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.
Objective To observe whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into the smooth muscle cells (SMCs) induced by bladder SMCs (BSMCs) conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system. Methods UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1 ∶ 1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium (induced group, group A) and complete medium (control group, group B), respectively; simple BSMCs served as positive control group (group C). The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of α-smooth muscle actin (α-SMA), Calponin, and smooth muscle myosin heavy chain (SM-MHC) of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days. Results The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of α-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of α-SMA could be observed in groups A and B; at 14 days, the positive expression of α-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of α-SMA, Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining. Conclusion UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.
Objective To evaluate the efficacy and safety of prophylactic octreotide for preventing complications after pancreas transplantation. Methods We searched The Cochrane Library (Issue 1, 2008), PubMed (1970 to January 2008), EMbase (1974 to January 2008) and CBM (1978 to January 2008). Six studies concerning prophylactic octreotide in preventing complications after pancreas transplantation were retrieved. Study selection and assessment, data collection and analyses were undertaken by two reviewers independently. Meta-analyses were done using The Cochrane Collaboration’s RevMan 4.2.10 software. Results Three RCTs, involving a total of 82 patients were included in the review. On the fifth postoperative day, the urinary amylase was significantly lower in patients in the octreotide group compared to the control group (SMD=–784.86, 95%CI –1464.24 to –105.49, P=0.02), and no significant difference was observed between the two groups in serum amylase (SMD=–12.26, 95%CI –56.53 to 32.06, P=0.59). No significant difference in the incidence of complications was noted between the two groups. The differences between the two groups in graft survival rate (90 days after operation) and patients’ 6-month survival rate were not statistically significant (RR=0.96, 95%CI 0.83 to 1.11, P=0.56; RR=1.17, 95%CI 0.86 to 1.58, P=0.32, respectively). As for safety, there were no reports of adverse effects of octreotide on CsA or FK506 absorption and no reports of other adverse reactions. Conclusion The evidence currently available shows that prophylactic octreotide is not capable of reducing dramatically the occurrence of pancreatitis, fistula, thrombosis and rejection after pancreas transplantation. And there is no obvious influence on graft survival rate and patient survival rate. Because the RCTs available for this systematic review are too small, further high-quality large-scale RCTs with long-term follow up are required to provide more reliable evidence.
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.
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 establish a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells. Methods The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24- effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry. Results After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P gt; 0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P lt; 0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% ± 8.6%; the cell activity was 93.3% ± 4.7%; and the level of FoxP3 was 74.2% ± 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25- Teffs. Conclusion MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.