Objective To explore the efficacy and safety of platelet-derived growth factor(PDGF)combined with allograft bone transplantation in the treatment of spinal tuberculosis.Methods A total of 177 patients with lumbar tuberculosis admitted to the 4th People's Hospital of Qinghai Province from August 2018 to August 2023 were selected as the research subjects.Patients were divided into control group(n=49)and observation group(n=128)based on the source of the transplanted bone.All patients underwent at least 2 weeks of standard quadruple anti-tuberculosis chemotherapy before surgery.Patients in the control group received PDGF combined with autograft bone transplantation,while patients in the observation group received PDGF combined with allograft bone transplantation.The surgical duration,intraoperative blood loss,and length of hospital stay of patients in the two groups were recorded;the erythrocyte sedimentation rate(ESR)and serum C-reactive protein(CRP)levels of patients in the two groups were compared before surgery and at 1,3,6 months after surgery.Preoperative CT and magnetic resonance imaging(MRI)examinations were performed,and postoperative CT and MRI were performed after bone fusion was completed to compare the changes in Cobb angle before and after surgery.The visual analogue scale(VAS)was used to assess the pain degree in the lumbar region before surgery and at 1,3,6 months after surgery.The VAS scores of patients in the two groups,VAS scores of male patients in the two groups,and VAS scores of female patients in the two groups were compared before and after surgery,respectively.Results There was no statistically significant difference in surgical duration and length of hospital stay between the observation group and the control group(P＞0.05).The intraoperative blood loss of patients in the observation group was significantly less than that in the control group(P＜0.05).There was no statistically significant difference in Cobb angle before and after surgery between the two groups(P＞0.05).The postoperative Cobb angle significantly decreased in both groups when compared to preoperative values(P＜0.05).The VAS scores of patients in both groups decreased sequentially before surgery and at 1,3,6 months after surgery,with statistically significant differences in intra-group pairwise comparisons(P＜0.05).The VAS scores of male patients in both groups decreased sequentially before surgery and at 1,3,6 months after surgery,with statistically significant differences in intra-group pairwise comparisons(P＜0.05).The VAS scores of female patients in both groups also decreased sequentially before surgery and at 1,3,6 months after surgery,with statistically significant differences in intra-group pairwise comparisons(P＜0.05).There was no statistically significant difference in VAS scores between the observation group and the control group before surgery and at 1,6 months after surgery(P＞0.05);the VAS score of patients in the observation group was significantly lower than that in the control group at 3 months after surgery(P＜0.05).There was no statistically significant difference in VAS scores between male patients in the observation group and male patients in the control group before surgery and at 1,3,6 months after surgery(P＞0.05);the VAS score of male patients in the observation group was significantly lower than that in the control group at 3 months after surgery(P＜0.05).There was no statistically significant difference in VAS scores between female patients in the observation group and female patients in the control group before surgery and at 1,6 months after surgery(P＞0.05);the VAS score of female patients in the observation group was significantly lower than that in the control group at 3 months after surgery(P＜0.05).The ESR of patients in both groups decreased sequentially before surgery and at 1,3,6 months after surgery,with statistically significant differences in intra-group pairwise comparisons(P＜0.05).The serum CRP levels of patients in both groups also decreased sequentially before surgery and at 1,3,6 months after surgery,with statistically significant differences in intra-group pairwise comparisons(P＜0.05).There was no statistically significant difference in ESR between the observation group and the control group before surgery and at 1,3,6 months after surgery(P＞0.05).There was no statistically significant difference in serum CRP level between the observation group and the control group before surgery and at 1,6 months after surgery(P＞0.05);the serum CRP level of patients in the observation group was significantly higher than that in the control group at 3 months after surgery(P＜0.05).Conclusion The effect of PDGF combined with allograft bone transplantation in the treatment of spinal tuberculosis is comparable to that of autograft bone transplantation,but PDGF combined with allograft bone transplantation can significantly reduce postoperative pain degree,improve patient comfort,avoid additional damage caused by autograft bone transplantation,and reduce the physical burden on patients.It can be considered a safe and reliable surgical method for bone grafting in lumbar tuberculosis surgery.

Objective:To establish an evaluation index system of hospital culture construction led by Party building in public hospitals, and to explore the relationships among Party building, hospital culture and patient satisfaction, and explain the mediating effect of hospital culture between Party building and patient satisfaction.Methods:Literature review and two rounds of Delphi method were used to create an evaluation index system of hospital culture construction led by Party building in public hospitals. On such basis, a questionnaire was designed and 429 staff from 33 public hospitals in Shanghai were surveyed through April and June 2021. Cronbach α coefficient method and exploratory factor analysis were used to test the reliability and validity of the indexes, and the regression analysis method was used to verify the mediating effects. Results:Following two rounds of Delphi survey run by 14 experts, a performance evaluation index system for the hospital culture construction was constructed, comprising 5 level-1, 13 level-2 and 29 level-3 indexes. The Cronbach α coefficients of overall indexes and 5 level-1 indexes fell in the range of 0.860-0.901( P<0.01), and the retest reliability of overall index and 5 level-1 indexes fell between 0.91-0.93( P<0.01). Based on the exploratory factor analysis, all of the characteristic roots of the factors generated by orthogonal rotation were>1, and the total variance explained contribution rate was 56.84%, proving the good validity of the indexes. The effect of Party building on patient satisfaction was significant( β1=0.139, P<0.01), and that on hospital culture effect significant as well( β2=0.168, P<0.01), while that on patient satisfaction was also significant( β3=0.115, δ=0.448, P<0.01)with hospital culture effect introduced as the mediating effect. Conclusions:The evaluation index system of hospital culture construction led by Party building proves highly scientific and effective, and hospital culture plays to some extent a mediating role between Party building and patient satisfaction. This proves that Party building presents a significant transmission effect on patient satisfaction through hospital culture building. The study provides a reference for strengthening Party building and hospital culture construction and improving patient satisfaction in public hospitals.

Objective To establish the cell line from specimens of resectable human gastrointestinal stromal tumors (GIST) and to verify the characteristics of cell biology in vitro. Methods The tissues from biopsies of human GIST were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. After growing to 90% confluence,the cells were detached for subculture and their characteristics,including morphology,growth kinetics,karyotype analysis,immunohistochemical analysis and tumorigenicity in nude mice were determined. Results GIST named GIST-H1 was successfully established. The cell line was passaged for more than 60 times 1 year. The characteristics demonstrated: The population doubling time calculated in the log phase of growth was 47.5 h. The cloning efficiency in the soft agar averaged 24.8%.Electronmicroscopically,there were rich ribosomes and mitochondrion in the cytoplasm. Immunohistochemical analysis showed CD117(+),SMA(+),dog-1(+),CD34(-),and S-100(-).Karyotype analysis illustrated aneuploidy with the modal chromosomal number 60-98.The GIST cells transplanted in nude mice had high tumorigenicity. Conclusion The immortalized GIST cells are devoloped in vitro and have specific characteristics of GIST.

OBJECTIVESTo identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-kappaB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).

METHODSA stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.

RESULTSLMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the kappaB site kappaB1 or kappaB5 was disrupted, whereas mutation of kappaB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.

CONCLUSIONLMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-kappaB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.

Apoptosis ; physiology ; Humans ; NF-kappa B ; physiology ; Nasopharyngeal Neoplasms ; physiopathology ; Protein Biosynthesis ; Signal Transduction ; physiology ; TNF Receptor-Associated Factor 1 ; Tumor Cells, Cultured ; Viral Matrix Proteins ; physiology

OBJECTIVETo clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.

METHODSTo determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.

RESULTSIn contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.

CONCLUSIONIn nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.

Gene Expression ; drug effects ; Herpesvirus 4, Human ; chemistry ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; NF-kappa B ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; pharmacology

OBJECTIVETo elucidate the regulation of the phosphorylation of epidermal growth factor receptor (EGFR) by the EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma cell line.

METHODSThe levels of EGFR expression and phosphorylation in pTet-on LMP1 HNE2 cell, a nasopharyngeal carcinoma (NPC) cell line, in the dynamic expression of LMP1 induced by different concentrations of doxycycline (Dox) were observed. The EGFR dominant negative mutant and LMP1 antisense expression plasmid were transiently transfected into pTet-on LMP1 HNE2 cells by lipofectamine, and the changes in EGFR phosphorylation were observed by immunocoprecitation and Western blot. The changes in EGFR phosphorylation were observed after EGF treatment.

RESULTSIn pTet-on LMP1 HNE2 cells, Dox-induced LMP1 upregulated EGFR expression and phosphorylation in a dose-dependent manner. After EGFR dominant negative mutant was transfected into pTet-on LMP1 HNE2 cells, the increase of EGFR phosphorylation was inhibited completely. When LMP1 antisense expression plasmid was transfected into pTet-on LMP1 HNE2 cells, the levels of EGFR phosphorylation were also inhibited significantly. Meanwhile, after EGF had been added into pTet-on LMP1 HNE2 cells, increase of EGFR phosphorylation was induced, but it was completely blocked by EGFR dominant negative mutant and the introduction of LMP1 antisense.

CONCLUSIONEB virus encoded LMP1 not only induces the dose-dependent expression of EGFR, but also the dose-dependent phosphorylation of EGFR. The phosporylation of EGFR may play a vital role in the development of nasopharyngeal carcinoma.

Blotting, Western ; Epidermal Growth Factor ; metabolism ; Herpesvirus 4, Human ; metabolism ; Humans ; Nasopharyngeal Neoplasms ; pathology ; virology ; Phosphorylation ; Receptor, Epidermal Growth Factor ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; metabolism

OBJECTIVETo observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.

METHODSThe morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.

RESULTSMorphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.

CONCLUSIONSNasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.

Cell Transformation, Viral ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Epithelial Cells ; physiology ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Nasopharynx ; cytology ; virology ; Tetradecanoylphorbol Acetate ; pharmacology

The Epstein-Barr virus latent membrane protein 1 (LMP1) oncopro tein causes multiple cellular changes, including activation of the NF-κB trans cription factor. To elucidate its possible mechanism, the interaction between LM P1 and the tumor necrosis factor receptor associated factor (TRAF) molecules was detected by the immunoprecipitation-Western blotting assay. Results showed tha t LMP1 was co-precipitated with TRAF1,2,3 in the LMP1-HNE2 cell line. In the m eantime, κB reporter gene analysis revealed that over expression of TRAF1 or TR AF2 augmented LMP1-mediated NF-κB activation from LMP1, suprisingly, overexpr ession of either TRAF3 or an dominant negative TRAF3 inhibited the NF-κB activ ation, indicating that TRAF1 or TRAF2 is a positive modulator of LMP1-mediated NF-κB activation, whereas,TRAF3 is a negative modulator. Rather both CTAR1 (carboxy-terminal activating region 1) and CTAR2 domains of LMP1 can independently activate NF-κB by interacting with TRAF proteins. These data indicate that LMP1 interacts TRAF1,2,3 which are important for LMP1-mediated N F-κB activation, and further suggest that signaling from TRAFs may be involved in the progression to malignancy in cells of epithelial origin such as nasophar yngeal carcinoma (NPC).

Abstract:Objective To elucidate the mechanisms by which Epstein-Barr virus-encoded latent membrane protein 1 activates NF-κB in nasopharyngeal carcinoma cells.Methods A tetracycline-regulated LMP1-expressing nasopharyngeal carcinoma cell line, Tet-on-LMP1-HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, IκBα and IκBβ, was analyzed by Western blotting. The subcellular localization of NF-κB (p65) was detected by indirect immunofluorescence assay. The NF-κB transactivity was studied by transient transfection and reporter gene assay. Results IκBα was phosphorylated and degraded after the inducible expression of LMP1, although the total protein levels remained stable. The steady-state level of total IκBβ protein may have resulted from the initiation of an autoregulation loop after the activation of NF-κB. No change in the IκBβ level was detected. NF-κB (p65) was translocated from the cytoplasm to the nucleus following degradation of IκBα. After the introduction of the dominant-negative mutant of IκBα (Del 71) into Tet-on-LMP1-HNE2 cells, both nuclear translocation and transactivation of NF-κB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, LMP1 activated NF-κB via phosphorylation and degradation of IκBα, but not IκBβ. The dominant-negative mutant of IκBα (Del 71) could completely inhibit both the nuclear translocation and transactivation of NF-κB induced by LMP1.

AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE 2 cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.