Objective:To explore the clinical significance of combined detection of the promoter methylation of plasma free Septin9, SDC2 and BCAT1 genes in peripheral blood for the diagnosis of colorectal cancer. Methods The data of patients admitted to the Department of Gastroenterology, Shanghai East Hospital Affiliated to Tongji University from January to September 2019 were retrospectively analyzed. They were divided into colorectal cancer group (62 cases of colon cancer, 59 cases of rectal cancer), precancerous lesions group (77 cases of colorectal adenoma, 5 cases of high-grade intraepithelial neoplasia), interference group (61 cases of colorectal cancer and advanced adenoma negative but suffered other intestinal lesions, 17 cases of non-colorectal cancer) and healthy group (94 cases). The methylation status of three genes (Septin9, SDC2 and BCAT1) in peripheral blood plasma was detected simultaneously by fluorescence PCR. The relationship between the positive rate of three genes detected jointly and the clinic pathological characteristics of colorectal cancer was analyzed and compared with serum carcinoembryonic antigen (CEA) positive rate. The colorectal cancer group was divided into stage Ⅰ, Ⅱ, Ⅲ and Ⅳ according to TNM stage, and the colorectal cancer group was analyzed and counted by grade. The diagnostic efficiency of detection methods was analyzed by receiver operating characteristic (ROC) curve, and the area under ROC curve (AUC) was compared.Results:The positive rate of combined detection of SDC2 and BCAT1 gene methylation was higher than other three groups (χ 2 =237.246, P<0.001). The positive rate of combined detection of plasma Septin9, SDC2 and BCAT1 gene methylation was higher than CEA in colorectal cancer group ( P<0.001). The positive rates of the combined detection of plasma Septin9, SDC2 and BCAT1 gene methylation in stage Ⅰ-Ⅳ of colorectal cancer group were 73%(16/22), 87%(34/39), 86%(30/35) and 96%(24/25), respectively. Compared with CEA group, the positive rate of combined detection of plasma Septin9, SDC2 and BCAT1 gene methylation in stage Ⅰ-Ⅲ of colorectal cancer group was higher than serum CEA ( P<0.001), but the positive rate of stage Ⅳ was not statistically significant compared with CEA group ( P>0.05). ROC curve analysis showed that the AUC of Septin9, SDC2 and BCAT1 was 0.857(95% CI 0.810-0.903),0.819(95% CI 0.768-0.871)and 0.862(95% CI 0.816-0.909), respectively. The AUC of combined detection of three gene methylations was 0.889 (95% CI 0.846-0.933), and the AUC of combined detection with serum CEA was 0.913 (95% CI 0.874-0.951). There was no significant difference in the positive rate of combined detection of Septin9, SDC2 and BCAT1 gene methylation among different gender, age and cancerous site of colon cancer patients (all P>0.05). Conclusion:The combined detection of the promoter methylation of plasma free Septin9, SDC2 and BCAT1 genes in peripheral blood plasma is helpful for the early diagnosis of colorectal cancer. The positive rate in stage Ⅰ-Ⅲ of colorectal cancer group is higher than serum CEA. The combined diagnosis of the three genes can improve the diagnostic efficiency.

To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Animals ; Coxsackievirus Infections ; drug therapy ; virology ; Down-Regulation ; Enterovirus B, Human ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics ; Virus Replication

Objective To investigate correlation between aspirin resistance(AR) and inflammatory factors. Methods One hundred and ten patients with coronary heart disease took aspirin 0.1 mg/d for 14 days.It was detected platelet aggregation function induced with adenosine disphosphate (ADP) and arachidonic acid (AA), and investigated correlation between AR and inflammatory factors. Interleukin-1? (IL-1?),interleukin-6 (IL-6) and high sensitive C-reaction protein (hs-CRP) levels. Results IL-6 level of patients with AR was significantly higher than that of aspirin sensitive (AS) patients. The other two index were not different between the two groups. Conclusion IL-6 levels could be used as predictor.

Objective To study the effect of Itk down regulation on PBMC cell proliferation and inflammatory cytokines production in a Coxsackievirus-induced myocarditis model.Methods BALB/c mice were injected via caudal vein with plasmid Itk-shRNA then infected with CVB3.The change of Itk protein expression,cell proliferation,cytokines production and mice survival rate of mice were observed in the fourth days after infected.Results Itk mRNA in groups of mice transfected with Itk-shRNA was reduced about 40％ in spleen cells,compared with that in control groups or shRNAnon groups (P＜0.05).PBMC proliferation and serum cytokines were significantly inhibited by transfected with Itk-shRNA.Conclusion Knocking down Itk expression can inhibit mice inflammatory reaction.

Objective To evaluate the possibility of short interfering RNA (siRNA) inhibiting Coxsackievirns B3 (CVB3) infection in vitro, and discover the mechanism initially. Methods We obtained proper effective dosage of siRNA by observing cytopathic effect (CPE). Estimate its antiviral activities and its pathway of siRNA by Western Blot assay and RT-PCR. Results Results showed that siRNA-3753 can be effectively transfected into HeLa cells, we can achieve a high transfection efficiency up to 98.77 % and its effect can last for 48 h stably in cells. 0.6 μmol/L siRNA-3753 got a high inhibiting effect of virus and didn't show any toxicity to cells. So we consider this concentration as the experimental concentration, siRNA-3753 can debase virus reproduction. The antiviral effect is sequence-specific and is not attributable to either interferon or the interferon response effectors protein kinase R (PKR). Conclusion The data confirmed that siRNA can effectively inhibit CVB3 infection in vitro, its antivirus effect was gained from specific debase of virus genome.

Objective To screen prescriptions for moxifloxacin hydrochloride tablets and optimize its preparation technology. Methods Taking the angle of repose,tap density,hardness,friability,disintegration time,tablet weight difference,and dissolution rate as indexes,the amount of each component,binder solvent,amount of binder,size of the mesh for granulation and particle drying process were investigated. The optimal formulation and process were determined based on the above results. Results With water as the binder solvent,binder volume of 6 ml,screen mesh number of 26 mesh,and finally drying 1 h at 50℃,the indicators of the tablet prepared met the quality requirements of tablet in the second part of the Pharmacopoeia of People′s Republic of China the 2015 ver-sion. And the dissolution profile was in good agreement with the commercially available preparation. Conclusion The quality of moxi-floxacin hydrochloride tablets prepared by the optimal formulation and process in this study is in accordance with the standard. The pre-scription and process can be used for the preparation of generic drugs of moxifloxacin hydrochloride tablets.

UNLABELLEDOBJECTIVE To observe the morphology and proliferation of epithelial cell rests of Malassez (ECRM) during tooth emergence and occlusal function, and to evaluate its roles.

METHODSCytokeratin 14 (CK14) was applied as special marker of ECRM cells. The morphology and distribution of ECRM were examined by light and transmission electron microscopy. PV two-step immunohistochemical method was used to detect the expression of CK14 and proliferating cell nuclear antigen (PCNA) in ECRM.

RESULTSECRM experienced instinct morphological changes during tooth emergence and occlusal function. They were observed as network of epithelial cells labeled by CK14, especially in furcation level regions of mouse molars and active cell proliferation during occlusion found period. Cell apoptosis was observed in many ECRM by transmission electron microscopy during late stage of the progess.

CONCLUSIONECRM may not only an accidental left-over of early embryonic development but rather play significant roles in occlusion found period.

Animals ; Apoptosis ; Cell Proliferation ; Epithelial Cells ; Mice ; Microscopy, Electron, Transmission ; Molar ; Periodontal Ligament ; Rest ; Tooth

OBJECTIVETo evaluate feasibility of inhibiting coxsackievirus B3 (CVB3) infection at cellular, protein and gene levels by using small interfering RNA (siRNA).

METHODSAntiviral activities of siRNAs were evaluated by observing cytopathic effect (CPE), using plaque reduction Western blotting assays and RT-PCR.

RESULTSEight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 percent inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30 percent inhibition of virus at 0.1 MOI, 70 percent inhibition at 0.01 MOI, 88 percent inhibition at 0.001 MOI, and 99 percent inhibition at 0.0001 MOI 48 hours after CVB3 infection.

CONCLUSIONSiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.

Coxsackievirus Infections ; therapy ; virology ; Cytopathogenic Effect, Viral ; drug effects ; Enterovirus ; genetics ; physiology ; HeLa Cells ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; Virus Replication ; drug effects

To study the inhibitory effect and function characteristics of small interfering RNA (siRNA) on cosxackievirus B3(CVB3) infection by RNA interference technique, siRNA-2B against the viral 2B region was synthesized and transfected into HeLa cell, which was then infected with CVB3. The efficiency of siRNA transfection was examined by FCM, the cell toxicity of siRNA-2B by MTT, and the antiviral ability of siRNA-2B by cytopathic effect (CPE), plaque reduction assay and RT-PCR. The results showed that siRNA-2B could be transfected efficiently into HeLa cell and lasted at least 48h. High concentration of siRNA-2B didn't show any sign of toxicity to cells. siRNA-2B exhibited a significant protective effect on cell viability by specific inhibition of viral replication. It showed a close relationship between the concentrations of siRNA-2B and the antiviral effects. siRNA-2B led to dramatical reduction of viral titers in supernatant of cell culture and weakened the reinfection ability of the virus. These data proposed that siRNA-2B, targeting 2B protein, can effectively inhibit CVB3 infection in HeLa cell and exhibits its transfection efficiency, viral inhibition specificity and adose-dependant manner, suggesting its potential role in prevention and treatement of CVB3 infection.
Enterovirus ; genetics ; growth & development ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Microscopy, Fluorescence ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Viral Nonstructural Proteins ; genetics ; Virus Replication ; genetics ; physiology

Objective By using the RNAi method to inhibit Itk protein expression specificity,to observe lymphocytes proliferation and cytokines production, verify its function as a drug target. Methods Designed siRNA aims at Itk sequence according to its sequence and solid structure, then electrotransfected into mouse spleen lymphocytes, We validated the decrease of Itk protein by Western-Blot, and detected the change of the cell proliferation by MTS and the change of inflammatory cytokines by ELISA. Results Itk protein can be suppressed by Itk-siRNA, there were significantly reduced compared to its control group on cell proliferation as well as cytokine secretion such as IL-2, IL-4, IL-5, IFN-T. They all have statistical differenc (P < O. 05). Conclusion Itk has animportant immunomodulatory effect in mouse spleen lymphocytes proliferation and secretion of inflammatory cytokines. This can supply an experimental basis to regard Irk as drug target for inflammation therapy.