Objective Design and synthesize short hairpin RNA (shRNA) expression vector of RNA for specific silencing of heparanase (HPA) gene,screened plasmid which silence effects is the best.Observe the function of ceil invasion after inhibiting the expression of HPA in cervical carcinoma cell lines (HeLa).Methods The genomic sequence of HPA gene was retrieved from GenBank database.Designed four pairs of specific oligonucleotide sequences and a negative control according to the shRNA design principles.They were inserted into the vector pYr-1.1,vectors,and transfected into HeLa cells via lipofectamine.Reverse transcription (RT)-PCR and immunofluorescence were employed to detect the expression of HPA gene in the transfected cells at the mRNA and protein levels,respectively.The plasmid were screened and transfected into HeLa cells,then transwell small room stromal invasion experiment were employed to observe the cervical carcinoma cell invasion.Results RT-PCR results of transfected HeLa cells shown that the mRNA amplification multiples were 0.54 ±0.05 in the HPA-592 group,0.89 ±0.18 in HPA-995 group,0.82 ±0.22 in the HPA-1351 group,0.91 ±0.47 in HPA-1658 group.While,they were 1.31 ±0.72 and 1.09 ±0.16 in negative control and blank control group,respectively.Green fluorescence was visible in the cytoplasm,which indicated that the HPA protein was expressed in the cytoplasm,of them the weakest green fluorescence in the HPA-592 group.The relative numbers of invasive cells among the HeLa cells were as follows:182 ±6 in the blank control group,258 ± 17 in the negative control group,and 44 ± 4 in the HPA-592-specific interference group(P ＜ 0.01).Conclusion Successfully screened shRNA vector targeting human HPA,efficiently inhibit expression of HPA gene when transfected into HeLa cells,and significantly reduced the invasion capacity of cervical carcinoma cells.

Objective To detect the expression of heparanase (HPA) in cervical cancer cells and investigate the impact of quercetin on the expression of HPA,and the molecular mechanism that quercetin inhibits the growth of cervical cancer cells.Methods The experimental groups included cervical cancer cell lines (HeLa and Caski) exposed to different concentrations of quercetin (20,40 and 80 μmol/L) in the culture medium.The control groups included a negative control group,which was cultured with RPMI 1640 only,and a positive control group,in which cervical cancer cells were transfected with HPA small interference RNA (siRNA) to silence HPA expression.The cellular expression levels of HPA were detected with fluorescence quantitative real-time PCR and western blot analysis at 24,48 and 72 hours after treatment.Results (1) HPA was significantly expressed in both cervical cancer cell lines (HeLa and Caski),and it exists both nucleus and cytoplasm.(2)The real-time PCR shows as follows:as the quercetin concentration increased (20,40 and 80 μmol/L),the mRNA expression level of HPA decreased (P ＜0.01),in which the inhibition of HPA expression was concentration dependent.In addition,the inhibition of HPA expression was also time dependent.As time growth,the expression level of HPA mRNA (24,48 and 72 hours) in HeLa and Caski cells decreased (P ＜ 0.01).Compared with negative control group,the expression level of HPA mRNA decreased in different concentrations of quercetin (40 and 80 μmol/L) in both HeLa and Caski cells (all P ＜ 0.05) ; Compared with positive control group,the expression level of HPA mRNA expressed no obvious difference in quercetin (80 μmol/L) group (P ＞ 0.05) in HeLa cells,while it was opposite in Caski cells(P ＜0.01).(3)The result of western blot shown that,as the quercetin concentration increased(20,40 and 80 μmol/L)and time growth (24,48 and 72 hours),the expression level of HPA protein decreased (P ＜ 0.01),and the inhibition of HPA protein expression was concentration and time dependent.Compared with negative control group,the expression level of HPA protein decreased in different concentrations of quercetin (40 and 80 μmol/L) in both HeLa and Caski cells (all P ＜ 0.05) ;Conpared with positive control group,the expression level of HPA protein expressed no obvious difference in quercetin (80 μmol/L) group (all P ＞ 0.05) in both HeLa cells and Caski cells (all P＞0.05).Conclusion Quercetin could inhibits the expression of HPA in cervical carcinoma cell lines,which inhibition is concentration and time dependent.

Objective To study the efficacy and safety of Gonadotropin-releasing hormone agonists (GnRH-a) combined with laparoscope conservative surgery in treatment of moderate or severe endometriosis.Methods From Jan.2007 to Jan.2010,68 patients with moderate or severe undergoing treatment in Renmin Hospital of Wuhan University were enrolled in this retrospective study.Three groups were classified,which were 25 patients in GnRH-a group,subcutaneous injection Leuprorelin on the second day of menstruation,every 4 weeks for 3 months.Twenty-three patients in Marvelon group,orally one marvelon tablet on the second day of menstruation,continuous 21 days for one period of treatment for 3 courses.Twenty patients in surgery group,without any medicine used preoperatively.All patients were followed by 12 months and compare their surgery time,blood loss,recovery,visual analog scale (VAS),and recurrence and so on.Results The operating time were (68 ± 18) min in GnRH-a group,(80 ± 21) min in Marvelon group and (90± 24) min in surgery group.The amount of bleeding were (118 ± 15) ml in GnRh-a group,(161 ± 18) ml in Marvelon group and (193 ± 13) ml in surgery group.There was significant lower in the operating time and amount of bleeding in GnRH-a group than those in other two groups (P ＜ 0.05).The activity time and the anus exhaust time were shorter in patients in GnRh-a group than those in the other two groups significantly(P ＜ 0.05).When followed up in 12 months after treatment,visual analogue scale had dropped from 3.8 (1.9-6.8) to 1.9 (1.1-2.8) in GnRh-a group,from 2.7 (1.3-5.5) to 1.8 (1.2-3.2) in Marvelon group and from 1.9(1.0-4.9) to 1.6(1.0-3.6) in surgery group.It was showed the most remarkable decreased VAS in GnRHa group when compared with the other two groups(P ＜ 0.05).The recurrence rates were 12％ (3/25) in GnRH-a group,22％ (5/23) in Marvelon group and 25 ％ (5/25) in surgery group.It was found that the most significant lower recurrence was in GnRH-a group when compared with the other two groups (P ＜ 0.05).Conclusions It was safe and efficacy that GnRH-a combined with laparoscopic conservative surgery were used in treatment of endometriosis.It could bring shorter operation time,less intraoperative blood loss,quick postoperative recover,the lower recurrence rate.

Objective To evaluate a chemically-modified chitosan anti-adhesion film in the prevention of intestinal adhesion after abdominal surgery.Method In this study 240 patients at the Department of Surgery, Fudan University Zhongshan Hospital undergoing abdominal surgery from Jan 2006 to Dec 2006 were randomly divided into two groups.In the research group, chemically-modified chitosan antiadhesion film was put both at the area of operation and under the incision before closing the abdomen.The recovery procedures were recorded including the recovery of gut movement, the degree and the lasting time of abdominal pain, complication after surgery, the abdominal pain and ileus within 1 year.Result Postoperative incision pain was less significant in research group.The gut function recovered quicker and dietary began earlier.The ratio of early ileus after the surgery decreased significantly.The abdominal adhesion symptom in 1 year after surgery ameliorated significantly.There was no significant difference in other postoperative complications in the two groups.Conclusion The use of chemicallymodified chitosan anti-adhesion film helps to prevent the intestinal adhesion after the abdominal surgery.

To study the effect of the osmotic stress in the microenvironment on the growth and metabolism of the encapsulated cells under aerobic condition, Osmo-sensitive yeast Y02724 and high-osmotic resistant yeast Hansel were used as models to explore the growth and metabolism state of the cells cultivated inalginate-chitosan-alginate (ACA) microcapsules. The changes of the yeast cells' specific growth rate, maximum product quantity and the secretion of ethanol and glycerol were analyzed. For Y02724, the yield of ethanol was increased in the ACA microenvironment compared to suspension cultivation. For Hansel, the maximum growth speed of microencapsulated cultivation had no obvious difference compared to the suspension cultivation. Moreover, after encapsulation, the production of glycerol was decreased for both Y02724 and Hansel compared to suspension cultivation. In conclusion, osmotic stress existed in the ACA microcapsules and affected the growth and metabolism of the cells.
Alginates ; metabolism ; Capsules ; metabolism ; Cell Culture Techniques ; methods ; Chitosan ; metabolism ; Osmosis ; physiology ; Osmotic Pressure ; Polylysine ; analogs & derivatives ; metabolism ; Saccharomyces cerevisiae ; growth & development ; metabolism ; Yeasts ; classification ; growth & development ; metabolism

OBJECTIVETo provide evidence for illustrating the molecular mechanism of nickel carcinogenesis, and to identify the differential expression of protein in crystalline NiS-induced neoplastic transformation of human bronchial epithelial cell by proteomics technology.

METHODSTwo dimensional electrophoresis (2-DE) and the ImageMaster 3.10 software were used to analyze the differential expression of protein, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) combined with database search was applied to identify protein peroxiredoxin 2 (PDX2) related to malignant transformation.

RESULTSThe good 2-DE pattern including resolution and reproducibility was obtained. Nearly 700 expressed proteins per 2-D gel were isolated with molecular weights (MW) ranging from 14,400 to 94,000 KD and pI 3 - 10. A protein PDX2 with MW 21,890 KD, pI 5.66, which was highly expressed in malignantly transformed cell, was identified using MALDI-TOF-MS.

CONCLUSIONPDX2 was involved in malignant transformation of human bronchial epithelial cell induced by crystalline nickel sulfide.

Bronchi ; cytology ; Cell Line ; Cell Transformation, Neoplastic ; chemically induced ; metabolism ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Nickel ; toxicity ; Peroxiredoxins ; metabolism ; Proteome

The aim of this paper is to report the synthesis of the mPEG-PCL-g-PEI copolymers as small interfering RNA (siRNA) delivery vector, and exploration of the siRNA delivery potential of mPEG-PCL-g-PEI in vitro. The diblock copolymers mPEG-PCL-OH was prepared through the ring-opening polymerization. Then, the hydroxyl terminal (-OH) of mPEG-PCL-OH was chemically converted into the carboxy (-COOH) and N-hydroxysuccinimide (NHS) in turn to prepare mPEG-PCL-NHS. The branched PEI was reacted with mPEG-PCL-NHS to synthesize the ternary copolymers mPEG-PCL-g-PEI. The structure of mPEG-PCL-g-PEI copolymers was characterized with Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The mPEG-PCL-g-PEI/siRNA nanoparticles were prepared by complex coacervation, and the nanoparticles size and zeta potential were determined, separately. The cytotoxicities of mPEG-PCL-g-PEI/siRNA nanoparticles and PEI/siRNA nanoparticles were compared through cells MTT assays in vitro. The inhibition efficiencies of firefly luciferase gene expression by mPEG-PCL-g-PEI/ siRNA nanoparticle at various N/P ratios were investigated through cell transfection in vitro. The experimental results suggested that the ternary (mPEG5k-PCL(1.2k))1.4-g-PEI(10k) copolymers were successfully synthesized. (mPEG(5k)-PCL(1.2k))1.4-g-PEI(10k) could condense siRNA into nanoparticles (50-200 nm) with positive zeta potential. MTT assay results showed that the cytotoxicity of (mPEG(5k)-PCL(1.2k))1.4-g-PEI(10k)/siRNA nanoparticles was significantly lower than that of PEI(10k)/siRNA nanoparticles (P < 0.05). The expression of firefly luciferase gene could be significantly down-regulated at a range of N/P ratio from 50 to 150 (P < 0.01), and maximally inhibited at the N/P ratio of 125. The mPEG-PCL-g-PEI polymers could delivery siRNA into cells to inhibit the expression of target gene with very low cytotoxicity, which suggested that mPEG-PCL-g-PEI could serve as a new type of siRNA delivery vector.
Cell Line, Tumor ; Cell Survival ; Drug Carriers ; Genes, Reporter ; Genetic Vectors ; Humans ; Luciferases ; metabolism ; Molecular Weight ; Nanoparticles ; Particle Size ; Polyesters ; chemistry ; Polyethylene Glycols ; chemistry ; Polyethyleneimine ; chemistry ; Polymers ; chemistry ; RNA, Small Interfering ; administration & dosage ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the clinicopathological significance of chromosome 17 polysomy in breast cancer.

METHODSRetrospective study of 200 cases of breast cancer including 106 cases of invasive ductal carcinoma and 94 cases of in-situ carcinoma was performed by fluorescence in-situ hybridization (FISH) to explore the relationship between chromosome 17 polysomy and age, nuclear atypia, lymphatic metastasis, HER2 gene amplification and HER2 protein expression.

RESULTSTwenty-six percent (52/200) of chromosome 17 polysomy was detected in 200 cases of breast ductal carcinoma, all of which were invasive ductal carcinoma. Overall 52. 8% (52/180) of invasive ductal carcinoma cases showed chromosome 17 polysomy, which was correlated to HER2 gene amplification (P = 0.000) and HER-2 protein expression (P=0.000), and to HER2 expression combined with HER2 gene amplification (P=0.001). Chromosome 17 polysomy with or without HER2 gene amplification was also associated with high-grade nuclear atypia (P = 0.012 or P = 0.010) and lymphatic metastasis (P = 0.002 or P = 0.009 ). However, chromosome 17 polysomy with or without HER2 gene amplification was not correlative with the age of patients (P = 1. 000 or P = 0. 415).

CONCLUSIONChromosome 17 polysomy may be related to the nuclear atypia, metastasis, HER2 gene amplification of invasive ductal carcinoma and thus a worse prognosis of the patients.

Breast Neoplasms ; genetics ; pathology ; Carcinoma, Ductal ; genetics ; Chromosome Aberrations ; Chromosomes, Human, Pair 17 ; genetics ; Gene Amplification ; Gene Dosage ; Gene Expression Regulation, Neoplastic ; genetics ; Genes, erbB-2 ; genetics ; Humans

BACKGROUNDY-27632 is a specific inhibitor of Rho-associated coiled kinase (ROCK) and has been shown to promote the survival and induce the differentiation of a variety of cells types. However, the effects of Y-27632 on adult human adipose tissue-derived stem cells (ADSCs) are unclear. This study aimed to investigate the effects of Y-27632 on the neuronal-like differentiation of ADSCs.

METHODSADSCs were isolated from women undergoing plastic surgery and cultured. ADSCs were treated with different doses of Y-27632 and observed morphological changes under microscope. The expression of nestin, neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2) in ADSCs treated with Y-27632 was detected by immunocytochemistry and Western blotting analysis.

RESULTSY-27632 had the potency to induce neuronal-like differentiation in ADSCs in a dose-dependent manner. Moreover, the differentiation induced by Y-27632 was recovered upon drug withdraw. ADSCs treated with Y-27632 expressed neuronal markers such as NSE, MAP-2 and nestin while untreated ADSCs did not express these markers.

CONCLUSIONSelective ROCK inhibitor Y-27632 could potentiate the neuronal-like differentiation of ADSCs, suggesting that Y-27632 could be utilized to induce the differentiation of ADSCs to neurons and facilitate the clinical application of ADSCs in tissue engineering.

Adipose Tissue ; cytology ; Adult ; Amides ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Humans ; Neurons ; cytology ; Pyridines ; pharmacology ; Stem Cells ; cytology ; drug effects

BACKGROUNDFenestration of the proximal anterior cerebral artery (ACA) A1 segment is a rare anatomic variation. The purpose of the this study was to report the incidence of fenestration in the proximal segment of the anterior cerebral artery and to delineate its configurations on cranial MR angiography.

METHODSMagnetic resonance angiography (MRA) was performed in 762 patients using 1.5 T imagers during the period July 2007 through September 2008. All images were obtained by the three-dimensional time-of-flight (3D TOF) technique. Volume rendering (VR) images in the horizontal rotation view were displayed stereoscopically. The presence of fenestration in the proximal segment of the anterior cerebral artery was identified and evaluated retrospectively by MRA.

RESULTSSix patients (four men and two women, 15 to 63 years of age, median age 50 years) had proximal ACA fenestration. The appearance rate of ACA fenestration was 0.8% (6/762). All 6 fenestrations were located at the A1 segment: three of them were with a slit-like shape and three were with a convex-lens-like shape, 5 of the right A1 segment, 1 of the left A1 segment.

CONCLUSIONRecognizing ACA fenestration is important to interpret cranial MR angiographys and helpful to make a plan for neurosurgical procedures or neurological intervention.

Adolescent ; Adult ; Anterior Cerebral Artery ; abnormalities ; Cerebral Angiography ; methods ; Cerebral Arterial Diseases ; diagnosis ; Female ; Humans ; Magnetic Resonance Angiography ; methods ; Male ; Middle Aged ; Young Adult