【作者】 林晖明；

【导师】 魏品康；

【作者基本信息】 第二军医大学 ， 中西医结合临床， 2010， 博士

【摘要】 背景代谢异常是恶性肿瘤重要并发症,是恶性肿瘤治疗困难、预后不佳的原因之一。改善代谢异常是提高患者生存质量的治疗关键。魏品康教授提出肿瘤“痰污染”假说,认为代谢异常的物质基础是痰。现代研究已证实肿瘤蛋白质代谢异常与炎性因子有关。我科以“消痰散结”为法则,创立消痰散结方应用于恶性肿瘤的临床治疗,取得了令人满意的疗效。因此,本实验通过观察消痰散结方对荷瘤大鼠炎性因子表达的调节作用,探讨消痰散结方调整恶性肿瘤蛋白质代谢异常的可能机制。目的通过建立Wistar大鼠皮下移植Walker256瘤模型,构建恶性肿瘤痰污染环境,运用ELISA、EnVision、实时PCR等方法,检测消痰散结方对痰浊中TNF-α、IL-6等炎性因子蛋白和mRNA表达的调控作用,探讨消痰散结方调整恶性肿瘤蛋白质代谢异常的作用机制,佐证肿瘤痰证理论的科学性以及肿瘤代谢异常的痰污染环境“痰浊”的物质存在性。方法30只Wistar大鼠,随机分为正常对照组、空白组、消痰散结方组；除正常对照组外,其余各组为荷瘤大鼠,皮下接种Walker256瘤,建立大鼠蛋白质代谢异常模型。24h后消痰散结方组灌胃给药(2ml／天,×4w)；正常对照组、空白组大鼠同期、同条件下一般饮食饲养；观察实验动物体重变化；第4周实验结束时,麻醉解剖所有动物,心脏取血后检测血清总蛋白、白蛋白水平,ELISA法检测血清TNF-α、IL-6表达。取胫骨前肌组织、肝组织行Envision、实时PCR等方法检测其中TNF-αIL-6、NF-Kb、泛素等的蛋白和mRNA表达。结果1.成功建立大鼠Walker256皮下移植瘤模型。移植术后7天左右可在皮下触及较硬肿块,后肿块逐渐增大。荷瘤鼠逐渐出现皮毛干枯、少食、消瘦、倦怠乏力、行动迟缓等衰竭症状。HE染色证实荷瘤鼠骨骼肌结构紊乱。2.荷瘤鼠因肿瘤而影响机体的正常生长,造模7天后各荷瘤鼠组体重增加值低于正常组(P<0.05)；造模21天后消痰散结方组体重增加值高于空白组(P<0.05)而低于正常对照组(P<0.05)。3.消痰散结方对血清总蛋白和白蛋白的影响：与正常组相比,空白组、消痰散结方组总蛋白未见明显降低(P>0.05)；各荷瘤鼠组白蛋白均较正常组有所下降(P<0.05),消痰散结方组高于空白组(P<0.05)。4.消痰散结方对血清TNF-α蛋白表达的影响：各荷瘤鼠组血清中TNF-α蛋白表达均较正常对照组明显升高(P<0.05)；消痰散结方组血清TNF-α蛋白明显低于空白组(P<0.05)消痰散结方对血清IL-6蛋白表达的影响：各荷瘤鼠组血清中IL-6蛋白表达均较正常对照组明显升高(P<0.05)；消痰散结方组血清IL-6蛋白明显低于空白组(P<0.05)。5.消痰散结方对肌肉组织TNF-α蛋白表达的影响：各荷瘤鼠组肌肉组织中TNF-a蛋白表达较正常对照组均明显升高(P<0.05)；消痰散结方组与空白组未见明显统计学差异(P>0.05)。消痰散结方对肌肉组织TNF-αmRNA表达的影响：各荷瘤鼠组肌肉组织中TNF-αmRNA表达较正常对照组均明显升高(P<0.05)；消痰散结方组与空白组未见明显统计学差异(P>0.05)。消痰散结方对肝脏组织TNF-α蛋白表达的影响：各荷瘤鼠组肝脏组织中TNF-α蛋白表达较正常对照组均明显升高(P<0.05)；消痰散结方组低于空白组(P<0.05)。消痰散结方对肝脏组织TNF-αmRNA表达的影响：各荷瘤鼠组肝脏组织中TNF-αmRNA表达较正常对照组均明显升高(P<0.05)；消痰散结方组低于空白组(P<0.05)6.消痰散结方对肌肉组织IL-6蛋白表达的影响：各荷瘤鼠组肌肉组织中IL-6蛋白表达较正常对照组均明显升高(P<0.05)；消痰散结方组与空白组差异无统计学意义(P>0.05)消痰散结方对肌肉组织IL-6 mRNA表达的影响：各荷瘤鼠组肌肉组织中IL-6 mRNA表达较正常对照组均明显升高(P<0.05)；消痰散结方组与空白组差异无统计学意义(P>0.05)消痰散结方对肝脏组织IL-6蛋白表达的影响：各荷瘤鼠组肝脏组织中TNF-α蛋白表达较正常对照组均明显升高(P<0.05)；消痰散结方组低于空白组(P<0.05)。消痰散结方对肝脏组织IL-6 mRNA表达的影响：各荷瘤鼠组肝脏组织中TNF-a mRNA表达较正常对照组均明显升高(P<0.05)；消痰散结方组低于空白组(P<0.05)。7.消痰散结方对肌肉组织NF-Kb蛋白表达的影响：各荷瘤鼠组肌肉组织中NF-Kb蛋白表达较正常对照组均明显升高(P<0.05)；消痰散结方组与空白组差异无统计学意义(P>0.05)消痰散结方对肌肉组织NF-Kb mRNA表达的影响：各荷瘤鼠组肌肉组织中NF-Kb mRNA表达较正常对照组均明显升高(P<0.05)；消痰散结方组与空白组差异无统计学意义(P>0.05)。8.消痰散结方对肌肉组织泛素蛋白表达的影响：各荷瘤鼠组肌肉组织中泛素蛋白表达较正常对照组均明显升高(P<0.05)；消痰散结方组低于空白组(P<0.05)。消痰散结方对肌肉组织泛素mRNA表达的影响：各荷瘤鼠组肌肉组织中泛素mRNA表达较正常对照组均明显升高(P<0.05)；消痰散结方组低于空白组(P<0.05)。结论1.建立的大鼠Walker256皮下移植瘤模型,制作方法简单,成瘤率高,具有与临床恶性肿瘤代谢异常类似的表现。2.消痰散结方可提高荷瘤大鼠的体重及血清白蛋白水平。3.荷瘤鼠血清、肌肉组织、肝脏组织中rNF-α、IL-6等炎性因子表达升高,证实炎性因子与肿瘤蛋白质代谢异常密切相关,4.消痰散结方可下调荷瘤大鼠肝脏组织中TNF-α、IL-6等炎性因子的表达,调控肝脏的蛋白合成,下调荷瘤大鼠肌肉组织中泛素的表达,调控骨骼肌ATP-泛素蛋白酶体途径,是其清化肿瘤痰污染环境,改善肿瘤代谢异常的可能机制之一。同时证明了肿瘤痰污染假说的科学性、“痰”的物质存在性。更多还原

【Abstract】 Background:Metabolic abnormalities which are the important complications of malignant tumor are one of the reasons for difficult treatment and bad prognosis. Adjusting metabolic abnormalities is the key to elevate patients’survival quality. Phlegm pollution hypothesis proposed by Professor Wei-Pinkang is said that the reason for metabolic abnormalities is muddy phlegm. It is already confirmed that tumor protein metabolic abnormalities are relevant to inflammation. Prof. Wei has established his theory of "dispersing phlegm and dissipating bind" in clinically treating patients with tumors. Therapeutic effect is satisfactory. So the purpose of this study is to discover the mechanism of Xiaotan Sanjie Decoction adjusting tumor protein metabolic abnormalities in a more comprehensive and convincing way by regulating the expressions of different inflammatory factors in the tumor-bearing rats.Objective:The study was designed to investigate the mechanism of Xiaotan Sanjie Decoction in tumor protein metabolic abnormalities and prove scientific nature of the theory of tumor phlegm syndrome and material existence of environment polluted by phlegm "muddy phlegm" promoting the tumor metabolic abnormalities. To construct environment polluted by phlegm by establishing subcutaneous model of Carcinosarcome Walker 256 in Wistar rats and by the methods of ELISA, Envision, realtime-PCR to detect the expressions of inflammatory factors’(TNF-a, IL-6, ect) mRNA and encoded protein regulated by Xiaotan Sanjie Decoction.Methods:30 Wistar mice were randomly divided into normal group, no-drugs group and Xiaotan Sanjie Decoction group. The rats were made subcutaneous model of Carcinosarcome Walker 256 except the rats in normal group to establish tumor protein metabolic abnormalities model. The rats in Xiaotan Sanjie Decoction group were applied with herbs 24 hours after the surgery (2ml/d,×4w); normal group and no-drugs group were raised in the same condition and with common food and drink. The weight was observed. The experiment was terminated on the 4th week.The rats were sacrificed after blood sampling collected from heart. The total protein and albumin were detected. The expressions of mRNA and encoded protein of TNF-a, IL-6, NF-Kb and Ubiquitin were detected by the methods of ELISA, Envision and Realtime-PCR.Results:1. We built subcutaneous model of Carcinosarcome Walker 256, the lump could be touched in the Subcutaneous 7 days after modeling and increas gradually. Rats bearing tumor emerged exhausted symptoms of dried-up coat, less food, athrepsy, lassitude and slowly action. The HE staining illustrated the structural disorder of skeletal muscle.2. Rats bearing tumor grew slowly because of tumor. Their weight were lower than normal group 7 days after modeling(P<0.05); Xiaotan Sanjie Decoction group’s was higher than that of no-drugs group (P<0.05) but was lower than normal group 21 days after modeling (P<0.05)3. Total protein and albumin exam showed:compared with normal group, total protein of no-drugs group and Xiaotan Sanjie Decoction group were not lower (P>0.05). Albumin of all rats bearing tumor was decreased (P<0.05). Albumin of Xiaotan Sanjie Decoction group’s was higher than that of no-drugs group (P<0.05).4. The results of Xiaotan Sanjie Decoction affecting the expressions of serum TNF-a protein showed:the expressions of serum TNF-a protein of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group was lower than no-drugs group (P<0.05)The results of Xiaotan Sanjie Decoction affecting the expressions of serum IL-6 protein showed:the expressions of serum IL-6 protein of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group was lower than no-drugs group (P<0.05)5. The results of Xiaotan Sanjie Decoction affecting the expressions of TNF-a protein in muscle tissues showed:the expressions of TNF-a protein in muscle tissues of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group had no difference with no-drugs group (P>0.05)The results of Xiaotan Sanjie Decoction affecting the expressions of TNF-a mRNA in muscle tissues showed:the expressions of TNF-amRNA in muscle tissues of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group had no difference with no-drugs group (P>0.05)The results of Xiaotan Sanjie Decoction affecting the expressions of TNF-a protein in livers showed:the expressions of TNF-a protein of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group was lower than no-drugs group (P<0.05)6. The results of Xiaotan Sanjie Decoction affecting the expressions of IL-6 protein in muscle tissues showed:the expressions of IL-6 protein in muscle tissues of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group had no difference with no-drugs group (P>0.05).The results of Xiaotan Sanjie Decoction affecting the expressions of IL-6 mRNA in muscle tissues showed:the expressions of IL-6 mRNA in muscle tissues of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group had no difference with no-drugs group (P>0.05)The results of Xiaotan Sanjie Decoction affecting the expressions of IL-6 protein in livers showed:the expressions of IL-6 protein of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group was lower than no-drugs group (P<0.05)7. The results of Xiaotan Sanjie Decoction affecting the expressions of NF-Kb protein in muscle tissues showed:the expressions of NF-Kb protein in muscle tissues of rats bearing tumor were higher than those of normal rats (P<0.05). Xiaotan Sanjie Decoction group had no difference with no-drugs group (P>0.05)The results of Xiaotan Sanjie Decoction affecting the expressions of NF-Kb mRNA in muscle tissues showed:the expressions of NF-κb mRNA in muscle tissues of rats bearing tumor were higher than those normal rats (P<0.05). Xiaotan Sanjie Decoction group had no difference with no-drugs group (P>0.05)8. The results of Xiaotan Sanjie Decoction affecting the expressions of Ubiquitin protein in muscle tissues showed:the expressions of Ubiquitin protein in muscle tissues of rats bearing tumor were higher than those normal rats (P<0.05). Xiaotan Sanjie Decoction group was lower than no-drugs group (P<0.05)The results of Xiaotan Sanjie Decoction affecting the expressions of Ubiquitin mRNA in muscle tissues showed:the expressions of Ubiquitin mRNA in muscle tissues of rats bearing tumor were higher than those normal rats (P<0.05). Xiaotan Sanjie Decoction group was lower than no-drugs group (P<0.05)Conclusion1. The subcutaneous model of Carcinosarcome Walker 256 in rats, which is made simply and has a high successful rate, is similar to the biological features of tumor protein metabolic abnormalities.2. Xiaotan Sanjie Decoction can conspicuously inhibit the weight lost of tumor-bearing rats.and Improve the level of serum albumin of tumor-bearing rats.3. They are confirmed that inflammation is correlated to tumor protein metabolic abnormalities, phlegm pollution hypothesis about tumor metabolic abnormalities is scientific and muddy phlegm exists corporeally, because the expressions of inflammatory factors increasing in serum, liver and muscle tissues.4. Xiaotan Sanjie Decoction can down-regulate the expressions of TNF-a and IL-6 in liver of tumor-bearing rats, regulation of liver protein synthesis. Down-regulate the expressions of ubiquitin in muscle tissues of tumor-bearing rats, and regulat skeletal muscle ATP-ubiquitin-proteasome. This may be one of the mechanisms of Xiaotan Sanjie Decoction adjusting tumor metabolic abnormalities and clearing phlegm polluted environment.更多还原