Anterior transversalis fascia approach versus preperitoneal space approach for inguinal hernia repair in residents in northern China: study protocol for a prospective, multicentre, randomised, controlled trial.

INTRODUCTION: Many surgical techniques have been used to repair abdominal wall defects in the inguinal region based on the anatomic characteristics of this region and can be categorised as 'tension' repair or 'tension-free' repair. Tension-free repair is the preferred technique for inguinal hernia repair. Tension-free repair of inguinal hernia can be performed through either the anterior transversalis fascia approach or the preperitoneal space approach. There are few large sample, randomised controlled trials investigating the curative effects of the anterior transversalis fascia approach versus the preperitoneal space approach for inguinal hernia repair in patients in northern China. METHODS AND ANALYSIS: This will be a prospective, large sample, multicentre, randomised, controlled trial. Registration date is 1 December 2016. Actual study start date is 6 February 2017. Estimated study completion date is June 2020. A cohort of over 720 patients with inguinal hernias will be recruited from nine institutions in Liaoning Province, China. Patient randomisation will be stratified by centre to undergo inguinal hernia repair via the anterior transversalis fascia approach or the preperitoneal approach. Primary and secondary outcome assessments will be performed at baseline (prior to surgery), predischarge and at postoperative 1 week, 1 month, 3 months, 1 year and 2 years. The primary outcome is the incidence of postoperative chronic inguinal pain. The secondary outcome is postoperative complications (including rates of wound infection, haematoma, seroma and hernia recurrence). ETHICS AND DISSEMINATION: This trial will be conducted in accordance with the Declaration of Helsinki and supervised by the institutional review board of the Fourth Affiliated Hospital of China Medical University (approval number 2015-027). All patients will receive information about the trial in verbal and written forms and will give informed consent before enrolment. The results will be published in peer-reviewed journals or disseminated through conference presentations. TRIAL REGISTRATION NUMBER: NCT02984917; preresults.

Hispidin induces autophagic and necrotic death in SGC-7901 gastric cancer cells through lysosomal membrane permeabilization by inhibiting tubulin polymerization.

Hispidin and its derivatives are widely distributed in edible mushrooms. Hispidin is more cytotoxic to A549, SCL-1, Bel7402 and Capan-1 cancer cells than to MRC5 normal cells; by contrast, hispidin protects H9c2 cardiomyoblast cells from hydrogen peroxide-induced or doxorubicin-induced apoptosis. Consequently, further research on how hispidin affects normal and cancer cells may help treat cancer and reduce chemotherapy-induced side effects. This study showed that hispidin caused caspase-independent death in SGC-7901 cancer cells but not in GES-1 normal cells. Hispidin-induced increases in LC3-II occurred in SGC-7901 cells in a time independent manner. Cell death can be partially inhibited by treatment with ATG5 siRNA but not by autophagy or necroptosis inhibitors. Ultrastructural evidence indicated that hispidin-induced necrotic cell death involved autophagy. Hispidin-induced lysosomal membrane permeabilization (LMP) related to complex cell death occurred more drastically in SGC-7901 cells than in GES-1 cells. Ca2+ rather than cathepsins from LMP contributed more to cell death. Hispidin induced microtubule depolymerization, which can cause LMP, more drastically in SGC-7901 cells than in GES-1 cells. At 4.1 µM, hispidin promoted cell-free tubulin polymerization but at concentrations higher than 41 µM, hispidin inhibited polymerization. Hispidin did not bind to tubulin. Alterations in microtubule regulatory proteins, such as stathmin phosphorylation at Ser16, contributed to hispidin-induced SGC-7901 cell death. In conclusion, hispidin at concentrations higher than 41 µM may inhibit tubulin polymerization by modulating microtubule regulatory proteins, such as stathmin, causing LMP and complex SGC-7901 cell death. This mechanism suggests a promising novel treatment for human cancer.

Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01.

Lactobacillus salivarius LI01, isolated from healthy humans, has demonstrated probiotic properties in the prevention and treatment of liver failure. Tolerance to bile stress is crucial to allow lactobacilli to survive in the gastrointestinal tract and exert their benefits. In this work, we used a Digital Gene Expression transcriptomic and iTRAQ LC-MS/MS proteomic approach to examine the characteristics of LI01 in response to bile stress. Using culture medium with or without 0.15% ox bile, 591 differentially transcribed genes and 347 differentially expressed proteins were detected in LI01. Overall, we found the bile resistance of LI01 to be based on a highly remodeled cell envelope and a reinforced bile efflux system rather than on the activity of bile salt hydrolases. Additionally, some differentially expressed genes related to regulatory systems, the general stress response and central metabolism processes, also play roles in stress sensing, bile-induced damage prevention and energy efficiency. Moreover, bile salts appear to enhance proteolysis and amino acid uptake (especially aromatic amino acids) by LI01, which may support the liver protection properties of this strain. Altogether, this study establishes a model of global response mechanism to bile stress in L. salivarius LI01. BIOLOGICAL SIGNIFICANCE: L. salivarius strain LI01 exhibits not only antibacterial and antifungal properties but also exerts a good health-promoting effect in acute liver failure. As a potential probiotic strain, the bile-tolerance trait of strain LI01 is important, though this has not yet been explored. In this study, an analysis based on DGE and iTRAQ was performed to investigate the gene expression in strain LI01 under bile stress at the mRNA and protein levels, respectively. To our knowledge, this work also represents the first combined transcriptomic and proteomic analysis of the bile stress response mechanism in L. salivarius.

Alterations and correlations of the gut microbiome, metabolism and immunity in patients with primary biliary cirrhosis.

We selected 42 early-stage primary biliary cirrhosis (PBC) patients and 30 healthy controls (HC). Metagenomic sequencing of the 16S rRNA gene was used to characterize the fecal microbiome. UPLC-MS/MS assaying of small molecules was used to characterize the metabolomes of the serum, urine and feces. Liquid chip assaying of serum cytokines was used to characterize the immune profiles. The gut of PBC patients were depleted of some potentially beneficial bacteria, such as Acidobacteria, Lachnobacterium sp., Bacteroides eggerthii and Ruminococcus bromii, but were enriched in some bacterial taxa containing opportunistic pathogens, such as γ-Proteobacteria, Enterobacteriaceae, Neisseriaceae, Spirochaetaceae, Veillonella, Streptococcus, Klebsiella, Actinobacillus pleuropneumoniae, Anaeroglobus geminatus, Enterobacter asburiae, Haemophilus parainfluenzae, Megasphaera micronuciformis and Paraprevotella clara. Several altered gut bacterial taxa exhibited potential interactions with PBC through their associations with altered metabolism, immunity and liver function indicators, such as those of Klebsiella with IL-2A and Neisseriaceae with urinary indoleacrylate. Many gut bacteria, such as some members of Bacteroides, were altered in their associations with the immunity and metabolism of PBC patients, although their relative abundances were unchanged. Consequently, the gut microbiome is altered and may be critical for the onset or development of PBC by interacting with metabolism and immunity.

Comparative genomic study of three species within the genus Ornithinibacillus, reflecting the adaption to different habitats.

In the present study, we report the whole genome sequences of two species, Ornithinibacillus contaminans DSM22953(T) isolated from human blood and Ornithinibacillus californiensis DSM 16628(T) isolated from marine sediment, in genus Ornithinibacillus. Comparative genomic study of the two species was conducted together with their close relative Ornithinibacillus scapharcae TW25(T), a putative pathogenic bacteria isolated from dead ark clam. The comparisons showed O. contaminans DSM22953(T) had the smallest genome size of the three species indicating that it has a relatively more stable habitat. More stress response and heavy metal resistance genes were found in the genome of O. californiensis DSM 16628(T) reflecting its adaption to the complex marine environment. O. scapharcae TW25(T) contained more antibiotic resistance genes and virus factors in the genome than the other two species, which revealed its pathogen potential.

[Short-term outcomes of radical resection of rectal carcinoma: hand-assisted laparoscopy versus traditional laparoscopic approach].

OBJECTIVE: To study the feasibility of hand-assisted laparoscopic radical resection of rectal carcinoma and compare the short-term outcomes of HALS versus traditional laparoscopy approach. METHODS: Clinical data of 42 cases of rectal carcinoma between January 2010 and March 2011 were enrolled in this study. Nineteen cases underwent HALS total mesorectal excision and 23 cases underwent traditional laparoscopy approach. RESULTS: All the operations were successfully accomplished without conversions to open surgery. The mean operation time of the HALS group was shorter than that of the traditional laparoscopic group (152 min vs. 168 min, P=0.009). Incision length was significantly longer in the HALS group (5.6 cm vs. 4.5 cm, P=0.000). The median overall costs were lower in HALS group (26 000 RMB vs. 29 000 RMB, P=0.008). The number of lymph nodes in resected specimen, intra-operative blood loss, length of hospital stay, time to passage of flatus were comparable between the two groups. CONCLUSIONS: Hand-assisted laparoscopic surgery has the advantages of laparoscopic surgery including minimal invasiveness, safety, and quicker postoperative recovery.

Effects of environmental hypothermia on hemodynamics and oxygen dynamics in a conscious swine model of hemorrhagic shock.

BACKGROUND: Hypothermia is associated with poor outcome in trauma patients; however, hemorrhagic shock (HS) model with anesthetized swine was different from that of clinical reality. To identify the effects of environmental hypothermia on HS, we investigated hemodynamics and oxygen dynamics in an unanesthetized swine model of HS under simulating hypothermia environment. METHODS: Totally 16 Bama pigs were randomly divided into ambient temperature group (group A) and low temperature group (group B), 8 pigs in each group. Venous blood (30 mL/kg) was continuously withdrawn for more than 15 minutes in conscious swine to establish a hemorrhagic shock model. Pulmonary arterial temperature (Tp), heart rate (HR), mean arterial pressure (MAP), pulmonary arterial pressure (PAP), pulmonary arterial wedge pressure (PAWP), central venous pressure (CVP), cardiac output (CO), hemoglobin (Hb), saturation of mixed venous blood (SvO2) and blood gas analysis were recorded at the baseline and different hemorrhagic shock time (HST). The whole body oxygen delivery indices, DO2I and VO2I, and the O2 extraction ratio (O2ER) were calculated. RESULTS: Core body temperature in group A decreased slightly after the hemorrhagic shock model was established, and environmental hypothermia decreased in core body temperature. The mortality rate was significantly higher in group B (50%) than in group A (0%). DO2I and VO2I decreased significantly after hemorrhage. No difference was found in hemodynamics, DO2I and VO2I between group A and group B, but the difference in pH, lactic acid and O2ER was significant between the two groups. CONCLUSION: Environmental hypothermia aggravated the disorder of oxygen metabolism after hemorrhagic shock, which was associated with poor prognosis.

Correlation of seven biological factors (Hsp90a, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3) with clinical outcomes of ALK+ anaplastic large-cell lymphoma.

OBJECTIVE: To explore correlation of seven apoptosis-related proteins (Hsp90a, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3) with clinical outcomes of ALK+ anaplastic large-cell lymphoma (ALCL). METHODS: Using immunohistochemistry and immunofluorescence double staining methods, the expressions of these seven apoptosis-associated proteins were studied to clarify their relationship with clinical outcomes of 36 ALK+ and 25 ALK-systemic ALCL patients enrolled between 1996 and 2006. The relationship of these apoptosis-regulating proteins with NPM-ALK status was also evaluated with the tyrosine inhibitor herbimycin A (HA) in vitro by immunocytochemistry, Western blotting and flow cytometric assays. RESULTS: The presence of Hsp90α-, MDM2-, Bax-, Cytochrome C, and Cleaved caspase3-positive tumor cells was found significantly different in ALK+ and ALK-ALCLs, which was correlated with highly favorable clinical outcome. The Bcl-2- and p53-positive tumor cells were found in groups of patients with unfavorable prognosis. Inhibition of NPM-ALK by HA could reactivate the p53 protein and subsequent apoptosis-related proteins and therefore induced apoptosis in ALK+ ALCL cells. CONCLUSION: Our results suggest that these seven proteins might be involved in apoptosis regulation and associated with clinical outcome of ALK+ systemic ALCLs. We also reveal a dynamic chain relation that NPM-ALK regulates p53 expression and subsequent apoptosis cascade in ALK+ ALCLs.

[Expression of ALK protein in large cell lymphoma with ALCL chromosome translocation in relation to prognosis].

The aim of this study was to investigate the expression of anaplastic lymphoma kinase (ALK) protein resulted from chromosome translocation in anaplastic large cell lymphoma (ALCL) and its relationship with the age and prognosis of patients with ALCL. The tissue microarray including 30 cases of ALCL and 2 normal control tissues were established, the expression of anaplastic lymphoma kinase (ALK) protein was detected by immunohistochemistry, the statistical analysis of detected results was carried out by SPSS software. The results showed that the ALK protein was expressed negatively in 2 cases of primary skin ALCL, but in 20 out of 28 cases of systematic ALCL the ALK protein was expressed positively and mainly located in cytoplasm and/or nucleus (71.4%). Clinically, the patients with ALK expression were younger than those without ALK expression (p < 0.05). The prognosis of patients with ALK expression was better than those without ALK expression (p < 0.05). It is concluded that there is a high incidence of ALK expression in ALCL, especially in younger group. ALK expression may be an useful and independent marker for the differential diagnosis and prognosis evaluation of ALCL.

[Nuclear microarray combined with fluorescence in situ hybridization for detecting ALK gene translocation in paraffin-embedded anaplastic large cell lymphoma and its significance].

OBJECTIVE: To compare the efficacy of nuclear microarray combined with fluorescence in situ hybridization (FISH) and immunohistochemistry in detecting ALK gene translocation and ALK fusion protein in anaplastic large cell lymphoma (ALCL). METHODS: ALK gene translocation and ALK fusion protein in 17 paraffin-embedded ALCL specimens were detected using nuclear microarray combined with FISH and immunohistochemical straining, respectively. RESULTS: The expression of ALK fusion protein was detected immunohistochemically with ALK antibody in 8 of the 17 specimens of systemic ALCL, including 4 with both nuclear and cytoplasmic positivity and 4 with only cytoplasmic positivity. Dual-color FISH identified 6 positive specimens, including the 4 specimens with both nuclear and cytoplasmic positivity as identified immunohistochemically, and 2 with immunohistochemical cytoplasmic positivity. FISH yielded negative results for the 2 specimens with immunohistochemical cytoplasmic positivity. CONCLUSION: Nuclear microarray combined with FISH eliminated the cytoplasmic interference of the results of conventional FISH and provides a high-throughput platform for clinical detection with greater specificity than immunohistochemistry.

[Detection of 3q27 chromosomal abnormality in diffuse large B cell lymphoma using FISH on cell microarray].

OBJECTIVE: To investigate the association of 3q27 chromosome rearrangement with bcl-6 gene amplification and the molecular classification, therapeutic efficacies, and clinical stages in diffuse large B cell lymphoma (DLBC). METHODS: The newly invented cell microarray was used to detect 3q27 chromosome rearrangement and bcl-6 gene amplification in 60 cases of DLBCL by fluorescence in situ hybridization (FISH). The molecular classification of germinal center B-cell-like (GCB) and non-germinal center B-cell-like (non-GCB) was investigated by analyzing the expression of CD20, CD10, bcl-6 and MUM1 simultaneously by immunohistochemical S-P method and tissue microarray. The information of therapeutic efficacies and clinical stages was obtained by analyzing clinical cases. The relationships among the factors were analyzed by statistics. RESULTS: In 60 cases of DLBCL, 48.3%(29/60) were GCB and 51.7%(31/60) were non-GCB. The 3q27 chromosome rearrangement and bcl-6 gene amplification were present in 15 and 22 cases respectively. In 15 cases with 3q27 rearrangement, bcl-6 protein expression was positive in 3(20.0%), which was significantly different from that in cases without 3q27 rearrangement (P=0.017). In 60 cases of DLBCL, bcl-6 gene amplification was present in 22 cases, in which 5(22.7%) were GCB and 17(77.3%) were non-GCB, which was significantly different from that in cases without bcl-6 gene amplification (P=0.003). In 36 cases undergoing the normal CHOP program treatment, bcl-6 gene amplification was present in 15 cases and the rates of the complete remission, partial remission and no change were 4(26.7%), 4(26.7%) and 7(46.7%) respectively, and again it was significantly different from that in cases without bcl-6 gene amplification (P=0.016). There were no statistical significances among bcl-6 gene, BCL-6 protein expression, and clinical stages. Cases with BCL-6 protein positive and negative expression were not correlated with therapeutic efficacies and clinical stages. CONCLUSION: There is lower expression of BCL-6 protein in cases with bcl-6 gene fragmentation. Cases with bcl-6 gene amplification are non-GCB with worse therapeutic results and later clinical stages. There may be other genes near chromosome 3q27 associated with DLBCL prognosis.

[Establishment of a whole-body visualization model of orthotopically implanted colorectal carcinoma and metastasis model in nude mice].

OBJECTIVE: To establish an animal model visualizing orthotopic growth and metastasis of colorectal cancer. METHODS: pEGFP-N1 plasmid was transfected into human colorectal carcinoma cell line SW480 so that the resultant SW480/EGFP cells emitted fluorescence that could be detected externally by fluorescence stereo microscope. SW480/EGFP cells were inoculated subcutaneously in nude mice, and the orthotopic tumor growth was evaluated in real time. Whole-body visualization models of orthotopically implanted colorectal carcinoma was established surgically, and the tumor growth and metastasis are evaluated by conventional pathological methods. RESULTS: SW480/EGFP cells stably expressed high-levels of enhanced green fluorescent protein. Subcutaneous injection of SW480/EGFP cells resulted in tumor growth in nude mice, and the emitted fluorescence could be quantitatively detected and imaged with fluorescence stereo microscope to visualize real-time tumor growth. Visualization animal model was established successfully with surgical orthotopic implantation (SOI) of the tumor, and all mice survived. After two weeks, all the mice developed colorectal carcinoma without metastasis, but 4 weeks later, 75%percnt; of the mice developed peritoneal tumor metastasis and 50% had liver metastasis. The whole-body visualization animal model was successfully validated by pathological detection. CONCLUSION: Whole-body visualization model of orthotopic and metastatic tumor growths provides a reliable means for observing the behavior of human colorectal carcinoma and can be helpful to study the growth and metastasis patterns of the cancer.

[Detection of t (14; 18) translocation and bcl-2 amplification in diffuse large B-cell lymphoma].

OBJECTIVE: To investigate the role of t (14; 18) chromosomal translocation and bcl-2 amplification in classification, clinical staging and prognostic evaluation of diffuse large B cell lymphoma (DLBCL). METHODS: Sixty cases of DLBCL were included in this investigation. Microdissection of the lymphoma tissue was performed. Tissue microarray and in-situ fluorescence hybridization technique were used to detect t (14; 18) and bcl-2 amplification. The phenotypes of either germinal center B-cell-like (GCB) or non-germinal center B-cell-like (non-GCB) were determined by immunohistochemistry including CD20, CD10, bcl-6 and MUM1 (S-P method) using the tissue microarray format. Clinical staging and therapeutic response were obtained by medical record review. The relationships among different parameters were analyzed by appropriate statistical methods. RESULTS: Among 60 cases of DLBCL, bcl-2/IgH was positive in 10 cases and bcl-2 gene amplification was detected in 18 cases. Overall, 29 (48.3%) cases were GCB and 31 (51.7%) cases were non-GCB. The t (14; 18) was seen in 8 (80.0%) cases of GCB and 2 (20.0%) of non-GCB. The difference was statistical significance (P = 0.031). Over-expression of bcl-2 was seen in all cases having both t (14; 18) and bcl-2 gene amplification. Of thirty-six patients who underwent routine CHOP treatment, bcl-2 gene amplification was seen in 13 cases. In these cases, the rates of complete remission, partial remission and no change were 3 (23.1%), 4 (30.8%) and 6 (46.2%) respectively, and the clinical stages were stage I - II (1 case, 7.7%) and stage III - IV (12 cases, 92.3%). The clinical stages and therapeutic response were significantly different between the bcl-2 amplification cases and those without (P = 0.046 and P = 0.019, respectively). CONCLUSIONS: T (14; 18) and/or bcl-2 gene amplification can lead to an over-expression of bcl-2 protein. The bcl-2 gene amplification correlates with worse therapeutic efficacies and advanced clinical stages. The reason for the correlation between bcl-2 over-expression and the prognosis is unclear, although it may be explained by different mechanisms that lead to bcl-2 over-expression. Detection of t (14; 18) chromosome translocation by FISH can be helpful in the classification of DLBCL.

A novel tissue array technique for high-throughput tissue microarray analysis -- microarray groups.

Tissue microarrays are ordered arrays of hundreds to thousands of tissue cores in a single paraffin block. We invented a novel method to make a high-throughput microarray group. Conventional smaller tissue microarrays were made first and then sectioned. Separate paraffin films were arrayed orderly onto a regular-sized glass slide to form a larger microarray group. Sections were not floated in a water bath but, rather, were cut singly using conventional microtome, arrayed orderly onto the glass slide with forceps instead of using a tape-based tissue transfer system, and then unfolded with warm water (46 degrees C) using a micropipette. This not only lowers the difficulty in sectioning but the overall tissue disks can be included in the same section. A microarray group of 2,534 small disks (theoretically, 2,560 disks can be made; 26 fell off during the procedure), the most up to now, was successfully made and may be used in immunohistochemistry, mRNA in situ hybridization, and flourescent in situ hybridization.

Construction and analysis of tree models for chromosomal classification of diffuse large B-cell lymphomas.

AIM: To construct tree models for classification of diffuse large B-cell lymphomas (DLBCL) by chromosome copy numbers, to compare them with cDNA microarray classification, and to explore models of multi-gene, multi-step and multi-pathway processes of DLBCL tumorigenesis. METHODS: Maximum-weight branching and distance-based models were constructed based on the comparative genomic hybridization (CGH) data of 123 DLBCL samples using the established methods and software of Desper et al. A maximum likelihood tree model was also used to analyze the data. By comparing with the results reported in literature, values of tree models in the classification of DLBCL were elucidated. RESULTS: Both the branching and the distance-based trees classified DLBCL into three groups. We combined the classification methods of the two models and classified DLBCL into three categories according to their characteristics. The first group was marked by +Xq, +Xp, -17p and +13q; the second group by +3q, +18q and +18p; and the third group was marked by -6q and +6p. This chromosomal classification was consistent with cDNA classification. It indicated that -6q and +3q were two main events in the tumorigenesis of lymphoma. CONCLUSION: Tree models of lymphoma established from CGH data can be used in the classification of DLBCL. These models can suggest multi-gene, multi-step and multi-pathway processes of tumorigenesis. Two pathways, -6q preceding +6q and +3q preceding +18q, may be important in understanding tumorigenesis of DLBCL. The pathway, -6q preceding +6q, may have a close relationship with the tumorigenesis of non-GCB DLBCL.

A new method to make nuclei or cell microarrays.

We invented a new method to make microarrays using nuclei extracted from paraffin-embedded tissues or cultured cells. A blank recipient paraffin block with 10 x 10 cores was constructed and sectioned to make the mold for the cell arrays. The sections of paraffin were mounted on poly-L-lysine-coated slides. Prepared nuclei or cells were injected into the cores of the paraffin mold. The slides were dried and dewaxed and nuclei or cell arrays were made. Using this method, we successfully made microarrays of nuclei extracted from diffuse large B-cell lymphoma paraffin-embedded tissues, nasopharyngeal cancer and lymphoma cell lines. This technique resulted in a paraffin-embedded cell preparation that yielded a cell density of approximately 500 to 1000 or 800 cells on average per 0.6-mm-diameter core. The microarrays were successfully used in fluorescence in situ hybridization, mRNA in situ hybridization, and cytohistochemical staining.

[Expression and cloning of mCD99L2 gene from mouse B lymphoma cell line A20 and construction of its eukaryotic expression vector].

OBJECTIVE: To detect and clone mCD99L2 gene from mouse B lymphoma cell line A20 and construct its eukaryotic expression vector pcDNA3.1-mCD99L2. METHODS: The expression of mCD99L2 mRNA in A20 cell line was detected by in situ hybridization. The total RNA of A20 cells was extracted to obtain the full-length cDNA of the coding region of mCD99L2 gene by RT-PCR, the product of which was ligated into pMD18-T vector and the DNA sequence of the insert was detected. The coding regions of mCD99L2 gene was amplified from pMD-mCD99L2 by PCR using primers containing EcoR I and Xho I sites and cloned into the eukaryotic expression vector pcDNA3.1/MycHis(+). RESULTS: In situ hybridization identified positive expression of mCD99L2 gene in the A20 cell line. The full-length cDNA of mCD99L2 coding region of A20 cell line was obtained by RT-PCR, which yielded a product of 712 bp as expected, and the DNA sequence was completely homologus to the mCD99L2 cDNA reported in GenBank. Restriction endonuclease digestion and DNA sequencing indicated that the eukaryotic expression vector pcDNA3.1(+)- mCD99L2 had been constructed successfully. CONCLUSION: mCD99L2 cDNA has been cloned from mouse B lymphoma cell line A20 and its eukaryotic expression vector pcDNA3.1(+)- mCD99L2 successfully constructed, which facilitates further functional study of mCD99L2 gene in mouse B lymphoma cell line A20.

[Cloning of human KIAA1173 gene and biological characterization of transfected 6-10B cells].

OBJECTIVE: To establish a 6-10B cell line with stable expression of KIAA1173 gene and study the biological behaviors of the cells. METHODS: The total RNA was extracted from normal skeletal muscular tissues for cloning of KIAA1173 gene by means of RT-PCR which was subsequently introduced into pcDNA3.1 (+) vector. The recombinant eukaryotic expression vector pcDNA 3.1(+)-KIAA1173 was constructed and identified by endonuclease digestion and sequencing before transfection into 6-10B cells via lipofectamine with the empty vector as the control. The positive cell clones were obtained by G418 selection. Stable expression of KIAA1173 gene in the transfected 6-10B cells was determined by RT-PCR, in situ hybridization and immunocytochemistry, and the biological behaviors of the transfected cells were observed by MTT assay, cell invasion assay and tumorigenesis assay in nude mice. RESULTS: High expression of KIAA1173 at both mRNA and protein levels was observed in the transfected 6-10B cells. The capability of proliferation, invasion and tumorgenicity of the KIAA1173-transfected cells in nude mice was lowered in comparison with those of the cells transfected with pcDNA3.1 (+) vector (P<0.05). CONCLUSIONS: KIAA1173 genes may function as a potential tumor suppressor of nasopharyngeal carcinoma both in vitro and in vivo. The 6-10B cell line expressing KIAA1173 has been obtained, which can be helpful for further study of KIAA1173 gene.

Expression of cell cycle regulator p57kip2, cyclinE protein and proliferating cell nuclear antigen in human pancreatic cancer: an immunohistochemical study.

AIM: To investigate the effects of p57(kip2), cyclinE protein and proliferating cell nuclear antigen (PCNA) on occurrence and progression of human pancreatic cancer. METHODS: The expression of p57(kip2), cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32 patients with pancreatic cancer was detected by SP immunohistochemical technique. RESULTS: The positive expression rate of p57(kip2) protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues (chi(2) = 5.317, P<0.05). p57(kip2) protein positive expression remarkably correlated with tumor cell differentiation (P<0.05), but not with lymph node metastasis (P>0.05). The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (chi(2) = 4.063, P<0.05). CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis (P<0.05). The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues (chi(2) = 5.189, P<0.05). PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis (P<0.05). CONCLUSION: The decreased expression of p57(kip2) and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer. p57(kip2), cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer.

[Detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma by hemi-nested PCR].

OBJECTIVES: To explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL), and verify the credibility of the established method. METHODS: bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced. RESULTS: bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1. CONCLUSION: There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.