Objective:To establish a stable primary culture method of human gingival epithelial cells, with a higher successful rate and shorter culture time.Methods:Nine patients who received “crown-lengthening surgery”with relatively healthy periodontal conditions were selected (n =9).Gingival sam-ples were collected from the 9 donors during gingivectomy.Gingival epithelial cells were isolated and cul-tured by both an advanced enzyme digestion method and a tissue explant method.In the advanced enzyme digestion culture process,2.5 g/L DispaseⅡwas used to separate the epithelial tissue part from the con-nective tissue part,which lasted for one night.Then the epithelial tissues were digested by 0.025% tryp-sin without EDTA for 10 minutes,and centrifuged by keeping the digested epithelial tissues that re-mained.This advanced method not only decreased the concentration and digesting time of the two above-mentioned enzymes,but also simplified the centrifugel process.The tissue explant method was not changed too much compared with the original method.Growing processes of the primary cells cultured by the two methods were observed and recorded respectively,and indirect immunocytochemical staining was used to identify the type of cultured cells.At the same time,successful rates and cell culture time were also compared between the two methods.Results:Human gingival epithelial cells with typical morpholo-gy could be cultured within a shorter period by the advanced enzyme digestion method with a successful rate of 88.9%,and proliferated rapidly as sheets.After 10 -14 d cells could be passaged,gradually turned to be like fibroblasts when passaged to the third generation,and eventually went to apoptosis.The primary culture time was longer by using the tissue explant method,and approximately after 17 -22 d cells could be passaged,although the successful rate was the same as the enzyme digestion method.Cy-tokeratin staining was both positive by indirect immunocytochemical staining of cells.Conclusion:Pri-mary human gingival epithelial cells cultured by the advanced enzyme digestion method could grow faster and be passaged to the second generation successfully,which could supply a stable origin for cellular ex-periments.

Objective To investigate the relationship between the expression of β-catenin and drug-resistance mechanism of choriocarcinoma according to the expression of β-catenin in JEG-3 cells (human choriocarcinoma cell line) and drug resistant JEG-3/VP16 cells.Methods The mRNA and protein expressions of β-catenin were analyzed with reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting.Flow cytometry was used to determine the percentages of β-catenin-positive cells in the two choriocarcinoma cell lines.Results Both drug resistant choriocarcinoma cells and drag sensitive cells were found to express β-catenin; but the expression of β-catenin mRNA (1.43 ±0.24) and protein(1.49 ±0.17)in drug resistant choriocarcinoma cells was found much higher than that in drug sensitive cells(0.65 ±0.14,0.66 ±0.16,P ＜0.01).And according to detect by flow cytometry,we found the number of β-catenin-positive cells in JEG-3/VP16 cells [(40.13 ±5.17) ％] was much more than that in JEG-3 cells [(13.15 ± 1.48) ％,P ＜ 0.01].Conclusions β-catenin was highly expressed in the drug resistant choriocarcinoma cell line (JEG-3/VP16).It indicates β-catenin might be involved in the drug resistance mechanism of choriocareinoma.

Objective To investigate the effects of granulocyte macrophage colony-stimulating factor (GM-CSF) combined with interleukin-12 (IL-12) genes on apoptosis of hepatoma cells.Methods The hepatoma cell lines were cultured in vitro and were divided into four groups: GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,negative control group (empty load group),respectively.The PIB-CMV3-GM-CSF and PIB-CMV3-IL-12 eukayotic expression vector was built,and 36 h after transfection,fluorescence microscope was used to detect the transfection effect;the expression level of IL-12,GM-CSF,p53,p38 and C-JUN mRNA were detected by RT-PCR,and Western blot was used to examine the expression level of IL-12,GM-CSF,p53,p38 and C-JUN protein.In addition,the flow cytometry was applied to detect cell apoptosis.Results Through fluorescence microscope,green fluorescence was observed in cells of GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,indicating that the plasmid has successfully transferred into cells.In addition,the expression of p53mRNA in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were 1.2±0.10,4.3±0.98,4.2±0.34,9.2±0.87,and the protein expression were 1.0±0.10,3.6±0.34,3.8±0.30,5.0±0.60.Compared with the empty load group,the expression level of p53 mRNA and protein were significantly increased in the three plasmid transfection groups (P<0.01).The expression of p53 mRNA and protein were significantly increased in co-transfection group than GM-CSF group and IL-12 group (P<0.01),while in the comparison with GM-CSF transfection group and IL-12 transfection group,the expression level of p53mRNA and protein in the co-transfection group could be improved to a higher degree(P<0.01).Meanwhile,p38 C-JUN mRNA expression levels in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were as follows: 7.5± 0.9,3.5±0.45,3.7±0.25,1.0±0.11,while p38protein expression levels were 10.1±1.03,6.1± 0.67,7.1 ± 0.61,1.0 ± 0.12,respectively,C-JUN mRNA expression levels were 11.2 ± 1.20,4.1 ± 0.19,3.3 ± 0.30,1.0 ± 0.01,separately,C-JUN protein expression levels were 2.25 ± 0.2,1.8 ± 0.13,1.4 ± 0.12,1.0 ± 0.09.P38, C-JUN mRNA and protein levels were significantly reduced in the three plasmid transfection groups compared with the empty load group (P<0.01).The expression of p38,C-JUN mRNA and protein were reduced to a lower degree in co-transfection group than in GM-CSF transfection group and IL-12 transfection group (P<0.01).Flow cytometer showed that the hepatoma cell apoptosis rate of the empty load group,GM-CSF transfection group,IL-12 transfection group,co-transfection group were (3.43±0.9)%,(5.87±1.02)%,(7.32±1.1)%,(17.47±2.11)%,the rates of the three plasmid transfection groups were significantly higher than that of the empty load group (P<0.01).And the apoptosis rate was significantly increased in the co-transfected group compared with other plasmid groups (P<0.01). Conclusion The combination of GM-CSF and IL-12 could significantly accelerate the apoptosis of hepatoma cells by up-regulating the expression of p53,and down-regulating the expression of p38 and C-JUN.

Objective:To review and analyze the prescriptions for Chinese herbal pieces in an integrative traditional Chinese and western medicine hospital to explore the trend and problems in clinical application for Chinese herbal medicines and provide reference for dispensing in pharmacy of traditional medicine and rational drug use. Methods: Totally 1200 prescriptions for Chinese herbal pieces in our hospital were surveyed from January to December in 2014. The irrational use was analyzed, and reasonable suggestions were provided for the clinical application. Results:The problems in the prescriptions for Chinese herbal pieces included uncompleted diagnosis,incompatible diagnosis and medication,nonstandard Chinese name,unclear footnote,irrational usage or dosage and incompati-bility. The prescriptions including above 15 varieties of Chinese herbal pieces in one accounted for 65. 70%, and those with average dose above 250 g accounted for 40. 25%. Conclusion:Regular inspection and quality supervision on the prescription for Chinese herb-al pieces should be strengthened to improve the quality of prescriptions and guarantee the safety and economy of the medication.

Objective To evaluate the significance of AFP-IgM, this is one of new tumor markers, in the diagnosis of primary hepatocellular carcinoma (PHC). Methods The contents of AFP-IgM and AFP in serum of 103 healthy subjects, 74 patients suffered primary hepatic carcinoma, 27 patients affected by liver cirrhosis and 63 patients affected by chronic hepatitis were detected by means of enzyme linked immunosorbent assay and electrochemiluminescence. No-PHC is comprised of liver cirrhosis,chronic hepatitis and health subjects as control group. Results The area under ROC curve of AFP was larger than that of AFP-IgM (0.85 vs 0.72, Z=3.21) and the best cut-off value of AFP-IgM and AFP was 3×105-AU/L and 10 ug/L respectively, which was determined by ROC curve. Under the cut-off value, the sensitivity of AFP- lgM and AFP for PHC were 64.9% and 79.7%, and the specificity were 75.6% and 80.3%, yet their efficacies were similar. However, for early diagnosis of liver cancer (stage Ⅰ and Ⅱ), the area under ROC curve of AFP-IgM was larger than that of AFP (0.91 vs 0.82,Z=1.73). The sensitivity of AFP-IgM andAFP were 94.4% and 72. 2%, and the specificity were 81.9% and 79.9%. The differences of AFP-IgMand AFP for early diagnosis of liver cancer were statistically significant. When both of the test results combined AFP-IgM with AFP are positive, it can be diagnosed as liver cancer. The specificity of combineddetermination of the two forms was 89.1%, and the efficacy was 79. 0%. Conclusions Both of thesensitivity and specificity of the AFP-IgM test were higher than that of the AFP for early diagnosis of livercancer. We also found that combined determination of the two forms significantly increased the specificityand the positive predictive value for the diagnosis of PHC, thus AFP-IgM was of especially significance forearly diagnosis of liver cancer.

OBJECTIVE: To improve the quality and efficiency of medication consultation and to promote rational drug use in clinic. METHODS: Totally of 520 cases of medication consultation records from the Affiliated Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of TCM during Oct. 2015-Oct. 2016 were selected according to random number table, and then summarized and analyzed statistically in respects of consultation content, age and type of consultants, department, consulted medicine types, the number of combination of TCM and Western medicine, satisfactory degree of consultants, etc. RESULTS: The main consultation contents included usage and dosage (41. 92％), combination of TCM and Western medicine (12. 88％), etc. Consultants mainly aged 50-70 years old (48. 27％), and were mainly the patients and their family members (95. 96％). Consultants were primarily diagnosed in cardiology department and endocrinology department. Top 3 consulted medicine types were cardiovascular system medicines, nervous system medicines, digestive system medicines, and types of Chinese patent medicines were mainly coordinating medicines and activating blood and removing stasis medicines. Types of TCM combined with Western medicine were less than or equal to 3 kinds in 420 cases (80. 77％). Most patients were very satisfied with outpatient medication consultation (93. 65％). CONCLUSIONS: Rational drug use consultation improves medication compliance of patients, meets the needs of patients on medication safety knowledge, and provides a strong guarantee for safe and rational drug use in clinic.

Objective To investigate potential causative associations between immunophenotype traits and cervical cancer by using two-sample Mendelian randomization(MR)analysis.Methods The genetic instrumental variables(IVs)of 731 immunophenotypes of peripheral blood were obtained from the GWAS Catalog database.The GWAS summary data of cervical cancer were obtained from FinnGen database.The inverse-variance weighted(IVW),weighted mode,weighted median,and MR Egger methods were used for evaluations.The sensitivity analysis and reverse Mendelian randomization analysis were conducted to eliminate bias and reverse causality.The MR Steiger directionality test was further used to ascertain the reverse causal relationship between immune cells and cervical cancer.Results A total of 71 immune cell subtypes associated with cervical cancer were identified,of which 31 had a strong association.The majority of the B cell panel was protective factors for cervical cancer.B-cell activating factor receptor(BAFF-R)was the most frequently expressed molecule in this analysis.It is expressed on several B cell subtypes.The CD20 on IgD+CD38+B cell(OR=1.887,95%CI:1.078-3.306,P=0.026)is the risk factor for cervical cancer.In cDC panels,the CD123 expression on plasmacytoid dendritic cell(OR=2.48,95%CI:1.229-5.003,P=0.011),CD123 expression on CD62L+plasmacytoid dendritic cell(OR=2.5,95%CI:1.231-5.077,P=0.011),CD80 expression on plasmacytoid dendritic cell(OR=2.62,95%CI:1.244-5.515,P=0.011),and CD80 expression on CD62L+plasmacytoid dendritic cell(OR=2.641,95%CI:1.246-5.596,P=0.011)were positively associated with the incidence of cervical cancer.All gynecological cancers in this study have no statistically significant effect on immune cells,according to reverse MR analysis.Conclusion This study emphasized the genetically predicted causality between immune cells and cervical cancer.In clinical practice,it is important to pay attention to the screening of peripheral blood immune cells for patients with cervical cancer.

Chimeric antigen receptor modified T (CAR-T) cell therapy is one of the important methods of tumor immunotherapy. The targeting, killing, proliferation and persistence of CAR-T cells are significantly enhanced than that of conventional T cells.After continuous improvement and evolution, CAR-T cell treatment has achieved excellent progress in hematological tumors and has received extensive attention. However, neurotoxicity arising from the treatment, also known as CAR-T cell relevant encephalopathy syndrome (CRES), has affected its clinical application. Exploring the pathogenesis of CRES and high-risk factors, and finding appropriate strategies is therefore critical for the prevention and treatment of CRES. Here, we take CD19-CAR-T cell treatment as example to review the symptoms and pathogenesis of CRES, discuss high-risk factors as well as coping strategies, in an effort to provide a reference for clinical treatment.

[摘 要] RUNX属于转录因子家族，是哺乳动物发育过程中不可或缺的调控因子，在哺乳动物的细胞增殖、分化、谱系发育、成骨和神经形成中发挥重要作用。围绕RUNX的研究已揭示了其功能的多样性以及在肿瘤发生发展过程中的功能。RUNX家族成员在不同类型的肿瘤中具有不一的作用，与肿瘤微环境中的各组分也有不同程度的关联。RUNX家族成员可以调节肿瘤微环境中CD4+辅助性T（Th）细胞和CD8+细胞毒性T细胞（CTL）的谱系发育分化，调控组织驻留T淋巴细胞的表型、分化和存活，驱动NK细胞的增殖活化和组织驻留。RUNX家族缺失会导致MDSC的增殖和成熟活化，从而诱导肿瘤免疫抑制微环境的产生。RUNX家族成员的表达也与肿瘤微环境中肿瘤相关成纤维细胞（CAF）的浸润程度、不同免疫细胞的浸润、免疫检查点基因表达和药物敏感性显著相关，它们可作为潜在的肿瘤预后标志物和免疫治疗靶点，与CAR-T细胞疗法的联用具有很大的应用前景。本文从RUNX的基本结构和功能研究及其参与调控肿瘤免疫和微环境中各类组分的作用进展进行综述，旨在为未来以RUNX为主要靶点的肿瘤免疫治疗提供新的视角。

Objective: To establish a nude mouse model of subcutaneous lung cancer using dual fluorescence reporting genes of luciferase (Luc) and near-infrared fluorescent protein (iRFP). Methods: The Luc and iRFP expressed lentiviral vector was constructed by Gateway method. After verified by sequencing, the lentivirus particle was prepared and infected into lung cancer A549 cells. Successfully infected A549 (mA549) cells were selected by puromycin and amplified. The expression of Luc and iRFP were observed under fluorescence microscope, and the expression of c-Met protein on the cell surface was detected by immunofluorescence. Twelve female nude mice were randomly divided into 2 groups, 6 in each group. A549 and mA549 cells were inoculated subcutaneously into the right forelimb of nude mice. The growth and fluorescence expression of the tumor were observed by in vivo imaging. The tumor formation was evaluated by hematoxylin-eosin (HE) staining and immunohistochemistry. Results: The Luc and iRFP stably expressed mA549 cell line was successfully constructed. The expressions of iRFP and Luc in mA549 cells were observed under fluorescence microscope. The results of immunofluorescence showed that c-Met protein expressed in both A549 cells and mA549 cells. The growth period of mA549 xenograft in nude mice was moderate and the tumorigenesis rate was 100%. The growth trend of mA549 cells in vivo was not significantly different from that of A549 cells (P>0.05). HE staining and immunohistochemistry results showed that the tumor issues displayed typical histopathological features of tumor. Immunohistochemistry results showed that both A549 and mA549 tumors expressed c-Met protein. Conclusion A stable, real-time monitoring model of iRFP-Luc-A549 lung cancer cell xenograft in nude mice was successfully constructed.