragastric administration and cisplatin solution by intraperitoneal injection which was the same as BSSG group and DDP group;all the treaments lasted14days. We observed the mental state、diet and activities of mice everyday, and measured body weight and tumor volume of mice every two days. At last,all the mice blood were collected by taking off the eyeballs on the fifteenth day, and tumor weight and numbers of spontaneous lung metastasis were also measured.The concentration of serum IL-2、IL-10、E2and T were measured by ELISA.Results:1、In vitro experiment, compared with model control group, low-dose group of BSSG(48h、72h)、middle-dose group、high-dose group and DDP group can inhibit the proliferation of A549cells(p<0.01),which has the dose effect and time effect. It is almost no difference between high-dose group and DDP group (p>0.05)2、In Vivo Experiment.Compared with model control group, the other treatment group could significantly inhibit tumor growth, and the difference was significant (p<0.01). Compared with BSSG group, the average tumor weight of BSSG combined with DDP group decreased significantly (p＜O.05).3、Compared with model control group, the other treatment group could significantly inhibit the spontaneous lung metastasis (p<0.05or p<0.01).4、After the experiment finished, compared with model control group, the weight of DDP group decreased (p<0.05); BSSG group and BSSG combined with DDP group were significantly increased (p<0.05), there was no difference between Chinese herbal medicine group and combined group (p>0.05). Compared with model control group, tumor volume of every treatment group was significantly smaller (p<0.05); compared with DDP group and BSSG group, though the tumor volume of combined group was smaller, the difference was not significantly(p>0.05).5、Compared with model group, the blood serum IL-2of all treatment groups increased significantly (p<0.01); compared with DDP group, BSSG combined with DDP group increased significantly (p<0.01). Compared with model control group, the blood serum IL-10of the treatment groups significantly decreased (p<0.01); compared with DDP group, the blood serum IL-10of BSSG group was significantly decreased (p<0.05).6、Compared with model control group, the blood serum E2of all treatment groups decreased significantly (p<0.01); compared with DDP group and BSSG group, the blood serum E2of BSSG combined with DDP group decreased significantly (p<0.01). Compared with model control group, the blood serum T of all treatment groups increased significantly (p<0.01); compared with DDP group and BSSG group, the blood serum T of BSSG combined with DDP group increased significantly (p<0.01). Compared with model control group, the blood serum E2/T of all treatment groups decreased significantly (p<0.01); compared with DDP group and BSSG group, the blood serum E2/T of BSSG combined with DDP group decreased significantly (p<0.01).Conclusion:1、Serum containing BSSG can inhibit the proliferation of A549cells, which has the dose effect and time effect.2、BuShenShuGan Formula could significantly inhibit growth and spontaneous lung metastasis of Lewis lung cancer, and there was synergistic effect with cisplatin.3、BSSG Formula can make an effect on the level of blood serum IL-2、IL-10、E2and T.It can improve the functions of immunity and regulate internal secretion, and this may be one of the mechanisms of the inhibition of tumor growth.4、BuShenShuGan Formula could improve the quality of the mice life, reduce the side effects caused by chemotherapy such as weight loss and so on.更多还原