本文選題：重癥急性胰腺炎 + 腸粘膜屏障�。� 參考：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:探討“柴黃清胰活血顆�！睂χ匕Y急性胰腺炎(severe acute pancreatitis,SAP)模型大鼠腸粘膜屏障的影響及作用機制。方法:選用雄性健康SD大鼠120只,采用完全隨機法分為5組:假手術組、SAP模型組、柴黃清胰活血顆粒低濃度組、柴黃清胰活血顆粒中濃度組、柴黃清胰活血顆粒高濃度組,每組各24只。通過向胰膽管內(nèi)逆行注射3.5%�；悄懰醽碚T導SAP模型。待造模成功的大鼠麻醉蘇醒后立即進行灌胃,每4小時1次,按1ml/100g體重給藥,高、中、低濃度組分別予高、中、低濃度柴黃清胰活血顆粒溶液灌胃(中濃度為0.1g/ml,高、中、低濃度比分別為4:2:1),模型組、假手術組予以等量生理鹽水灌胃。造模第6h、12h、24h每組各取8只大鼠麻醉后開腹,觀察胰腺出血壞死、腸積氣情況;取腹主動脈血制備血清,分別檢測血淀粉酶、二胺氧化酶(diamine oxidase,DAO)、D-乳酸、內(nèi)毒素;取胰腺、十二指腸組織予以HE染色后在光鏡下觀察;并運用免疫組化法檢測小腸黏膜緊密連接蛋白Occludin的表達。結果:1、建立模型:大鼠造模6h后可見胰腺組織出現(xiàn)片狀出血和紫黑色壞死;HE染色光鏡下可見胰腺間質水腫、點片狀出血、炎細胞浸潤、灶性變性壞死;血淀粉酶有不同程度升高。上述觀察結果均符合SAP改變,提示造模成功。2、肉眼觀察:SAP模型組大鼠胰腺組織出現(xiàn)片狀出血和紫黑色壞死;胃腸道積氣明顯,腸管管壁變薄,透亮;大網(wǎng)膜、腸系膜上有較多皂化斑。而高、中、低濃度組大鼠胰腺壞死及腸道積氣較模型組減輕,大網(wǎng)膜、腸系膜上有較少皂化斑。3、血淀粉酶:與假手術組比較,模型組及高、中、低濃度組的血淀粉酶在第6、12、24小時均升高(p0.05)。高、中、低濃度組第12、24小時血淀粉酶較模型組均降低(p0.05);第12小時中、低濃度組的血淀粉酶比高濃度組高(p0.05)。第24小時血淀粉酶高濃度組低濃度組中濃度組(p值均0.05)。4、通過對大鼠血清dao、d-乳酸、內(nèi)毒素檢測,模型組及高、中、低濃度組的d-乳酸、dao、內(nèi)毒素均較假手術組升高(p0.05),說明sap造模成功后,血清d-乳酸、dao、內(nèi)毒素增加。第12小時高、中、低濃度組的內(nèi)毒素組間比較均有差異(p0.05),第12小時的d-乳酸低濃度組分別與高、中濃度組比較有差異(p0.05),第12小時低濃度組的dao比模型組高(p0.05),第24小時中濃度組的dao比模型組高(p0.05),除第24小時低濃度組的dao比高濃度、中濃度組低(p0.05)外,其它濃度組組間未見明顯差異。提示:柴黃清胰活血顆粒可減少sap大鼠血d-乳酸、血漿內(nèi)毒素的產(chǎn)生,降幅由大到小依次為高濃度組、中濃度組、低濃度組。本次實驗未觀察到該制劑對sap大鼠血dao明顯的量效、時效關系。5、胰腺組織he染色光鏡下顯示:模型組胰腺組織受損嚴重,且隨著時間遞增逐漸加重,第6h即可見胰腺間質水腫、點片狀出血,炎細胞浸潤,灶性變性、壞死。高、中、低濃度組第6h與模型組比較未見明顯改變,而第12h、24h與模型組比較損傷有所減輕。高、中、低濃度組同時間點對比,胰腺組織損傷好轉幅度由大到小依次為高濃度組、中濃度組、低濃度組。胰腺病理學評分:第24小時高、中、低濃度組病理學評分較模型組有顯著變化,且呈降低趨勢(p0.05);第24小時高、中、低濃度組的病理學評分組間比較均有統(tǒng)計學差異(P0.05)。提示:柴黃清胰活血顆粒可降低SAP大鼠胰腺病理學評分,降幅由大到小依次為高濃度組、中濃度組、低濃度組。6、大鼠十二指腸HE染色光鏡下顯示:模型組腸粘膜受損嚴重,且隨著時間遞增逐漸加重,第6h即可見腸絨毛充血水腫,大量中性粒細胞浸潤、杯狀細胞減少,頂端上皮壞死、脫落,潰瘍形成。第12h、24h高、中、低濃度組與模型組比較損傷均有所減輕。高、中、低濃度組同時間點對比,腸粘膜損傷好轉幅度由大到小依次為高、中濃度組低濃度組。提示柴黃清胰活血顆�？蓽p少SAP大鼠腸粘膜損傷程度,且腸粘膜損傷改善程度為高、中濃度組低濃度組。7、大鼠十二指腸腸粘膜緊密連接蛋白Occludin的表達顯示:模型組及高、中、低濃度組的細胞漿、細胞膜表達水平均較假手術組顯著下降,提示SAP大鼠造模成功后,腸黏膜組織中Occludin的表達減弱。而高、中、低濃度組與同時間點的模型組比較,Occludin的表達逐漸恢復,且隨著時間遞增表達逐漸增強。提示柴黃清胰活血顆�？苫謴蚐AP大鼠緊密連接蛋白Occludin表達,且恢復程度為高、中濃度組低濃度組。結論:早期應用柴黃清胰活血顆粒能夠改善重癥急性胰腺炎模型大鼠胰腺組織及腸道黏膜損傷,其機制可能與降低大鼠血清乳酸、血漿內(nèi)毒素含量,上調(diào)Occludin蛋白的表達有關,從而起到改善腸黏膜屏障的作用。
[Abstract]:Objective: To investigate the effect and mechanism of "Chai Huang Qingyi Huoxue Granule" on intestinal mucosal barrier of severe acute pancreatitis (SAP) model rats. Methods: 120 male healthy SD rats were selected and divided into 5 groups by complete random method: sham operation group, SAP model group, Chai Huang Qing pancreas Huoxue granule low concentration group, Chai Huang Qingyi The concentration group in Huoxue granule, the high concentration group of Chahuang Qingyi Huoxue granule, 24 in each group. The SAP model was induced by retrograde injection of 3.5% taurocholic acid into the pancreatic duct. After the rats were awakened, the rats were irrigated immediately after the anesthesia was awakened, and every 4 hours and 1 times, the high, middle, and low concentration groups were given high, middle, and low concentration of Chai Huang Qing respectively. Pancreas Huoxue granule (0.1g/ml, high, medium and low concentration ratio was 4:2:1). The model group and sham operation group were given the same amount of normal saline. 8 rats in each group were anaesthetized to open the abdomen to observe the hemorrhagic necrosis of the pancreas and the condition of intestinal accumulation. The blood serum was prepared from the abdominal aorta, and the blood amylase and two amine were detected respectively. Diamine oxidase (DAO), D- lactic acid, endotoxin, pancreatic and duodenal tissue were observed under light microscopy after HE staining; and immunohistochemical method was used to detect the expression of tight connexin Occludin in small intestinal mucosa. Results: 1, the model was established: after the rat model 6h, the pancreatic tissue appeared flaky bleeding and purple black necrosis; HE staining light was found. The pancreatic interstitial edema, point bleed, inflammatory cell infiltration, focal degeneration and necrosis were seen under the microscope, and the blood amylase was elevated in different degrees. The results of the above observation all conformed to the changes of SAP, suggesting that the model was successful.2, the naked eye was observed in the SAP model group, the pancreas tissue appeared flaky bleeding and purple black necrosis, gastrointestinal tract accumulation was obvious, the intestinal tube wall became thinner. There were more saponification spots on the greater omentum and mesentery. The pancreas necrosis and intestinal accumulation in the high, middle and low concentration rats were less than that in the model group. The greater omentum and the mesentery had less saponification spots.3 and blood amylase: compared with the sham operation group, the blood amylase in the model group and the high, middle and low concentration groups increased (P0.05) at the 6,12,24 hour. High, medium and low concentration In group 12,24 hours, the blood amylase was lower than that in the model group (P0.05); in the twelfth hour, the blood amylase in the low concentration group was higher than that in the high concentration group (P0.05). The concentration group in the low concentration group of the high concentration group of blood amylase (P value was 0.05).4, through the serum Dao, d- lactic acid, endotoxin test, the model group and the d- lactic acid in the high, middle and low concentration group, Dao, Endotoxin was higher than that of sham operation group (P0.05), indicating that after the success of SAP model, serum d- lactic acid, Dao, endotoxin increased. Twelfth hours high, middle and low concentration of endotoxin group were different (P0.05), Twelfth hours low concentration of d- lactate group were respectively higher than medium concentration group (P0.05), Twelfth hours low concentration group Dao ratio model group High (P0.05), the concentration of Dao in the twenty-fourth hour concentration group was higher than the model group (P0.05), except the high concentration of Dao in the twenty-fourth hour low concentration group and the lower concentration group (P0.05), there was no obvious difference between the other concentration groups. It suggested that the Chai Huang Qing pancreas Huoxue granule could reduce the blood d- lactic acid and the plasma endotoxin production in the SAP rats, and the decrease from large to small was the high concentration group. In the medium concentration group and low concentration group, the dose effect of this preparation on the blood Dao of SAP rats was not observed, the aging relationship was.5, and the pancreas tissue HE staining light microscope showed that the pancreas tissue of the model group was seriously damaged and gradually increased with the time, and the 6h was the pancreatic edema, the dot bleed, the inflammatory cell infiltration, the focal degeneration and necrosis. High, middle, low concentration group 6h and model group no significant change, but 12h, 24h and model group, compared with the model group, compared with the time point, the group of pancreatic tissue damage improved from large to small in order of high concentration group, middle concentration group, low concentration group. The pancreatic pathology score: twenty-fourth hours high, middle, low concentration group disease Compared with the model group, there was a significant change in the score of the model group (P0.05), and there was a statistically significant difference between the twenty-fourth hours, middle and low concentration groups. It was suggested that Chai Huang Qing pancreas Huoxue granule could reduce the pathological score of pancreas in SAP rats, and the decrease from large to small was high concentration group, middle concentration group and low concentration group.6. HE staining in the rat duodenum showed that the intestinal mucosa of the model group was seriously damaged and increased with time. 6h was seen in the intestinal villus congestion and edema, a large number of neutrophils infiltrated, the goblet cells decreased, the epithelia of the apical necrosis, exfoliation, and ulcers formed. The injury of the group 12h, 24h, low concentration and the model group were all lightened. The lower concentration group compared with the time point, the intestinal mucosal injury improved from large to small in order of high, middle concentration group low concentration group. It suggests that Chai Huang Qing pancreas Huoxue granule can reduce the degree of intestinal mucosal injury in SAP rats, and the improvement of intestinal mucosa damage is high, middle concentration group low concentration group.7, rat duodenal intestinal mucosa close connexin Occludi The expression of N showed that the expression level of cell membrane in the model group and the high, middle and low concentration groups was significantly lower than that in the sham operation group. It suggested that the expression of Occludin in the intestinal mucosa was weakened after the successful model of SAP rats. The high, middle and low concentration groups were compared with the model group at the same time point, and the expression of Occludin was gradually restored with the increase of time. It is suggested that Chai Huang Qing yhuoxue granules can restore the expression of tight connexin Occludin in SAP rats, and the degree of recovery is high and the concentration group is low in concentration group. Conclusion: the early application of Chai Huang Qing pancreas Huoxue granule can improve the injury of pancreatic tissue and intestinal mucosa in rats with severe acute pancreatitis, and its mechanism may reduce the blood of rats. Clear lactic acid, plasma endotoxin content, upregulated the expression of Occludin protein, thereby playing a role in improving intestinal mucosal barrier.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5;R-332

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