Objective:To prepare a monoclonal antibody against human angiopoietin-related protein 2(ARP2).Methods: Human spleen ARP2 gene was obtained by RT-PCR.A pET32a-ARP2 plasmid was constructed and was incorporated into(E.coli.) The products were purified and were used to immunize 6-week-old BALB/c female mice.Hybridoma secreting anti-ARP2 monoclonal antibody was obtained by standard procedure.Mass production was carried out after specificity identification with Western blotting.Results: The fusion protein obtained by pET32a system had a relative molecular weight of about 570 000,which was in accordance with the theoretical value.The purity of the protein was more than 90% after purification.The antibody titer was 110~(4)in the hybridoma culture supernatant and 110~(7)-10~(8) in the ascites.The IgG2a type antibody had a relative molecular weight of about 570 000 by Western blot analysis.Immunohistochemistry method showed that the antibody bond with human ARP2.Conclusion: The prepared anti-human ARP2 monoclonal antibody in this study can be used for identification of ARP2 protein.

Objective To investigate the features and risk factors associated with lymph node metastasis in colorectal neuroendocrine neoplasm.Methods Clinical and pathological data of 99 patients with colorectal neuroendocrine neoplasm treated between January 2009 to January 2015 were analyzed retrospectively.Comparisons of categorical data and univariate analysis of risk factors of lymph node metastasis were conducted.Results Of the 99 patients,the rate of regional lymph node metastasis was 30.3％ (30/99)with 56.7％ limited to para-intestinal lymph nodes in 17 cases,26.7％ limited to mesenteric lymph nodes in 8 cases,and 16.7％ limited to mesenteric root central lymph nodes in 5 cases.No metastasis exceeding central lymph nodes was observed.Multivariate regression analysis revealed that tumor size,invasion of lymphatic vessel and pathological grading were independent risk factors for lymph node metastasis in colorectal neuroendocrine neoplasm (P ＜ 0.05).Conclusions Colorectal neuroendocrine neoplasm with larger tumor size,invasion of lymphatic vessel or higher grade (G2,G3) has high risk of regional lymph node metastasis.

Objective To investigate the feasibility of DNA sequencing analysis in molecular diagnosis for spinal muscular atrophy (SMA). Methods Two pairs of primers were utilized to amplify the region including 5 different bases in SMA-causative gene SMN1 and its homologue copy SMN2 by polymerase chain reaction (PCR). The first primer amplified a fragment 501 bp long spanning from SMN intron 6 to intron 7 targeting four different bases (g.31957, 32006, 32154 and 32269). The second primer reversely amplified a 189 bp long fragment within SMN exon 8 including one base-pair differ-ence (g.32734). PCR procedure was followed by Sanger sequencing technique to identify the 5 different bases. SMA patients caused by SMN1 homozygous deletion were distinguished from carriers or normal controls by absence of SMN1 specific bas-es in sequence chromatograms. This assay was performed in 7 SMA suspected patients and their parents. The specimens were also detected by PCR- restriction fragment length polymorphism (RFLP) method. Results It was found that 6 of 7 SMA suspected patients showed only SMN2 specific bases at the 5 different base positions among the region from intron 6 to exon 8, which meant the patient displaying only SMN2-specific nucleotide a, T, g, g and A at g.31957, 32006, 32154, 32269 and 32734, while their parents (carriers) showed a/g, T/C, g/a, g/a and A/G at the same sites. SMN1 gene was deleted in the patient, and the deletion region was inferred from intron 6 to exon 8. Because carriers had both SMN1 and SMN2 genes, they can be discriminated from the SMN1 deleted patient. One of 7 patients yield an unique sequence chromatogram of a, T, g, g and A/G, indicating that exon 8 of SMN1 was not deleted in this patient. Conclusion DNA sequencing analysis is an alter-native simple method for detecting SMA caused by homozygous deletion of SMN1. We recommend to replace the widely used PCR-RFLP method with DNA sequencing assay.

OBJECTIVE:To investigate Smad3 and Smad7 expression in cardiac tissues in myocardial infarction (MI) rats and the effect of simvastatin (SIM) on expression of Smads.METHODS:36 rats were randomized into SIM (40 mg?kg-1,n=12) group,MI group (normal saline,n=12) and SHAM group (normal saline,n=12).Rats in first two groups were induced MI model.Rats of 3 groups were sacrificed after four weeks administration.The mRNA expressions of Smad3 and Smad7 in non-infarction regions were measured by RT-PCR.Smad3 and Smad7 protein expressions were measured by Western blotting assay.RESULTS:As compared with SHAM group,the expressions of Smad3 in MI group and SIM group were increased while the expressions of Smad7 were decreased.As compared with MI group,the mRNA expression and protein expression of Smad3 in SIM group was decreased while that of Smad7 increased (P

Objective: To prepare a monoclonal antibody against human angiopoietin-related protein 2 (ARP2). Methods: Human spleen ARP2 gene was obtained by RT-PCR. A pET32a-ARP2 plasmid was constructed and was incorporated into E. coli. The products were purified and were used to immunize 6-week-old BALB/c female mice. Hybridoma secreting anti-ARP2 monoclonal antibody was obtained by standard procedure. Mass production was carried out after specificity identification with Western blotting. Results: The fusion protein obtained by pET32a system had a relative molecular weight of about 570 000, which was in accordance with the theoretical value. The purity of the protein was more than 90% after purification. The antibody titer was 1: 104 in the hybridoma culture supernatant and 1: 107-108 in the ascites. The IgG2a type antibody had a relative molecular weight of about 570 000 by Western blot analysis. Immunohistochemistry method showed that the antibody bond with human ARP2. Conclusion: The prepared anti-human ARP2 monoclonal antibody in this study can be used for identification of ARP2 protein.

Objective To construct the replication-deficient recombinant adenovirus coding ARP2 gene before the studies of gene transfection to ischemic myocardium.Methods From Dec.2004 to Oct.2005,in the Department of Cardiology,Changhai Hospital of the Second Military Medical Univesity,the ARP2-pAxCAwt was constructed by inserting the cDNA of interest into the SwaI site of pAxCAwt.Results The right direction of the insert was confirmed by restriction.Conclusion The direction of the insert must be confirmed by restriction analysis,and the DNA package protein is much helpful during the transfection of the recombinant cosmid to E.coli.

MicroRNAs (miRNAs) are small non-coding RNAs that contain 22 to 25 nucleotides and play important roles in post-transcriptional regulation of target genes. MiRNAs are involved in cell proliferation, differentiation, apoptosis, inflammation, and cancer development. Alveolar bone resorption is the main clinical manifestation of periodontitis. Osteoclasts are unique cells regulated by osteoblasts and inflammatory cytokines and are responsible for bone resorption in periodontitis. Recently, miRNAs have emerged as an important regulator of osteoclast differentiation and function. In this study, we review the recent research progress on the effects of miRNAs on osteoclast differentiation and function, particularly the mechanisms of miRNAs-mediated osteoclast formation and bone loss in periodontitis.
Bone Resorption ; Bone and Bones ; Cell Differentiation ; Cell Proliferation ; Humans ; MicroRNAs ; Osteoblasts ; Osteoclasts

Objective Through analyzing benign and malignant pleural effusion samples by proteomic techniques, finding protein biomarkers to provide help and new clues for effusion differential diagnosis.Methods Two-dimensional electrophoresis(2-DE) and matrix-assisted laser desorption ionization-time of flight-mass spectrometry(MALDI-TOF-MS) were used to search and identify protein biomarkers, enzyme linked immunosorbent assay(ELISA) was to validate the biomarkers.Results By comparing malignant group with benign group, 43 significantly different protein spots(Up or down regulated≥2 times) were found.Including 9 up regulated spots and 34 down regulated spots.And 7 spots were identified(Up or down regulated≥3 times) by MALDI-TOF-MS and validated 2 spots immunoglobulin λ(Igλ) and haptoglobin(Hp) by ELISA.The results showed that Igλ showed no statistical significance between two groups, while Hp showed the statistical significance(P<0.05).The diagnostic sensitivity and specificity of Hp in malignant pleural effusion were 75.00% and 52.38% at diagnostic cut-off point of 389.02 μg/L.Conclusion The application of proteomics technology has a great help with protein biomarkers searching in pleural effusion.HP has a certain value for the differential diagnosis of benign and malignant pleural effusionand and worthy of further study.

Objective To investigate the effect and potential mechanism of pigment epithelium derived factor(PEDF) acting upon SK-MES-1 cell and human umbilical vein endothelial cells(HUVECs).Methods CCK-8 was used to detect the effect of varying concentrations of PEDF upon HUVECs and SK-MES-1 cell, measuring the degree of cell proliferation and inhibition effect across varying times.The flow cytometry tests were carried out to invest gate the apoptosis of these two kinds of cells when exposed to varying concentration of PEDF.qRT-PCR were carried out to assess the vascular endothelial growth factor(VEGF) gene expression level in these two kinds of cells after treatment of PEDF.Results CCK-8 results revealed that PEDF had a concentration-dependent and time-dependent cell proliferation inhibition effect on SK-MES-1 cell and HUVECs(P<0.05);Flow cytometry showed that the apoptosis of the cells in the treatment group were higher than that of control group(P<0.05), and the apoptosis rate of high concentration group was higher than that of the low concentration group(P<0.05);qRT-PCR results showed that PEDF was able to inhibit expression of mRNA of VEGF in both HUVECs and SK-MES-1 cell compared with control samples(P<0.05).Conclusion The antitumor properties of PEDF is mainly related to the inhibition of tumor angiogenesis and direct effects on tumor cells, the effect of PEDF on HUVECs and SK-MES-1 cell maybe related to the effects of PEDF on downregulating expression of VEGF.

Oxytocin ( OT ) is a cyclic neuropeptide containing nine amino acids residues, in addition to the traditional roles of uterine contraction and lactation, it also plays important roles in the central nervous system and other peripheral organs, such as improving schizophrenia, autism - related psychiatric and psy-chological symptoms. Oxytocin exhibits its physiological func-tions by binding to its receptor (oxytocin receptor,OTR). Cur-
rently researchers are manipulating OT system by developing new OTR ligand ( agonists and antagonists ) , hoping to prevent and treat OTR related diseases. This paper reviews the latest devel-opment of OTR agonists, antagonists and its physiological roles in central nerve system and peripheral organs.