BACKGROUND: The diversity of purification procedures resulting in various purities of olfactory ensheathing cells (OECs) used for grafting is considered to be relevant in the effectiveness of OECs transplant. It is important to develop a well-defined method which produces OECs of great purity and is easy to unify for the future standardization of research involving OECs.OBJECTIVE: To establish a method being easy to unify for purifying OECs to acquire highly and uniformly enriched population of OECs for standardized studies on cell transplantation.DESIGN: Randomized and controlled experiment.SETTING: Department of Orthopaedics, Affiliated Zhongda Hospital of Southeast University School of Clinical Medicine;Central Laboratory of Southeast University School of Clinical Medicine; Experimental Animal Center of Southeast University School of Clinical Medicine.MATERIALS: This experiment was carried out in the Central Laboratory of Southeast University School of Clinical Medicine from February to August 2006. Twenty-eight adult female SD rats weighing 200-250 g were selected in this study. The main reagents were detailed as follows: DMEM/F-12 (GIBCO); 2.5 g/L trypsin (GIBCO); poly-L-lysine (SIGMA); bovine pituitary extract (BPE, SIGMA); fetal bovine serum (FBS, Sijiqing Biological Agent Co., Ltd., Hangzhou);rabbit anti-low-affinity nerve growth factor receptor (anti-P75, SIGMA); biotinylated goat anti-rabbit IgG (Boster Bioengineering Co., Ltd., Wuhan); methyl thiazolyl tetrazolium (MTT) kit (SIGMA).METHODS: Primary cultures of OECs were separated from adult SD rats olfactory bulbs. At day 8 in vitro, the primary cultures were divided randomly into 4 groups, namely differential adhesion method group, immunoadsorption method group,the modified method group,and control group.①The cell suspension in the modified method group was seeded into uncoated flasks and incubated at 37 ℃ in 0.05 volume fraction of CO2 for 1 hours. The supematants were seeded into flasks that had been prepared as follows. The bottoms of these flasks were moistened with anti-P7s (1 mg/L) and were made to dry at 37 ℃, and then they were washed one time with DMEM/F-12. The supernatants were incubated on the anti-p75-treated flasks for 45 minutes at 37 ℃, 0.05 volume fraction of CO2. For removing unbound cells, the flasks were washed five times with DMEM/F-12. The bound cells were detached from the flasks with a cell scraper, centrifuged,and resuspended in D/F-10S with 105 U/L penicillin/streptomycin and 20 mg/L BPE. The cell suspension in differential anchoring method group or immunoadsorption method group was purified as previously described by Nash or Ramo'n-Cueto respectively. Three groups of cell suspensions resulted from the above three methods were seeded respectively onto poly-L-lysine-coated 24-well cell culture chambers and incubated for 14 days at 37 ℃ in 0.05 volume fraction of CO2. Without purification, the cell suspension in control group was also resuspended in D/F-10S with 105 U/L penicillin/streptomycin and 20 mg/L BPE and seeded onto poly-L-lysine-coated 24-well cell culture chambers and incubated under the same culture condition as the other groups.②Purity comparisons for the four groups were made at 2, 5, 8, 10, 12 and 14 days after the end of their respective manipulation to evaluate the effectiveness of the modified method. At per time point in each of the four groups, fifteen visual fields of cultures were selected randomly to count the At the day of 14, viabilities of OECs in the four groups were assessed by MTT assays.MAIN OUTCOME MEASURES: OECs purities at per time point and viabilities of OECs at the day of 14 in each of the four groups after the end of their respective manipulation.RESULTS:① The purities of OECs in the modified method group at each time point were greater (P＜0.05-0.01) than counterparts in the other three groups. OECs purities decreased with culture prolongation in all groups, but the changes of purities over the whole period of observation in the modified method group were the least. The last purities of OECs yielded from the modified method were still extremely great (92.1±1.2)%, whereas the parallels in the others were no more than (85.2±2.2)%.② There was no significant difference in viabilities of OECs between the modified method group and any of the others at the day of 14 (P=0.895).CONCLUSION: The modified method for purifying OECs from the adult rat olfactory bulb is highly effective without extra impairment on the viability of OECs and will be beneficial to the future standardization of research involving OECs.

Objective:To screen and characterize the common epitopes recognized by monoclonal antibody 3A8,which binds several kinds of Gram negative bacteria,which were recognized by monoclonol antibody 3A8 agaisnt different strains of LPS.Methods:Phage clones binding to 3A8 were screened from C7C phage displayed peptide library,the attached phage clones were eluted by LPS2630(O111:B4).After 3 rounds of panning,the positive phage clones were identified by ELISA,and the amino acid sequences of these positive clones were deduced from DNA sequences.In order to predict how these peptide mimics interaction with 3A8,peptides(A2 and A5) based on conserved sequences of positive clones were synthesized and conjugated with KLH for further study.The binding of A2-KLH and A5-KLH to 3A8 were identified by ELISA.Results:15 of 33 phage clones were identified as positive clones and shown specific binding with 3A8.LPS2630 potently inhibited the binding of phage clone to 3A8.In analysis of the amino acid sequence,there were seven kinds of sequences containing highly hydrophilic residues,and Ser Pro Pro/Pro X Pro was the conserved sequence.The peptides A2 and A5 could bind to 3A8 specially.Conclusion:The conserved sequences containing Ser Pro Pro/Pro X Pro are obtained,which are recognized by mAb 3A8 against broad spectrum Gram negative bacteria and several strain LPS.The synthetic peptides based on the conserved sequence can bind to 3A8,indicating that the peptides can mimic a common epitope of Gram negative bacteria,which be expected as candidate epitope for vaccinization.

Objective To describe the pathological unit and octagonal en bloc resection for the treatment of ossification ligamentum flavum(OLF)in thoracic spine with spondylotic myelopathy.Methods Ninety-five patients from January 2002 to January 2007 were diagnosed as thoracic OLF,61 males and 34 females with an average age of 53.9 years(range,31-78 years).There were upper thoracic spine OLF in 32 cases,middle thoracic spine OLF in 24 cases and lower thoracic spine OLF in 39 cases.Single-segment OLF was found in 53 cases,double segments OLF was found in 38 cases and three segments OLF was found in 4 cases.CT scan multiplanar co-localized reconstruction was employed to detect the structure of spine with OLF.The Japanese Orthopaedic Association(JOA)lower limb motor function score,sphincter function score and motor function improvement rate were used to evaluate the outcomes.Results CT scan was engaged to observe 141 OLF pathological unite.The OLF pathology unit was defined as all the spine structures between the extension lines of the lower margin of the OLF two adjacent pedicles.Each OLF associates with an OLF pathology unit.The mean follow up duration was 38.3 months(range,24-60 months).Among 86 patients with sensations disturbance before operation,67 totally recovered and 19 relieved after operation.Trunk restrictions in 69 cases before operation were completely recovered after operation.Postoperative JOA sphincter function score was 2.651±0.334,comparing with preoperation score(2.262±0.561),and the difference was statistically significant.Postoperative JOA motor function score was 3.694±0.429,which was significantly increased than preoperative score 1.539±0.873,and motor function recovery rate was 87.57%.There was excellent in 71 cases,good in 17 cases and fair in 5 cases.The excellent and good rate was 94.74%.Conclusion The octagonal en block resection is relative safe for treatment thoracic OLF with myelopathy.Pathological unit of OLF in thoracic spine is more accurate to summarize the pathological contents and features of the OLF and its adjacent structure.

OBJECTIVE:To systematically evaluate therapeutic efficacy of modified Shenqi dihuang decoction in the treatment of chronic glomerular nephritis. METHODS:Retrieved from Wanfang database,CNKI,VIP,CBM,Cochrane Library,Medline, EMBase and PubMed,randomized controlled trials(RCTs)about modified Shenqi dihuang decoction combined with conventional therapy and (or) other Chinese patent medicine (trial group) vs. conventional therapy and (or) other Chinese patent medicine (control group)in the treatment of chronic glomerular nephritis were collected. Meta-analysis was carried out by using Rev Man 5.3 software after literature scanning,data extraction and quality evaluation. Stata 14.0 software was used to conduct Egger test to evaluate publication bias. If publication bias existed,the effect of publication bias on outcome was further evaluated by scissor compensation. TSA v0.9 software was used for trial sequential analysis(TSA)of total efficency. RESULTS:A total of 10 RCTs were included,involving 707 patients. The results of Meta-analysis showed that total response rate of trial group [RR＝1.40,95%CI(1.22, 1.61),P＜0.001] was significantly higher than that of control group; the level of urine protein [SMD＝－1.97,95%CI(－2.92,－1.03),P＜0.001] and Scr [MD＝－28.41,95%CI(－38.95,－17.87),P＜0.001] in trial group were significantly lower than control group,with statistical significance. Publication bias test of total response rate was conducted,and results of it showed that there was publication bias,but bias did not affect the results of this study. TSA analysis showed that the evidence of Meta-analysis was false positive. CONCLUSIONS:Therapeutic efficacy of modified Shenqi dihuang decoction for chronic glomerular nephritis need to be proved by more stringent RCTs.

Objective To evaluate the clinical results of indirect reduction and fixation with the self-manufactured external fixator as a viable alternative in the surgical treatment of intraarticular calcaneal fractures.Methods From May 2006 to May 2009,a total of 30 patients undergone surgical treatment of intraarticular calcaneal fractures were analyzed,including 20 males and 10 females with an average age of 36 years (range,15-53).According to Sanders classification based on the computed tomography scan of intraarticular calcaneal fractures,16 patients were classified as type-Ⅲ,and 14 type-Ⅳ in this series.All fractures were treated first with the external fixator as indirect reduction and fixation device on the whole,which can enlarge the interspace of the subtalar joint significantly.Then,posterior articular facet of calcaneus was exposed and reduced through a small lateral incision.The calcaneal's length,breadth,thalamus height,maximum vertical displacement of the post-articular surface,and B(o)hler angle were measured preoperatively,3 days and 6 months after operation in X-ray film.Reduction results were evaluated by CT scan according to the standard of Buckley.Results The average follow-up time of all patients was 29 months (range,4-45).Lateral and axial roentgenograms showed satisfactory restoration of the calcaneal's anatomical structure.There were significant differences between preoperative values and those 3 days or 6 months postoperatively.There were no significant differences between values 3 days postoperatively and those 6 months postoperatively.The reduction results of posterior articular facet were evaluated by CT scan.Twenty-seven patients obtained anatomical reduction,3 patients obtained uneven articular facet within 2 mm.Conclusion This selfmanufactured external fixator is a vialbe alternative in the treatment of intraarticular calcaneal fractures,which has advantages of minimal invasion,practicality and less complications.

The aim of this study was to investigate the effect of transmembrane 9 superfamily protein member 2 (TM9SF2) in proliferation and migration of triple negative breast cancer cell line MDA-MB-231.The expression of TM9SF2 in triple negative breast cancer cell line MDA-MB-231 and nontumorigenic mammary epithelial cell line MCF-10A were measured by Western blot. MDA-MB-231 cells were treated with siRNA-TM9SF2 to knock-down the expression of TM9SF2. The effect of silencing TM9SF2 was measured with Western blot.The proliferation of cells was tested by MTS,and the migration was measured with Transwell and wound-healing assay.Proteins related to proliferation (PI3K,AKT,SRC and ERK) and migration (Snail,Slug and N-cadherin) were measured with Western blot.Protein expressions of TM9SF2 was better improved in triple negative breast cancer MDA-MB-231 cell line than MCF-10A.Compared with the control group, the siRNA-TM9SF2 infected group had lower expressions of PI3K, Snail, Slug and N-cadherin, and at the same time phosphorylation of AKT was decreased. The results suggest TM9SF2 can promote the proliferation and metastasis of triple negative breast cancer MDA-MB-231 cell line.

BACKGROUND: AND PURPOSE: Previous studies have explored the association between retinal vascular changes and cognitive impairment. The retinal vasculature shares some characteristics with the cerebral vasculature, and quantitative changes in it could indicate cognitive impairment. Hence, a comprehensive meta-analysis was performed to clarify the potential relationship between retinal vascular geometric changes and cognitive impairment. METHODS: Relevant databases were scrupulously and systematically searched for retinal vascular geometric changes including caliber, tortuosity, and fractal dimension (FD), and for cognitive impairment. The Newcastle-Ottawa Scale was used to evaluate the methodological quality of included studies. RevMan was used to perform the meta-analysis and detect publication bias. Sensitivity analyses were also performed. RESULTS: Five studies that involved 2,343 subjects were finally included in the meta-analysis. The results showed that there was no significant association between central retinal artery equivalents (Z=1.17) or central retinal venular equivalents (Z=1.74) and cognitive impairment (both p>0.05). Similarly, no significant difference was detected in retinal arteriolar tortuosity (Z=0.91) and venular tortuosity (Z=1.31) (both p>0.05). However, the retinal arteriolar FD (mean difference: −0.03, 95% CI: −0.05, −0.01) and venular FD (mean difference: −0.03, 95% CI: −0.05, −0.02) were associated with cognitive impairment. CONCLUSIONS A smaller retinal microvascular FD might be associated with cognitive impairment. Further large-sample and well-controlled original studies are required to confirm the present findings.

OBJECTIVETo observe sperm motion parameters in pre-freeze and post-thaw semen samples using computer-assisted sperm analysis (CASA) system.

METHODSSemen analyses of 238 samples before freezing and after thawing were separately performed by Hamilton-Thorne Sperm Analyzer.

RESULTSSperm motility in post-thaw samples was significantly decreased. There was significant correlation and difference between pre-freeze and post-thaw samples in sperm motion parameters, including average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), straightness (STR) and linearity (LIN), except heat cross frequency (BCF). The percentage of sperm movement velocity parameters (VAP, VSL and VCL) and moving pattern parameters (ALH) significantly decreased, while that of LIN and STR significantly increased in post-thaw samples.

CONCLUSIONCASA system is of clinically applied value and is a useful tool for evaluating sperm motion parameters in pre-freeze and post-thaw semen samples.

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le(b)) and Le(y) antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the β-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.
Binding Sites ; Blood Group Antigens ; chemistry ; immunology ; Caliciviridae Infections ; immunology ; virology ; Crystallography, X-Ray ; Epitopes ; chemistry ; immunology ; Evolution, Molecular ; Humans ; Lewis Blood-Group System ; chemistry ; immunology ; Norovirus ; chemistry ; immunology ; pathogenicity ; Protein Binding ; Viral Proteins ; chemistry ; immunology

α-cells, which synthesize glucagon, also support β-cell survival and have the capacity to transdifferentiate into β-cells. However, the role of α-cells in pathological conditions and their putative clinical applications remain elusive due in large part to the lack of mature α-cells. Here, we present a new technique to generate functional α-like cells. α-like cells (iAlpha cells) were generated from mouse fibroblasts by transduction of transcription factors, including Hhex, Foxa3, Gata4, Pdx1 and Pax4, which induce α-cell-specific gene expression and glucagon secretion in response to KCl and Arg stimulation. The cell functions in vivo and in vitro were evaluated. Lineage-specific and functional-related gene expression was tested by realtime PCR, insulin tolerance test (ITT), glucose tolerance test (GTT), Ki67 and glucagon immunohistochemistry analysis were done in iAlpha cells transplanted nude mice. iAlpha cells possess α-cell function in vitro and alter blood glucose levels in vivo. Transplantation of iAlpha cells into nude mice resulted in insulin resistance and increased β-cell proliferation. Taken together, we present a novel strategy to generate functional α-like cells for the purposes of disease modeling and regenerative medicine.
Animals ; Blood Glucose ; Fibroblasts* ; Gene Expression ; Glucagon ; Glucose Tolerance Test ; Immunohistochemistry ; In Vitro Techniques ; Insulin ; Insulin Resistance ; Mice* ; Mice, Nude ; Polymerase Chain Reaction ; Regenerative Medicine ; Transcription Factors