BACKGROUND: Laryngeal cancer can cause hoarse voice, dyspnea, and dysphagia, for which common surgical approaches or biotherapy hardly results when nodal metastases are present. Identification of the factors associated with laryngeal cancer metastases is a crucial task in the study of the malignancy, as such a revelation may help establish molecular markers for accurate assessment of metastases and develop biological agents to restrain the invasion and metastases.OBJECTIVE:To study the association of MMP-2, MMP-9, CD44v6, PCNA,nm23, VEGF, p53, Cath-D and E-cadherin with the metastasis of laryngeal cancer so as to provide evidence for clinical treatment of laryngeal cancer.DESIGN: An experimental study ofclinical specimens.PARTICIPANTS: All specimens of primary laryngeal cancer were obtained from patients receiving surgical treatment in the Department of Otolaryngology,Fourth Affiliated Hospital of Harbin Medical University from February 2002 to November 2003. Immunohistochemical examination of the specimens was conducted in the Third Affiliated Hospital of Sun Yat-sen University.INTERVENTIONS: The expressions of MMP-2, MMP-9, CD44v6, PCNA,nm23, VEGF, p53, Cath-D and E-cadherin were examined immunohistochemically in the specimens from 70 patients of laryngeal cancer. Logistic stepwise regression model was used to analyze the influence of the 9 clinicopathologic factors on lymph node metastasis.MAIN OUTCOME MEASURES: The expressions of MMP-2, MMP-9,CD44v6, PCNA, nm23, VEGF, p53, Cath-D and E-cadherin in laryngeal cancer specimens.RESULTS: Using the established equation to estimate lymph node metastasis, the frequency of lymph node metastasis was highest in patients with positive MMP-9 expression(Wald = 10. 350 1, P ＜ 0.05) . Lower E-cadherin expression was accompanied by higher frequency of lymph node metastasis, and nm23 expression in patients with lymph node metastases was significantly decreased in comparison with patients without metastases (Wald=6.189 6 and 6. 863 2, P ＜0.05).CONCLUSION: The expressions of MMP-9, E-cadherin and nm23 are useful indicators for preoperative prediction of lymph node metastasis of laryngeal cancer and provide valuable information for prognostic evaluation of the interventions.

BACKGROUND: Matrix metalloproteinases-7 (MMP-7) plays an important role in the invasion and metastasis of cancer and is related to prognosis of many carcinomas.OBJECTIVE: To investigate the significance of MMP-7 expression in the invasion and metastasis of laryngeal cancer and explore the relationship between MMP-7 expression and the prognosis of laryngeal squamous cell cancer (LSCC).DEGIGN: An open experimental study based on the cells.SETTING: Two otolaryngology departments of two university hospitals and one otolaryngology department of a municipal hospital.MATERIALS: The experiment was conducted in the Center Laboratory of Second Affiliated Hospital of Harbin Medical University from April 2002 to January 2003. The materials were laryngeal cancer and corresponding neighboring noncancerous tissues, MMP-7 antibody and immunohistochemical kit.METHODS: The expression of MMP-7 protein in 70 samples of paraffin-embedded LSCC and corresponding neighboring noncancerous tissues was detected with immunohistochemical technique. Reverse transcription-polymerase chain reaction(RT-PCR) and Western blot technique were used to examine the expression of MMP-7 in 35 frozen specimens of LSCC and corresponding neighboring noncancerous tissues.MAIN OUTCOME MEASURES: The relationship of MMP-7 mRNA expression with the invasion depth(T grade), neck nodal metastasis and prognosis of LSCC.RESULTS: Immunohistochemical staining of MMP-7 was observed in 77.1% (54/70) of cancer tissues and in 5.7% (4/70) of neighboring noncancerous tissues( P ＜ 0.01) . The mRNA expression of MMP-7 was detected in 74.3% (24/35) of cancer tissues and in 5.7% (2/35) of neighboring noncancerous tissues( P ＜ 0.01 ). The expression of MMP-7 was higher in T3-T4 group than that in T1-T2 group(P ＜ 0.01), and was positively correlated with nodal metastasis( P ＜ 0. 01 ). Western blot showed that both 28 ku (latent form of MMP-7) and 19 ku(active form of MMP-7)bands had close relationship with T grade and neck nodal metastasis. Kaplan-Meier survival curve showed that the group with high MMP-7protein expression had poorer prognosis than the group with weak or negative MMP-7 protein expression(Log-rank = 4. 755 9, 8. 951 3; P = 0. 029 2,0.002 8).CONCLUSION: MMP-7 is closely correlated with the invasion, metastasis,and prognosis of LSCC, and it may serve as a marker in estimating the invasive and metastatic potency and prognosis of LSCC.

Objective　To study the probability of using hepatitis D virus (HDV) ribozyme as a kind of anti-hepatitis-C-virus (HCV) gene thera-py drugs. Methods　The natural HDV genomic ribozyme′s stem Ⅳ was optimized and its substrate-binding region reconstructed, thus three recombinant HCV-specific HDV genomic ribozymes RzC1, RzC2 and RzC3 were obtained. HCV RNA 5'-noncoding region and 5'-fragment of C region (HCV RNA5'-NCR-C) were transcribed from plasmid pHCV-neo by T7 phage RNA polymerase in vitro, and radiolabelled at its 5'-end. The trans-cleaving reaction was performed by mixing the ribozymes and substrate at mol ratio 100∶1 under conditions as follows: 37℃, pH7.5, Mg2+ 20 mmol/L and deionized formamide 2.5 mol/L. Percentage of trans-cleaved products were calculated at different time points and used as the activity indicator of the three ribozymes. Results　RzC1, RzC2 trans-cleaved more substrate when the time extended, and got to 24.9%,20.3% after reac-ting for 90 minutes respectively; RzC3 was not able to trans-cleave its substrate. Conclusion　Recombinant HDV genomic ribozymes have the ability to trans-cleave HCV RNA, but the appropriate target sequence should be selected.

Isolated freshly rat islets were transferred to 24-well plates and incubated with different concentrations of glucose or resveratrol for 1 or 24 h.The results showed that resveratrol dose-dependently inhibited glucose-stimulated insulin secretion from isolated rat islets after 1 h incubation,with 10%,35%,and 80% (P<0.05 or P<0.01) decrease at the concentrations of 1,I0,and 100 μmol/L.10 μmol/L resveratrol decreased the intracellular calcium concentration by 60% (P<0.05).After incubation for 24 h,resveratrol increased palmitatesuppressed insulin secretion to 75% (P<0.01) of control.These results suggest that resveratrol acutely inhibits insulin secretion from primary pancreatic islet via regulating intracellular calcium ion concentration,and in the long run resveratrol may protect β-cells from lipotoxicity.

The incidence of type 1 diabetes(T1DM)has increased significantly worldwide and has become a major public health problem.At present, it is believed that the occurrence of T1DM is related to various factors such as genetic susceptibility, immunity and environment, but the specific cause and exact mechanism are still not fully explained.In recent years, with the understanding and research of gut micro-ecology, abnormalities in the composition or functionality of gut microbiota lead to a variety of diseases, for instance, gastrointestinal diseases, obesity, cardiovascular disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 2 diabetes and so on.Yet there are evidences indicating that an imbalance of gut microbiota is associated with the development of type 1 diabetes mellitus, and in the T1DM children, the ecology of gut microbiota is different from healthy children.However, there are few studies on the gut microbiota in children with diabetes.This article will review the progress of gut microbiota of children with type 1 diabetes.

The effect of troglitazone on glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and its mechanism were investigated.10 μmol/L troglitazone had no effect on basal insulin secretion,but significantly decreased GSIS and stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylations (all P<0.01).These reactions were completely reversed by AMPK inhibitor compound C,suggesting that the troglitazone acutely inhibits insulin secretion via stimulating AMPK activity in beta cells.

Objective To detect the serum level of vitamin D in infants with atopic dermatitis (AD),and to investigate the relationship between the serum level of vitamin D and severity of AD in infants.Methods Clinical data were collected from patients with moderate to severe AD (AD group)through a questionnaire survey in Children's Hospital of Shanxi from February to April in 2016,and the severity of AD was evaluated by the SCORing atopic dermatitis (SCORAD) score.A total of 95 health checkup examinees served as the control group.Enzyme-linked immunosorbent assay (ELISA)was performed to detect the serum level of 25 (OH) D3 in the AD group and control group,as well as the total serum IgE level in the AD group.Blood cell analyzer was used to determine the proportion of blood eosinophils in the AD group.Results A total of 97 patients with AD were enrolled into the study,including 43 (44.3 ％) patients with moderate AD and 54 (55.7％) patients with severe AD.The serum level of 25 (OH) D3 was significantly lower in the AD group than in the healthy control group ([66.71 ± 21.07] nmol/L vs.[85.43 ± 14.87] nmol/L,P ＜ 0.01),as well as in the patients with severe AD than in the patients with moderate AD ([47.54 ± 29.36] nmol/L vs.[63.89 ± 26.67] nmol/L,P =0.006).The proportion of blood eosinophils was significantly higher in the severe AD group than in the moderate AD group (0.124 ± 0.094 vs.0.061 ± 0.060,P ＜ 0.001).There was no significant difference in the total serum IgE level between the moderate AD group and severe AD group (P =0.375).Among the patients with AD,the serum level of 25 (OH) D3 was negatively correlated with the proportion of blood eosinophils (r =-0.336,P ＜ 0.05),but there was no correlation between the serum level of 25 (OH)D3 and total serum IgE level (r =-0.174,P ＞ 0.05).The serum level of 25 (OH)D3 was significantly associated with breastfeeding and vitamin D supplementation (P ＜ 0.05),but unrelated to age,gender,course of disease and acute exudative phase (all P ＞ 0.05).Conclusion The serum level of 25 (OH) D3 is evidently decreased in infants with AD,and vitamin D deficiency is closely related to the severity of AD in infancy.

Objective: To investigate the analgesic mechanism of Tuina (Chinese therapeutic massage) by observing the effect of the N-methyl-D-aspartate receptor subunit 2B (NR2B)/postsynaptic density-95 (PSD-95) pathway on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation. Methods: Fifty Sprague-Dawley rats were randomly divided into a blank group, a model group, a Tuina group, a blocker agent group, and a blocker agent + Tuina group. The sciatic nerve chronic constriction injury (CCI) model was prepared by the sciatic nerve ligation method. From the 4th day after modeling, rats in the Tuina group and the blocker agent + Tuina group were subject to daily Tuina intervention, and those in the blocker agent group and the blocker agent + Tuina group were daily intrathecally injected with NR2B blocker agent (MK-801). The spontaneous pain score was used to observe the pain behavior of all rats. The expression levels of NR2B and downstream PSD-95 were measured by immunohistochemistry, and the dendritic structure changes were observed by Golgi staining for rat spinal cord dorsal horn after 14 d of continuous intervention. Results: Compared with the blank group, the degree of rat spontaneous pain after CCI was elevated in both the model and the Tuina groups (P<0.01) and was reduced in the Tuina group after the Tuina intervention compared with the model group (P<0.05). Compared with the model group, the rat spontaneous pain level after blocking NR2B was reduced in both the blocker agent group and the blocker agent + Tuina group (P<0.05). The NR2B and PSD-95 protein levels were significantly higher in the model group compared with the blank group (P<0.01); the total number of dendritic branches was increased (P<0.01), and the total dendritic length became longer (P<0.01) in the spinal cord dorsal horn. The rat NR2B and PSD-95 protein levels were significantly decreased in the Tuina group compared with the model group (P<0.01); the total dendritic branch number was reduced (P<0.01) and the total length was shortened (P<0.01) in the spinal cord dorsal horn. After blocking NR2B, the expression levels of NR2B and downstream PSD-95 protein were significantly lower in both the blocker agent group and the blocker agent + Tuina group compared to the model group (P<0.01). The total branch number was significantly reduced (P<0.01), and the total length was significantly shortened (P<0.01) of the dendrites in the spinal cord dorsal horn. Conclusion: Tuina may exert an analgesic effect by remodeling the dendritic structure in the spinal cord dorsal horn in rats with lumbar disc herniation, and its mechanism may be related to the inhibition of NR2B/PSD-95 signaling pathway.

Objective:To investigate the relationship of plasma cholinesterase (ChE) with triglyceride (TG) levels in newly diagnosed patients with type 2 diabetes (T2DM).Methods:Clinical data and biochemical parameters of 321 patients with newly diagnosed T2DM admitted to the Department of Endocrinology of People′s Hospital of Shanghai Putuo from January 2018 to June 2020 were retrospectively collected. The patients were classified into four groups based on the plasma ChE level: Q1group ( n=81, <6 915 U/L), Q2 group ( n=80, 6 916-8 268 U/L), Q3 group ( n=80, 8 269-9 578 U/L), and Q4 group ( n=80, ≥9 579 U/L). The correlation of plasma ChE with TG level was analyzed. Results:With the increased ChE level, TG level significantly increased ( P<0.001). Correlation analysis showed that ChE was positively correlated with body weight, body mass index (BMI), TG, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), uric acid (UA), alanine aminotransferase (ALT), aspartate aminotransferase (AST)( r=0.35, 0.39, 0.35, 0.31, 0.27, 0.24, 0.25, 0.11, P<0.05, P<0.01 or P<0.001)while negatively correlated with age, systolic blood pressure, blood urea nitrogen (BUN), creatinine, and glycated albumin (GA)( r=-0.46、-0.14、-0.20、-0.14、-0.21, P<0.05 or P<0.001). Multivariate stepwise regression analysis showed that ChE was an independent risk factor for TG ( β=0.270, P<0.001). Logistic Multivariate regression analysis showed that after adjustment for sex, age, body weight, hemoglobin, leukocytes, total protein, albumin, globulin, ALT, BUN, creatinine, uric acid, smoking history, drinking history, HbA 1C, GA, TC, and LDL-C, the risk of hypertriglyceridemia in Q4 was 6.024 folds higher than Q1 group ( P=0.011). With the TG (1.70 mmol/L) as cut-off value, the optimal cut-off point of the ChE was 7 801 U/L, as calculated by receiver operating characteristic(ROC) curve analysis of ChE and hypertriglyceridemia. Conclusions:ChE level is positively correlated with TG in newly diagnosed patients with T2DM.

ObjectiveTo investigate the effect of icariin （ICA）-mediated vitamin D system on peripheral blood dendritic cells （DCs） and helper T cells 17 （Th17）/regulatory T cells （Treg） balance in myocardial remodeling model of Dahl salt-sensitive rats. MethodFifty SPF Dahl salt-sensitive rats were divided into model group， vitamin D group （3×10^-5 mg·kg^-1·d^-1）， and high-， medium-， and low-dose ICA groups （120， 60， 30 mg·kg^-1·d^-1）， and 10 Dahl salt-resistant rats were used as normal group. The myocardial remodeling model was established by feeding rats with a high-salt diet containing 8% NaCl. After six weeks of modeling， the normal group and the model group were given an equal volume of ultrapure water by gavage， and other groups were continuously administrated for six weeks. Cardiac echocardiography， hematoxylin-eosin （HE） staining， and Masson staining were used to observe the pathological changes in cardiac structure and fibrosis. The levels of serum 25（OH）D₃， B-type N-terminal pro-brain natriuretic peptide （NT-ProBNP）， interleukin （IL）-17， transforming growth factor （TGF）-β₁， IL-12， and IL-10 were detected by enzyme-linked immunosorbent assay （ELISA）. The phenotype of peripheral blood DCs and the ratio of Th17/Treg cells of rats were detected by flow cytometry. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the mRNA and protein expressions of vitamin D receptor （VDR），1α-hydroxylase （CYP27B1）， and 24-hydroxylase （CYP24A1） in peripheral blood DCs of rats. ResultCompared with the control group， the rats in the model group had pathological changes such as disordered arrangement of myocardial cells and cytoplasmic hypertrophy and swelling. Myocardial collagen fibers proliferated significantly， and the arrangement of myocardial fibers was disordered. The levels of serum 25（OH）D₃ and IL-10 were significantly decreased， and the levels of serum IL-17， TGF-β₁， IL-6， IL-12， and NT-ProBNP were significantly increased （P<0.05）. The costimulatory molecules CD40， CD80， CD86， and MHC-Ⅱ were highly expressed in the peripheral blood DCs， and the expression of CD11 and CD11b was lower （P<0.05）. The proportion of Th17 cells in the peripheral blood was significantly increased， and the proportion of Treg cells was decreased. The ratio of Th17/Treg was increased （P<0.05）. The mRNA and protein expressions of CYP24A1 in peripheral blood DCs increased， and the mRNA and protein expressions of CYP27B1 and VDR decreased （P<0.05）. Compared with the model group， the arrangement of myocardial fibers in each drug administration group was relatively regular， and the swelling of myocardial cells was significantly reduced. The pathological morphology of myocardial tissue was improved to varying degrees. The pathological changes in myocardial tissue were improved and alleviated to varying degrees. The drug could reduce the serum levels of NT-ProBNP， IL-17， TGF-β₁， IL-6， and IL-12 and increase the level of serum 25（OH）D₃ and IL-10 （P<0.05）. The expression of costimulatory molecules CD40， CD80， CD86， and MHC-Ⅱ in the peripheral blood DCs of rats was decreased， and the expression of CD11 and CD11b molecules was increased （P<0.05）. The drug could reduce the proportion of Th17 cells in peripheral blood and the ratio of Th17/Treg cells and increase the proportion of Treg cells （P<0.05）. It could decrease the mRNA and protein expressions of CYP24A1 in peripheral blood DCs of rats and elevate the mRNA and protein expression of VDR and CYP27B1 （P<0.05）. ConclusionICA can regulate the phenotype of peripheral blood DCs and the ratio of Th17/Treg cells by regulating the vitamin D system and play a role in improving myocardial remodeling from the perspective of immune balance.