Objective To study the roles of regulated upon activation normal T cell expressed and secreted(RANTES) and monocyte cbemoattractant protein-1 (MCP-1) in human immunodeficien- cy virus-1(HIV 1) infected patients.Methods RANTES and MCP-1 in HIV-1 infected patients, including treated and untreated groups,and healthy control group were determined by enzyme-linked immunosorbent assay(ELISA).The recombinant plasmids,hMCP-pcDNA3.1,bRANTES- peDNA3.1 and hMCP/bRANTES-pcDNA3.1,were constructed and transfected into CHO cells to overexpress the corresponding recombinant proteins,whose chemoattract function was then studied. Results The level of RANTES was (164.3?21.3) pg/mL in healthy control group,(1 224.1?62.0) pg/mL in untreated group and (475.3?36.2) pg/mL in treated group.The level of MCP-1 was (90.6?28.5) pg/mL in healthy control group,(335.0?30.3) pg/mL in untreated group and (807.2?62.6) pg/mL in treated group.In HIV-1 infected patients,the levels of RANTES and MCP-1 were significantly increased.After highly active antiretroviral therapy (HAART),the level of RANTES declined,but MCP-1 increased further.Western blot assay revealed that the three recombinant proteins could be recognized by monoclonal antibodies respectively.All of them could chemoattract human peripheral blood mononuclear cells(PBMCs).And the chemoattractant potency of MCP/RANTES fusion protein was stronger.When the recombinant proteins were used with con- centrations as 50,200,400 and 800 pg/mL,respectively,the number of PBMCs chemoattracted by MCP/RANTES fusion protein was 52?10~4/mL,102?10~4/mL,132?10~4/mL and 184?10~4/mL; the number of PBMCs chemoattracted by RANTES was 27?10~4/mL,51?10~4/mL,65?10~4/mL and 96?10~4/mL;the number of PBMCs chemoattracted by MCP-1 was 18?10~4/mL,44?10~4/mL, 54?10~4/mL and 74?10~4/mL.Conclusion RANTES and MCP-1 may both be involved in the HIV infection process and host immunological reaction against HIV.

Objective To investigate the effect of globular adiponectin on the high expression of monocyte chemotactic protein-1 (MCP-1) induced by high glucose in rat renal tubular epithelial cells (NRK52E),and its relationship with adiponectin receptors and p38MAPK.Methods NRK52E cells were cultured in vitro and divided into six groups:normal glucose group (NG,5.6 mmol/L glucose),high glucose group(HG,25 mmol/L glucose),gAd groupl (HG+gAd 2 mg/L),gAd group2 (HG+gAd 5 mg/L),gAd group3 (HG+gAd 10 mg/L),p38MAPK antagonist group:(SB,HG+SB203580 10 μmol/L).The protein expression of phosphorylated p38MAPK (p-p38MAPK),total p38MAPK (t-p38MAPK),MCP-1 and AdipoR1/AdipoR2 were examined by western blotting.The mRNA expression of MCP-1 and AdipoR1/AdipoR2 were detected by RT-PCR and real-time PCR respectively.Results Compared with NG group,the mRNA and protein expression of MCP-1 increased significantly in HG group (all P＜ 0.05).The phosphorylation of p38MAPK increased (P＜ 0.05) with no change in t-p38MAPK protein.The addition of gAd or SB203580 inhibited the unregulation of MCP-1 and p-p38MAPK induced by HG.Two kinds of adipoR,adipoR1 and adipoR2,were all detectable in NG group,and mRNA and protein expression of adipoR1 was higher than that of adipoR2 (P＜ 0.01).Compared with NG group,the expression of adipoR decreased in HG group,but the difference had no statistical significance(P ＞ 0.05).Compared to HG group,the mRNA and protein expression of adipoR1 increased in gAd groups (all P ＜ 0.01).Conclusion The gAd can dose-dependently attenuate the overexpression of MCP-1 induced by high glucose,and this protective effect may be mediated by adipoR1 and p38MAPK.

Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.
Gene Targeting ; Green Fluorescent Proteins ; genetics ; MicroRNAs ; genetics ; Oryza ; genetics ; Plasmids ; Protoplasts ; metabolism ; RNA, Plant ; genetics ; Transfection

Objective To explore the characteristics of infection after cardiac-lung transplantation.Methods March 23th 2006,one patient received orthotopic heart-lung transplantation,the clinical data were observed and analyzed.Results Depend on etiology,antibioti and antifungul were selected,the patient recoved.Conclusion It is very important to early etiology diagnosis and reasonable selection of antibiotic.

AIM: To determine whether the viartrils could provide a beneficial effect on the prevention of early/middle stage osteoarthritis(OA) and affect the proliferation of chondrocytes. METHODS: An OA model was produced with severing the anterior, posterior cruciate ligaments of the knee in 24 adult New Zealand rabbits. The animals were then randomly divided into viartrils group and control group. After surgical operation, viartrils (mainly contains glucosamine sulphate) 2 pills per day were administered to the animals in viartrils group. The animals were sacrificed and specimens were taken from the weight-bearing portion of the femoral condylar seven weeks after operation. Each case was evaluated according to a modified histological-histochemical grading system(HHGS) using HE and safranin O/fast green staining slides, and immunohistochemical method was used to detect the proliferation of chondrocytes in articular cartilage. RESULTS: The method of severing the anerior, posterior cruciate ligaments of the knee could successfully induce the early/middle stage model of OA. The pathological remark in control group was significantly higher than that in the viartrils group (P

ObjectiveTo investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues.Methods Genomic DNA,extracted from K-ras mutant cell lines SW480 (homozygous,c.35G ＞ T), DLD-1 (heterozygous,c.38G ＞ A) and wild-type HT-29,was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2％,3％,5％,10％,20％,30％ and 50％,the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived fromclinicalformalin-fixed andparaffinembedded(FFPE)tissues.ResultsCancer cell lines with known K-ras mutations ( SW480 and DLD-1 ) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5％ and 10％ content,the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3％ (4/12) and 58.3％ (7/12) respectively,whereas the mutation rates detected by pyrosequencingbased assay were 91.7％ (11/12) and 100％(12/12) respectively,there were significant differences between those two sequencing methodology ( P ＜0.05).Furthermore,we found 10 patients with K-ras exon 2 point mutations at codons 12 and 13 by pyrosequencing-based assay from 30 colorectal cancer FFPE tissues,the point mutation rate was 33.3％ (10/30) and all of the mutations determined were heterozygous.The codon 12 was most frequently affected [30％ (9/30)].Mutations with the highest frequency were G ＞ A transitions [ 50％ ( 5/10 ) ],followed by G ＞ T transversions [ 30％ ( 3/10 ) ].Conclusion The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens,thereby allowing the selection of the cancer treatment in clinical individualized practice.

The clinical data and routine biochemical parameters of 64 maintenance hemodialysis (MHD)patients and 20 controls were analyzed in the study.Serum cystatin C levels were detected by latex particle enhalice inununo-turbidimetry:and the cardial structure and function were assessed by echocardiography.As MHD time extended,the levels of semm cystatin C increased gradually,accompanied with high incidence of left ventricular hypertrophy(LVH).The LVH-positive patients had higher systolic blood pressure and left veHtricular mass index,and had higher serum cystatin C than tllose in LVH-negative patients.The serum cystatin C levels were positively correlated with left yentricular mass index(r=0.633,P<0.01)and systolic blood pressure(r:0.397,P<0.01).The results suggest that serum cystatin levels may be an influencing factor for long-term cardiacvascular complication in MHD patients.

Objective To study the effect of mechanical stretch on the expression of human beta-defensin-3 (HBD-3) in alveolar epithelial cells(A549 cells) elicited by interferon-gamma(IFN-γ) and to investigate the role of HBD-3 in the pathogenesis of ventilator-associated pneumonia (VAP) . Method A549 cells cultured in vitro were treated with mechanical stretch (group S), 10 ng/ml IFN-γ (group I) ,and 10 ng/ml IFN-γ with mechanical stretch (group IS), respectively. Cells without treatment served as controls (group C). Cells were stretched by 20% amplitude of stretch at 30 cycles/mm by Flexercell-4000[TM]Unit for 2 h, 4 h, and 6 hours. The HBD-3 mRNA expression was determined by real-time RT-PCR after treatment. After 6 hours, treatment, cells were cultured for 24 hours and the expression of HBD-3 was examined by laser scanning confocal microscope. The experimental data were statistically analyzed by using one-way ANOVA analysis and q-test. Results The expression of HBD-3 mRNA in A549 cells could not significantly be changed by mechanical stretch alone. Compared with group C,the HBD-3 mRNA expression after treatment with 10 ng/ml IFN-γ for 2 hours,4 hours and 6 hours increased significantly by (2.63 C,the HBD-3 mRNA expression after treatment with 10 ng/ml IFN-γ and mechanical stretch for 2 hours,4 hours and 6 hours increased by (1.54 were significantly lower than those in group I (P < 0.01). The HBD-3 expression in group IS after mechanical stretch for 6 significantly different from than in group C. Conclusions Mechanical stretch can significantly suppress the up-regulation of HBD-3 in alveolar epithelial cells elicited by IFN-γ, and this may be one of the explaina-tions that patients under mechanical ventilatiori(MV) have a higher risk of VAP.

Objective To study the sonographic features of papillary thyroid carcinoma (PTC) associated with cervical lymph nodes metastasis for early diagnosis and prediction of the invaded cervical lymph nodes. Methods The sonographic features of 170 patients with pathologically confirmed PTC in First Afifliated Hospital of Harbin Medical University between 2011 and 2013 were retrospectively reviewed. There were 59 cases with neck lymph nodes metastases and 111 cases without neck lymph nodes metastases. Receiver operating characteristic (ROC) curve was aaplied to analyze the cut-off values of resistance index (RI) and peak systolic velocity (PSV) for judging the presence or absence of cercical lymph node metastasis. The Chi-square test and rank sum test were used to compare the different sonographic features between each group. The Logistic regression analysis was used to obtain the relevant factors of PTCs with cervical lymph node metastasis. Results ROC curve analysis showed that the cut-off values of RI and PSV were 0.735,13.95 cm/s. The primary tumor diameter, the existence of halo, the involvement of thyroid upper pole, the microcalciifcation, the blood suply classiifcation and the RI, PSV were statistically signiifcantly different between PTCs with and without cervical lymph node metastasis, whereas no statistical signiifcance was detected between the primary tumor echo pattern, boundary and the longitudinal/transveral ratio between the metastatic and nonmetastatic group. Logistic regression analysis showed that the PTC primary tumor diameter and PSV were independent factors coorelated with cervical lymph node metastasis. Conclusion Some sonographic features of PTC are closely correlated with lymph nodes metastasis, which are valuable in predicting the cervical lymph nodes metastasis in patients with PTC pre-operatively.

Human kerotinocyte growth factor (hKGF) gene amplified by PCR was inserted into the shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hKGF, which was linearized with Pme I and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 to obtain the recombinant adenoviral plasmid pAdEasy-hKGF. The recombinant adenoviral plasmid was then transfected into HEK-293 cell lines via Lipofectamine 2000 to package and amplify the recombinant adenovirus containing hKGF gene detected by PCR. The recombinant adenovirus produced could effectively infect HaCat cells. The result of Western blotting showed that HaCat cells infected with the recombinant adenovirus expressed and secreted hKGF protein.