Idiopathic Refractory Nephrotic Syndrome (IRNS) has been an intractable problem in clinical treatment .Anti CD20 monoclonal antibody is a new type of immunosuppressive agent , which can induce the lysis and apoptosis of B cells .Significant-ly, it improves the prognosis of IRNS patients .In recent years, a number of case studies and clinical trials have been conducted on the effectiveness of anti CD20 monoclonal antibody in the treatment of children with IRNS .In this paper, the mechanism, clinical applica-tion, adverse effects and problems in the study of anti CD 20 monoclonal antibody in IRNS will be reviewed .

Objective:To investigate the protective effect and potential mechanism of cordycepin on renal proximal tubular cells injury induced by lipopolysaccharide (LPS).Methods:Renal proximal tubular cells NRK-52E were incubated on a cell culture plated at a density of 1×10 5/mL for experiment, then divided into control group (Ctrl group), LPS group (cells were stimulated with 1 mg/L LPS), 10 μmol/L or 20 μmol/L cordycep in intervention groups (LPS+C 10 group and LPS+C 20 group). Cell viability was measured using cell counting kit-8 (CCK-8) reagent. The level of intracellular reactive oxygen species (ROS) was detected by 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining. The protein expressions of inflammatory factors intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), interleukin-1β (IL-1β), and nuclear factor-κB (NF-κB) were detected by Western blotting. Results:Compared with the Ctrl group, LPS significantly inhibited NRK-52E cell viability, increased intracellular ROS, and up-regulated the expressions of ICAM-1, VCAM-1, IL-1β and NF-κB. Compared with LPS group, after treated with 10 μmol/L or 20 μmol/L cordycepin, NRK-52E cell viability was significantly increased (Ctrl group as 1: 0.717±0.017, 0.916±0.036 vs. 0.554±0.046) and intracellular ROS level was significantly decreased (Ctrl group as 1: 1.527±0.165, 1.098±0.168 vs. 2.543±0.127), meanwhile the expressions of ICAM-1, VCAM-1, IL-1β and NF-κB were significantly down-regulated [Ctrl group as 1, ICAM-1/GAPDH: 2.364±0.097, 1.561±0.074 vs. 3.101±0.121; VCAM-1/GAPDH: 2.866±0.135, 1.920±0.098 vs. 4.170±0.119; IL-1β/GAPDH: 2.358±0.107, 1.563±0.179 vs. 3.301±0.210; phosphorylation NF-κB p65 (NF-κB p-p65)/GAPDH: 2.559±0.166, 1.596±0.148 vs. 3.183±0.098], the differences were statistically significant (all P < 0.05). Compared with the LPS+C 10 group, the cell activity of LPS+C 20 group was more significant (0.916±0.036 vs. 0.717±0.017, P < 0.01), and the expressions of ICAM-1, VCAM-1, IL-1β, NF-κB were down-regulated more significantly (ICAM-1/GAPDH: 1.561±0.074 vs. 2.364±0.097, VCAM-1/GAPDH: 1.920±0.098 vs. 2.866±0.135, IL-1β/GAPDH: 1.563±0.179 vs. 2.358±0.107, NF-κB p-p65/GAPDH: 1.596±0.148 vs. 2.559±0.166, all P < 0.05).Conclusion:Cordycepin could significantly increase the survival rate of NRK-52E cells, reduce intracellular ROS level, and inhibit inflammation, and the anti-inflammation effect can be related with NF-κB pathway.

Objective To evaluate puncture technique in thoracic endovascular aortic repair with abdominal aortic aneurysm and assess the feasibility and safety of using a pre-close technique for puncture and closure of femoral access sites. Methods From May 2010 to August 2013, the pre-close technique which involved two 6 F per-close ProGlide devices deployed in the femoral artery before upsizing to a 18-25 F sheath and one or two deployed before upsizing to a 14-16 F sheath were applied to 42 patients with abdominal aortic aneurysm (group A). Forty-seven patients using surgical femoral cutdown from December 2006 to April 2010 were enrolled into group B. The rate of technical success, time from procedure to the aortic delivery, operation time, low limb braking time, local complication, time from procedure to discharge, local vascular diameter after 3 months was evaluated and compared between two groups. Results There was no significant difference in endograft external diameter between two groups ( P>0.05). The rate of technical success was 97.62%(41/42) in group A and 95.74%(45/47) in group B, and there had no significant difference (P>0.05). Time from procedure to the aortic delivery, operation time and time from procedure to discharge in group A were significantly shorter than those in group B: (21.79 ± 5.79) min vs. (41.37 ± 11.79) min, (127.66±37.83) min vs. (157.84±42.71) min, (6.59±1.89) d vs. (9.14±2.57) d, P<0.05. The incidence rate of local complications, low limb braking time, and local vascular diameter after 3 months had no significant difference between two groups:7.14%(3/42) vs. 8.51%(4/47), (8.51± 1.83) h vs. (8.38±1.79) h, (1.05 ±0.36) mm vs. (0.98 ±0.31) mm, P>0.05. Conclusion The puncture technique with per-close ProGlide is safe and effective in percutaneous endovascular aortic repair which can be adopted as an alternative technique of surgical femoral cutdown approach in patients with abdominal aortic aneurysm.

Objective To investigate the clinical significance of thyroglobulin antibody (ATG) and thyroid peroxidase antibody (ATPO) in patients with vitiligo. Methods Venous blood samples were obtained from 87 patients with vitiligo and 90 age- and sex-matched normal human controls. Chemiluminescence was applied to measure the serum levels of ATG, ATPO, free triiodothyronine, free tetraiodothyronine and thyroid stimulating hormone (TSH). Results There was a significant increase in the positivity rates of ATG (23.0% vs 6.7%, P < 0.01) and ATPO (24.1% vs 7.8%, P < 0.01) as well as the serum level of TSH (3.4 ± 2.4 vs 2.4 ± 1.2 pmol/L, P < 0.05) in the patients with vitiligo compared with the normal human controls. It is worth mentioning that all patients positive for ATG or ATPO were diagnosed with vitiligo vulgaris. The positivity rates of ATG and ATPO in patients with vitiligo aged from 11 to 20 years and 21 to 40 years were significantly higher than those in age-matched normal controls (all P < 0.05). Also, female patients had a higher positivity rate of ATG and ATPO than female controls did (34.1% vs 8.5%, χ2 = 8.90, P < 0.01; 34.1% vs 10.6%,χ2 = 7.29, P < 0.05). The highest positivity rates of both ATG and ATPO were 53.3%, which were observed in vitiligo patients aged from 11 to 20 years, followed by patients from 21 to 40 years (ATG 34.5%, ATPO 34.5%). In patients with vitiligo positive for both ATG and ATPO, the occurrence of autoimmune thyroid disease was 70% (14/20), significantly higher than that in ATG- and ATPO- positive healthy controls (16.7%, χ2 = 5.4, P < 0.05). Conclusions ATG and ATPO were observed in young female patients with vitiligo vulgaris, and they may be associated with the development of autoimmune thyroid diseases.

Objective To prepare monoclonal antibody(McAb) against human tissue kallikrein (HK) and develop an ELISA kit allows for the in vitro quantitative determination of human tissue kallikrein in urine. Methods To generate a monoclonal antibody specific for TK, the synthetic TK peptide consisting of 12 amine acids(12P), was fused to keyhole limpet hemocyanin(KLH) and used for immunization. Using hybridoma screening, monoclonal secreting cell lines were identified and used to generate ascites in BALB/c mouse. Antibody was purified by affinity column chromatography. 12% SDS-PAGE and Western blot were used to visualize the purified antibody. This kit employs indirect competitive ELISA technique and BiotinAvidin System. 12P was fused to bovine serum albumin(BSA) and has been pre-coated onto a microplate at first. Standards and samples were added to the appropriate microplate wells with a biotin-conjugated McAb croplate well. A TMB substrate solution is added to each well. The enzyme-substrate reaction is terminating by the addition of a sulphuric acid solution and the color change is measured spectrotometrically at a wavelength of 450 nm. The concentration of tissue kallikrein in the samples is then determined by comparing the O.D. of the samples to the standard curve. Results 8 hybridoma cell lines secreting mAbs special to HK,SDS-PAGE and Western blot demonstrated successful preparing and purification of McAb( 100% ). The linearity of this ELISA kit is demonstrated(r =0. 990). The range of detection of the assay is 0.008 μg/ml to 0. 5 μg/ml. The assay remained stable, with no change in the values measured, over five cycles of freezing and thawing. Conclusion 8 McAbs against HK have been prepared successfully and possess high titer and specificity. The development of an ELISA kit for detecting HK can meet the needs of detection of HK in urine samples.

Objective To explore the regulation mechanism of autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3), via c-Jun in methotrexate resistant human choriocarcinoma JEG-3 cell lines. Methods Human choriocarcinoma JEG-3 cell lines, and methotrexate resistant choriocarcinoma JEG-3 (JEG-3/MTXR) cell lines were used in our present study. Phosphorylation c-Jun (p-c-Jun) was evaluated after exposure to 0.02 ng/ml methotrexate for 72 hours in both cells by western blot. c-Jun gene was knockdown by small interference RNA (siRNA) in JEG-3/MTXR cells, and LC3 was evaluated by western blot and reverse transcription-PCR. The binding of LC3 promoter with c-Jun protein was detected via chromatin immunoprecipitation assay (ChIP) with or without 0.02 ng/ml methotrexate exposure. Results The results showed that p-c-Jun was up-regulated after methotrexate treatment for 72 hours (1.99±0.20, versus 0.20±0.06 at 0 hour;P<0.05) by western blot analysis in JEG-3/MTXR cell lines. Further investigation demonstrated that c-Jun-siRNA could inhibit the up-regulation of LC3 formation and after methotrexate exposure (LC3 mRNA:1.24±0.17 versus 3.03±0.43;LC3 protein:0.52±0.07 verus 1.20± 0.15; all P<0.05). The binding of LC3 promoter by c-Jun protein was up-regulated after methotrexate treatment by the method of ChIP in methotrexate resistant JEG-3/MTXR cells [(2.95 ± 0.35) times]. Conclusion Autophagy-related gene LC3 expression regulated by c-Jun protein may be involved in the effect mechanism of the development of methotrexate resistance in choriocarcinoma JEG-3 cells.

While the medicine pattern of biomedicine turn to biological-psychology-society, the medical trouble communication becomes more and more important in the medical service. Good medical trouble communication ability is the essential condition of doctor. As oral cavity clinicians, only by gasping the principle of communication can we appropriately utilize some skills of communication exchange,establish the good medical trouble relations with the patient and achieve the good treatment result finally.

Objective To obtain recombinant human Smith D1 (Sm D1) antigen and establish detecting assay.Methods Human Smith D1 antigen was synthesized by PCR using human Leukemic cDNA. The prokaryotic expression vector pGEX-ST-Sm D1 was constructed and transformed into E.coli.BL21 cell.Protein expressed under the induction of IPTG.We established DIGFA for detecting anti-Sm D1 antibodies with purified Sm D1 antigens.Results Sequence and restriction analysis revealed Sm D1 gene was cloned in frame into pGEX-5T,SDS-PAGE profile showed a clear protein band with a relative molecular weight of 39 000 and western blotting indicated that the expressed product specifically reacted to polyclonal anti-human Sm D1 genes.There was no significant difference between DIGFA and IB.The agreement between DIGFA and IB was 91.7% as calculated by Kappa statistical method.The sensitivity and specificity of DIGFA were 100% and 83.3% repectively.Conclusions Human Sm D1 gene is successfully cloned、 expressed and purification.The DIGFA,using purified Sm D1 antigens,is as good as IB,rather simpler, more rapid and reliable assay.

To clone, express and primarily use human autoantigen Sm D1 in methylotrophic yeast Pichia Pastoris. The gene Sm D1 was cloned by PCR.The PCR product was inserted into the vector pPIC9k. The recombinant plasmid pPIC9k- Sm D1 was transformed into yeast SMD1168 by electroporation. The positive clones were screened in MD plates. The high copy number transformants were rapidly selected by using G418 and were induced by methanol. Supernatants after induction were analyzed by SDS-PAGE and im-munodot. The PCR product was showed about 360 bp in size which was in accordance with predicted. The pPIC9k-Sm D1 showed the same seqencing result with GenBank’s report and restriction enzyme analysis confirmed our prediction. The pPIC9k-Sm D1 positive clone produced an about 16 kD protein which had natural immunogenicity of human autoantigen Sm D1 by SDS-PAGE and immunodot. The sensitivity and specificity of immunodot were 96% and 100%, respectively. The agreement between immunodot and im-munoblot was 98%. Successfully cloning and high-level expression of human autoantigen Sm D1 in methy-lotrophic yeast Pichia pastoris laid a foundation for further research work.

Objective To study the gene mutations and clinical features of mandibuloacral dysplasia with type A lipodystrophy (MADA) in a Chinese family. Methods The information of 5 family members including 2 siblings suspected atyp-ical progeria was assembled. Genomic DNA was extracted from peripheral blood of 5 family members, the 12 exons of LMNA gene were ampliifed by PCR and then the PCR products were directly sequenced and analyzed by using Blast software online. The SIFT and PolyPhen-2 software were used to predict the harmfulness of mutations. Results The 2 siblings were clinically diagnosed as MADA. Heterozygous c.1579C>T (p.Arg527Cys) and c.1583C>T (p.Thr528Met) mutations were detected in this family. The father carried c.1583C>T (p.Thr528Met) mutation, the mother carried c.1579C>T (p.Arg527Cys) mutation, and their normal daughter were all heterozygous carriers with c.1583C>T (p.Thr528Met) mutation. Compound heterozygous c.1579C>T (p.Arg527Cys) and c.1583C>T (p.Thr528Met) mutations in 2 siblings led to MADA. The MADA showed an autosomal re-cessive inheritance pattern in this family. Conclusions The 2 siblings with MADA in this family were caused by compound heterozygous mutations in LMNA gene.