Acta Academiae Medicinae Sinica

Objective To investigate the regulation of epithelium growth factor receptor (EGFR), pan-Ras, and extracellular regulated protein kinase (ERK) with both a ras homologue member I (ARHI) suppression and epithelium growth factor (EGF) stimulation. Methods After identification and implication, the constructed plasmid pIRES2-EGFP-ARHI was transfected into Panc-1. The untransfected cell was also explored as controls. The growth curve was drawn to indicate the proliferation effect of ARHI. EGFR-ELISA was performed to investigate the expression of EGFR. Western blot analysis was used to investigate the expression of protein MAPK/ERK1/2, pan-Ras in Panc-1. Results The proliferation rate of Panc-1 was inhibited by ARHI compared with both empty plasmid and untransfected cell. The amount of EGFR was parallel in both transfected and untrasfected cell but affected by EGF stimulation. The amount of pan-Ras was decreased after ARHI transfection. The optimum concentration of EGF effect on P-ERK was 50 ng/ml. Conclusion Both ARHI and EGF play roles in the EGF-EGFR-Ras-Raf-MAPK/ERK1/2 pathway.

Objective To study the role of the feeder layer cells as niche in the process of expansion of late endothelial progenitor cell in vitro. Methods We cultured mononuclear cells (MNC)from human peripheral blood (PB)on the plate with the feeder layer cells which were irradiated late endothelial progenitor cells(EPC)or human umbilical vein endothelial cells (HUVEC) by EGM-2. After 21 days, the numbers of obtained late EPC colonies were counted separately, and their surface antigen of the late EPC was verified by fluorescence-activated cell sorter (FACS) analysis, and their ability of forming vessel structure with Matrigel in vitro. The differentiation of single stem cell on the feeder layer cell was traced by video-microscopy. Results After 21 days of culture,（40.0±3.9）and（39.3±3.1）late EPC colonies that MNC of a handred milliliter PB were cultured, respectively, on the feeder layer cells of EPC and HUVEC were much more than (2.0±1.3) colonies cultured on without the feeder layer cells （all P <0.05）. These cells also expressed CD31，CD34，eNOS,FLt-1，P1H12，Sendo，VEcadherin，and CD117, as shown by FACS analysis. Furthermore, they formed vessel structure with Matrigel in vitro. The video-microscopy showed the asymmetric cell division was participated by the feeder layer cell during the expansion of single stem cell. Conclusion The massive expansion of late EPC can be achieved by the provision of the feeder layer cells, which may be involved in the stem cell asymmetric cell division.

Objective To study the role of osteocyte in bone remodeling due to mechanical loading in vitro. Methods MLO-Y4 osteocyte-like cells were exposed to fluid flow-induced shear stress（12dyn/cm²）for 0, 1, 2, 4, 6, 12, and 24 hours. Osteocyte exposed to shear stress at different time points were used in co-culture system for 9 days, and then the cells were stained with tartrate-resiseant acid phosphatase on the 9^th day and the amount of positively stained osteoclasts were counted and compared. The expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa (RANKL) were detected by semi-quantitative reverse transcription polymerase chain reaction (semi-quantitative RT-PCR). Results Compared with bone cells without stimulation with fluid flow-induced shear stress, the amount of osteocytes significantly decreased at all time points after the application of fluid flow-induced shear stress (all P<0.05). The OPG expression at mRNA levels was significantly up-regulated in the first 12 hours （P<0.001）, the RANKL mRNA expression was significantly down-regulated in the first 4 hours (P<0.05), and the RANKL/OPG ratio significantly decreased within 12 hours (P<0.01). However, all these indicators showed no significant difference at 24 hours when compared with the pre-stimulation level. Conclusion Osteocytes may act as mechanosensors that are able to inhibit bone resorption after mechanical loading; however, such effect shows certain adaptation ability to shear stress as time goes.

Objective To study the expression of gene associated with retinoid-interferon-induced mortality-19（GRIM-19) in preimplantation embryo of mice and explore its role in embryonic development. Methods The protein and mRNA expressions of GRIM-19 in 2-cell, 4-cell, 8-cell, morula, and blastocyst phases of mice preimplantation embryo were detected by Western blot analysis and Real-time polymerase chain reaction (PCR). Results GRIM-19 was continuously expressed in every stage of preimplantation embryo of mice. Western blot analysis and Real-time PCR demonstrated a gradual increase of GRIM-19 expression from 2-cell, which reached a peak in 8-cell phase and then decreased progressively. Conclusions The expression of GRIM-19 in mouse preimplantation embryos changes as at different developmental phases．GRIM-19 may play an important role during embryonic development.

Objective To explore the effect of the electroconvulsive shock (ECS) on the glutamate level and the hyperphosphorylation of tau protein in depressed rats. Methods The depression rat models whose olfactory bulbs were removed were established. Using the analysis of variance of factorial design, we set up two intervention factors including electric current (three levels: 25, 50, and 75 mA) and duration (three levels: 3, 6, and 9 times), which constituted 9 combinations (n=6). Fifty-four adult depression rat models whose olfactory bulbs were removed were randomly divided into nine experimental groups (n=6 in each group). The hippocampus was removed within 12 hours after the ECS finished. The level of glutamate in the hippocampus was detected by high-performance liquid chromatography, and that of Tau protein, which includes p-PHF-1^Ser396/404, p-AT8^Ser199/202, and p-12E8^Ser262, in the hippocampus with Western blot analysis. Results The glutamate level and the hyperphosphorylation of tau protein in the hippocampus of depressed rats remarkably increased. The changes of the hyperphosphorylation of tau protein were correlated with the electric current and duration of ECS, and these two factors showed an synergic effect. Conclusion ECS enhances the hyperphosphorylation of tau protein in the hippocampus of depressed rats by up-regulating the glutamate level.

Objective To observe the effects of different concentrations of propofol on brain regions activated by mechanical stimuli, and then to investigate the analgesic effect of propofol. Methods Twenty healthy male volunteers were randomly divided into two groups: light anesthesia group (group L) (BIS 60-80) and deep anesthesia group (group D)(BIS 40-60). Propofol was administrated by target controlled infusion system in pilot study. The target effect site concentration (ESC) of propofol was defined as the average of the ESC from BIS 80 to 60 or BIS 60 to 40 in group L or group D respectively. Mechanical stimuli were applied using von Frey filaments at the center of the left foot, and the pain threshold and VAS scores were evaluated. fMRI examinations were taken 1 week after pilot study with the following sequences: structure imaging+ functional imaging: functional imaging=stimulus sequence+propofol sequence, in which the stimulus sequence was 6×(20 s on＋20 s off). This sequence was repeated after propofol sequence. Results As shown by fMRI, in group L, active brain regions of (the second stimulation-the first stimulation, P2-P1) were seen in cingulate gyrus, thalamus, and cerebellum, while active brain regions of (P1-P2) were seen in temporal lobe, frontal gyrus, and occipital lobe. In group D, the active brain region of (P2-P1) was only seen in cerebellum, while active brain regions of (P1-P2) were seen in cingulate gyrus and thalamus. Active brain regions of (deep-low) with propofol infusion in response to vFFs stimulation were observed in cerebellum. Conclusions Propofol at different concentrations has different effect on the activation of brain reions. It may exert its analgesic effect via different mechanisms.

Objective To investigate the clinicopathological characteristics of gastrointestinal tract involvement of anaplastic large cell lymphoma (ALCL). Methods The clinicopathological features of four patients with ALCL that involved gastrointestinal tract were retrospectively analyzed using immunohistochemical study, T-cell receptor gene rearrangement analysis, and evaluation for Epstein Barr virus infection status. Results Most tumor cells in all these four cases are large and highly pleomorphic, and all four cases were classified as the “common pattern” ALCL. Tumor cells in all four tumors expressed CD30, and expressed at least one cytotoxic maker. Two patients were confirmed to be with anaplastic lymphoma kinase (ALK)-positive ALCL, and four patients were negative during in situ hybridization for Epstein-Barr virus-encoded RNA but showed clonal T-cell receptor gene rearrangement. Conclusion Gastrointestinal tract involvement of ALCL has the unique clinicopathological features.

Objective To evaluate the CD4+CD25+ regulatory T cells (Treg) and other lymphocyte subsets in the peripheral blood of patients with advanced lung adenocarcinoma. Methods Peripheral blood samples were obtained from 64 patients with advanced lung adenocarcinoma (case group) and analyzed by flow cytometry. The ratios of CD4+CD25+Treg T cells and other T lymphocyte subsets in peripheral blood were compared with those from 33 healthy controls (control group). Results The percentages of CD3+ and CD3+CD4+ were (66.5±11.0)% and (37.7±10.6)% respectively in the peripheral blood of the case group, which were significantly lower than those ［(72.0±6.0)% and (42.0±6.4)%］ in the control group (t=-3.2,-2.4; P=0.020, 0.015, respectively). The ratio of CD4+ CD25+ Treg cells in case group (10.5±4.0)% was significantly higher than that ［(8.4±3.5)%］ in the control group (t=-2.2, P= 0.013). CD4+/CD8+ value of case group (1.4±0.8) was significantly lower than that (1.8±0.7) in control group(t=-2.2, P=0.029). CD3+CD8+, CD8+CD28-, and CD8+CD28+ showed no significant differences (all P>0.05). Smoking, differentiation grade, and size of the tumor showed no association with the function damage of T lymphocyte subsets, while the carcino-embryonic antigen level did. Conclusions In patients with advanced lung adenocarcinoma, Treg increases and CD4+/CD8+ decreases, suggesting remarkably suppressed immune functions. However, more research is warranted to validate the association of T cells subset dysfunction with smoking, differentiation grade, and size of tumor.

Objective To evaluate the effect of peroxisome proliferator-activated receptor (PPAR)α agonist fenobibrate on the secretion of endothelium-derived ontracting factors in hypertensive rats. Methods The changes of vascular tension in SHR rats after having been incubated with 0.1, 1.0, or 10.0 μmol/L fenobibrate or 10.0 μmol/L fenobibrate and PPARα antagonist MK866 or PPARγ antagonist GW9662 for one hour were observed, and the findings were compared with those in WKY rats (control group). The serum levels of vascular endothelial contraction factor prostacyclin (PGF) 1α, 2α, and thromboxane B2 (TXB2) were determined by enzyme-linked immunosorbent assay (ELISA). The expression of COX-1 protein was determined by Western blot analysis. Results Compared with the control group, fenobibrate significantly reduced the vasoconstriction ability of the SHR rats(P=0.013). PPARα antagonist MK866 significantly improved the vascular contractility of SHR rats that had been incubated with 10.0 μmol/L fenobibrate (P=0.021). PPARγ antagonist GW9662 had no significant effect on the vascular contractility of SHR rats after having been incubated with 10.0 μmol/L fenobibrate (P=0.071). The serum levels of PGF1α(P=0.014), 2α(P=0.023), and TXB2 (P=0.017) in SHR rats incubated with 10.0 μmol/L fenobibrate were significantly lower than in the control group. With the presence of vascular endothelium, the expression of COX-1 in SHR rats incubated with fenobibrate was significantly lower than that in SHR rats incubated without fenobibrate (P=0.027). Conclusion Fenobibrate reduces the secretion of endothelium-dependent contracting factors in SHR rats through lowering the expression of COX-1.

Objective To search for novel tumor associated antigens (TAA) in esophageal squamous cell carcinoma (ESCC). Methods The proteins extracted from tissues of ESCC were separated by two dimensional polyacrylamide gel electrophoresis and transferred to PVDF membrane. Sera from ESCC patients and healthy individuals were used for primary antibodies for Western blot analysis. The differential spots were excised for trypsin hydrolysis and the tryptic peptides were analyzed by matrix-assisted laser disorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The identified TAA of ESCC was validated by immunohistochemical staining (IHC). Results Sera from ESCC patients yielded multiple positive spots, and one 28 800 Da protein that exhibited positive reactivity with 60% (12/20) sera of ESCC patients and only 5% (1/20) sera of healthy controls (P<0.01). The 28 800 Da protein was identified as phosphoglycerate mutase 1 (PGAM1) by MALDI-TOF-MS. Immunohistochemical analysis showed that PGAM1 was located in both cytoplasm and nucleus, and had a higher expression in cancer tissues. Conclusion PGAM1 maybe a candidate of ESCC.

Objective To evaluate the accuracy of marginal and internal fit of the zirconium copings manufactured by two different computer-aided design（CAD）/computer-aided manufacturing（CAM） system on the implant abutment. Methods Using different scanning mode,five Procera^® zirconium copings and five Lava zirconium copings were fabricated on the same implant abutment, and then compared with five precious metal copings fabricated by traditional method. Fifteen abutment replica were made with die-stone and the copings were randomly cemented on them, then they were sectioned and invested. The marginal, shoulder, occlusal, and axial fit of each sample was measured by scanning electron microscopy (SEM). Results There was no significant difference in marginal and axial fit among the three groups（P>0.05). Significant difference in occlusal fit was found among the three groups（P<0.05）: Lava group showed better fit than the others（P<0.05） and Procera^® group showed better fit than the control（P<0.05). Conclusions These two types of zirconium coping have clinically acceptable marginal and internal fit. The internal fit of zirconium coping may be affected by different manufacturing techniques.

Objective To explore the relationship between the T cell subsets and glucose level and first-phase insulin secretion function in patients with type 2 diabetes mellitus (T2DM). Methods We determined the oral glucose tolerance (OGTT), insulin release test(IRT), body mass index(BMI), glycohemoglobin A1c (HbA1c), T lymphocyte subsets （CD4⁺,CD8⁺）, and activity of natural kill(NK) cell and ⊿I₃₀/⊿G₃₀ in 78 newly diagnosed T2DM patients, 60 impaired glucose tolerance (IGT) patients, and 60 normal controls. Results DM and IGT patients had significantly lower levels of CD4⁺, CD4⁺/CD8⁺ratio, activity of NK cell, and ⊿I₃₀/⊿G₃₀ and significantly higher levels of HbA1c and CD8⁺compared with normal controls（all P＜0.05）. Patients in DM group had significantly lower level of CD4⁺,⊿I₃₀/⊿G₃₀ and significantly higher levels of FBG and HbA1c compared with IGT group. There was no significant difference in terms of CD8⁺, CD4⁺/CD8⁺ratio, and activity of NK cell between IGT and DM groups, whereas CD4⁺ T cells were negatively correlated with FBG and HbA1c and positively with ⊿I₃₀/⊿G₃₀ . Multipul regression stepwise analysis showed that CD4⁺ was independently associated with HbA1c and ⊿I₃₀/⊿G₃₀. Conclusion T2DM patients tends to have disorders in cellular immunity, which is correlated with blood glucose level and the insulin secretion function.

Objective To investigate the time to disappearance of thyroglobulin antibodies (TgAb) and its influencing factors in papillary thyroid carcinoma (PTC) patients with positive TgAb after radioiodine (¹³¹I) ablation. Methods We retrospectively analyzed the clinical data of 64 PTC patients who had undergone a total thyroidectomy and subsequent ¹³¹I therapy. Patients were divided into two groups according to the presence of concurrent Hashimoto thyroiditis (HD). TgAb1 (TgAb levels determined before ¹³¹I therapy) were above the normal thresholds in all patients. The method of survival analysis (including Life tables and Cox Regression analysis) was used to assess the time to disappearance of TgAb and its influencing factors. Results The median time to disappearance of positive TgAb (9.0 months vs. 10.4 months, P=0.923) showed no significant difference between these two groups. Cox Regression analysis showed that, among the potential influencing factors including TgAb ［partial regression coefficient (B)=-0.004, P=0.001］, age(B=-0.018, P=0.362), sex (B=0.644, P=0.320) and HD (B=0.341, P=0.372), only TgAb1 was correlated with the TgAb negative conversion time. Conclusions TgAb1 has remarked influence on the time to disappearance of positive TgAb in PTC patients. Patients with a higher TgAb need longer time to achieve negative conversion.

Objective To assess the validity of World Health Organization-University of California-Los Angeles Auditory Verbal Learning Test (WHO-UCLA AVLT) in the diagnosis of Alzheimer's disease (AD). Methods Using WHO-UCLA AVLT, we assessed 183 AD patients (AD group) , 1283 subjects with normal cognitive status (normal control group), and 134 individuals suffered from other diseases easy to be confused with AD (confused control group). Results The AVLT score was 40.9±0.3 in normal control group, 30.7±0.9 in confused control group, 16.6±1.0 in mild AD group, and 10.2±1.2 in moderate AD group after adjustment for educational level, age, sex, and rural/urban residence (all P<0.05). With the 5th percentage of the overall score as the cutoff point, this tool showed a sensitivity of 86.3% and a specificity of 93.3%. Conclusion WHO-UCLA CVLT is highly sensitive and specific in the diagnosis of AD.

Rho-associated coiled-coil protein kinase (ROCK) is a serine/ threonine kinase that belongs to AGC family of kinases. By inducing the formation of stress fibers and reorganizing the cytoskeleton, it is involved in many biological behaviors of cells including cell contraction, cell migration, cell division, and morphological changes, and thus exerts important roles in regulating the multiple functions of cells.

Polycomb group (PcG) proteins are a family of epigenetic regulators responsible for the repression of genes in proliferation and differentiation of stem cells. PcG protein complex consists of two important epigenetic regulators: PRC1 (polycomb repressive complex 1) and PRC2 (polycomb repressive complex 2). In order to further understand the functions of PcG proteins in stem cell growth and differentiation, we review the PcG protein composition, PcG protein localization in the target gene, PcG protein recruitment, and the functions of PcG proteins in the development of stem cells.

TAR DNA binding protein-43(TDP-43) and fused in sarcoma/translocated in liposarcoma protein (FUS/TLS) have been found to be associated with two neurodegenerative diseases - amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in TDP-43 and FUS/TLS lead to abnormal protein expressions, which result in altered RNA processing. The pathological changes of TDP-43 and FUS/TLS-associated ALS and FTD are similar. Although the interactions between ALS and FTD remain unknown, it is speculated that TDP-43 and FUS/TLS-associated neurodegenerative diseases may share similar pathogenesis.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited disease associated with germ-line mutations in mismatch repair genes and microsatellite instability. This article reviews the molecular biology and clinical pathology of HNPCC.

Clinically nonfunctioning pituitary adenomas are the most common types among pituitary adenomas. These tumors are usually diagnosed in their later stages due to the absence of clinical symptoms and detectable hormonal hypersecretion. Although these tumors are benign, they are hard to be completely removed during neurosurgery due to the massive invasion into the surrounding tissues at diagnosis. Furthermore, relapse is common. In recent years, medical treatment of pituitary adenomas has witnessed a rapid development. New medications have shown certain effectiveness in reducing the tumor size and improving the clinical symptoms.

Post-operative adhesions are a common complication of pelvic and abdominal surgeries. Many approaches for preventing post-operative adhesions have been developed. This review summarizes the recent advances in this topic.