Objective: To construct subtracted cDNA libraries of stomach tumors and normal stomach tissue using suppression subtractive hybridization(SSH).Methods: cDNA Library subtraction was performed using the protocol described in the Clontech PCR-Select cDNA Subtraction Kit. cDNA was synthesized from 2 μg of poly A+RNA from the tumor and normal tissues using AMV reverse transcriptase. The tester and driver cDNAs were digested with RsaⅠ, a four-base-cutting restriction enzyme that yields blunt ends. The tester cDNA was then subdivided into two portions, and each was ligated with different cDNA adaptor. Two hybridizations were performed. In the first, an excess of driver was added to each sample of tester. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together without denaturing and thus the templates were generated from differentially expressed sequences for PCR amplification. Using suppression PCR, only differentially expressed sequences were amplified exponentially and after second PCR amplification the background was reduced and differentially expressed sequences were further enriched. The cDNAs were then directly inserted into a T/A cloning vector to generate a stomach tumor-specific subtracted cDNA library. Results: The amplified library contained 800 positive clones. Plasmid inserts were PCR amplified and showed 250－700 bp inserts. Conclusions: The successfully constructed subtracted cDNA library of gastrocarcinoma and normal tissue enables us to compare two populations of mRNA and obtain clones of genes that expressed in one population but not in the other.The characterization of these genes will allow them to be exploited for their diagnostic and therapeutic potentials.

Hippo signaling pathway plays an important role in the regulation of organ growth,tissue regeneration,tumor occurrence and development.Transcriptional co-activator with PDZ-binding motif (TAZ)is the downstream transcription factor of Hippo signaling,participates in the regulation of the entire signaling path-way.In recent years,many studies have found that over-expression of TAZ has a close relationship with the oc-currence,development and prognosis of breast cancer.Discussing the relationship of Hippo-TAZ with breast cancer and breast cancer stem cells may provide a new way for breast cancer diagnosis and treatment.

OBJECTIVETo observe clinical effects of posterior short-segment fixation with undermining decompress by posterior ligament complex for the treatment of upper lumbar burst fractures.

METHODSFrom October 2010 to March 2013,23 patients with upper lumbar burst fractures (Denis B type) were treated by posterior short-segment fixation with undermining decompress by posterior ligament complex. There were 18 males and 5 females aged from 26 to 64 years old with an average of 45.7 years old. Twelve patients were caused by falling down, 5 cases were caused by traffic accident, 4 cases were the bruise injury caused by heavy object and 2 cases were caused by other injury. Fourteen patients were L1 fracture and 9 patients were L2 fracture. Thirteen patients were combined with nerve injuries (degree D according to ASIA classification). Internal fixation were removed from 12 to 20 months with an average of 14.3 months. JOA scores and imaging changes were recorded and compared at different time points.

RESULTSAll patients were followed up from 18 to 24 months with an average of 20.4 months. Thirteen patients with nerve injuries were completely recovered at 3 to 6 months after operation. JOA score at 1 year after operation was 20.63 ± 0.92, and 20.38 ± 1.06 at 3 months after removal of internal fixation,which were improved obviously than 9.90 ± 2.73 at 3 months after operation. (P > 0.05) Anterior height of injured vertebrae, vertebral body angle and local Cobb angle was (95.0 ± 0.53)%, (2.78 ± 1.36) and (2.43 ± 1.52) °respectively, and improved obviously than that of before operation (P < 0.05). There was no statistical significance in JOA scores at 3 months after removal of internal fixation and 1 year after operation (P > 0.05).

CONCLUSIONposterior short-segment fixation with undermining decompress by posterior ligament complex for the treatment of upper lumbar burst fractures has advantages of minimally invasive, could effective recover vertebrae height, maintain stability of spine, decrease low back pain. It is a safe and effective operative method.

Adult ; Decompression, Surgical ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Lumbar Vertebrae ; injuries ; Male ; Middle Aged ; Spinal Fractures ; surgery

OBJECTIVETo explore the application value of morphology assessment of sperm from fresh semen in routine in vitro fertilization (IVF).

METHODSWe analyzed the morphology of the sperm from fresh or optimized semen samples and, based on the sperm morphology of the raw semen, allocated 908 IVF cycles due to the pure tubal factor to different groups: morphologically normal sperm (MNS) ≤ 4%, > 4% - ≤ 15%, and > 15% in Trial 1 and MNS ≤ 1%, > 1% - ≤ 2%, > 2% - ≤ 3%, and > 3%-- ≤ 4% in Trial 2. We compared the rates of fertilization, cleavage, high-quality embryo, -blastocyst formation, and pregnancy among different groups.

RESULTSThe total fertilization rate was significantly lower in the MNS ≤ 4% than in the MNS > 4% - ≤ 15% and >15% groups (74.40% vs 78.61% and 80.03%, P < 0.01). Compared with the MNS ≤ 1%, > 1% - ≤ 2%, and > 2% - ≤ 3% groups, the MNS > 3% - ≤ 4% group showed remarkably increased rates of 2PN normal fertilization (77.23%, 78.97% and 78.99% vs 85.47%, P < 0.01), cleavage (95.71%, 96.01% and 97.27% vs 98.73%, P < 0.05), and blastocyst formation (53.85%, 49.01% and 49.55% vs 63.41%, P < 0.01). No statistically significant differences were observed in the rates of clinical pregnancy, implantation, early abortion, live birth, or malformation at birth among different groups (P > 0.05).

CONCLUSIONMNS ≤ 4% affected the total rate of fertilization while MNS ≤ 3% reduced the rate of normal fertilization in IVF. However, even MNS ≤ 1% did not result in fertilization disorder or failure. Therefore, teratozoospermia alone was not an indicator of ICSI and sperm mor- phology assessment had no obvious value for predicting the rates of embryo quality, clinical pregnancy, and live birth in IVF.

Female ; Fertilization in Vitro ; Humans ; Male ; Pregnancy ; Pregnancy Outcome ; Spermatozoa ; cytology

OBJECTIVETo optimize the methods of isolating human eccrine sweat gland cells in vitro so as to get efficiently primary human sweat glands.

METHODSThe fresh and normal skin tissue was cut into pieces of microskin about 1mm3 and the following 3 group digestion buffer was applied to isolated gland cells. The digestion buffer of group A was the equivoluminal mixture of Trypsin-Ethylene Diamine Tetraacetic Acid (EDTA) and collagenase-II (2 mg/ml). The digestion buffer of group B was collagenase-II (2 mg/ml) traditionally and group C was Trypsin-EDTA. These three groups were placed into an incubator simultaneously and the emerging time of dissociated sweat glands was calculated. Sweat glands were sorted out and then placed in culture dish. The adherence and the growth of cells were observed. The proliferation index was detected by flow cytometry. The identification of cultured cells was performed by immunocytochemical staining.

RESULTSAfter digesting 30 min in group A and C, a very few of dissociated sweat glands were emerging. But after digesting for 2 h, there were lots of dissociated sweat glands emerging in group A rather than in group C. The emergence of dissociated sweat glands in group B would require at least 6 hours. After seeded in culture dishes, the sweat glands in group C couldn't adhere to the wall of dish, but the sweat glands in group A and B adhered very well and even grew like paving stones after 9 days. In addition, the proliferation index were (18 ± 4) % and (17 ± 6) % respectively, there was no statistical difference. The results of immunocytochemical staining showed that the cells expressed carcino-embryonic antigen (CEA) and cytokeratin 7(CK7) in group A and B.

CONCLUSIONTrypsin-EDTA combined with collagenase-II can shorten the time of isolating sweat gland cells and have no effect on cell activity and proliferation.

Cell Separation ; methods ; Cells, Cultured ; Eccrine Glands ; cytology ; Humans ; In Vitro Techniques

Objective To understand the hemagglutination inhibition antibody level in patients with influenza A H1N1.Methods Sera from 28 patients with influenza A H1N1 at different time points after illness onset were collected and measured by hemagglutination inhibition assay.Results The serum hemagglutination inhibition antibody tilers at 1,5,15,22,37,49 and 58 days after illness onset were 5.36、9.39、39.02、57.99、137.92、55.19 and 57.99 respectively.The top geometric mean tiler of hemagglutination inhibition antibody was 148.55.The antibody seroconversion rate and seroprotection rate were occurred in 96.4％ (27/28)of patients.Conclusion The patients with influenza A H1N1 have effective immune response.

OBJECTIVETo demonstrate molecular characterization of a newly isolated enterovirus.

METHODSVirus were isolated from patient with unknown-causing disease and tested by reverse transcription-polymerase chain reaction (RT-PCR) and 5'3'RACE (rapid amplification of cDNA ends, RACE), in an attempt to obtain the sequence of this newly isolated enterovirus.

RESULTSSequence analysis showed that this newly isolated enterovirus shared 83%-94% nucleotide identity and 91%-100% amino acid identity with enterovirus 89. Phylogenetic analysis indicated that it was probably a new subtype of enterovirus 89.

CONCLUSIONThis newly isolated enterovirus in the stool specimen from patient has the same serotype with enterovirus 89, but it was probably a new subtype of enterovirus 89.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Enterovirus ; classification ; genetics ; isolation & purification ; Feces ; virology ; Genome, Viral ; genetics ; Humans ; RNA, Viral ; genetics ; Sequence Analysis, DNA

OBJECTIVETo study the expression of miRNA-106a gene in esophageal squamous cell carcinoma (ESCC) and its association with clinicopathological features and prognosis of ESCC patients.

METHODSReal-time fluorescence quantitative polymerase chain reaction (PCR) assay was used to determine the expression of miRNA-106a gene in esophageal cancer tissue and corresponding normal mucosa of 81 cases. Immunohistochemical technique was applied to detect the expression of p53, human epidermal growth factor receptor 2 (HER-2), DNA topoisomerase II (Topo II) and multidrug resistance-associated protein (MRP). The association of miRNA-106a expression with clinicopathological features, expression of related proteins, and prognosis of the patients was analyzed.

RESULTSAmong the 81 cases, under-expression of miRNA-106a gene was found in 48 cases (59.3%), normal expression in 22 cases (27.2%), and overexpression in 11 cases (13.6%). The expression of miRNA-106 gene was significantly associated with lymph node metastasis, pathological stage, and nerve invasion (all P < 0.05), significantly associated with expression of p53 (P = 0.006), and not significantly associated with expressions of HER-2, Topo II and MRP proteins (all P > 0.05). The expression of miRNA-106a gene was also significantly associated with progression-free survival (PFS, P = 0.032), but not significantly with overall survival (OS, P = 0.486). The results of Cox multivariate regression analysis showed that the PFS of ESCC patients was significantly correlated with lymph node metastasis (P = 0.029), but not correlated with the age, gender, tumor length, T stage, degree of differentiation, nerve invasion, and miRNA-106a expression (all P > 0.05).

CONCLUSIONSIn esophageal squamous cell carcinomas, the miRNA-106a gene is under-expressed, with tumor suppressor function, and may be regarded as a biological marker to assess the prognosis in patients with esophageal squamous cell carcinoma.

Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Topoisomerases, Type II ; metabolism ; Disease-Free Survival ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; MicroRNAs ; metabolism ; Middle Aged ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Invasiveness ; Neoplasm Staging ; Proportional Hazards Models ; Real-Time Polymerase Chain Reaction ; Receptor, ErbB-2 ; metabolism ; Survival Rate ; Tumor Suppressor Protein p53 ; metabolism

Objective Transcription factor forkhead box L 2 (FOXL2) is a key regulator of granulosa cells (GCs) estrogen syn-thesis and function maintenance .However, the FOXL2 protein expres-sion and function regulation mechanism are unknown .We explored how DNAJB11 regulates estrogen synthesis of granulosa cells with immunoprecipitation , immunofluorescent staining and luciferase re-porter gene. Methods The expression and localization of DNAJB 11 was detected by immunohistochemistry staining in isolated mouse ovary tissues .we use immunoprecipitation , immunofluorescence staining and luciferase reporter gene assay to investigate the mechanism of DNAJB11, a member of the endoplasmic reticulum Hsp 40 /DnaJ family, regulating the estrogen synthesis in granulosa cells . Results DNAJB11 is expressed in the mouse ovary and granulosa cells .Follicle-stimulating hormone (FSH) promotes DNAJB11 ex-pression in a time and concentration dependent manner and induces endogenous DNAJB 11 protein translocation from the ER to the nu-cleus in KGN cells.Moreover, Adenovirus-mediated overexpression of DNAJB11 did not affect the proliferation of granulosa cells .How-ever, the concentration of estrogen in granulosa cells was affected by concentration -dependent and subcellular localization-dependent manner (10749±801.7 pg/mL vs 14217±1218.0 pg/mL P<0.01).Immunoprecipitation assay confirmed that DNAJB11 binds to FOXL2 in granulosa cells .When overexpressed in the nucleus of granulosa cells , DNAJB11 could significantly enhance the stability of FOXL2 (P<0.05) and promote FOXL2-mediated activity of Cyp19A1 promoter (P<0.01), while the expression of DNAJB11 in the nucleus increased the expression of Cyp 19A1 protein by 1.5 times ( P<0.05) . Conclusion These results demonstrate that DNAJB 11 was a new binding molecule of transcription factor FOXL 2 and regulated FOXL 2 protein stability and transcription activity .

AIM To observe the clinical effects and safety of Zibei Zhike Granules (Asteris Radix et Rhizoma,Ardisiae japonicae Herba,Fritiliariae cirrhosae Bulbus,etc.) for acute broncho-bronchitis with remained toxicity lingering lung.METHODS A multi-center,randomized,double-blinded,double-simulation and positive drug parallel controlled trial was adopted.Two hundred and forty cases of patients with the 1 ∶ 1 ratio were assigned to treatment and control group.The treatment group were treated with Zibei Zhike Granules and the control group were treated with Zikebao Tablets (Asteris Radix et Rhizoma,Citri rubrum exocarpium,Platycodi Radix,etc.).The treatment course lasted five days.RESULTS The total effective rate of acute broncho-bronchitis in the treatment group was 73.04％,and 54.78％ in the control group.There was statistical significance between the total effective rate of the two groups (P ＜ 0.01).The total effective rate of traditional Chinese medicine syndrome in the treatment group was 73.91％ and 60.86％ in the control group,and there was statistical significance between the total effective rate of the two groups (P ＜ 0.05).The treatment group showed better clinical effects in improving individual symptoms of cough and spitting sputum.CONCLUSION Zibei Zhike Granules has good clinical effects on acute broncho-bronchitis.