脂肪干细胞条件培养缓解牛磺胆酸钠和胰蛋白酶诱导的胰腺氧化应激损伤的研究

doi:10.16431/j.cnki.1671-7236.2023.02.037

摘要/Abstract

摘要： 【目的】探究脂肪干细胞条件培养基(adipose-derived-stem cells conditioned medium,ADSCs-CM)缓解牛磺胆酸钠和胰蛋白酶诱导的胰腺氧化应激损伤的保护作用机制。【方法】试验选择1只中华田园犬用来分离培养脂肪干细胞(adipose-derived stem cells,ADSCs)以制备条件培养基(conditioned medium,CM),另选9只中华田园犬随机分为对照组(CON组)、急性胰腺炎(acute pancreatitis,AP)模型组(AP组)和CM治疗组(CM组),AP组和CM组经胰胆管逆行注射牛磺胆酸钠(5% 0.1 mL/kg)、胰蛋白酶(3 500 U/kg)混合溶液,建立犬AP模型,CON组注射等量生理盐水。术后CM组静脉注射ADSCs-CM(0.1 mL /kg),CON组和AP组注射等量生理盐水,24 h后通过B型超声波检查胰腺回声情况和形态,采集胰腺组织样本进行病理组织学检测,采集血液样本进行犬胰腺特异性脂肪酶(cPL)、血气、血常规、生化指标检测,比色法检测血浆及胰腺总一氧化氮合酶(total nitric oxide synthase,T-NOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase,i-NOS)、谷胱甘肽(glutathione,GSH)及谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)水平。【结果】B型超声波显示AP组及CM组胰腺回声不均匀,周围脂肪呈高回声。病理组织切片显示AP组胰腺肿胀出血、周围脂肪变性、十二指肠肠壁出血严重,胰腺出现间质水肿,大量炎性细胞浸润,多量细胞坏死崩解,严重出血。CM组胰腺及周围组织变化、细胞肿胀、炎性细胞浸润和出血较AP组轻微;cPL检测均为阳性。与CON组相比,AP组Cl^-、白蛋白(albumin,ALB)、球蛋白(globulin,GLB)、总蛋白(total protein,TP)含量均显著降低(P＜0.05),淀粉酶(amylase,AMY)、脂肪酶(lipase,LIPA)、血浆T-NOS、胰腺i-NOS活性以及总胆固醇(total cholesterol,TCHO)、胰腺GSH含量均显著升高(P＜0.05);CM组Na⁺、Cl^-、GLB、TP含量均显著降低(P＜0.05),AMY、胰腺GPx活性以及胰腺、血浆GSH含量均显著升高(P＜0.05)。与AP组相比,CM组总CO₂(total CO₂,TCO₂)、Na⁺、Cl^-、ALB、TCHO含量以及血浆T-NOS活性均显著降低(P＜0.05),胰腺GPx活性、血浆GSH含量均显著升高(P＜0.05)。【结论】ADSCs-CM能减轻AP模型犬胰腺损伤程度,其发挥保护作用的机制与血浆中T-NOS活性降低及胰腺GPx和血浆GSH水平增高有着密切关系,本研究可为犬AP的治疗及ADSCs-CM的应用提供理论依据。

关键词: 犬; 急性胰腺炎; 脂肪干细胞条件培养基; 氧化应激

Abstract: 【Objective】 The aim of this study was to explore the protective mechanism of adipose-derived stem cells conditioned medium (ADSCs-CM) in alleviating pancreatic oxidative stress injury induced by sodium taurocholate and trypsin.【Method】 One Chinese garden dog was selected to isolate and culture adipose-derived stem cells (ADSCs) and prepare conditioned medium (CM). Nine dogs were randomly divided into control group (CON group),AP model group (AP group) and CM treatment group (CM group).The AP and CM groups were retrogradely injected with a mixture of 5% sodium taurocholate (0.1 mL/kg) and trypsin (3500 U/kg) through the pancreaticobiliary duct to establish a AP model,The CON group was injected with the same volume of normal saline.After surgery, CM group was intravenously injected with ADSCs-CM(0.1 mL/kg),the CON group and AP group were injected with the same volume of normal saline.Blood and pancreatic tissue samples were collected 24 h after surgery,histopathology,B-ultrasound,canine pancreas-specific lipase (cPL),blood routine,biochemical indexes were detected 24 hours after operation,and the levels of total nitric oxide synthase (T-NOS),inducible nitric oxide synthase (i-NOS),glutathione (GSH) and glutathione peroxidase (GPx) in pancreas and serum were detected by colorimetry.【Result】 B-ultrasound showed that the pancreas echo in AP group and CM group was uneven,the surrounding fat was hyperechoic. Pathological tissue sections showed that the pancreatic swelling and hemorrhage,peripheral steatosis,severe duodenal hemorrhage,interstitial edema,a large number of cell necrosis and inflammatory cell infiltration,and severe hemorrhage of pancreas were occurred in the AP group.The histopathological changes of pancreas and surrounding tissues in the CM group were milder than that of in the AP group. The cPL tests were all positive.Compared with the CON group,the contents of Cl^-,ALB,GLB and TP in the AP group decreased significantly (P＜0.05),while the activities of AMY,LIPA,plasma T-NOS,pancreatic i-NOS and T-CHO,GSH contents increased significantly (P＜0.05).In the CM group,the contents of Na⁺,Cl^-,GLB and TP decreased significantly (P＜0.05),while the activities of AMY,pancreatic GPx,and the contents of plasma GSH,pancreatic GSH increased significantly (P＜0.05).Compared with the AP group,the contents of TCO₂,Na⁺,Cl^-,ALB,TCHO and plasma T-NOS activity in the CM group decreased significantly (P＜0.05),pancreatic GPx activity and plasma GSH content increased significantly (P＜0.05).【Conclusion】 ADSCs-CM could reduce the degree of pancreatic injury in AP model dogs,and the protective mechanism was closely related to the decrease of plasma T-NOS activity and the increase of pancreatic GPx and plasma GSH levels.This study could provide a theoretical basis for the treatment of AP and the application of ADSCs-CM.

Key words: canine; acute pancreatitis; adipose-derived stem cell conditioned medium; oxidative stress

中图分类号:

S829.2

刘博, 李美林, 刘旭, 梁双赢, 牟宝龙, 赵雯婷, 葛延松. 脂肪干细胞条件培养缓解牛磺胆酸钠和胰蛋白酶诱导的胰腺氧化应激损伤的研究[J]. 中国畜牧兽医, 2023, 50(2): 798-806.

LIU Bo, LI Meilin, LIU Xu, LIANG Shuangying, MU Baolong, ZHAO Wenting, GE Yansong. Effects of Adipose-derived Stem Cell Conditioned Medium Alleviates Oxidative Stress Injury Induced by Sodium Taurocholate and Trypsin[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 798-806.

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