DBTC诱发大鼠慢性胰腺炎模型的建立及其胰腺炎纤维化发生机制研究

发布时间：2018-04-15 22:21

本文选题：慢性胰腺炎 + SD大鼠　；参考：《中南大学》2008年博士论文

【摘要】： 第一部分DBTC诱发慢性胰腺炎动物模型的建立目的应用DBTC诱导建立SD大鼠CP模型,并加以两种作用点不同的药物干预,研究CP发生发展过程中病理学变化、纤维化程度、血请AMS变化及浸润炎性细胞的作用。方法实验组大鼠一次性尾静脉注入0.8 mg/kg DBTC的80%乙醇溶液,2天后再随机分为A组30只,不用任何药物;B组26只,按6mg/kg.d行每天一次腹腔内注射己酮可可碱注射液(PTX);C组37只,每周腹腔内注射曲古霉素A(TSA)1ml(1μg/ml);对照组尾静脉注入等量80%乙醇溶液。实验组鼠分别于第14天、28天和56天处死,对照组鼠于第56天处死。处死鼠均行大体肉眼观察及胰腺、肝、肺、肾镜下病理学观察及胰腺病理学评分;血清AMS含量由全自动生化分析仪测定,胶原纤维染色为VG染色法,肥大细胞为甲苯胺蓝染色法;巨噬细胞、CD_4~+和CD_8~+染色均为EnVision免疫组化法。结果 (1)大体观察:A、B和C三组胰腺在14天、28天和56天处死鼠均出现不同程度的水肿、包膜张力增加、局部胰腺坏死出血及与周围组织有粘连,28天和56天鼠部分胰腺出现局限性硬结节形成,以上表现B组和C组比A组轻。对照组胰腺均正常。实验组胰腺外部分肝、肺出现出血、坏死及脓肿形成。 (2)镜下观察及评分:对照组胰腺组织学结构正常。实验组中56天处死鼠病理学评分、纤维化评分及CP发生率均明显高于14天处死鼠(P＜0.05或P＜0.01)。实验组之间CP发生率无明显差异,但A组28天和56天处死鼠中至重度CP发生率明显高于B和C组(P＜0.05)。实验组胰腺外肝、肺镜下表现相似于肉眼表现。 (3)血清AMS:各实验组血清AMS含量均明显高于对照组(P＜0.01),但各组之间无明显差异(P＞0.05)。 (4)炎性细胞计数:各实验组56天处死鼠巨噬细胞和肥大细胞计数明显高于14天处死鼠(P＜0.05)和对照组(P＜0.01);B和C组56天处死鼠CD_4~+计数明显低于14天处死鼠(P＜0.01),但CD_8~+计数则相反(P＜0.01);实验组之间CD_4~+/CD_8~+比值无明显差异,但均明显低于对照组(P＜0.05或P＜0.01)。 (5)相关性分析:实验组病理学评分、纤维化评分、巨噬细胞计数和肥大细胞计数之间均呈密切正相关(P＜0.01);A组和C组病理学评分与CD_4~+计数均呈密切负相关(P＜0.05或P＜0.01);A组纤维化评分与CD_8~+计数呈密切负相关(P＜0.01)。结论 (1) DBTC一次性尾静脉注射能成功地建立SD大鼠CP模型,该模型具有操作简单、建模时间短、CP发病率高及费用低等优点。但DBTC对肝、肺等主要脏器有一定非致死性毒副作用。(2)该模型较符合人类CP病理形态学特征及其血清AMS含量改变。(3)胰腺组织内浸润的巨噬细胞、肥大细胞及CD_8~+细胞在该模型CP发生发展过程中起了重要作用。第二部分大鼠慢性胰腺炎纤维化发生机制研究目的探讨大鼠CP发生发展与炎性细胞、PSC及细胞生长因子的关系以及PTX、TSA干预大鼠CP的作用。方法实验组和对照组大鼠胰腺组织经4%甲醛固定后制作石蜡包埋切片。α-SMA和desmin染色为EnVision二步法,PDGF-BmRNA、TGF-β1mRNA、CTGFmRNA染色为原位杂交法。结果 (1)炎性细胞计数见第一部分;(2)各实验组不同时间点活化-PSC(a-PSC)计数均明显高于对照组(P＜0.01);A组28天和56天处死鼠a-PSC计数明显高于14天处死鼠(P＜0.05),B和C组56天处死鼠a-PSC计数明显高于14天处死鼠(P＜0.05)。(3)实验组各时间点静止-PSC(s-PSC)计数均明显低于对照组(P＜0.05),但三组各时间点之间均无明显差异(P＞0.05)。(4) B、C两组28天和56天处死鼠CTGFmRNA和PDGF-BmRNA表达阳性率及其评分均明显高于对照组(P＜0.01);A组56天处死鼠TGF-β1mRNA表达阳性率及其评分均明显高于对照组(P＜0.05或P＜0.01);A组和B组56天处死鼠TGF-β1mRNA表达阳性率明显高于14天处死鼠(P＜0.05);A组28天和56天处死鼠CTGFmRNA和PDGF-BmRNA表达阳性率及其评分均明显高于14天处死鼠(P＜0.05或P＜0.01)。(5)实验组a-PSC、s-PSC计数及PDGF-BmRNA、TGF-β1mRNA、CTGFmRNA表达阳性率和评分之间均无明显差异(P＞0.05)。(6)轻、中和重度CP a-PSC计数及TGF-β1mRNA、CTGFmRNA、PDGF-BmRNA表达评分和计数均高于正常组(P＜0.01);中度和重度CP a-PSC计数及CTGFmRNA、PDGF-BmRNA表达评分和计数均明显高于轻度CP(P＜0.05或P＜0.01);轻度和中度CP s-PSC计数明显低于正常组织(P＜0.01)。(7)实验组中a-PSC计数及TGF-β1mRNA、CTGFmRNA、PDGF-BmRNA表达评分均与病理评分、纤维化评分呈密切正相关(P＜0.05或P＜0.01);实验组中a-PSC计数与TGF-β1mRNA、CTGFmRNA、PDGF-BmRNA表达评分均呈密切正相关(P＜0.01)。结论 (1)a-PSC参与了CP的纤维化过程,PDGF-BmRNA、TGF-β1mRNA、CTGFmRNA三种细胞因子能活化PSC及与胰腺纤维化关系密切。(2)肥大细胞和巨噬细胞与活化PSC有较密切关系。(3) CP发病机制复杂,单独作用于某个环节的药物难以对其发病进行控制,如PTX和TSA。
[Abstract]:Establishment of Animal Model of Chronic Pancreatitis Induced by the First Part of DBTC

Purpose

The CP model of SD rats was induced by DBTC , and two different drug interventions were used to study the changes of pathology , degree of fibrosis , AMS changes and infiltration inflammatory cells in the development of CP .

method

The rats in the experimental group were injected with 0.8 mg / kg DBTC 80 % ethanol solution , 2 days later , 30 rats were randomly divided into group A . Twenty - seven rats were given intraperitoneal injection of pentoxifylline injection ( PTX ) once a day .

Results

( 1 ) The rats of A , B and C showed different degrees of edema on 14 days , 28 days and 56 days . The results showed that there were localized hard nodules on the pancreas in both group A , B and C .

In the experimental group , the incidence of CP was significantly higher than that in control group ( P < 0.05 or P < 0.01 ) , but the incidence of CP in the experimental group was significantly higher than that in group B and C ( P < 0.05 ) .

( 3 ) AMS : AMS in the experimental group was significantly higher than that in the control group ( P < 0.01 ) , but there was no significant difference between the groups ( P > 0.05 ) .

( 4 ) Inflammatory cell count : The counts of mouse macrophage and mast cells were significantly higher than those in control group ( P < 0 . 05 ) and control group ( P < 0 . 01 ) , but CD _ 4 ~ + / CD _ 8 ~ + ratio was not significantly different between experimental group and control group ( P < 0 . 05 or P < 0 . 01 ) .

( 5 ) Correlation analysis : There was a close correlation between pathological score , fibrosis score , macrophage count and mast cell count in experimental group ( P < 0 . 01 ) ; the pathological score of group A and C was negatively correlated with the count of CD _ 4 ~ + ( P < 0 . 05 or P < 0 . 01 ) ; the fibrosis score of group A was negatively correlated with the count of CD _ 8 ~ + ( P < 0.01 ) .

Conclusion

( 1 ) The CP model of SD rats can be successfully established by DBTC single - use tail vein injection , and the model has the advantages of simple operation , short modeling time , high CP incidence and low cost .

Study on the mechanism of fibrosis in rats with chronic pancreatitis in the second part

Purpose

To investigate the relationship between the development of CP and inflammatory cell , PSC and cell growth factor in rats and the effect of PTX and TSA on CP in rats .

method

The paraffin - embedded sections were prepared by 4 % formaldehyde fixation in the experimental group and control group . 伪 - SMA and Desin were stained with EnVision two - step method , PDGF - B mRNA , TGF - 尾1 mRNA and CTGFmRNA were stained by in situ hybridization .

Results

( 4 ) The positive rate of expression of TGF - 尾1 mRNA and PDGF - BmRNA in group A and group B were significantly higher than those in control group ( P < 0.05 or P < 0.01 ) .

Conclusion

( 1 ) a - PSC is involved in the fibrotic process of CP , PDGF - BmRNA , TGF - 尾1 mRNA and CTGFmRNA can activate PSC and closely related to pancreatic fibrosis .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R657.5;R-332

【引证文献】