益气健脾抗癌法对胃癌脾虚本质的影响及抗肿瘤作用实验研究

【作者】 丁涛；

【导师】 殷东风；

【作者基本信息】 辽宁中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：胃癌是一种严重影响人们生命健康的恶性肿瘤，全球每年新发胃癌100余万例，中国占42%，死亡约80万，中国占35%，中国是胃癌发病率和死亡率最高的国家之一，发病率和死亡率均是世界平均水平两倍多。对胃癌的诊断及治疗是医学界关注的重要内容。在临床实践中，我们发现胃癌患者大多有中医脾虚证的表现，有疲乏、无力、食欲不振、抵抗力差、消瘦等表现。这是由于胃癌本身可以影响脾的运化和胃的受纳功能，而脾虚本身也降低了机体的抗邪能力，不利于控制胃癌，脾虚证和胃癌之间互相影响。前期临床研究已经初步证明，在“益气健脾抗癌”原则下形成的方剂治疗胃癌、肠癌，具有抗癌、改善患者的症状、减轻放化疗毒性等作用。胃癌的脾虚本质理论正是对胃癌和脾虚紧密相连关系的反映。在临床实践中，益气健脾抗癌法在治疗胃癌中具有重要的地位，通过实验研究揭示胃癌的脾虚本质以及益气健脾抗癌中药的作用机理具有重要的理论与实际意义。本实验研究正是基于这种胃癌脾虚本质的理论，建立人胃癌SGC-7901移植瘤裸小鼠模型，采用益气健脾中药及益气健脾抗癌中药干预胃癌鼠模型，观察对脾虚相关指标：瘤鼠体重、食量、饮水量、胃残留率、肠推进率、生长抑素、脾脏系数、脾脏蛋白激酶C（PKC）的影响；对肿瘤相关指标：瘤重、瘤体细胞凋亡及瘤体表皮生长因子（EGF）、血管内皮生长因子（VEGF）表达的影响，揭示胃癌的脾虚本质，及益气健脾抗癌法的抗癌作用机理。材料与方法：胃癌细胞株为人胃癌细胞株SGC-7901，实验动物为裸小鼠。药物：益气健脾中药组成：太子参、茯苓、白术、甘草、半夏、陈皮、木香、砂仁。抗癌中药组成：山慈菇、土茯苓、浙贝母、白花蛇舌草。建立人胃癌细胞SGC-7901裸鼠皮下移植瘤模型：首先将人胃癌SGC-7901细胞株体外培养，选择对数生长期的肿瘤细胞，在裸鼠腋下接种肿瘤细胞悬液0.1ml，接种15天后形成癌瘤。每个实验分为空白组、模型组、益气健脾组、益气健脾抗癌组，每组10只。空白组给予生理盐水、模型组给予生理盐水、益气健脾组给予益气健脾中药、益气健脾抗癌组给予益气健脾合用抗癌中药。各组均灌胃14天。第一部分实验是益气健脾抗癌法对胃癌小鼠消化吸收功能影响的实验研究，测定的指标为：小鼠体重变化、食量、饮水量、胃残留率、肠推进率，用放射免疫法测定小肠及血浆中生长抑素含量。第二部分实验为益气健脾抗癌法对胃癌小鼠脾脏PKC影响的实验研究，测定的指标为：脾脏系数、采用Western blot法测定脾脏PKC蛋白表达。第三部分实验为益气健脾抗癌法抑制胃癌小鼠肿瘤生长及对EGF、VEGF表达影响的实验研究，测定指标：瘤重、抑瘤率、采用TUNEL法测定瘤体细胞凋亡、采用Western-blot法测定瘤体EGF、VEGF表达。结果：1.体重增加量：空白组最多为5.99±0.24g，明显多于其它三组（p＜0.05），模型组体重增加最少为2.50±0.27g，而益气健脾组体重增加为4.21±0.35g，益气健脾抗癌组体重增加量为4.62±0.40g，均好于模型组（p＜0.05）；益气健脾组与益气健脾抗癌组对比则无统计学意义，但后者有好于前者的趋势。在食量与饮水量方面，表现出了与体重变化相同的结果，空白组最好，依次为益气健脾抗癌组、益气健脾组和模型组，其它三组均明显好于模型组。2.胃残留率及小肠推进率：空白组胃内残留率为31.2±11.4%、小肠推进率为65.4±9.6%，在各组中为最好，表现为胃内残留率最小，小肠推进率最大，其它三组与之比较有明显差别（p＜0.05），其中益气健脾抗癌组与空白组差别最小。模型组的胃内残留率为48.3±12.6%、小肠推进率为44.8±11.3%，在各组中为最差，其它三组与之比较有明显差别（p＜0.05）。益气健脾组胃内残留率为39.7±11.2%、小肠推进率为56.3±10.4%,益气健脾抗癌组胃内残留率为35.5±10.7%、小肠推进率为60.1±10.2%，给药两组比较无统计学差异，但益气健脾抗癌组有好于益气健脾组的趋势。3.生长抑素：生长抑素含量在空白组最低为，小肠和血浆含量分别为143±32ng/g及84±32ng/L；在模型组最高，分别为182±51ng/g、125±39ng/L；益气健脾组为177±46ng/g、110±42ng/L益气健脾抗癌组为160±42ng/g、102±38ng/L。各组和空白组比较有明显差异（p＜0.05）。给药两组和模型组比较有明显差异（p＜0.05）；益气健脾组与益气健脾抗癌组对比，后者表现出了好于前者的趋势，但无统计学意义。4.脾脏系数：空白组最大为1.17±0.12%，模型组脾脏系数最小为0.71±0.24%，益气健脾组为0.94±0.18%，益气健脾抗癌组为1.06±0.27%。和空白组比较，模型组和益气健脾组均小于空白组，有统计学意义（p＜0.05）；益气健脾抗癌组和空白组比较，脾脏系数略小，但无统计学差异；与模型组对比，其它三组的脾脏系数均明显大于模型组，有统计学意义（p＜0.05）。5.脾脏PKC表达：空白组最高为0.557±0.028，其它各组依次为益气健脾抗癌组0.509±0.010、益气健脾组0.442±0.017、模型组0.419±0.021。和空白组相比，模型组PKC表达降低最明显（P<0.01），益气健脾组及益气健脾抗癌组PKC表达也降低（P<0.01、P<0.05）；和模型组比较，益气健脾组和益气健脾抗癌组PKC表达高于模型组，且有明显统计学意义（P<0.01），其中益气健脾抗癌组最高，但仍低于空白对照组。6.瘤重和抑瘤率：模型组、益气健脾组、益气健脾抗癌组瘤重为分别为3.82±0.73g、2.76±0.25g、2.44±0.37g，药物治疗两组的肿瘤生长明显小于模型组，分析有统计学意义（p＜0.05）；益气健脾抗癌组的瘤重小于益气健脾组，对比有统计学意义（p＜0.05）。抑瘤率方面，益气健脾组和益气健脾抗癌组分别为28%、36%，益气健脾抗癌组明显高于益气健脾组。7.凋亡细胞数：模型组、益气健脾组、益气健脾抗癌组的凋亡细胞数分别为0.80±0.79、7.30±1.57、18.00±2.45个，给药两组多于模型组，结果有统计学意义（p＜0.01）。益气健脾组与益气健脾抗癌组对比，后者的凋亡细胞明显多于前者，二者差别有统计学意义（p＜0.01）。8. EGF、VEGF表达：模型组中EGF、VEGF表达最高，益气健脾组表达较之略低，但无统计学意义；益气健脾抗癌组EGF、VEGF表达明显低于前两者，结果有统计学意义（p＜0.05）。结论：1.胃癌能影响脾的运化功能，应用益气健脾中药治疗后，脾的功能有一定程度的改善，可以加强胃排空和小肠推进，但益气健脾联合抗癌中药后的健脾作用更加明显。2.胃癌能抑制脾脏本身的生长，并对脾脏中的PKC表达有抑制作用，应用益气健脾抗癌中药治疗后，这种抑制作用得到了减弱。3.益气健脾抗癌中药与单用益气健脾中药相比，其对肿瘤的抑制作用更加明显，不仅具有抗癌作用，而且对肿瘤生长相关的两个重要因素EGF、VEGF的表达有明显的抑制作用。4.胃癌能影响脾的功能，应用益气健脾抗癌中药治疗后，能达到健脾和抗癌双重治疗目的。因此，在临床治疗上，在应用益气健脾中药的同时，应该联合应用具有抗癌作用的中药，这种联合用药的原则体现了中医辨病与辨证治疗结合的治疗理念，值得进一步推广。更多还原

【Abstract】 Purpose：Gastric cancer is a serious impact on people’s lives and health ofmalignant tumor, around the world each year there were more than one millionnew cancer cases, China accounted for42%, killed about800thousands, Chinaaccounted for35%, China was one of countries with the highest morbidity andmortality of gastric cancer, incidence and mortality rate was more than two timesof the world average level. The diagnosis and treatment of gastric cancer wasthe important content of medical attention. In clinical treatment, we found thatmost gastric cancer patients had spleen-qi deficiency syndromes. They often hadthe symptoms of fatigue,weakness,loss of appetite, poor body resistance, weightloss and other clinical manifestations. Gastric cancer could affect theascending function of spleen and transport function of stomach. The spleendeficiency also reduced the body’s resistance to disease. These aspects madeit difficult to control gastric cancer. The spleen deficiency and stomach cancerinfluence each other. Preliminary clinical studies have proved that theformation of "Yiqi Jianpi anticancer" principle had the effect of anti-cancerin the treatment of digestive system tumor. It can improve the symptoms, reducethe toxicity of chemotherapy. The theory of the spleen deficiency essence ofgastric cancer reflects this relationship. In clinical practice, Yiqi Jianpianticancer method plays an important role in the treatment of gastric cancer.Through the experimental study reveals the spleen deficiency essence of gastriccancer, the mechanisms of Yiqi Jianpi anticancer drugs has important theoreticaland practical significance. This study is based on the theory of the spleendeficiency essence of gastric cancer, establishing human gastric cancer SGC-7901model in nude mice. Treat the gastric cancer mouse model with the traditionalChinese medicine of Yiqi Jianpi and Yiqi Jianpi anti-cancer. Observe relevantindexes of spleen deficiency: mice weight, food intake, water intake, gastricresidual rate, intestinal propulsion, somatostatin, spleen coefficient, spleen protein kinase C (PKC) effect; the parameters related to tumor growth and tumorbody: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF)expression of gastric cancer with deficiency of the spleen, revealing the spleendeficiency essence of gastric cancer and the mechanism of Yiqi Jianpi anti-cancerdrugs.Material and method：1. Choose human gastric carcinoma cell line SGC-7901, the experimental animalwas nude mice.2. Drugs: Chinese medicine Yiqi Jianpi include: Radix Pseudostellariae, Poria,Atractylodes, licorice root, pinellia, dried orange peel, Amomum villosum,costas. Anticancer medicine composition include: tulip, tuckahoe, ZhejiangFritillaria, Oldenlandia diffusa.3.To establish the model of human gastric cancer cell SGC-7901xenografts innude mice: the culture of human gastric cancer cell line SGC-7901in vitro,selection of tumor cells in logarithmic growth phase, nude mice inoculated with0.1ml tumor cells, the tumor will formate15days after inoculation.4. Administration: experiment animals were divided into blank group, model group,Yiqi Jianpi group, Yiqi Jianpi cancer group. There were10rats in each group.The blank control group were treated with normal saline; model group were givensaline, Yiqi Jianpi group were given Yiqi Jianpimedicine; Yiqi Jianpi anticancergroup were given Yiqi Jianpi anticancer medicine. The blank group and model group weretreated with normal saline for14days. The drug groups were given correspondingmedicine for14days.5. Indicators: The first part of the experiment was the experimental study YiqiJianpi anticancer effects on gastric cancer in mice by absorption digestion,the indexs: nude mice weight, food intake and water intake, gastric residualrate, intestinal propulsion rate, determination of the content of somatostatinin small intestine and in plasma by radioimmunoassay. The second study was theeffects of Yiqi Jianpi anticancer on the PKC of mice spleen, the indexes:expression of PKC protein was detected by Western blot method.The third part of the experiment was observation on gastric cancer tumor growth in mice andtumor EGF, VEGF expression, the index: tumor weight, inhibition rate of tumor,and tumor cell apoptosis was determined by TUNEL method, the expression of thetumor EGF, VEGF were determined by Western-blot method.Results：1．Amounts of weight gain: blank group was5.99±0.24g, significantly more thanthe other three groups (P <0.05), model group was2.50±0.27g, and Yiqi Jianpigroup was4.21±0.35g, Yiqi Jianpi anticancer group was4.62±0.40g, the otherthree groups were better than the model group (P <0.05). No statisticalsignificance of Yiqi Jianpi group and Yiqi Jianpi anticancer group was observed,but the latter was better than the former trend. In the aspect of food intakeand water intake, showed the same results with the weight change, blank groupwas the best, followed by Yiqi Jianpi anticancer group, Yiqi Jianpi group andmodel group, the other three groups were better than model group.2．gastric residual rate and intestinal propulsive rate: in the blank group,the gastric residual rate was31.2±11.4%and small intestinal propulsion ratewas65.4±9.6%, was the best in all groups, had the minimum gastric residualrate and the maximum small intestinal propulsion rate, there were significantdifference (P <0.05) compared with the other three groups, the difference ofYiqi Jianpi anticancer group and the blank group was minimum. In model group,the gastric residual rate was48.3±12.6%and small intestinal propulsion ratewas44.8±11.3%, was the worst in all groups, there were significant difference(P <0.05) compared with the other three groups. Yiqi Jianpi group, gastricresidual rate was39.7±11.2%, small intestinal propulsion rate was56.3±10.4%,Yiqi Jianpi anticancer group, gastric residual rate was35.5±10.7%, smallintestinal propulsion rate was60.1±10.2%, there was no difference between thetwo groups, but the Yiqi Jianpi anticancer group had a better trend to Yiqi Jianpigroup.3. Somatostatin. the amounts of somatostatin were lowest in the blank group,the small intestine and the plasma levels were143±32ng/g and84±32ng/L. the amounts of somatostatin were the highest In the model group, were182±51ng/gand125±39ng/L; In Yiqi Jianpi group were177±46ng/g and110±42ng/L. In YiqiJianpi anticancer group were160±42ng/g and102±38ng/L. There were obviousdifferences between groups and the blank group (P <0.05). compared with themodel there was significant difference between Yiqi Jianpi group and the modelgroup (P <0.05), there was significant difference between Yiqi Jianpi anticancergroup and the model group also(P <0.05). Compared with the Yiqi Jianpi group,the Yiqi Jianpi anticancer group showed a good trend, but without statisticalsignificance.4. Spleen coefficient: the spleen coefficient in blank group was1.17±0.12%,model group was0.71±0.24%, Yiqi Jianpi group was0.94±0.18%, Yiqi Jianpianticancer group was1.06±0.27%. Compared with the blank group, model groupand Yiqi Jianpi group were less than the blank group, there were statisticallysignificant (P <0.05); the Yiqi Jianpi anticancer group were little less thanthe blank group, but there was no significant difference between the two groups.Compared with the model group, spleen coefficient of the other three groups weresignificantly higher than model group, there were statistical significance (P<0.05).5. The expression of PKC in the spleen: blank group was0.557±0.028, other groupswere Yiqi Jianpi anticancer group0.509±0.010, Yiqi Jianpi group0.442±0.017,model group0.419±0.021. Compared with the blank group, in the model group,the expression of PKC decreased most significantly (P<0.01), In Jianpi Yiqi groupand Yiqi Jianpi anticancer group, the expression of PKC also decreased (P<0.01,P<0.05). Compared with the model group, the expression of PKC were higher inJianpi Yiqi group and Yiqi Jianpi anticancer group,there were statisticalsignificance (P<0.01), the expression of PKC of Yiqi Jianpi anticancer groupwas highest in the three groups, but still lower than the blank group.6.tumor weight and inhibition rate of tumor: the tumor weight of model group,Yiqi Jianpi group and Yiqi Jianpi anticancer group were3.82±0.73g,2.76±0.25gand2.44±0.37g, the tumor growth of the two groups were less than that in model group, there were statistical significance (P <0.05); the tumor weight of YiqiJianpi anticancer group was less than Yiqi Jianpi group, there was statisticallysignificant (P <0.05). The tumor inhibition rate of Yiqi Jianpi group amd YiqiJianpi anticancer group were28%,36%, there was obvious statisticalsignificance.7. The number of apoptotic cells: the number of apoptotic cells of model group,Yiqi Jianpi group and Yiqi Jianpi anticancer group were0.80±0.79,7.30±1.57and18±2.45, Compared with the model group, the Yiqi Jianpi group and the YiqiJianpi anticancer group were higher, there were statistically significant (P<0.01). Compared with the Yiqi Jianpi group, the number of apoptotic cells ofthe Yiqi Jianpi anticancer group, was significantly more than the former, therewere statistically significant difference (P <0.01).8. The expression of EGF, VEGF: model group, the expression of EGF, VEGF of modelwere the highest, the expression of Yiqi Jianpi groups were lower than the modelgroup, but there were no statistically significant; the expression of EGF, VEGFof Yiqi Jianpi anticancer group were significantly lower than that of the formertwo, there were statistically significant (P <0.05).Conclusion：1. Gastric cancer can affect the metabolic function of the spleen, the Yiqi JianpiChinese medicine can improve the spleen function, can strengthen gastricemptying and small intestinal propulsion, when the Yiqi Jianpi Chinese medicinewere used with the anticancer Chinese medicine together, the improve effect canbe more obvious.2. Gastric cancer can inhibit the growth of spleen, inhibit the expression of spleenPKC, application of Yiqi Jianpi anticancer treatment, this inhibitory effectwas weakened.3. Yiqi Jianpi anticancer treatment compared with the single Yiqi Jianpitreatment, the inhibitory effect on the tumor is more obvious, not only hadanticancer effects, but also can inhibit the expression of the EGF and VEGF,these were important genes related to tumor growth. 4. Gastric cancer can affect the function of the spleen, the application of YiqiJianpi anticancer treatment can achieve the dual goal of strengthening the spleenfunction and anticancer. Therefore, in clinical treatment, in the applicationof Yiqi Jianpi medicine, at the same time, the anticancer medicine should becombined, this combination principle embodies the combination of disease andsyndrome differentiation and treatment of TCM treatment philosophy, worthy offurther promotion.更多还原