This study is to investigate the antitumor activity of CIP-36 on multidrug resistant human oral squamous carcinoma cell line (KBV200 cells) in vitro and the possible anticancer mechanisms. MTT assay, Hoechst fluorescein stain, RT-PCR and immunohistochemistry were carried out on KBV200 and KB cells. The growth of many tumor cells was obviously inhibited by CIP-36, especially the multidrug resistant cells KBV200. Obvious apoptosis could be observed in the Hoechst 33342 staining experiments. The results of RT-PCR showed that the levels of p53, p21, caspase-3 and bax mRNA increased, and meanwhile the expression of mdr-1 and bcl-2 mRNA decreased in a dose-dependent manner. The data were significantly different from that of vehicle. The expression of P-gp significantly decreased with the increasing dosage of CIP-36 examined by immunohistochemistry. It can be concluded that CIP-36 could change resistance-related genes and proteins to overcome multidrug resistance in the KBV200 cell line.

Objective To investigate the efficacy and safety of lauromacrogol foam sclerosant in the treatment of peripheral venous malformations.Methods 21 patients with peripheral venous malformations were treated with foam sclerotherapy.The sclerosing foam was produced by Tessari's method using 1％ lauromacrogol.When necessary,the injections were repeated at an interval of four weeks,and this process contained 3 to 5 injections.Therapeutic outcomes and safety were established by evaluating size of lesion,symptom,duration of treatment and side-effects of treatment before and after treatment.Results 21 patients were followed up for 3 to 18 months.10 cases showed markedly shrinked or even disappeared of treated malformations,9 cases showed a reduction in size over 50 ％,and 2 cases showed a reduction in size less 50 ％.Local swelling occurred in almost per session,pyrexia in 5 patients,which resolved spontaneously within several days to 1 week.No major complication occurred.Conclusions Foam sclerotherapy using lauromacrogol seems to be a safe and effective therapeutic method for peripheral venous malformations.

Objective To establish a model for cutaneous squamous cell carcinoma by irradiation of SKH-1 hairless mice with solar-simulated ultraviolet (solar UV), and to explore the biological characteristics of the model. Methods A total of 91 SKH-1 hairless mice were randomly divided into seven experimental groups (n = 10) and seven control groups (n = 3). The mice in experimental groups were irradiated with minimal erythema dose of solar UV 4 times per week for various durations (4, 8, 12, 16, 20, 24, 28 weeks), while the control mice received no irradiation. The general status and skin appearance of mice were observed during the treatment process. Mice were killed immediately after the last irradiation at different time points and pathological examination was carried out to observe the histological changes of skin lesions. Results Papules measuring equal to or more than 1 mm in diameter began to develop in some mice in experimental group 10 weeks after the first irradiation; tumors began to appear in 39.3% (11/28) of the remaining mice in experimental group on week 20, and in 100% (10/10) of the remaining mice on week 28. The cumulative dose approximated to 26.99 J/cm2 for UVB and 242.91 J/cm2 for UVA after 28-week irradiation. No tumor was observed in the control mice. Pathological examination revealed characteristic changes of squamous cell carcinoma in 30% of the mice on week 12, 33.3% on week 16, 60% on week 20, 87% on week 24, and 100% on week 28. Conclusions Ultraviolet could induce the hyperplasia of skin in SKH-1 hairless mice, and even cause the development of cutaneous squamous cell carcinoma after prolonged irradiation.

Objective To investigate the application value for predicting human cytomegalovirus(HCMV) infection with viral load in urinary epithehal cell (EC).Methods Peripheral blood and urine specimens from 82 infants with HCMV latent infection and 84 infants with HCMV active infection were collected respectively.Plasma HCMV DNA load and the levels of HCMV lgM/IgG antibody were detected by real-time fluorescence quantitative polymernse chain reaction (FQ-PCR) and chemiluminescence immunsassay.HCMV pp65 antigen in peripheral blood polymorphonuclear leukocytes (PMNLs) was detected by indirect immunofluorescence assay.The urinary EC count and HCMV DNA load were detected by UF-100 automated urine sediment analyzer and FQ-PCR,respectively.HCMV DNA load in urinary EC was calculated accordingly.At the same time,the sensitivity and specificity for diagnosis of active HCMV infection were evaluated by receiver operating characteristic curve (ROC).Results The positivity of HCMV DNA in urinary EC was 94.58% (157/166),which was the highest among the urinary EC from 166 cases of HCMV infection.HCMV DNA load ranged from 5.67×102to 1.31×107 copies/103 EC There was no significantly statistical difference among urine specimens from different periods of time(P>0.05).HCMV DNA load in active infection group [5.13±0.99(copies/103EC,lg)]is significantly higher than that in latent infection group [3.92±0.82 (copies/103 EC,lg),t = 8.52,P < 0.01];According to ROC curve analysis,its sensitivity and specificity were 71.4% and 75.2% respectively when cut-off value was 4.55.The virus load was significantly decreased in urinary EC in post-treatment infants as compared with pre-trestment(t=5.44,P<0.01).Conclusion Detection of HCMV DNA load in the urinary EC is a cost-effective method and can be used to predict HCMV active infection in infants and monitor treatment of HCMV infection.

Objective To evaluate the effect of 8% emulsified isoflurane preconditioning on renal ischemia-reperfusion (I/R) injury in rats.Methods Thirty-two male Sprague-Dawley rats,aged 10-13 weeks,weighing 220-300 g,were randomly divided into 4 groups (n =8 each):sham operation group (group S); I/R group;emulsified isoflurane preconditioning group (group E) ; intralipid preconditioning group (group I).Renal ischemia was induced by occlusion of the left renal pedicle for 45 min with atraumatic microclips followed by 3 h reperfusion.8 % emulsified isoflurane and 30 % intralipid 4 ml· kg-1· h-1 were infused intravenously for 30 min followed by 15 min washout before renal UR in groups E and I,respectively.Arterial blood samples were taken at 3 h of reperfusion to determine the concentrations of serum creatinine (Cr),cystatin C (Cys C),TNF-α,IL-6 and IL-10.The animals were then sacrificed and left kidneys were removed and stained with hematoxylin-eosin for microscopic examination and assessment of necrosis of renal proximal convoluted tubules (0 =normal,4 =necrosis of whole segment of proximal convoluted tubules).Results Compared with group S,the serum Cr,Cys C,TNF-α,IL-6 and IL-10 concentrations and severity of necrosis of renal proximal convoluted tubules were significantly increased in groups I/R,E and I (P ＜ 0.05).The serum Cr,Cys C,TNF-α and IL-6 concentrations and severity of necrosis of renal proximal convoluted tubules were significantly lower,while the serum IL-10 concentration was higher in group E than in groups I/R and I (P ＜0.05).There was no significant difference in the indexes mentioned above between groups L and I/R (P ＞ 0.05).The damage to renal tissues was less serious in group E than in groups I/R and L.Conclusion Preconditioning with 8 % emulsified isoflurane can attenuate renal I/R injury by inhibiting inflammatory responses in rats.

Objective To investigate the local immune response in condyloma acuminatum treated with aminolevulinic acid-photodynamic therapy (ALA-PDT).Methods In vitro and in vivo studies were performed.A previously established keratinocyte cell line human papilloma virus (HPV) 16E7/HaCaT which stably expresses HPV16E7 protein was used in this study.Peripheral blood mononuclear cells (PBMCs) were separated from 10 healthy volunteers.After pretreatment with ALA-PDT,HPV16E7/HaCaT cells were cocultured with the PBMCs for 3 hours in a Transwell chamber followed by the observation of chemotactic migration of PBMCs.Tissue samples were obtained from the lesions of 10 patients with condyloma acuminatum before,and at 1,2,3 and 48 hours after the first session of ALA-PDT.Immunohistochemistry was conducted to determine the number of CD4+ T cells,CD8+ T cells and CD68+ macrophages as well as CD4/CD8 T-cell ratio in the tissue samples.Results After 3-hour coculture with HPV16E7/HaCaT cells pretreated by ALA-PDT,PBMCs showed apparent chemotactic migration.Immunohistochemistry revealed a statistical increase in the number of CD4+ T cells,CD8+ T cells and CD4/CD8 T-cell ratio at 48 hours (all P ＜ 0.05),as well as in the number of CD68+ macrophages at 3 hours and 48 hours (both P ＜ 0.05) after the first session of ALA-PDT.Conclusion ALA-PDT may induce local antiviral immune response in condyloma acuminatum.

Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting (HRM) and study its preliminary clinical application.Methods Two common mutations [ IVS-2-654 ( C ＞ T ) and -28 ( A ＞ G ) ]of β-thalassemia in Wenzhou city population were selected.The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls.A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity,sensitivity and repeatability were methodologically evaluated.One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children's Hospital of Wenzhou Medical College were enrolled into this study.The genomic DNA was extracted from whole blood cells and detected by HRM method.The results were compared with the direct sequencing data.Results HRM method could detect the mutations [ IVS-2-654( C ＞ T) and -28 ( A ＞ G ) ]of β-thalassemia and the results did not show any non-specific amplified fragments.All within-run and between-run coefficients of variation for different DNA types' Tm were smaller than 0.1％.And minimum 103 copies of DNA of each assay and 10％ mutation could be determined by this method.One hundred and seventeen patients with clinically suspected β-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing.There were 45 IVS-2-654 ( C ＞ T)heterozygous mutation and 9-28 ( A ＞ G)heterozygous mutation and none homozygous mutation.Conclusion The method of rapid identification of β-thalassemia gene mutations based on HRM analysis is successfully established,which is a convenient,rapid,specific,sensitive and accurate technique for screening gene mutations in β-thalassemia as well as a general technical platform to identify other gene mutations.

Objective To investigate the clinical and pathological features of Uurich congenital muscular dystrophy (UCMD). Methods The clinical aspects of 3 patients with UCMD, 2 with Duchenne muscular dystrophy (DMD) and 1 with congenital muscular dystrophy 1A (MDC1A) were analyzed. And the muscle specimens from these patients were studied using immunohistochemistry and immunofluorescence staining. Results UCMD was clinically characterized by neonatal hypotonia with proximal contracturos and distal hyperlaxity at birth or early infancy. Histochemical staining revealed muscle frber hypoplasia andinterstitium proliferation. Immunohistochemistry staining with anti-collagen Ⅵ antibody revealed complete(1/3) or partial (2/3) deficiency of collagen Ⅵ in the sarcolemma and interstitial matrix. Partial deficiency was better demonstrated by immunofluorescence staining. Conclusions The proximal contractures and distal hyperlaxity is the clinical hallmark of UCMD. Collagen Ⅵ immunolabelling can confirm the diagnosis of UCMD.

Penicillium decumbens T. is an important filamentous fungus for the production of cellulases to effectively degrade lignocellulose for second generation biofuel production. In order to enhance the capability of Penicillium decumbens to produce cellulases, we constructed a creB (a deubiquitinating enzyme encoding gene) deletion cassette, and generated a creB knockout strain with homologous double crossover recombination. This mutation resulted in a detectable decrease of carbon catabolite repression (CCR) effect. The filter paper activity, endoglucanase activity, xylanase activity and exoglucanase activity of the deltacreB strain increased by 1.8, 1.71, 2.06 and 2.04 fold, respectively, when comparing with the parent strain Ku-39. A 2.68 fold increase of extracellular protein concentration was also observed. These results suggest that the deletion of creB results in CCR derepression. These data also suggest that CREB influences cellulase production of Penicillium decumbens. In generation, this study provides information that can be helpful for constructing cellulase hyper-producing strain.
Cellulase ; biosynthesis ; Endopeptidases ; genetics ; metabolism ; Gene Knockout Techniques ; Lignin ; metabolism ; Mutant Proteins ; metabolism ; Penicillium ; enzymology ; genetics ; Recombination, Genetic ; Ubiquitinated Proteins ; genetics ; Ubiquitination

OBJECTIVE: To clarify the molecular signaling mechanism underlying the inhibitory effect of metformin on transforming growth factor-β1 (TGF-β1)-stimulated collagen I production in rat biliary fibroblasts. METHODS: Primary biliary fibroblasts were isolated under aseptic condition from 50 Sprague-Dawley rats (half male and half female), and microscopic observation identified no obvious difference in the morphology or viability of the cells from rats with different sexes or body weight. The cells were treated with TGF-β1 (10 ng/mL), Smad3 siRNA+TGF-β1, CTGF siRNA+TGF-β1, metformin (10 mmol/L)+ TGF-β1, or Compound C (10 μmol/L)+metformin+TGF-β1. The expressions of CTGF and collagen I in the treated cells were determined using ELISA kit or Western blotting; the phorsphorylated and total Smad3 and AMPK expressions were detected using immunoblotting. RESULTS: TGF-β1 time- and dose-dependently induced collagen I production in rat biliary fibroblasts. The activated AMPK by metformin dose-dependently inhibited TGF-β1-induced collagen I production. Pre-incubation of cells with the AMPK inhibitor Compound C restored the inhibitory effect of AMPK on TGF-β1-induced collagen I secretion ( < 0.01). Activation of AMPK by metformin significantly reduced TGF-β1-induced collagen I production by suppressing Smad3-driven CTGF expression ( < 0.01), and the application of Compound C reversed such changes in the fibroblasts ( < 0.01). CONCLUSIONS Metformin inhibits TGF-β1-stimulated collagen I production by activating AMPK and inhibiting Smad3- driven CTGF expression in rat biliary fibroblasts.
Animals ; Cells, Cultured ; Collagen ; Female ; Fibroblasts ; Male ; Metformin ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad3 Protein ; Transforming Growth Factor beta1