【作者】 胡小兰；

【导师】 陈先祥；

【作者基本信息】 湖北中医药大学 ， 中西医结合临床， 2010， 硕士

【摘要】 目的：本实验采用二乙基二硫代氨基甲酸盐(DDC)致大鼠胰腺纤维化模型,比较加入黄芩苷后胰腺组织TGF-β1、CTGF、Fn蛋白表达量的变化,并观察大鼠胰腺组织病理情况,从胰腺组织TGF-β1、CTGF、Fn的蛋白表达量及病理学改变两方面探讨黄芩苷干预实验研究大鼠胰腺纤维化形成的机制与作用。方法：47只SPF级雄性SD大鼠随机分为5组(A组：空白组、B组：模型组、C组：小剂量黄芩苷组、D组中剂量黄芩苷组、E组大剂量黄芩苷组),B、C、D、E组用二乙基二硫代氨基甲酸盐(DDC)每周(周一、周四)腹腔注射两次,每次剂量为750mg/kg,共注射8次。A组每周两次注射等剂量的生理盐水,共注射8次。造模后第3周C、D、E组每日用不同剂量(25、50、100 mg/kg)的黄芩苷腹腔注射,B组注射生理盐水作为对照,造模及给药期间各组均予常规饲料。于造模开始后第6周实验结束前一天将各组大鼠禁食12个小时,心脏采血后处死,打开腹腔收集新鲜胰腺组织经生理盐水漂洗,肉眼观察胰腺组织改变后用4%的多聚甲醛液固定,常规石蜡包埋切片。放射免疫法和酶动力法检测血清透明质酸(HA)和淀粉酶,HE染色观察大鼠胰腺组织的病理学变化,免疫组化分析大鼠胰腺组织中的转化生长因子TGF-β1、CTGF、Fn含量,光镜下观察照相。结果：1、黄芩苷对大鼠一般情况的影响空白组大鼠一般情况良好,饮食、活动、精神均正常,3周后体重明显增加。模型组大鼠造模2周后大鼠多饮、少食、多尿、体重有所减轻,精神倦怠,反应较迟钝,鼻腔内有出血,皮毛疏松无光泽,大便糖稀,少数腹腔积液明显,2只死于腹部积液量过多。各黄芩苷组早期进食均减少,精神不佳,鼻腔内均有出血,大便溏稀。给药3周后进食无明显减少,精神、活动较正常,大便性状无明显改变。其中小、中剂量组分别有一只大鼠死于腹部注射部位撕咬,大剂量组一只大鼠造模后腹水过多经抽腹水抗感染治疗无效后死亡。2、胰腺组织病理形态学改变：HE染色镜下改变。空白组：胰腺腺泡呈嗜碱性,腺细胞为锥体形,泡浆丰满,细胞核呈圆形位于基底部,细胞大小一致,排列整齐,胰腺小叶轮廓清晰,小叶间结缔组织中可见胰导管及血管等,胰岛圆润饱满。模型组：胰管扩张,胰腺腺泡变形,水肿。小叶间纤维组织增生,大量炎细胞浸润,小叶结构消失,有的形成炎性包裹,部分腺体萎缩坏死代之以纤维瘢痕。小剂量黄芩苷组：可见胰腺腺泡水肿,但见散在坏死腺泡及炎性浸润,小叶内、小叶间腺泡组织局限性纤维化或无改变。中剂量组：可见水肿的腺泡,小叶间少量炎细胞浸润,小叶内、小叶间腺泡组织少有纤维化改变。大剂量组：可见水肿的腺泡,浸润的炎性细胞,部分小叶结构消失,小叶内、小叶间腺泡组织局限性纤维化或无改变。3、血清生化：淀粉酶各给药组与模型组比较P<0.05,各组与空白组比较P<0.05,各给药组见两两比较无差异。透明质酸各组与空白组比较P<0.01,各给药组与模型组比较P<0.05,各给药组之间相互比较无差异。4、各组TGF-β1、CTGF、Fn蛋白表达量变化：TGF-β1、CTGF免疫组化标记后,光镜下可见模型组TGF-β1主要表达于间质的纤维组织、成纤维细胞、巨噬细胞及血管内皮细胞胞浆和细胞膜,呈棕黄或棕褐色。而空白组大鼠不表达或仅微弱地表达于间质细胞内,呈淡黄色。CTGF在胰腺组织中主要分布于邻近间质区的部分腺上皮细胞的细胞质和间质的成纤维细胞的细胞质中,以细胞浆染成棕黄或棕褐色为阳性表达。而空白组大鼠无或仅有极少量的CTGF在腺上皮细胞的细胞质中表达。采用CMIA系列-多功能彩色病理图像分析TGF-β1、CTGF表达程度,黄芩苷处理各组TGF-β1、CTGF蛋白表达量与模型组相比差异均有统计学意义(P<0.05),黄芩苷处理组间相互比较无差异。Fn免疫组化标记后,光镜下可见模型组Fn主要表达在小叶间及腺泡周围纤维组织中,呈树枝状分布呈网格状包绕腺泡,在小血管周围和导管周围也有少量表达,呈棕黄或棕褐色,并随间质病变程度的增加而增加。空白组大鼠较弱地表达于间质中,呈淡黄色。半定量分析其表达程度,黄芩苷处理各组Fn蛋白表达量与模型组相比差异均有统计学意义(P<0.05),黄芩苷各处理组间相互比较无差异。结论：1、腹腔内反复注射DDC后通过氧化刺激使胰腺组织内氧自由基增高而激活胰腺星状细胞(PSCs)制造大鼠慢性胰腺炎胰腺纤维化模型。2、造模后胰腺外分泌功能下降,而黄芩苷处理组淀粉酶和模型组比较明显升高表明黄芩苷有改善胰腺外分泌功能的作用。造模后透明质酸含量明显升高提示胰腺组织损伤并有纤维增生改变,且黄芩苷处理后透明质酸含量下降,显示黄芩苷有改善组织纤维化程度的作用。3、黄芩苷可能通过下调TGF-β1水平,抑制胰腺星状细胞活化增值,降低CTGF蛋白表达,减少纤维连接蛋白(Fn)而具有抗大鼠胰腺纤维化作用。更多还原

【Abstract】 Objective:To establish the rats model of chronic fibrosing pancreatitis with intraperitoneal injection of DDC and To study the efect and potential mechanism of baicalin on pancreatic fibrotic ratsMethods:Fourty seven male Sprague-Dawley rats were randomly divided into 5 equal groups:normal control group(A group), model control group(B group),low-dose baicalin-treated group (C group), mediate-dose baicalin-treated group(D group), andhigh-dose baicalin-treated group (E group). The rats of the later 4 groups underwent intraperitoneal injectin of 750mg/kg body weight (BW) of DDC twice a week for 4 weeks into the pancreatic duct so as to establish models of pancreatic fibrosis. At the third week the baicalin-treated rats were intraperitoneal injectin with different doses of baicalin(25, 50, and 100 mg/kg body weight) every day, while the normal and model control groups received 0.9% sodium chloride solution instead. lasting 4 weeks, the day befor the last day, the rats were fasted 24 hours and then blood samples were colected from rats’heart. Immediately the rats were put to death and their pancreases were taken out. The levels of hyaluronic acid (HA) and diastase were determined by radioimmunoassay and enzyme dynamia. Pancreatic tissue was embedded by paraffin and cut. The histopathological alterations of pancreatic tissue with HE staining were studied by optical microscopy.immunohistochemistry was used to detect the protein expression of transforming growth factor-β1 (TGF-β1) and CTGF and Fn, and measure the expression of TGF-β1 and Fn and CTGF for the observation by tramission electron microscope. Results:The rats’common conditions of group A、C、D、E、were obviously better than that of model group B. The levels of diastase of the low-dose, mediate-dose, and high-dose baicalin-treated groups were all higher than that of the model control group (P<0.05). The serum level of HA of the low-dose, mediate-dose, and high-dose baicalin-treated groups were all significantly lower than that of the model control group (P< 0.05). Comparison of HA and diastase with the each group used baicalin was nonsense. The degrees of fibrosis of the baicalin-treated groups were ameliorated in comparison with the model control group. The protein content of TGF-β1 and fibronectin (Fn) and CTGF of the low-dose, mediate-dose, and high-dose baicalin-treated groups were all lower than that of the model control group (P<0.05). Comparison with the each group used baicalin was nonsense. Comparison of TGF-β1 and CTGF and Fn with the each group used baicalin was nonsense.Conclusions:The models of chronic pancreatitis rats can be induced with in 4 weeks by intraperitoneal injection of DDC twice a week. Baicalin can improve the common conditions of chronic pancreatitis rats; decrease the levels of HA; improve the level s of amylase; Anti-fibrosis effect of baicalin on pancreatic fibrosis maybe related to its elimination oxygen free radical effect and the content of TGF-β1 and CTGF protein.更多还原