ObjectiveTo establish a better immunofluorescence protocol to detect co-localization of p53 and mitochondria which may benefit studies aiming to detect mitochondrial expression of proteins.MethodsHeLa cells were treated with hypoxia and the expression of p53 was detected by immunoblotting. HeLa cells were fixed with methanol, methanol: acetone (1: 1, v/v) mixture, and 4% paraformaldehyde, respectively; the former two groups were not permeable, while the latter was penetrated with 0.1% Triton-X 100 and stained with p53 and mitochondria at the same time. After HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 was reduced to 0.05%, 0.025%, 0.01%, and 0.005%. After the HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 decreased to 0.01% and 0.005% for the first time, then, after staining with p53, the mitochondria were stained with 0.1% Triton-X 100 for the second time.ResultsThe expression of p53 was up-regulated (P<0.01) after hypoxia, which could be used in the following immunofluorescence experiment. The co-localization of p53 and mitochondria was observed in the nucleus and cytoplasm in both the methanol group and the mixed solution group. The co-localization of p53 was the most obvious in the mixed solution group. After using 0.1% Triton-X 100, the p53 signal was mainly in the nucleus, but no co-localization was observed. After fixation with 4% paraformaldehyde, to some extent, the reduced concentration of 0.05% and 0.025% Triton-X 100 weakened the p53 signal in nucleus and enhanced the co-localization signal. However, the signal in nucleus was still stronger than that in cytoplasm. When it was reduced to 0.01% and 0.005%, p53 signal was detected in cytoplasm but not in nucleus, suggesting that the nuclear membrane was not penetrated under this condition, but it also failed to penetrate the mitochondrial membrane, leading to the failure of mitochondrial labeling. The second permeability completely avoided the p53 signal in nucleus, and successfully labeled mitochondria, and the co-localization of p53 and mitochondria was detected.ConclusionsCo-localization of p53 and mitochondria is detectable in cells fixed by methanol or methanol and acetone mixture which brings out better results. Penetrating twice with Triton X-100 of different concentrations following paraformaldehyde fixation help avoid signals in nuclei and falicitate co-localization detection.
【摘要】 目的 探讨同种异基因骨髓间充质干细胞（bone mesenchamal stem cells，BMSC）静脉输注对大鼠到小鼠胰岛移植物的功能保护和小鼠糖尿病状态改善。 方法 全骨髓培养法获得C57BL/6小鼠BMSC。不连续梯度离心法分离纯化Sprague-Dawley（SD）大鼠胰岛，将300胰岛当量的胰岛单独或与BMSC联合移植入链脲菌素诱导的糖尿病BALB/c小鼠肾包膜下，并通过尾静脉在移植后0、3和5 d注射CM-DiI标记的BMSC 5×105/只，对照组给于磷酸盐缓冲溶液。移植后监测血糖，第9天处死小鼠，取肝、脾、胸腺、淋巴结和移植胰岛的肾脏，冰冻切片，荧光显微镜观察CM-DiI标记细胞的组织分布；免疫荧光法观察移植物中胰岛素和胰高血糖素表达，评价胰岛的功能。 结果 BMSC静脉输注后主要分布于胸腺，其次是脾脏和淋巴结，肾和肝组织中未观察到BMSC；BMSC联合胰岛移植组血糖控制水平优于其他组，且在第7天的口服糖耐量实验优于单纯胰岛移植组。 结论 与胰岛联合移植的BMSC对受者免疫器官和组织有明显的趋向性，且对胰岛细胞的体内存活有一定保护作用。【Abstract】 Objective To research on the protection function by the allogeneic rat bone mesenchymal stem cells (BMSC) on rat to mouse islet transplantation and the improvement of diabetic state in mouse. Methods BMSC were prepared from C57BL/6 mouse bone marrow cells and identified by flow cytometry (FCM). Islets were isolated from Sprague-Dawley (SD) rats with Ficoll discontinuous centrifugation. CM-DiI labeled BMSC at 5×105 for one mouse were intravenously infused into STZ induced diabetic BALB/c mice after rat to mouse islet transplantation at day 0, 3 and 5. Mice with PBS intravenously infused after islet transplantation were set as the negative controls. Blood glucose was monitored every day at the first 3 days after transplantation, and then monitored every two days. At day 9 after transplantation, spleen, thymus, lymph nods, liver and islets recipient kidney were harvested. Ice slices were prepared and CM-DiI labeled cells were investigated with fluorescence microscope. Results CM-DiI-labeled BMSC were mainly distributed in thymus followed by spleen and lymph nodes. In liver and kidney, there was no red fluorescence observed. The blood sugar control for combined BMSC infusion group was superior to other groups, and the control level of islet combined BMSC infusion group were better than single islet transplantation group in OGTT at day 7. Conclusion Allogeneic BMSC can sustain the insulin secretion of islets in vivo and tend to distribute in immune organs or adenoid tissues after infusion.
ObjectiveTo explore the suitable method for isolation and maintenance of primary cultures of human gallbladder epithelial cells (GECs) for establishing the basis of research works in physiological function of gallbladder and its related diseases.MethodsGECs were isolated with collagenase type Ⅳ and blunt separation.The dishes were coated with fibronectin, laminin and polyDlysine respectively.Additional 10 ng/ml epidermal growth factor was added to DMEM medium containing 20% fetal calf serum.The cells were studied under light and electron microscope to determine their shape and distribution.ResultsEach gallbladder yielded approximately (1-5)×107columnar epithelial cells,greater than 95% of which were viable by trypan blue exclusion.The cells grew vigorously within one week which was flat and multangular in shape. CK19 expressed positive.Electron microscope showed typical gallbladder epithelia with microvilli,tight junctions and mucus droplets.ConclusionCombination of collagenase type Ⅳ，mechanical blunt separation and twostep attachment is of great benefit for separating and harvesting GEC.Fibronectin coated culture dish and DMEM medium containing 20% calf serum and 10 ng/ml hEGF is of great benefit for culturing gallbladder epithelial cells.
Objective To review research progress of pancreas stellate cells in pancreas fibrosis and understand characteristics and activation of pancreas stellate cells and its mechanism on pancreas fibrosis. Method The relevant literatures about pancreas stellate cells and its studies in pancreas fibrosis were reviewed. Results The activation of pancreatic stellate cell is associated with fibrosis of pancreatitis and end stage of pancreas transplantation, but its effect and regulation mechanisms for the extracelluar and intracellular molecular network need to be further investigated. Conclusion Elucidation of activation of pancreas stellate cells will facilitate understanding of pancreas fibrosis and searching new target in treatment of pancreas fibrosis.
Objective To evaluate the impact of portal or systemic venous pancreas graft drainage on patient and graft outcomes following simultaneous pancreas kidney transplantation (SPK). Methods We searched The Cochrane Library (2008, Issue 1), PubMed (1970 to Feb 2008) and EMBASE (1974 to Feb 2008) to find studies concerning the effect of systemic versus portal venous pancreas graft drainage on patient and graft outcomes. Meta-analyses were conducted using The Cochrane Collaboration’s RevMan 4.2 software. Results Three RCTs involving 401 simultaneous pancreas kidney transplants were included in our meta-analysis. Statistically significant differences were only observed in 3- and 5-year pancreas graft survival rates (P=0.03 and P=0.05). No significant difference was noted in patient or kidney graft survival rates. Conclusion Currently available evidences from RCTs does not support the effectiveness of portal drainage in preventing thrombosis, rejection or infection after SPK. Large-scale, long-term and appropriately designed RCTs are required to conclude whether portal and systemic drainage in pancreas transplantation are equivalent in terms of patient and graft survival.
Objective To investigate the research base and current understanding of the mechanism of ischemia-reperfusion injury (IR) to intrahepatic cholangiocytes after l iver transplantation, so as to identify the key points of the mechanism and provide references for cl inical practice. Methods We searched PubMed (1970 to 2007) and CBM(1979 to 2007). Qual ity assessment and data collection were performed by two reviewers independently. Since the baseline supplied and the measure were very different, we decided to provide a descriptive summary only. Results The earliest study on liver IR was publ ished in 1970. A total of 65 papers were included. There were 13 on cl inical studies, 35 on basic research studies; and 17 review articles. Most basic studies focus on injury mechanism: ① The physiology of bile ducts and Intrahepatic Bil iary Duct Cells(IBDC); ②the IR caused injury mechanism of IBDC during or after liver transplantation; ③ the basic injury mechanisms include: cold ischemia, warm ischemia, reperfusion, injury of bile and bile salts. Most clinical studies focused on preventive measures, including surgical and non-surgical approaches. Based on the evidence from basic research, changing the composition and perfusion methods of perfusate and protecting the specific blood supply to biliary ducts and cholangiocytes during the operation were important in preventing or reducing such an injury. Conclusion ① The heterogeneity of morphology, function, status and the special blood supply in large and small IBDC are important material base. ② Our own study indicated that simple IR or H/R was able to change the expression of MHC, MIC, DR4, DR5 and other adhesion molecules. ③ Compared to hepatic cells, hIBDC can’ t resist cold ischemia and even worse in tolerating reperfusion injury. ④ Hydrophobic bile salts will could increase the harm to bile ducts during organ preservation. ⑤ Due to the low quantity and limited quantities of clinical researches, the power of evidence was low. The evaluation indexes and baseline conditions are not unified. So the conclusions are for reference only.