Objective To investigate the expression of cyclooxygenase-2(COX-2)and Ki-67 in rectal cancer tissues and their predictive value for the sensitivity to neoadjuvant chemoradiotherapy(NAC).Methods The clinical data of 87 patients receiving chemoradiotherapy before rectal cancer surgery at Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine(the Second Hospital of Nanjing)from June 2021 to September 2022 were retrospectively analyzed.In addition,40 normal rectal tissue samples were selected from the Department of Pathology of Nanjing University of Chinese Medicine(the Second Hospital of Nanjing)as control.The expression levels of COX-2 and Ki-67 in tumor and adjacent tissues of patients with rectal cancer as well as in normal rectal tissues were detected by using the immunohistochemical method.The patients were divided into chemoradiotherapy-sensitive group(n=62)and chemoradiotherapy-resistant group(n=27)according to whether they were sensitive to chemoradiotherapy.The correlation between the expression levels of COX-2,Ki-67 in tumor tissues and adjacent tissues and the sensitivity to chemoradiotherapy was analyzed.The relative factors affecting the effect of chemoradiotherapy on rectal cancer patients were analyzed by using the logistic regression model.The receiver operating characteristic(ROC)curve was drawn,and the area under the curve(AUC)was used to evaluate the predictive value of COX-2 and Ki-67 expression levels in tumor tissues of rectal cancer patients for the sensitivity to NAC.Results Among the 87 patients with rectal cancer,60 patients were sensitive to chemoradiotherapy,with a sensitivity rate of 68.97％.The positive expression rates of COX-2 and Ki-67 in tumor tissues and adjacent tissues were significantly higher than those in normal rectal tissues(x2=53.187,7.131,53.047,14.613;P＜0.05).The positive expression rates of COX-2 and Ki-67 in tumor tissues were significantly higher than those in adjacent tissues(x2=72.572,67.616;P＜0.05).The positive expression rates of COX-2 and Ki-67 in tumor tissues of patients in the chemoradiotherapy-sensitive group were significantly lower than those in the chemoradiotherapy-resistant group(x2=3.965,6.264;P＜0.05).Logistic regression analysis showed that the positive expression of COX-2 and Ki-67 in tumor tissues were factors affecting the efficacy of NAC in rectal cancer patients(P＜0.05).ROC curve analysis results showed that the sensitivity of COX-2 and Ki-67 expression and their combination for predicting sensitivity of patients to NAC was 100.00％,100.00％,and 100.00％,respectively;while the specificity was 13.33％,20.00％,and 31.67％,respectively;and the AUC was 0.567,0.600,and 0.658,respectively.Conclusion The positive expression of COX-2 and Ki-67 in tumor tissues are factors affecting the efficacy of NAC in rectal cancer patients,and the combined detection of COX-2 and Ki-67 expression has a high predictive value for the sensitivity of NAC.

Adjuvants enhance the efficacy of the vaccine by regulating or enhancing the immune response to antigens,helping to reduce the amount of antigen and improve the vaccine safety.With the deepening of adjuvant researches,it has been found that the combination of two or more adjuvants can produce synergistic effects by activating multiple immune mechanisms.Highly effective vaccines are urgently needed for the group with pathogens that are difficult to remove and with low immune system function.This requirement can be met by the combination of immunologic adjuvants.In this paper,the progress of some new adjuvants and the applications of combined adjuvants were reviewed including liposomes,immunostimulating complexes,Montanides,polycationic peptide IC31,nanoemulsion and AS adjuvant systems.

Immunological checkpoints are a mechanism evolved by human beings to control the intensity and duration of immunoreaction and minimize the excessive inflammatory responses and autoimmune diseases caused by overactive immune responses.Compared to radiotherapy,chemotherapy and other traditional treatments,immunotherapy has fewer side effects on normal cells,and has become an emerging technology in tumor treatments.As the focus on tumor treatment research,immunological checkpoint therapy can damage the tumor cells by breaking the immune tolerance and activating the body's own immune system,which make it a promising treatment method.In this paper,the mechanisms of immune activation,immune regulation and immune evasion were described.The action mechanism and clinical application of anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death receptor-1 (PD-1) were summarized.The application prospect of immune checkpoint inhibitors was discussed.

In recent years,cancer immunotherapy has developed rapidly due to its significant advantages compared with the traditional cancer treatment methods.Tumor immunotherapy aims at mobilizing or stimulating the body's own immune function,thereby inhibiting and killing cancer cells.With the development of nanotechnology,biological nano-carrier materials provide a new insight into the vaccine development.Nano-vaccines are therapeutic or prophylactic vaccines based on nanotechnology including exogenous antigens for inducing immune responses,vectors delivering antigens,and adjuvants for enhancing immunogenicity and accelerating and prolonging the availability of cancer vaccines.Nano-delivery vectors have good biocompatibility as well as unique physical and chemical properties.They can effectively deliver the antigens,and further activated the immune response of antigenspecific cellulars based on the activation of the body's humoral immunity by regulating the presentation pathways in the antigen-presenting cells.In this paper,the applications of nano-delivery systems in cancer vaccine research were summarized.

Objective To prepare stable aqueous dispersions of chitosan/multi-walled carbon nanotubes (CS/MWCNTs) composites,and observe the effects of CS/MWCNTs on the growth of human umbilical vein endothelial cells (HUVEC).Methods CS/MWCNTs composites were prepared by electrostatic interactions between negatively charged MWCNTs and positively charged low-molecular-weight CS.The prepared CS/MWCNTs were characterized by transmission electron microscopy and Zetasizer nano-analyser.The cellular uptake of the fluorescently labeled CS/MWCNTs was observed by laser confocal microscopy after incubating with HUVEC for 24 h at different concentrations.In vitro cytotoxicity and cellular reactive oxygen were also detected.Results When the mass ratio of low-molecular-weight CS to MWCNTs was equal or greater than 10∶1,the CS/MWCNTs can be stabilized in solution.Cellular uptake experiments showed that the CS/MWCNTs could enter into the cells and locate mainly in the cytoplasm.Cytotoxicity study showed that the CS/MWCNTs composites was less toxic than MWCNTs alone at high concentration (10 and 20 μg/ml).However,there was no significant differencein the level of cellular reactive oxygen between the two groups (P＜0.05).Conclusions CS/MWCNTs composites showed low cytotoxicity and high stability,which would be a promising carrier for drug delivery.

Objective To investigate the change of serum osteoprotegerin level and relationships between serum osteoprotegerin level and coronary heart disease (CHD) in patients with type2 diabetes mellitus (DM) complicated with CHD.Methods One hundred and eight patients with type 2 DM complicated with CHD were selected as DM with CHD group,including 68 cases with acute coronary syndrome and 40 cases with stable angina pectoris.In addition,60 type 2 DM patients without CHD (DM without CHD group) and 40 healthy people (control group) were selected.Serum osteoprotegerin level was measured by enzyme linked immunosorbent assay.Results The serum osteoprotegerin level in DM with CHD group was significantly higher than that in DM without CHD group and control group [(4.12 ± 0.71)ng/L vs.(2.69 ± 0.52) and (2.14 ± 0.37) ng/L],and there were statistical differences (P＜ 0.05 or ＜ 0.01).In DM with CHD group,the serum osteoprotegerin level in acute coronary syndrome was significantly higher than that in stable angina pectoris [(4.56 ±0.92) ng/L vs.(3.61 ±0.76) ng/L],and there was statistical difference (P ＜ 0.05).The serum osteoprotegerin level in patients with Gensini score ＞ 40 scores (41 cases)was significantly higher than that in patients with Gensini score 20-40 scores (53 cases) and patients with Gensini score ＜ 20 scores (14 cases) [(4.92 ± 0.89) ng/L vs.(4.08 ± 0.75) and (3.39 ± 0.85) ng/L],and there were statistical differences (P ＜ 0.01 or ＜ 0.05).The serum osteoprotegerin level in patients with Gensini score 20-40 scores was significantly higher than that in patients with Gensini score ＜ 20 scores,and there was statistical difference (P ＜0.05).The serum osteoprotegerin level in single-vessel lesion (16 cases),double-vessel lesion (57 cases) and three-vessel lesion (35 cases) [(3.52 ± 0.82),(4.54 ± 0.68),(4.75 ±0.93) ng/L] were significantly higher than those in control group,and there were statistical differences (P ＜ 0.05); and the serum osteoprotegerin level in double-vessel lesion and three-vessel lesion were significantly higher than those in single-vessel lesion,and there were statistical differences (P ＜ 0.05) ;there was no statistical difference between three-vessel lesion and double-vessel lesion(P＞ 0.05).The results of Logistic regression analysis showed that glycosylated hemoglobin,total cholesterol,high sensitivity C reactive protein and osteoprotegerin were independent risk factors of type 2 DM complicated with CHD.Conclusions Serum osteoprotegerin level in patients with type 2 DM complicated with CHD is significantly increased.There is a significant association between serum osteoprotegerin level and the presence and severity of CHD,and the serum osteoprotegerin level is independent risk factors of type 2 DM complicated with CHD.

Objective To evaluate the effects of different storage periods at room temperature on domestic cisatracurium-induced neuromuscular (N-M) block.Methods One hundred and twenty ASA Ⅰ or Ⅱ patients,aged 18-64 yr,scheduled for elective operations under general anesthesia,were randomly divided into 3 groups (n =40 each):cisatracurium stored for 60 days at 4-8 ℃ group (group LT),cisatracurium stored for 30 days at room temperature group (group RT30) and cisatracurium stored for 60 days at room temperature group (group RT60).Anesthesia was induced with iv injection of midazolam and target-controlled infusion of propofol (target plasma concentration 3 μg/ml) and remifentanil (target effect-site concentration 3-5 ng/ml).A bolus of cisatracurium 0.2 mg/kg was given intravenously over 5-10 s as soon as the patients lost consciousness.N-M block was monitored with TOF-Watch SX (Organon,Netherlands).Single stimulation was applied to the ulnar nerve at wrist.The maximal degree of N-M block,onset time,clinical duration,recovery index and 75 ％ recovery time were recorded.The patients were intubated and mechanically ventilated when N-M block reached the maximal degree.The intubation condition was evaluated.Hypotension,bradycardia and skin allergy were recoded.Results Compared with group LT,no significant change was found in the onset time,clinical duration,recovery index and 75％ recovery time in group RT30 (P ＞ 0.05),and the onset time was significantly prolonged,clinical duration and 75％ recovery time were shortened in group RT60 (P ＜ 0.05).The onset time was significantly longer and clinical duration was shorter in group RT60 than in group RT30 (P ＜ 0.05).The intubation condition was excellent or good in the three groups and there was no significant difference among the three groups.There was no significant difference in the incidence of hypotension and bradycardia among the three groups (P ＞ 0.05).No patients developed skin allergy.The maximal degree of N-M block was 100％ in groups LT,RT30 and RT60 except one case with 95％ in group RT60.Conclusion No significant change is found in the N-M block induced by domestic cistracurium when stored for 30 days at room temperature,however,the N-M block is significantly attenuated when stored for 60 days at room temperature.

ObjectiveTo isolate and identify endothelial progenitor cells (EPCs) from human umbilical cord,and to study the cell proliferation and gene transfection of green fluorescent protein plasmid in vitro.MethodsEPCs were isolated from human umbilical cord in enzyme digestion method.The biological characteristics of EPCs were identified by flow cytometry and laser confocal microscope.The enhanced green fluorescent protein (EGFP) gene transfection mediated by EPCs was investigated using Lipofectamine 2000 as transfection reagent.ResultsEndothelial progenitor cells isolated from umbilical cord formed typical endothelial cell colony 9 days later.These cellsdisplayed an improved positive expression of CD133 and kinase insert domain receptor (KDR).The endotheliallineage characteristics of expanded cells were confirmed by fluorescein isothiocyanate (FITC)-UEA-1 binding and DiI-ac-LDL uptake assay with the aid of laser confocal microscope.The transfection results demonstrated high expression of EGFP taking EPCs as host cell.ConclusionEndothelial progenitor cells isolated from umbilical cord can be propagated and induced to differentiate into endothelial cells in the appropriate culture conditions.EPCs demonstrated to be an ideal carrier for gene and cell therapy.

ObjectiveConjugation of fluorescent dye onto plasmid DNA was investigated in order to monitor delivery process of plasmid DNA.MethodsPlasmid was activated with bromine,stored for different timeintervals at 4 ℃ or room temperature,and subsequently coupled with 1,10-diaminodecane to prepare aminemodified plasmid DNA.Amine-modified plasmid was then reacted with isothiocyanate (FITC) for fluorescent labeling,and the labeling ratio was calculated after purification.The effect of storage conditions (time/temperature) of bromine-actived plasmid (BP) on fluorescent labeling efficacy was estimated,and the cell transfection efficiency of fluorescent plasmid-lipofectamine complex was observed.The fluorescent plasmid delivered by lipofectamine 2000 in A10 cells was observed by laser scanning confocal microscope (LSCM) and flow cytometry.ResultsThe experimental data showed that prolonged storage time of bromine-activated DNA had a negative effect on the labeling ratio,and lower storage temperature had a positive effect on the labeling ratio.It also demonstrated that FITC modification had no effect on the transfection efficiency of plasmid-lipofectamine complex as compared with that of unlabeled plasmid-lipofectamine complex,and FITC modified plasmid had enough fluorescent intensity to monitor cell uptake with flow cytometer and sub-cellular distribution with LSCM.ConclusionA facile method for conjugating fluorescent dye onto plasmid was established in the study,and could be utilized to trace the plasmid delivery for investigating the transfection mechanism.

ObjectiveTo explore the endocytic pathway of TAT-LHRH modified chitosan/DNA nanoparticle (TLCDN) that exhibits high transfection efficiency and targeting to HepG2.MethodsPlasmid DNA was labeled with fluorescein,and the resulting fluorescent DNA was complexed with chitosan or TAT-LHRH modified chitosan to form chitosan/DNA nanoparticle (CDN) and TLCDN by the complex coacervation method.Internalization of TLCDN or CDN by HepG2 cells were measured in the presence of three kinds of inhibitors of endocytic pathway,Chlorpromazine,Filipin or Dynasore,using High-Content Analyzer to collect and analyze the data.ResultsChlorpromazine led to more decreased uptake of CDN than that of TLCDN,although not statistically significant.Filipin demonstrated significant inhibitory effect on the uptake of TLCDN while promoted the uptake of CDN.Dynasore resulted in a similar decrease in the uptake of both nanoprticles.ConclusionIt was demonstrated that CDN was taken up by HepG2 cells mainly through the clathrin-dependent endocytic pathway and TLCDN was more likely to be internalized by HepG2 cells through the caveolin-mediated endocytic pathway although the clathrin-dependent endocytic pathway was also involved.