1、 肝纤维化论文：羟基喜树碱对大鼠肝纤维化防治作用及部分机制的研究【中文摘要】观察 HCPT 对肝纤维化大鼠肝功能、纤维化程度等影响,研究 HCPT 对肝纤维化大鼠模型的防治作用；检测肝组织中Bax、Bcl-2 基因表达、a-SMA 蛋白表达、TUNEL 染色等指标,初步探讨 HCPT 抗大鼠肝纤维化的作用机制。为肝纤维化的治疗探索新途径提供实验基础；为拓宽 HCPT 的临床应用范围提供理论依据。方法：64 只 SD 大鼠随机分为正常组、模型组、低剂量 HCPT 治疗组、中剂量 HCPT 治疗组、高剂量 HCPT 治疗组共 5 组。采用 40% CCl4 花生油溶液 0.2ml/100g,2 次
2、/周诱导大鼠肝纤维化模型。正常组给予生理盐水 1 ml/kg 腹腔注射,每周 2 次；模型组仅给予 CCl4 造模；其余3 组造模同时分别给予 0.25mg/kg、0.5mg/kg、1.0mg/kgHCPT 腹腔注射每周 3 次,共 8 周。各组分别在实验的第 8 周末进行以下指标检测：采用全自动生化仪检测各组大鼠血清ALT、AST、ALB、TP、A/G；采用全自动血球分析仪检测大鼠血白细胞、血小板；取肝脏相同部位用于石蜡切片,分别进行 HE 染色、Masson 三色染色,对病理标本进行纤维化程度分期；对肝脏组织石蜡切片进行 a-SMA 免疫组织化学检测,并进行 TUNEL 染色；取肝脏相同部
3、位采用 RT-PCR 技术检测 Bax、Bcl-2 mRNA 表达以及Bax/Bcl-2mRNA 比值的变化。结果：1.各组肝功能：模型组的ALT、AST 与正常组相比均明显升高(P 值均 0.05)；PLT 检测仅正常组与模型组有统计学差异(P0.05)。3.各组肝脏大体形态观察：正常组大鼠肝脏外观红润光滑,边缘锐利,质地柔软；模型组肝脏体积显著缩小,与肝周组织粘连严重,表面凹凸不平,肝叶边缘粗顿,质地较硬；各 HCPT 治疗组肝脏体积均大于模型组,表面略有灰暗,未见有明显增生结节,质地尚柔软。4.病理切片 HE 及 Masson 染色和肝纤维化病理分期：病理组织学观察：正常组肝小叶结构完整
4、,无纤维增生,无炎症细胞侵润；模型组肝小叶结构紊乱,小叶被纤维分割,较多假小叶形成,并有较多炎症细胞侵润,同时可见肝细胞气球样变及大泡性脂肪变性；各剂量 HCPT 治疗组较模型组明显改善,肝小叶结构较清晰,纤维组织不同程度增生以汇管区明显,无明显假小叶形成,仅于汇管区周围见部分肝细胞脂肪变性,为大小泡混合性脂肪变性,可见少量炎症细胞侵润。各组肝纤维化病理分期：模型组的肝纤维化程度与正常组相比明显升高(P0.05)。5. RT-PCR 检测肝组织Bax、Bcl-2mRNA 表达：模型组的 Bax、Bcl-2mRNA 与正常组相比均明显升高(P 值均 0.05)。7.肝组织 TUNEL 染色：TU
5、NEL 染色显示阳性着色细胞主要集中于肝脏组织汇管区,以纤维间隔中最为明显,其分布情况与 a-SMA 蛋白免疫组织化学染色区域一致；而肝细胞未见明显染色。结论：HCPT 对四氯化碳诱导的肝纤维化大鼠模型具有防治作用；HCPT 作用机制可能为 HCPT 抑制 a-SMA 蛋白的表达,抑制HSCs 活化增值；上调 Bax/ Bcl-2mRNA 比值,诱导 HSCs 凋亡,从而改善了肝纤维化进程,达到了抗肝纤维化的治疗效果；同时本研究采用HCPT 的给药剂量、给药途径、给药间隔等是安全、可行的；对 HCPT防治其他组织器官纤维化(如肺纤维化等)的研究具有借鉴意义。【英文摘要】:To observe
6、the preventive and therapeutic effect of hydroxycamptothecin (HCPT) in CC14-induced liver fibrosis in rats by detect the Liver function and the degree of fibrosis; Preliminary study the mechanism of the HCPT by detect the Bax、Bcl-2mRNA level,the a-SMA expression and the TUNEL stain in liver. To expl
7、ore new ways for the treatment of liver fibrosis and to provide theoretic evidence for expanding the clinical apply of the drug.Methods:64 SD rats were randomly divided into 5 groups:Normal group, Model group, Low dose treatment group, Intermediate-dose group and high-dose group.Hepatic fibrosis was
8、 induced in rat by abdominal cavity injection of CC14,each rat give 40%CC14 (0.2 ml/kg bodyweight).twice a week,8 weeks total. The normal group inject saline only,the low-dose group inject HCPT(0.25mg/kg bodyweight) 3 times a week,the intermediate-dose group and the high-dose group each apply 0.5mg/
9、kgN 1 mg/kg instead,8 weeks total. Each group remained 10 rats in the eighth week.We check the following sample:etected the ALT、AST、ALB、TP and A/G levels on serum by using automatic biochemical analyzer;Detected the WBC and PLT levels on serum by using automatic blood cell analyzer;Take the same pos
10、ition for the paraffin sections of livers then make the HE stain and masson stain to observe the degree of hepatic fibrosis;ake the paraffin sections of liver tissue for a-SMA immunohistochemistry and TUNEL stain;ake the same position for the RT-PCR test of livers to observe hepatic Bax/Bcl-2mRNA le
11、vels and the Bax/Bcl-2mRNA ratioResults:1. Liver function in each group:The ALT and AST levels on serum of the model group were much higher than the normal group (P0.05).The normal group was the only one which has significant difference to the model group (P0.05)3. Gross morphology of livers in each
12、 group:The appearance of normal groups livers are red and smooth, they have sharp edges and soft texture.The livers of the model group were reduced Significantly, Severe adhesion with the adjacency tissues,have uneven surface, hard texture and rough edges.The livers of the 3 therapy groups were bigg
13、er than the model group. The livers have slightly dark surface,still soft texture but have not see hyperplastic nodules significantly.4. HE and Masson staining pathology and hepatic fibrosis index:Histopathology: Has integrity Lobular structural, no fibrosis and no inflammatory cell infiltration of
14、the normal group.The model group have a lot of pseudolobuli, inflammatory cell infiltration,hepatocyte ballooning and steatosis of bullous.However, the 3 therapy groupslobular structure are clear, Proliferation of fibrous tissue in varying degrees of portal area,fewer Pseudolobules,but a lot fatty d
15、egeneration of liver cells Near the portal area,and few inflammatory cells infiltration can be seen,The histopathological instalment:The statistics of liver fibrosis show that The histopathological instalment of the model group were much higher than the normal group (P0.05)7. TUNEL staining of liver
16、 tissue:TUNEL staining showed positive staining liver cells mainly in the liver portal area, especially in interval in the fiber like the immunohistochemical staining region for the a-SMA protein. No obvious staining of liver cellsConclusion:HCPT could significantly inhibit hepatic fibrosis in CC14-
17、induced in rats. The mechanism is that HCPT may inhibit the expression of a-SMA protein and the proliferation of HSCs.To induce apoptosis of activated HSCs by changing the ratio between the Bax/Bcl-2mRNA, improving the process of liver fibrosis, reaching the therapeutic effect of liver fibrosis.At t
18、he same time, we found the dosage, route of administration, dosing interval of HCPT in this study are safe and feasible.This results also are sense to prophylaxis and treatment other tissues and organs of fibrosis (eg pulmonary fibrosis, etc.) by the HCPT.【关键词】肝纤维化 肝星状细胞 羟基喜树碱 Bax Bcl-2【英文关键词】hepati
19、c fibrosis Hepatic stellate cells hydroxycamptothecine Bax Bcl-2【目录】羟基喜树碱对大鼠肝纤维化防治作用及部分机制的研究 摘要 3-5 ABSTRACT 5-7 第 1章 前言 11-13 第 2 章 材料与方法 13-23 2.1 材料 13-14 2.1.1 实验动物与饲料 13 2.1.2 主要试剂 13 2.1.3 主要仪器 13-14 2.2 研究方法 14-22 2.2.1 主要溶液的配置 14-15 2.2.2 实验动物模型的制备、分组以及药物处理 15 2.2.3 标本获取与处理 15-16 2.2.4 检测大鼠肝
20、功能、白细胞、血小板指标 16 2.2.5 观察大鼠肝脏大体形态、肝组织病理组织学以及肝纤维化的病理分期 16-17 2.2.6 免疫组织化学检测肝组织 -SMA 表达 17-18 2.2.7 肝脏组织切片 TUNEL 染色 18-20 2.2.8 RT-PCR 检测肝组织 Bax/Bcl-2 mRNA 表达 20-22 2.3 统计方法 22-23 第 3 章 结果 23-37 3.1 HCPT 对肝纤维化大鼠肝功能的影响 23-25 3.1.1 HCPT 对肝纤维化大鼠 ALT、AST 的影响 23-24 3.1.2 HCPT 对肝纤维化大鼠 ALB、TP、A/G 的影响 24-25 3.
21、2 HCPT 对大鼠血液白细胞、血小板的影响 25-26 3.3 HCPT 对大鼠肝脏大体形态、病理组织学以及病理分期的影响 26-31 3.3.1 HCPT 对大鼠肝脏大体形态的影响 26-27 3.3.2 HCPT 对大鼠肝脏病理组织学的影响 27 3.3.3 HCPT 对大鼠肝组织纤维化分期的影响 27-31 3.4 HCPT 对肝纤维化大鼠肝组织 -SMA 表达影响 31-33 3.5 肝组织 TUNEL 染色 33-35 3.6 HCPT 对肝纤维化大鼠肝组织 Bax、Bcl-2 mRNA 表达的影响 35-37 第 4章 讨论 37-43 4.1 羟基喜树碱与纤维化疾病 37 4.2 HCPT 给药途径、给药剂量、给药间隔及安全性探讨 37-39 4.3 HCPT 对肝纤维化大鼠模型的防治作用 39-40 4.4 HCPT 抗实验性肝纤维化的机制探讨 40-43 第 5 章 结论与展望 43-45 5.1 结论 43 5.2 展望 43-45 致谢 45-46 参考文献 46-49 攻读学位期间的研究成果 49-50 综述 50-58 参考文献 55-58
