【作者】 陈婧华；

【导师】 陈垦；

【作者基本信息】 广东药学院 ， 临床医学， 2011， 硕士

【摘要】 【目的】探索原代培养胰腺腺泡细胞方法并鉴定所培养细胞的性质，为急性胰腺炎(acute pancreatitis, AP)体外模型的构建创造条件，通过促红细胞生成素(Erythropoietin，EPO)对胰腺腺泡细胞和肺组织保护作用的研究，为今后EPO治疗AP的临床应用提供理论依据。【方法】分离提纯胰腺腺泡细胞，将其置于DMEM-F12培养液中培养，24h后电子显微镜下观察胰腺腺泡细胞的超微结构变化，并运用PaA抗体鉴定培养细胞性质。90只Sprague-Dawley(SD)大鼠分为假手术组(sham-operation group, SO)，SAP(Severeacute pancreatitis, SAP)组，EPO1000组，EPO3000组，EPO5000组，每组18只，分别按以下时间24h、48h、72h随机处死6只。逆行胰胆管注射5%牛磺胆酸钠(sodiumtaurocholate, STC)（0.1ml/100g)制备SAP大鼠模型。测大鼠肺、胰腺干湿比重及体重系数，人工碘比色法测血清AMS，Elisa试剂盒测IL-1、IL-6和TNF-α，分析3种炎性因子的相互关系；HE染色观察胰腺和肺组织病理变化，免疫组化法检测胰腺组织中EPOR的表达，Elisa法检测EPOR表达水平。【结果】①新鲜分离的大鼠胰腺腺泡细胞悬浮在培养液中，形态不规则，细胞质与细胞核比例较大。分离纯化后腺泡细胞绝大多数呈圆形或椭圆形，细胞核呈圆形，偏于一侧，细胞间杂质较少。②24h后可见细胞数有所增加，细胞明显聚集，2～3天换液后观察细胞，细胞活性好，细胞数目明显增多，传代良好。③经台盼蓝染色测定细胞活性达96%以上，符合进一步实验要求。④电镜下，大量的酶原颗粒位于细胞顶部的胞质内，细胞内可见发达的粗面内质网，在酶原颗粒聚集的区域可见高尔基复合体。⑤经PaA抗体检测腺泡细胞在免疫荧光镜下呈现大片绿色荧光。⑥SAP组大鼠肺脏干湿比重大于SO组(P<0.05)，经EPO治疗后，肺、胰腺水肿减轻；SAP组大鼠体重系数亦大于SO组和EPO组(P<0.05)；⑦SAP组与EPO组血清AMS、IL-1、IL-6、TNF-α水平均明显高于SO组，经EPO治疗24h和48h后，测血清AMS明显降低（P<0.05），SAP组和EPO组各时间点的血清炎症因子(IL-1、IL-6、TNF-α)含量均高于SO组, SAP组升高显著，48h达到峰值，72h有所回落，经EPO治疗后各指标均有下降，EPO疗效呈时间依赖性，三者之间呈正相关性(r=0.534, p=0.02; r=0.584, p=0.04; r=0.820，p=0.00)，有相互促进表达作用。⑧SO组胰腺和肺组织均无明显病理变化；SAP组大鼠胰腺和肺组织出现不同程度水肿、渗出、炎症细胞浸润，部分组织出现出血、坏死、空泡形成，细胞间结构模糊，甚至连接成片；EPO治疗组胰腺和肺组织病理损伤程度明显改善，病理评分显著低于SAP组（P<0.05）。组间比较显示：除EPO3000和EPO5000间无统计学意义外，其他各组间差异均有统计学意义。⑨EPO治疗组、假手术组大鼠胰腺和肺组织中，可见散在的EPOR阳性表达，染色位置位于胞浆；不同剂量EPO治疗组中EPOR在胰腺和肺组织中均呈现大片深染的阳性表达。⑩ELISA法测EPOR表达量示：假手术组、SAP组EPO1000组中EPOR表达量的变化不显著，差异无统计学意义，EPOR表达量随EPO剂量的增加而增加。【结论】①本实验方法可成功原代培养胰腺腺泡细胞，培养细胞纯度及存活率高，符合体外实验要求；②PaA抗体可成为鉴定胰腺腺泡细胞的一种便捷、可行的方法，其特异性高，可推广应用于鉴定胰腺腺泡细胞；③SAP组大鼠血清中炎症介质IL-1、IL-6、TNF-α水平呈逐渐升高趋势；EPO可有效减轻胰腺和肺组织病理损伤，降低SAP大鼠血清AMS、IL-1、IL-6、TNF-α水平；④ELISA法及免疫组化法检测出胰腺腺泡细胞上EPOR的表达，其表达量随EPO剂量的增加而增加，但与治疗时间无明显相关性，由此推测EPO可与EPOR结合后，激活下游的细胞信号转导通路，从而起到抗炎的作用。更多还原

【Abstract】 [Objective] To explore a new method on primary culture of pancreatic acinar cells and indentify the nature of the cells, it is the preparation for acute pancreatitis (AP) in vitro. To investigate the protective effects of Erythropoietin (EPO) on pancreatic acinar cells and lung, this will be useful for researching the clinical theoretical basis.[Methods] Pancreatic acinar cells were placed in DMEM-F12 medium after separated and purified. 24 hours later, the ultrastructural changes of pancreatic acinar cells were observed by electron microscopy. PaA antibody was used to identify the properties of the cultured cells. After that, Sprague Dawley (SD) rats were randomly divided into sham operation group(SO), SAP (acute pancreatitis, SAP), EPO1000 group, EPO3000 group, EPO5000 group, Each group contained 18 rats and executed at the time of 24h, 48h and 72h. SAP rats were prepared by retrograde pancreatic duct injection of 5% sodium taurocholate (STC)(0.1ml/100g). Dry-Wet proportion and weight coefficient of lung were measured at the different time points by electronic scale. The levels of amylase (AMS) in serum were tested by artificial iodine colorimetry. The levels of interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in serum were tested by ELISA kits,and then the relationship would be tested among them. The pathological changes of pancreas and lung were observed by HE staining. The expressions of EPOR were tested by immunohistochemical method and ELISA kits.[Results]①Fresh isolated pancreas acinar cells suspended in the medium, the shape of the cells were irregular. It was large of the proportion of cytoplasm and nuclear. Most of the separated and purified acinar cells were round or oval.②The nuclear was round, leaned to one side and impurities in the intercellular were less. The number of the cells was increased after 24 hours. It was obviously gathered together. After 2~3 days, changed the medium and the observed the activities of the cells. The number of the cells was significantly increased and passaged well.③The viability of the acinar cells was greater than 96% as determined by trypan blue assay and results were in accordance with experimental requirements.④Under the electron microscopy, there are a large number of zymogen granules in the cytoplasm were at the top of the cells, which were rich in rough endoplasmic reticulum. It could be seen the golgi complexes in the region of zymogen granules gethering.⑤It could be found the highly expression of green fluorescence in acini using the antibodies against PaA by immunocytochemistry.⑥The ratio of lung Drought-Wet weight in SAP group was much higher (P < 0.05) than in SO group. After the treatment of EPO, lung and pancreas edema could be easer than before. Lung index of rats were much higher than SO and EPO group (P < 0.05).⑦The levels of AMS and IL-1、IL-6、TNF-αin serum were significantly higher in SAP groups and EPO groups than that in SO groups, however, AMS and IL-1、IL-6、TNF-αwas declined significantly when given EPO intervention for 24h and 48h(P<0.05), but there were no statistics significant difference between EPO3000 and EPO5000 groups. The effects of EPO was time-dependent, it was positive correlation (r=0.534, p=0.02; r=0.584, p=0.04; r=0.820,p=0.00), there is a mutual role in promoting expression.⑧T here are no significant pathological changes or only slight edema of pancreas and lung tissues in SO group. However, varying degrees of edema, exudation, and inflammatory cell infiltration have happed in pancreatic and lung tissues of rats in SAP group, partial pancreatic or lung tissues had happened hemorrhage and necrosis, the structure of which had changed into blur, and even into a slice. The treatment of EPO could improve the pathological damages and the pathological score was significantly lower than SAP group (P<0.05).⑨I t could be seen that EPOR-positive stanining cells, located in the cytoplasm, were scattered in the pancreatic tissue in SO group. The expression of EPOR in pancreas and lung tissue showed large deeply stained EPOR-positive staining cells in different doses of EPO treatment groups.⑩There is no significant EPOR expression changes in SO, SAP, EPO1000 groups measured by ELISA kits. The quantities of the expression of EPOR increased with the increasing doses of EPO. [Conclusion]①Primary pancreatic acinar cells can be successfully cultured by this method, the purity and viability of acinar cells were in line with the vitro experiments.②PaA antibody may be a convenient ang feasible method to identify the pancreatic acinar cells with its high specificity. It can be widely used for identification of pancreatic acinar cells.③It was gradually increased of the levels of AMS, IL-1、IL-6、TNF-αin serum in SAP group. The treatment of EPO may effective reduce the pathological pancreas and lung injury and obviously decreased the levels of AMS, IL-1、IL-6、TNF-αin serum.④Using ELISA and Immunohistochemical methods can detect the expression of EPOR on pancreatic acinar cells. The expression of EPOR increased with the increasing doses of EPO, but there is no significant correlationship with the timing of treatment. We may speculate that EPO might bind with EPOR and activate the pathways of downstream signal transduction, which may play an critical role on anti-inflammatory and anti-apoptosis.更多还原