BACKGROUND: Blood stem cells (BSCs) are the most primitive cells in the immune system and can be differentiated into many kinds of cells. As the regulatory cells of immune response, dendritic cells (DCs) have been attracted more and more attention in the field of autoimmune diseases. Due to different resources of precursor cells, DCs have different cytokines, ideal cytokine matching,applying orders, and experimental cultured conditions. Furthermore, development, phenotypic expression, and mature degree are still different.OBJECTIVE: To investigate the influence of tumor necrosis factor- α (TNF- a ) and interleukin-4 (IL-4) on the culture system and provide improved method for inducing functional DCs in vitro, derived from cord blood CD34+ hematopoietic precursor cells (HPC).DESIGN, TIME AND SETTING: Observational study, which was performed in the Department of Microbiology and Immunology, Nanjing Medical University from March 2005 to November 2005.MATERIALS: The cord blood was collected from neonatal umbilical cord in the 81 Hospital of Nanjing City. CD34 monoclonal antibody coated magnetic bead system (MACS) was provided by Miltenyi Biotec Company, Germany; human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF), human recombinant interleukin-4 (rhlL-4), and human recombinant tumor necrosis factor-α (rhTNF-α) by Pepro Tech Company, USA.METHODS: CD34+HPC were isolated and purified from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Then the cells were cultured with different culture medium which contained different combinations of cytokines:GM-CSF and TNF-α (GT) or GM-CSF, TNF-α and IL-α (GTI) in order to be induced to differentiate into dendritic cells (DCs).The DCs derived from CD34+HPC were identified for their morphology and phcnotype by FACS and laser scanning con_focal fluorescence microscopy, and also for their abilities of inducing proliferation of allogenic T cells by 3H-TdR incorporation assay.RESULTS: The purity of selected CD34+ cells with MACS was more than 90%. DCs could be obtained from CD34+HPC by the culture in presence of GM-CSF and TNF-α or GM-CSF, TNF- o and IL-4. With the time of culture lasting, the cells expressed lower level surface antigen of CD34 and HLA-DR (P ＜ 0.05), and possessed the phenotypes of DCs characterized by higher expression of CD80, CD86, CD83 and CDIa. At 13-15 days, the cells possessed higher level of phenotypes of DCs compared to 7-9 days and 10-12 days. DCs induced with GTI culture system expressed higher levels of surface antigen CD80, CD86, CD83,CD 1 a and a lower level of CD 14 than those induced with GT culture system. DCs displayed typical morphology and property and expressed higher level surface antigen of CD86 and CD80, especially expressed CD86 when they were induced with proper cytokines of GM-CSF and TNF- α added at 0 hour, and of IL-4 added at 48 hours.CONCLUSION: DCs can be generated from CD34+HPC by proper culture system, the design of GM-CSF + TNF- α + IL-4(GM-CSF and TNF- α were added at 0 hour, IL-4 was added at 48 hours) is preferred.

Objective To research a new type of gastrointestinal anastomat -- anastomosis device with three rows of staples (Pa-tent No .2012200583213) ,and clarify its associated functions through esophagogastric anastomosis operations on pigs ,so that we could provide experiences for its clinical application in future .Methods Compared with domestic anastomat with two rows of sta-ples ,we designed and manufactured a new type of anastomat with three rows of staples and researched its function .Choosing 12 do-mestic pigs ,about 60 kg ,gastroesophageal anastomosis was taken twice with each case by anastomat with three or two rows of sta -ples randomly in sequence .According to the different types of anastomats ,cases were divided into two groups :group A ,used anas-tomat with three rows of staples ,including 12 cases of anastomosis ;group B ,used anastomat with two rows of staples ,including an-other 12 cases of anastomosis .Results Compared with group B ,cases of group A have less bleeding sites (t = 7 .00 ,P < 0 .01) . Without reinforcement and with 0 .5 kg of tension ,fewer of outermost staples exposed(t= 6 .17 ,P< 0 .01) .And the shape of used staples of group A is double circles ,which has bigger mechanical strength than that of group B (t= 6 .57 ,P < 0 .01) .Conclusion The function of anastomat with three rows of staples surpasses that of traditional anastomat with two rows of staples in pig esopha -gogastrostomy surgery .

Objective To establish and assess the utility of 99Tcm-sulfur colloid (SC) salivary imaging in the routine evaluation of pulmonary aspiration in adult patients with respiratory tract diseases.Methods Eight patients (7 men,1 woman; age range 68 to 80 years,mean age (76 ± 4) years) with respiratory tract disease and history of aspiration by clinical assessment were evaluated prospectively by 99Tcm-SC salivary imaging from April to July 2012.A dose of 74.0 MBq 99Tcm-SC was added to 20 ml saline,mixed well,and administered orally to patients.Dynamic imaging was acquired with posterior projection for 30 min at a rate of 30 s per frame.Two experienced physicians assessed all examination results and reached consensus for final diagnosis.Radioactivity detected at either the bronchi or within the lung fields was reported as positive for aspiration.This study was approved by the institutional review board of Hospital Ethical Committee,and the written informed consent was obtained from patients or their guardians.Results All patients were positive for aspiration by 99Tcm-SC salivary imaging (8/8).Aspiration into bilateral main bronchus was seen in 2 cases,right main bronchus and branch in 4 cases,and left main bronchus and branch in 2 cases.Aspirated tracer could be visualized as early as 3 min,latest at 24 min,and the median was 19 min.Conclusion 99Tcm-SC salivary imaging is useful for the detection of aspiration in adult patients with respiratory tract diseases.

Aim To explore the effects of propofol on learning and memorizing ability and the effects of com-bination of propofol and phenobarbital sodium on epi-leptic rats.Methods Thirty-six epileptic rats were di-vided into epileptic model group (EP),normal saline group (NS),lipid emulsion +epileptic group (LE), phenobarbital sodium +epileptic group (PB),propofol+epileptic pattern (Prof),and combination of propo-fol and phenobarbital sodium +epileptic group.Each group had 6 rats.Tests of Morris water maze were giv-en to the rats to evaluate their learning and memorizing ability.The protein expression of toll-like receptor 4 (TLR4 )was examined by ELISA.Results There were no effects of saline and lipid emulsion on learning and memorizing ability and the expression of TLR4 pro-tein in hippocampus in epileptic rats (P >0.05 ). Propofol could increase the incubation period in epilep-tic rats obviously,shorten the plateau period,and in-crease the expression of TLR4 protein in hippocampus (P <0.05 ).Phenobarbital sodium could shorten the plateau period in epileptic rats,and increase the ex-pression of TLR4 protein in hippocampus (P <0.05), but it had no effect on the incubation period (P >0.05).Compared with PB,combination of propofol and phenobarbital sodium +epileptic group had a lon-ger incubation period and a shorter plateau period with an increase of the expression of TLR4 protein in hippo-campus (P <0.05 ).Compared with propofol group, combination of propofol and phenobarbital sodium +ep-ileptic group had a shorter plateau period (P <0.05) with an obvious increase in the expression of TLR4 pro-tein in hippocampus (P <0.05),but it had no effect on incubation period (P >0.05 ).Conclusions Propofol damages the learning and memorizing ability of epileptic rats.Phenobarbital sodium had no obvious effect on the learning ability in epileptic rats,but harms the memorizing ability in epileptic rats.Combi-nation of propofol and phenobarbital sodium affects the learning and memorizing ability of epileptic rats.Hip-pocampus TLR4 may be involved in the effect of propo-fol and phenobarbital sodium on the learning and mem-orizing ability of epileptic rats.

Objective To observe the characteristics of hepatic progenitor cells(HPCs)activation in liver tissues of patients with hepatitis B cirrhosis,and to investigate the relationship between the number of HPCs and HBV infection.Methods Cytokeratin 7(CK7)-was stained immunohistochemically in liver tissues of 16 patients with hepatitis B cirrhosis.HPCs and duetular reactions were quantitively analyzed.The expression of HBsAg and HBcAg were also detected to evaluate its relationship with HPCs activation.Results HPCs were extensively activated and marked duetular reactions can be observed in cirrhotic liver tissues.Tlle expression of HBsAg was positively correlated with HPCs activation.Conclusions HPCs are extensively activated in cirrhotic liver tissues,and HBV infection may facilitate its activation.

In order to identify rat ovarian germ cells, we expressed and purified rat RVLG protein in Escherichia coli cells and prepared a mouse anti-rat RVLG polyclonal antibody. The rat RVLG cDNA was obtained from rat testicle tissue by RT-PCR and was cloned into the vector pMD19-T. Sequence analysis proves that the cloned RVLG cDNA fragment was 60 bp longer than that released in the GenBank (NM_001077647), resulting from an alternative splicing of the RVLG pre-mRNA. The RVLG cDNA was double digested with the restriction endonucleases BamH I and EcoR I, and then was extracted from gel and inserted into the prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmid pGEX-RVLG was verified for successful construction and then was transformed into Escherichia coli BL21(DE3) for induction to express the GST-RVLG fusion protein by IPTG. The GST-RVLG fusion protein was expressed in Escherichia coli BL21 (DE3) at a high level which accounts for more than 10% of the total bacterial cellular protein. The purified RVLG protein was used as an antigen to immunize KM mouse for the production of polyclonal antibody in ascetic fluid followed by celiacly injecting the mouse with S180 cells. The mouse anti-rat RVLG antibody was analyzed by ELISA, Western blotting and immunohistochemistry for its specificity and titer. The antibody could recognize RVLG protein specifically and its titer was about 1:20 000. These results confirm that the mouse anti-rat RVLG polyclonal antibody with high affinity and specificity has been prepared successfully, and lay a foundation for our ongoing research on the specific expression of RVLG in rat ovary.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Base Sequence ; Cloning, Molecular ; DEAD-box RNA Helicases ; biosynthesis ; genetics ; immunology ; DNA, Complementary ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Female ; Mice ; Molecular Sequence Data ; Ovary ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Objective To investigate the efficacy and safety of indocyanine green fluorescence imaging in the lymph node dissection of radical thyroidectomy.Methods Radical thyroidectomy was performed using indocyanine green fluorescence imaging technology for two patients at the Department of General Surgery of Chinese People's Liberation Army (PLA) General Hospital in July 2018.Indocyanine green was injected into the thyroid glands after bilateral thyroid glands were exposed during operation.Bilateral total thyroidectomy plus central lymph node dissection was performed in case 1,and bilateral total thyroidectomy plus central area and left lateral area(area Ⅱ a,Ⅲ,Ⅳ) lymph node dissection was performed in case 2.Both operations were performed under the guidance of real-time fluorescence imaging system.The total number of lymph nodes detected,the number of small lymph nodes (diameter less than 3 mm),the level of parathyroid hormone(PTH),the incidence of complications such as hypocalcemia,hoarseness and short-term recurrence were observed.Results After excitation by the near-infrared light of the fluorescence detector probe,the display showed that the parathyroid gland and surrounding tissues were not visualized,and the thyroid glands and lymph nodes were brightly illuminated.The number of lymph nodes dissected in the central region of the two patients was 20 (13 with diameter less than 3 mm) and 10(6 with diameter less than 3 mm),respectively.For case 2,13 lymph nodes were dissected in the left lateral area (area Ⅱ a,Ⅲ,Ⅳ),and 8 lymph nodes with diameter less than 3 mm were dissected.There were no complications such as hypocalcemia and hoarseness after operation.The levels of parathyroid hormone and serum calcium were normal on the first day and 3 months after operation.There was no recurrence or metastasis of the tumors by ultrasonography 3 months after operation.Conclusion Indocyanine green fluorescence real-time imaging technology can help to identify lymph nodes specifically during radical thyroidectomy,and can achieve real-time dynamic imaging,which can make lymph node dissection more thorough and can be used as a new method for lymph node tracing in thyroid cancer surgery.

Objective To evaluate the relationship between endothelin-1 (ET-1) and p38 mitogenactivated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathways during mechanical stretch-induced enhancement of adhesion of rat pulmonary microvascular endothelial cells (PMVECs).Methods Rat PMVECs were seeded in the culture plate at a density of 0.5×105 cells/ml (2 ml/well) and divided into 5 groups (n=24 each) using a random number table:control group (group C),mechanical stretch group (group MS),mechanical stretch plus specific PI3K inhibitor LY294002 group (LY group),mechanical stretch plus specific p38 MAPK inhibitor SB203580 group (SB group),and mechanical stretch plus selective ETA receptor blocker BQ123 group (BQ group).Cells were exposed to 20％ cyclic stretch at 0.3 Hz for 4 h using a sine wave.In LY,SB and BQ groups,LY294002,SB203580 and BQ123 at the final concentration of 10 μmol/L were added,respectively,after mechanical stretch,cells were incubated for 10 min,and then extracted and purified rat polymorphonuclear neutrophil leukocytes (PMNs,5× 105 cells/well) were added and co-incubated with PMVECs for 30 min and then washed out.The concentrations of ET-1 and interleukin-6 (IL-6) in the culture medium were determined using enzyme-linked immunosorbent assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated Akt (p-Akt) was detected by Western blot.Adhesion of PMNs was measured by immuno-histochemistry,and the adhesion rate was calculated.The expression of P-selectin mRNA was detected using real-time polymerase chain reaction.Results Compared with group C,the concentrations of IL-6 and ET-1 in the culture medium were significantly increased,the expression of p-p38 MAPK,p-Akt and P-selectin mRNA was up-regulated,and the adhesion rate of PMNs was increased in the other four groups (P＜0.05).Compared with group MS,the concentration of IL-6 in the culture medium was significantly decreased,the expression of p-Akt and P-selectin mRNA was down-regulated,and the adhesion rate of PMNs was decreased in LY,SB and BQ groups,the concentration of ET-1 in the culture medium was significantly decreased in group BQ,and the expression of p-p38 MAPK was significantly down-regulated in SB and BQ groups (P＜0.05).Conclusion The signaling mechanism underlying ET-1-mediated enhancement of rat PMVEC adhesion may be related to activating p38 MAPK and PI3K/Akt signaling pathways.

Objective:To investigate the antibacterial effect of iodophor on Staphylococcus aureus biofilm (BBF).Methods:Staphylococcus aureus were cultured in vitro and 480 pieces of titanium alloy plates were selected. On the surface of titanium plates, in vitro models of Staphylococcus aureus biofilms were established at days 7, 14, 21 and 28 respectively with 120 pieces of titanium plates at each time points. The biofilms at each time point were assigned to no iodophor immersion (PBS group), 5 g/L iodophor immersion for 5 minutes (5-min group) and 5 g/L iodophor immersion for 10 minutes (10-min group), according to the random number table method. FITC-ConA, propidium iodide (PI) and SYT09 were used to dye Staphylococcus aureus in PBS group. After dyeing, confocal laser scanning microscopy and scanning electron microscopy were used to observe the morphological structure of bacterial biofilms, and the Colony forming unit (CFU) was counted by the viable count method. In the other two groups, PI and SYT09 were applied to dye Staphylococcus aureus, and then confocal laser scanning microscopy and scanning electron microscopy were used to observe the changes of biofilms and bacterial viability after iodophor immersion. The antibacterial effect of iodophor was evaluated by the viable count method.Results:After dyeing Staphylococcus aureus with FITC-ConA and PI in PBS group, confocal laser scanning microscopy showed that the extracellular polymers of the bacteria increased gradually with the extension of culture time. The space structure of biofilm was gradually mature, changed significantly at day 21 and became mature at day 28. After staining Staphylococcus aureus with PI and SYT09 in PBS group, confocal laser scanning microscopy showed that the number of bacteria increased, and had a mountain-like shape. Scanning electron microscopy showed that the number of bacterial extracellular polymers increased gradually with the extension of culture time and a structured microenvironment was formed and gradually matured. In 5-min and 10-min groups, all bacteria were killed at days 7 and 14 [0(0, 0)CFU/ml], the antibacterial effect was weakened at 21 days, but the antibacterial effect of iodophor immersion in 10-min group [100 (100, 125)CFU/ml] was better than that in 5-min group [300 (275, 425)CFU/ml] ( P<0.05). There was no significant difference in iodophor immersion in 5-min group [500 (375, 700)CFU/ml] and 10-min group [250 (175, 400)CFU/ml] at 28 days ( P>0.05). Conclusions:The maturation of biofilm is the overall maturation of bacteria and bacterial extracellular polymers and the formation of a spatialized microenvironment. Bounded by the 21st day, biofilms are divided into young biofilms and mature biofilms. The main difference between them lies in the maturation of extracellular polymers and microenvironment. For the bacterial biofilm with culture time less than 21 days, the antibacterial effect of the iodophor immersion for 10 min is better than that of 5 min. However, for the bacterial biofilm with culture time greater than 21 days, there is no significant difference in the antibacterial effect of the bacterial biofilm of prolonged iodophor immersion time.

We retrieved the PDTC patient medical record in our center who have received multi-disciplinary comprehensive treatment in March 2019. By reviewing his treatment process, we hope to improve the recognition of this disease and provide reference for individualized programs.