小牛脾提取物注射液抗肝癌和免疫调节作用的研究
发布时间：2018-11-28 15:46
【摘要】：恶性肿瘤是目前造成人类死亡的三大杀手之一,严重威胁人类的身体健康。而肝细胞癌是世界上最常见的肿瘤之一,每年肝癌导致全球662000人死亡,其中大约一半患者出现在中国。另外,肝癌治疗还具有预后不良、易复发和易转移等特点。目前,肝癌的治疗手段主要有化学治疗、放射治疗、手术治疗、基因治疗、经皮治疗和肝移植等六种方法,但这些手段不仅费用高昂还容易引起极大的毒副作用。小牛脾提取物注射液作为多组分生化提取物具有多靶点、低毒性的特点,但目前还没有系统的研究阐述其抗肿瘤活性及相关机制。本文主要研究小牛脾提取物注射液的抗肝癌活性及其相关机制,并对其免疫调节作用进行评价。本研究主要包括以下几个方面:1.小牛脾提取物注射液体外抗肝癌活性及其机制的研究考察小牛脾提取物注射液(CSEI)对多种癌症细胞的抑制率,其中,以肝癌细胞系Hep G2和SMMC-7721为主要研究对象考察小牛脾提取注射液对肝癌细胞系的促凋亡作用。同时,采用生化分析手段考察注射液促肝癌细胞凋亡的机制。实验结果显示,小牛脾提取物注射液在体外可以抑制人肝癌、乳腺癌和肺癌等细胞系的生长,其中注射液对肝癌细胞系SMMC-7721和Hep G2的抑制效果最明显,而且注射液可以增加肝癌细胞中ROS的产生、改变线粒体膜电位、激活caspase级联反应等。此外,采用Western blot检测细胞和肿瘤中凋亡相关蛋白PARP、cleaved-PARP、Bcl2、P38、phospho-P38、JNK、phospho-JNK1、ERK1/2、phospho-ERK1/2、cleaved-caspase3、cleaved-caspase 8和cleaved-caspase 9等的表达情况,同时显著降低氧化应激相关蛋白Nrf2和Catalase在肝癌细胞和肿瘤中的表达。综上所述,小牛脾提取物注射液抑制癌细胞增殖并通过ROS相关的线粒体途径诱导肝癌细胞的凋亡。2.小牛脾提取物注射液体内抗肝癌活性及其机制的研究为了进一步确定小牛脾提取物注射液的抗肝癌作用,建立Hep G2肝癌异位裸鼠模型。小牛脾提取物注射液治疗14天内考察整个治疗期间小鼠体重和肿瘤体积。结束治疗后,取肿瘤进行Western blot实验分析抗肿瘤及氧化应激相关蛋白质表达情况。实验结果证明,小牛脾提取物注射液能够使Hep G2肝癌异位模型中裸鼠的肿瘤体积显著下降,并对小鼠体重没有影响。与此同时,Western blot结果与体内实验一致。以上结果说明小牛脾提取物注射液具有良好的抗肝癌活性能,其通过ROS/MAPKs依赖的线粒体途径诱导肝癌细胞凋亡。3.小牛脾提取物注射液免疫调节活性的评价本文通过建立由环磷酰胺诱导的免疫低下模型研究小牛脾提取物注射液的免疫调节活性。经过14天的治疗,小牛脾提取物注射液(在临床使用剂量范围内)可以逆转由环磷酰胺诱导的免疫力低下,显著增加动物脾脏指数并恢复血清中IL-2,6,10,12和IFN-α,γ以及TNF-α等免疫和炎症相关细胞因子的水平,提高NK细胞杀伤活力和T淋巴的细胞的转化能力,同时通过增加动物脾脏中IKKβ的磷酸化作用于NFκB通路来调节动物机体内免疫和炎症水平。综上所述,CSEI是一种安全有效的抗肝癌药物,同时具有调节机体免疫力的活性,其通过ROS/MAPKs依赖的线粒体通路诱导人肝癌细胞凋亡。本研究为小牛脾提取物注射液作为肝细胞癌和其他原发肿瘤的潜在治疗剂提供药理学证据。
[Abstract]:Malignant tumor is one of the three major killers that currently cause human death, which is a serious threat to human health. Hepatocellular carcinoma is one of the world's most common tumors, leading to a global 662,000 deaths each year, of which about half of the patients are in China. in addition, that treatment of the liver cancer has the characteristics of poor prognosis, easy recurrence and easy transfer, and the like. At present, the treatment of liver cancer mainly includes six methods such as chemotherapy, radiation therapy, surgical treatment, gene therapy, skin treatment and liver transplantation. As a multi-component biochemical extract, the extract of calf-spleen extract has the characteristics of multi-target and low toxicity, but there is no systematic study on its anti-tumor activity and related mechanism. In this paper, the anti-liver cancer activity and its related mechanism of the extract of calf-spleen extract were studied, and the immunoregulation effect was evaluated. The study mainly includes the following aspects: 1. The inhibitory rate of calf-spleen extract injection (CSEI) on various cancer cells was studied in vitro. The effect of calf-spleen extraction injection on the apoptosis of liver cancer cell line was studied with the liver cancer cell line Hep G2 and SMMC-7721 as the main research object. At the same time, the mechanism of the apoptosis of the liver cancer cells was examined by means of biochemical analysis. The experimental results show that the extract of calf spleen can inhibit the growth of cell lines such as human liver cancer, breast cancer and lung cancer in vitro, and the effect of injection on the SMMC-7721 and Hep G2 of the liver cancer cell line is most obvious, and the injection can increase the generation of ROS in the liver cancer cell, the mitochondrial membrane potential is changed, the caspase cascade reaction is activated, and the like. In addition, Western blot was used to detect the expression of apoptosis-related proteins PARP, clear-PARP, Bcl2, P38, phospho-P38, JNK, phospho-JNK1, ERK1/ 2, phospho-ERK1/ 2, clear-caspase3, clear-caspase-8, and clear-caspase-9 in the cells and tumors. In conclusion, calf spleen extract injection inhibits the proliferation of cancer cells and induces apoptosis of the liver cancer cells through the ROS-related mitochondrial pathway. The anti-liver cancer activity and its mechanism in the extract of calf-spleen extract were studied in order to further determine the anti-hepatoma effect of the extract of calf-spleen extract. The body weight and tumor volume of the mice during the course of the treatment were examined within 14 days of treatment with the calf spleen extract injection. After the treatment, the expression of anti-tumor and oxidative stress-related proteins was analyzed by Western blot. The results of the experiment show that the tumor volume of the nude mice in the Hep G2 liver cancer heterotopic model can be significantly reduced, and the weight of the mice is not affected. At the same time, the results of Western blot were consistent with in vivo experiments. The above results show that the extract of calf spleen extract has good anti-liver cancer activity, and it can induce the apoptosis of liver cancer cells through the mitochondrial pathway dependent on ROS/ MAPKs. The immunomodulatory activity of the extract of calf-spleen extract was evaluated by establishing an immune-low model induced by cyclophosphamide. After 14 days of treatment, the calf-spleen extract injection (in the clinical use dose range) can reverse the hypoimmunity induced by the cyclophosphamide, significantly increase the animal spleen index and restore the IL-2, 6, 10, 12 and IFN-1 in the serum, The levels of immune and inflammatory-related cytokines, such as TNF-1, and the ability of NK cell killing and T-Lymphatic cells are improved, and the level of immunity and inflammation in the body of an animal is regulated by increasing the phosphorylation of IKK in the spleen of the animal in the NF-B pathway. In conclusion, CSEI is a safe and effective anti-liver cancer drug, and has the activity of regulating the immunity of the body, and the mitochondrial pathway, which is dependent on the ROS/ MAPKs, induces the apoptosis of human liver cancer cells. The present study provides pharmacological evidence for calf spleen extract injection as a potential therapeutic agent for hepatocellular carcinoma and other primary tumors.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2363348
[Abstract]:Malignant tumor is one of the three major killers that currently cause human death, which is a serious threat to human health. Hepatocellular carcinoma is one of the world's most common tumors, leading to a global 662,000 deaths each year, of which about half of the patients are in China. in addition, that treatment of the liver cancer has the characteristics of poor prognosis, easy recurrence and easy transfer, and the like. At present, the treatment of liver cancer mainly includes six methods such as chemotherapy, radiation therapy, surgical treatment, gene therapy, skin treatment and liver transplantation. As a multi-component biochemical extract, the extract of calf-spleen extract has the characteristics of multi-target and low toxicity, but there is no systematic study on its anti-tumor activity and related mechanism. In this paper, the anti-liver cancer activity and its related mechanism of the extract of calf-spleen extract were studied, and the immunoregulation effect was evaluated. The study mainly includes the following aspects: 1. The inhibitory rate of calf-spleen extract injection (CSEI) on various cancer cells was studied in vitro. The effect of calf-spleen extraction injection on the apoptosis of liver cancer cell line was studied with the liver cancer cell line Hep G2 and SMMC-7721 as the main research object. At the same time, the mechanism of the apoptosis of the liver cancer cells was examined by means of biochemical analysis. The experimental results show that the extract of calf spleen can inhibit the growth of cell lines such as human liver cancer, breast cancer and lung cancer in vitro, and the effect of injection on the SMMC-7721 and Hep G2 of the liver cancer cell line is most obvious, and the injection can increase the generation of ROS in the liver cancer cell, the mitochondrial membrane potential is changed, the caspase cascade reaction is activated, and the like. In addition, Western blot was used to detect the expression of apoptosis-related proteins PARP, clear-PARP, Bcl2, P38, phospho-P38, JNK, phospho-JNK1, ERK1/ 2, phospho-ERK1/ 2, clear-caspase3, clear-caspase-8, and clear-caspase-9 in the cells and tumors. In conclusion, calf spleen extract injection inhibits the proliferation of cancer cells and induces apoptosis of the liver cancer cells through the ROS-related mitochondrial pathway. The anti-liver cancer activity and its mechanism in the extract of calf-spleen extract were studied in order to further determine the anti-hepatoma effect of the extract of calf-spleen extract. The body weight and tumor volume of the mice during the course of the treatment were examined within 14 days of treatment with the calf spleen extract injection. After the treatment, the expression of anti-tumor and oxidative stress-related proteins was analyzed by Western blot. The results of the experiment show that the tumor volume of the nude mice in the Hep G2 liver cancer heterotopic model can be significantly reduced, and the weight of the mice is not affected. At the same time, the results of Western blot were consistent with in vivo experiments. The above results show that the extract of calf spleen extract has good anti-liver cancer activity, and it can induce the apoptosis of liver cancer cells through the mitochondrial pathway dependent on ROS/ MAPKs. The immunomodulatory activity of the extract of calf-spleen extract was evaluated by establishing an immune-low model induced by cyclophosphamide. After 14 days of treatment, the calf-spleen extract injection (in the clinical use dose range) can reverse the hypoimmunity induced by the cyclophosphamide, significantly increase the animal spleen index and restore the IL-2, 6, 10, 12 and IFN-1 in the serum, The levels of immune and inflammatory-related cytokines, such as TNF-1, and the ability of NK cell killing and T-Lymphatic cells are improved, and the level of immunity and inflammation in the body of an animal is regulated by increasing the phosphorylation of IKK in the spleen of the animal in the NF-B pathway. In conclusion, CSEI is a safe and effective anti-liver cancer drug, and has the activity of regulating the immunity of the body, and the mitochondrial pathway, which is dependent on the ROS/ MAPKs, induces the apoptosis of human liver cancer cells. The present study provides pharmacological evidence for calf spleen extract injection as a potential therapeutic agent for hepatocellular carcinoma and other primary tumors.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
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