BACKGROUND: At present,enhanced green fluorescent protein(EGFP)is proved to be the best labeled molecule,with unique advantages,such as high fluorescence specificity,easy to be detected,and so on.Recombined retroviral vector EGFP-pLNCX2,which can stably express EGFP,can be construct using gene-engineering technique.Transfecting allogenic chondrocytes is indeed very useful to investigate the target gene expression and process of constructing tissue engineered cartilage in vivo.OBJECTIVE:To construct recombined retroviral vector EGFP-pLNCX2 which can stably express EGFP,and investigate optimal conditions for retrovirus transfection of chondrocytes.DESIGN:Randomized controlled observation.SETTING:Shanghai Jiao Tong University.MATERIALS:Thirty New Zealand rabbits of either gender,1 week of age,were purchased from Experimental Animal Center of Chinese Academy of Sciences.Amphotropic retrovirus package cell line PT67 pLNCX2 and pEGEP-C1 were purchased from Clontech Company;NIH 3T3 cell line was purchased from ATCC Company;DH5a Bacterium coli was preserved by the laboratory of Shanghai Jiao Tong University;Retroviral vector pLNCX2 was purchased from Clontech Company;pEGFP-C1 plasmid with EGFP were donated by professor Cong Xiao-qian from Chinese Academy of Sciences.METHODS:This experiment was carried out in the Shanghai Jiao Tong University in August 2005.Chondrocytes of New Zealand rabbits were isolated and cultured.The recombinant retroviral vector EGFP-pLNCX2,which can stably express EGFP,was constructed to transfect cultured chondrocytes from New Zealand rabbits by using gene engineering technique.Transfection results were observed under fluorescence microscope.Altogether 6×105 chondrocytes incubated in 10 cm-diameter flat plate were used to transfect retrovirus-EGFP immediately and at 12,24 and 48 hours after inoculation.One week later,EGFP expression efficiency was measured with flow cytometer,and best occasion for retrovirus transfecting primary chondrocytes.Enzyme-digested chondrocytes were inoculated for 24 hours to transfect retrovirus.After 250 mg/L G418 was added,chondrocytes were screened on the 2nd,3rd,4th,5th and 6th days separately.After phosphate buffer solution(PBS)washings,the transfection efficiency of chondrocytes was detected by flow cytometer,and the best occasion for G418 screening was observed.MAIN OUTCOME MEASURES:①EGFP-pLNCX2 transfection efficiency.②The best occasions for retrovirus transfecting primary chondrocytes and G418 screening.RESULTS: ①Retroviral vector EGFP-pLNCX2 transfected primary chondrocytes of rabbits,and high expression of transfected chondrocytes could be obtained through preliminary screening of G418.After being screened and expressing EGFP,chondrocytes kept normal morphology,with pseudopod adhering to the wall and matrix secreting vigorously.②The best occasion for retrovirus transfecting primary chondrocytes was at 24 hours after cell inoculation.The transfection efficiency determined with flow cytometer was 19.14% on the 5th day.The best occasion for G418 screening was on the 5th day after culture.Transfection efficiency of G418 screening was 55.75% on the 7th day.CONCLUSION:Recombinant retroviral vector EGFP-pLNCX2 can effectively transfect chondrocytes.The best occasion for retrovirus transfecting primary chondrocytes is at 24 hours after inoculation, and the best occasion for G418 screening is on the 5th day after culture.

Objective To investigate the expression and prognostic significance of Interleukin-36α (IL-36α) in colorectal cancer.Methods The expression of IL-36α was tested by immunohistochemical staining in 329 cases of colorectal cancer.According to the intensity and the proportion of positive tumor cells,all the patients were divided into IL-36α low and high expression groups.The clinicopathological factors and prognosis of patients between IL-36α low high expression groups were compared.Results Significant differences were observed in the number of patients in tumor differentiation and pM classification between patients in the IL-36α low and high expression groups (P ＜ 0.05).The 5-year overall and tumorfree survival rates of patients were 79.3％ and 77.2％ in IL-36α low expression group,and 66.3％ and 65.3％ in IL-36α high expression group (P ＜0.05).COX proportional hazard regression model revealed that high expression of IL-36α was associated with short overall survival time and tumor-free survival time of colorectal cancer patients (P ＜ 0.05).Multivariate analysis identified IL-36α expression in colorectal cancer as an independent prognosticator (P ＜ 0.05).Conclusions High expression of IL-36α was correlated with tumor differentiation and pM classification of colorectal cancers,and it is an independent predictor of poor survival for patients with colorectal cancer.

Objective To explore the value of practicing PBL which was guided by MDT in teaching diagnoses and treatments of war wound and trauma caused by compound agents.Methods An emulational war wound case caused by compound factors was designed and a multi-disciplinary team been organized,then totally 45 interns who would graduate in 2017 were divide into two groups randomly,experimental group which had 23 cases received PBL model combined with MDT followed six step procedure which included case preparation by teachers,independent analysis and group discussion by interns,problem extraction by teachers and division of solution by interns before class,then answering and debating problems by interns in class,conclusion and reporting after class in the end,while the control group which had 22 cases received traditional teaching model in accordance with common case discussion in class including characteristics of patient,diagnosis and diagnostic basis,examinations needed to carry out,first aid measures and professional treatments.The effect of new teaching model was evaluated by assessment in class and questionnaire after class.The data was analyzed through Chi-square test by SPSS 22.0.Results Class assessment showed 18 (78.3％) interns in experimental group displayed good abilities of proposing,analyzing and resolving medical problem,as well as good presentation and speech skills.Questionnaire survey showed that not only clinical teaching of war wound and trauma could meet the demands of talent training in battle-field rescue,but also displayed new model,which could help interns to enhance abilities of first-aid and treatment of war wound on future war field,reinforce the consciousness of military medical support and service.In addition,it could clear confusion more professionally and strengthen teamwork and overall importance.Conclusion Applying PBL combined with MDT in teaching diagnoses and treatments of war wound and trauma can not only help interns to review and retain important knowledge of related subjects,but also improve the abilities of interns in clinical diagnoses and treatments,and moreover,it merges the medical knowledge and war wound cure together organically.In short,the new model is well worth applying in clinical teaching and military medical education because of its excellent effects.

Objective To investigate the clinical value of coronary artery Z-scores on echocardiography in diagnosing coronary artery abnormalities. Methods The echocardiography results of 612 patients with Kawasaki disease (KD) at the acute and recovery phase were retrospectively studied. Coronary artery luminal diameters were converted to body-surface-area-adjusted Z-scores. According to coronary Z-scores classiifcation, all the subjects were divided to four groups:415 cases with no dilation (ND), 133 cases with small coronary artery abnormalities (SCAAs), 47 cases with large coronary artery abnormalities (LCAAs), and 17 cases with giant coronary artery abnormalities (GCAAs). Clinical features (gender, age, typical clinical manifestations, fever duration) and laboratory results (CRP, ESR, WBC, PLT) were compared among all the four groups. Coronary artery diameters and the Z-scores were compared between acute and convalescence phase. Results Along with the increase of coronary Z-score, fever duration was prolonged [ND group:(7.75±3.12) d, SCAAs group (8.50±4.12) d, LCAAs group: (8.57±3.58) d, GCAAs group: (11.88±4.33) d, F=22.375, P<0.05]. With coronary Z-score increasing, PLT also increased (F=22.029, P=0.000), and the highest PLT was observed in GCAAs group. There were no significant differences in the CRP, ESR and WBC among all the four groups (F=0.236, 1.116, 0.121, all P>0.05). No significant different coronary diameters were found in ND cases between recovery and acute phase [(2.24±0.34) mm vs (2.33±0.36) mm, t=1.926, P > 0.05]. But there were significant difference in the coronary Z-scores of ND patients between recovery and acute phase (0.41±0.82 vs 1.17±0.75, t=8.332, P < 0.05). The coronary Z-scores in SCAAs group (1.32±0.89 vs 3.40±0.62, t=11.073, P < 0.05), LCAAs group (3.12±2.27 vs 6.20±1.28, t=4.579, P<0.05) and GCAAs group (11.88±6.77 vs 20.4±9.70, t=3.480, P<0.05) at recovery phase were smaller than values at acute phase. Conclusions The KD coronary Z-scores are the body-surface-area-adjusted standard value, and not subject to the influence of children growth and development. Therefore, it may accurately evaluate the severity of coronary artery abnormalities and its recovery process. Accurate quantitative of the coronary artery luminal dimensions is important in KD clinical management and prognosis prediction.

Objective Comparing the efficacy of endoscopic balloon dilatation and endoscopic stricteroto-my for postoperative anastomotic stenosis of colorectal cancer. Methods Retrospectively analyzed the clinical data of patients with postoperative anastomotic stenosis of colorectal cancer that underwent anastomotic dilatation from 2013 to 2016,and analyzed the anastomotic stenosis before and after treatment,and compared the efficacy of the two groups of dilatation methods. Results There was no statistically significant difference between the two groups in baseline characteristics. Balloon dilatation was effective in 3 cases(23.1%),ineffective in 10 cases(76.9%). 7 cases(63.6%)were effective in the stricterotomy group,4 cases(36.4%)were ineffective,the difference was statistically significant(P=0.045).Two groups of patients were not bleeding after surgery,infection and perfora-tion and other complications. Conclusion Endoscopic stricterotomy of postoperative anastomotic stenosis of colorectal cancer is more effective than conventional endoscopic balloon dilatation

OBJECTIVETo investigate the expression and clinical significance of G protein-coupled receptor 31 (GPR31) in colorectal cancer tissue.

METHODSCancer tissues and adjacent normal tissues of 321 cases with colorectal cancer confirmed by pathology and undergoing resection in the First Affiliated Hospital of Sun Yat-sen University from January 1996 to December 2008 were collected. The expression of GPR31 was examined by immunohistochemical staining. According to the expression level of GPR31 (A value=0.051), all the patients were divided into low GPR31 expression group and high GPR31 expression group. Clinicopathology and prognosis between the two groups were compared. Risk factors affecting prognosis were investigated.

RESULTSGPR31 expression was significantly higher in colorectal cancer tissues compared to adjacent normal tissues (mean A, 0.063±0.014 vs. 0.045±0.020, P<0.001). A total of 197 cancer tissue samples were defined as low expression and 124 as high expression. Significant difference was observed in the number of patient in pM classification between the two groups (P=0.007). High expression group had obviously higher distant metastasis rate than low expression group [12.1% (15/124) vs. 4.1% (8/197), P=0.007]. The 5-year survival rate and tumor-free survival rate were 84.3% and 82.2% in the low expression group, and both 59.7% in high expression group (all P<0.05). Multivariate analysis revealed elderly, abnormal CEA, lymphatic metastasis, distant metastasis and up-regulated GPR31 expression were independent risk factors of overall survival and disease-free survival in colorectal cancer patients (all P<0.05).

CONCLUSIONSGPR31 expression is significantly up-regulated in colorectal cancer tissues. High GPR31expression indicates poor prognosis of colorectal cancer, and may be used as a predictive marker.

Aged ; Biomarkers, Tumor ; metabolism ; Colorectal Neoplasms ; diagnosis ; metabolism ; Disease-Free Survival ; Humans ; Lymphatic Metastasis ; Prognosis ; Receptors, G-Protein-Coupled ; metabolism ; Risk Factors ; Survival Rate ; Up-Regulation

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been infected rapidly and is generally susceptible to population. Moreover, it has become the most serious public health problem in the world. In the process of coronavirus disease 2019 (COVID-19) treatment, the traditional Chinese medicine (TCM) intervention has achieved positive efficacy and which is widely recognized. However, the COVID-19 epidemic is still very serious, especially due to the characteristics of asymptomatic infection such as concealment, limitations and subjective symptoms, which has increased the difficulty of prevention and control. In view of asymptomatic infection, isolation is the main management. There is little mention of specific treatment options in each version of COVID-19 treatment plan. Through systematic study of TCM theory, we explored the way of diagnosis and treatment of asymptomatic infections. Based on the theory of latent evil, clearing away latent pathogens to truncate the course of disease and reduce the incidence. Based on the theory of prevention of disease, strengthen the lung and stomach to protect the place from pathogen and prevent the transmission of disease evil. It is supplemented by accurate treatment according to individual, timing and local conditions, in order to provide reference for the treatment of asymptomatic infection.

OBJECTIVETo investigate the role of miR-26b in the invasion and metastasis of colorectal cancer.

METHODSData of public chip databases were extracted to analyze the relationship between miR-26b expression and lymph node metastasis. Two types of colorectal cancer cell lines, Caco2 and DLD1, were selected, and the miR-26b-high colorectal cancer cell line was constructed using the method of lentivirus infection. The effects of up-regulating miR-26b expression on the invasion and metastasis of colorectal cancer cells were analyzed by Transwell migration and invasion experiment and wound healing assay. The effect of up-regulating miR-26b expression on stem cell phenotype of colorectal cancer cells was analyzed by sphere-formation assay.

RESULTSThe microarray detection results showed that the expression of miR-26b in tumor tissues of patients with lymph node metastasis was significantly higher than those without lymph node metastasis[(12.04±0.20) vs. (11.31±0.19), t=2.646, P = 0.010]. In the in vitro experiment section, the Transwell experiment results showed that the number of invasive cells [(16.40±1.36) vs. (3.80±0.86), t=7.814, P=0.000] and migrating cells [(33.40±2.93) vs. (8.80±2.40), t=6.505, P=0.000] in miR-26b-high colorectal cancer cells was significantly higher as compared to miR-26b-low cells(all P<0.05). Would healing assay also confirmed that the migration speed of miR-26b-high colorectal cancer cells was significantly accelerated. Both the rate and the density of sphere formation were higher in miR-26b-high colorectal cancer cells than those in miR-26b-low colorectal cancer cells [Caco2:(168.3±11.7) vs. (54.2±10.8), t=7.185,P=0.002; DLD1:(4 076.0±409.8) vs.(1 613.0±210.1), t=5.349, P=0.006].

CONCLUSIONmiR-26b may promote the invasion and metastasis of colorectal cancer by accelerating the migration and invasion of colorectal cancer cells and enhancing the stem cell phenotype of tumor cells.

Caco-2 Cells ; Cell Line, Tumor ; Cell Movement ; physiology ; Cell Proliferation ; Colorectal Neoplasms ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs ; genetics ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis

OBJECTIVETo investigate the influence of CCL21 on the invasion and metastasis of colorectal cancer (CRC).

METHODSCCL21 over-expressing CRC cell line was constructed by lentivirus infection and CCL21 low-expressing CRC cell line was constructed by lipofection. The effects of CCL21 on the invasion and metastasis of CRC cells and the stem cell-like phenotype were investigated by Transwell migration, invasion assay, wound healing assay and sphere formation assay.

RESULTSReal-time quantitative PCR and western blot confirmed that the expression of CCL21 was up-regulated by lentiviral transfection and down-regulated by siRNA liposome transfection. In vitro, Transwell assays showed that the invasion and migration in CCL21 over-expressing CRC cells decreased significantly as compared to those of CCL21 low-expressing cells. In wound healing assay, the CCL21 over-expressing CRC cells showed a significantly lower rate of migration. In addition, the sphere formation rate and density of CCL21 over-expressing CRC cells were lower than those with low-expression of CCL21.

CONCLUSIONCCL21 can suppress the migration and invasion of CRC cells and weaken their stem cell-like phenotype.