背景:研究表明大黄游离蒽醌能够保护肠黏膜细胞结构和功能的稳定, 但尚未有将其与脂肪干细胞联合治疗大鼠急性胰腺炎的报道.目的:观察并探讨大黄游离蒽对脂肪干细胞移植治疗大鼠急性胰腺炎的影响及其机制.方法:体外快速复苏并培养购自中国医学科学院的冻存鼠脂肪干细胞, 随后采用MTT比色法测定脂肪干细胞的细胞存活率并行PKH-26标记.将北京维通利华动物实验技术有限公司提供的SD大鼠随机平均分为4组, 病理模型组大鼠采用肠壁穿刺逆行胰胆管注射体积分数5％牛黄胆酸钠建立急性胰腺炎病理模型, 然后尾静脉注射0.5 mL的L-DMEM完全培养液;大黄游离蒽组建模后给予大黄游离蒽醌200 mg/kg灌胃;脂肪干细胞组建模后尾静脉注射0.5 mL细胞浓度1×107 L-1脂肪干细胞;联合治疗组建模后同时给予以上2种干预.各个实验组中所有干预治疗措施每日进行1次且连续治疗3 d.结果与结论: (1) 与理模型组的大鼠相比, 大黄游离蒽醌组和脂肪干细胞组大鼠血清淀粉酶活性以及白细胞介素6水平显著降低 (P <0.05), 胰蛋白酶原激活肽水平显著升高 (P <0.05);经过脂肪干细胞移植和大黄游离蒽醌灌胃处理联合治疗, 上述3指标水平较大黄游离蒽醌组和脂肪干细胞组进一步降低 (P<0.05); (2) 苏木精-伊红染色结果显示, 此2个单独治疗组大鼠胰腺组织中脂肪变性、出血、细胞坏死及炎症细胞浸润等病理变化程度均有明显减轻, 联合治疗组中大鼠的病理变化的缓解程度则更为明显; (3) 荧光显微镜下观察可见, PKH-26标记的阳性细胞数联合治疗组最多, 脂肪干细胞组次之, 而在大黄游离蒽组及病理模型组则未见 (P <0.05); (4) TUNEL法检测可见, 与病理模型组比较, 2个单独治疗组中胰腺组织中的细胞凋亡数明显降低, 联合治疗组进一步降低 (P <0.01); (5) RT-PCR和Western blot法检测显示, 病理模型组大鼠胰腺组织转化生长因子β1及Smad2/3基因和蛋白表达最高, 联合治疗组最低 (P <0.01), 而2个单独干预组均较病理模型组显著较低 (P <0.05); (6) 结果证实, 大黄游离蒽干预联合脂肪干细胞移植治疗大鼠急性胰腺炎可以有效改善模型大鼠急性胰腺炎的血液生化指标水平;并可明显地缓解急性胰腺炎模型大鼠炎症反应程度和胰腺组织形态学病变以及胰腺细胞的凋亡, 这可能与降低转化生长因子β/Smad信号通路有关.%BACKGROUND: Studies have shown that rhubarb-free anthraquinone can protect the stability of the intestinal mucosa cell structure and function. However, the combination of rhubarb-free anthraquinone with adipose-derived stem cells to treat acute pancreatitis in rats has not yet been reported. OBJECTIVE: To investigate the effects of rhubarb-free anthraquinone on the treatment of acute pancreatitis by fat stem cell transplantation in rats. METHODS: Cryopreserved mouse adipose-derived stem cells (provided by the Chinese Academy of Medical Sciences) were rapidly recovered and cultured in vitro. The survival cells were labeled using PKH-26, and then the cell survival rate of adipose-derived stem cells was determined by MTT colorimetry. Sprague-Dawley rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. were equally randomized into four groups. Animal models of acute pancreatitis were made in all rats through intestinal wall puncture for retrograde administration of 5％ sodium taurocholate to the pancreaticobiliary tube, and then the model rats were given tail vein injection of L-DMEM complete medium (0.5 mL) in the model group, intragastric administration of 200 mg/kg rhubarb-free anthraquinone in the rhubarb-free anthraquinone group, tail vein injection of adipose-derived stem cells (1×107 cells/L, 0.5 mL) in the cell transplantation group, and intragastric administration of 200 mg/kg rhubarb-free anthraquinone plus tail vein injection of 1×107 adipose-derived stem cells (0.5 mL) in the combined treatment group. All interventions in each group were performed once a day, for 3 continuous days. RESULTS AND CONCLUSION: (1) The levels of serum amylase and interleukin-6 were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group compared with the model group (P < 0.05), while the level of trypsinogen activation peptide significantly increased (P < 0.05). In the combined treatment group, the levels of three indicators mentioned above were significantly decreased compared with the rhubarb-free anthraquinone group and cell transplantation group (P < 0.05). (2) The results of hematoxylin-eosin staining showed that the pathological changes in the pancreatic tissue of rats were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, such as fatty degeneration, hemorrhage, cell necrosis and inflammatory cell infiltration. The remission of pathological changes was more obvious in the combined treatment group. (3) The number of positive cells labeled by PKH-26 was the highest in the combined treatment group, followed by the cell transplantation group, while there were no PKH-26-positive cells in the rhubarb-free anthraquinone group and model group (P < 0.05). (4) Compared with the model group, the number of apoptotic cells in the pancreatic tissue was significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, and lowest in the combined treatment group (P < 0.01). (5) The expression of transforming growth factor-β1 and Smad2/3 at gene and protein levels was highest in the model group, followed by the rhubarb-free anthraquinone group and cell transplantation group (P < 0.05), and lowest in the combined treatment group (P < 0.01). To conclude, the combined intervention of rhubarb-free anthraquinone and adipose-derived stem cell transplantation could effectively improve the blood biochemical index level in the rats with acute pancreatitis, significantly relieve the degree of inflammatory response, pancreatic tissue morphology, and apoptosis of pancreatic cells, which may be related to the reduction of the transforming growth factor-β/Smad signal pathway.
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