Objective To construct the RNAi expression vector of hTERT gene,and to investigate the inhibitory effects of the vector on the growth of mammary adenocarcinoma cell MCF-7 and the activity of telomerase.So as to present a new approach to the gene therapy for breast cancer which aim at inhibiting the telomerase activity.Methods An interference target sequence TGTTCAGCGTGCTCA ACTA which aimed at hTERT gene was designed and the siRNA expression plasmid pGenesil-hTERT was constructed by the means of recombination.Meanwhile a negative control recombinant-pGenesil-HK that did not aim at any gene was constructed by the same method.The recombinants expression vectors pGenesil-hTERT and pGenesil-HK were identified by electrophoresis after digestion and DNA sequencing.The two recombinants were transfected into the MCF-7 cells by lipofectamineTM 2000.The telomerase activity was tested by telomerase repeat sequence amplification and polyacrylamide gel electrophoresis.Flow cytometry was used to detect the cell apoptosis.Results The results of analysis and the sequencing identification confirmed that the target sequence was inserted into the predicted site precisely and the recombining plasmids were successfully constructed.The PAGE results showed that the mark traps of telomerase were obviously decreased and the telomerase activity of pGenesil-hTERT was obviously inhibited.The apoptotic rate of the pGenesil-hTERT group was significantly higher than that of the pGenesil-HK group(P

Objective To explore the expressions and.significancse of interleukin (IL)-10 and IL-18 in serum and tissues of patients with cervical intraepithelial neoplasias (CIN) and cervical cancer,and to analyze their relationships with clinical pathological characteristics and prognosis.Methods One hundred and eight patients' tissues and clinical blood samples (68 cases of CIN and 40 cases of squamous cell carcinoma) were enrolled in this study,including 25 with low-grade cervical intraepithelial neoplasias (CIN Ⅰ),43 with high-grade cervical intraepithelial neoplasias (CIN Ⅱ-Ⅲ),and 40 with invasive cervical squamous cell carcinoma (SCC);meanwhile,20 cases normal cervical tissues were enrolled.The expressions of IL-10 and IL-18 in cervical tissues and serum of the studied patients were measured by enzyme-linked immunosorbent assay and polymerase chain reaction.Meanwhile,second generation hybrid capture was used to detect human papilloma virus(HPV) DNA in cervical smears obtained from the studied patients with the different lesions.The relationships among IL-10 and IL-18 with HPV DNA and clinical prognosis were analyzed.Results The expressions of IL-10 and IL-18 in normal control serum were (212.75 ± 62.09),(187.84 ± 81.03)pg/ml respectively.The expressions of IL-10 in CIN Ⅰ,CIN Ⅱ-Ⅲ and SCC serums were (324.71 ±75.87),(397.43 ±68.56),(482.77 ± 104.05) pg/ml,with statistically significant difference (F =17.657,P =0.001).The expressions of IL-18 in CIN Ⅰ,CIN Ⅱ-Ⅲ and SCC serums were (305.53 ± 64.08),(392.74 ± 87.38),(499.86 ± 92.04) pg/ml,with statistically significant difference (F =13.309,P =0.003).The relative expressions of IL-10 in normal control,CIN Ⅰ,CIN Ⅱ-Ⅲ and SCC tissues were 0.99 ±0.01,3.24 ±0.68,7.32 ±0.99,13.24 ± 1.03,with statistically significant difference (F =21.694,P =0.000).The relative expressions of IL-18 in normal control,CIN Ⅰ,CIN Ⅱ-Ⅲ and SCC tissues were 0.98 ± 0.01,2.02 ± 0.84,7.01 ± 1.59,14.38 ± 2.10,with statistically significant difference (F =19.912,P =0.001).The expressions of IL-10 and IL-18 were positively associated with HPV infection (r =0.696,P =0.001;r =0.852,P =0.001).The median survival time of patients with high expressions of IL-10 was 9.74 months,which obviously shorter than those patients with low expressions of IL-10 (24.47 months),with statistically significant difference (x2 =21.363,P =0.001).The median survival time of patients with high expressions of IL-18 was 12.32 months,which obviously shorter than those patients with low expressions of IL-18 (22.88 months),with statistically significant difference (x2 =7.457,P =0.006).Conclusion High expressions of IL-10 and IL-18 are observed in patients with CIN and SCC,which can be used as biomarkers for predicting early cervical lesions.

Objective To evaluate the clinical effect of the treatment for calcaneal fractures by locking compression plate.Methods From January 2008 to December 2011,30 patients with 40 calcaneal fractures were operated with locking compression plate.There were 25 males (33 feet) and 5 females (7 feet) with an average age of 44.8 years (range,22-60 years).Fifteen feet were on the left side,and 25 feet were on the right side.All patients had closed fractures,with complicated spinal injury in 2 patients.According to Sanders classification,20 feet were type Ⅱ[fractures,15 feet were type Ⅲ,and 5 feet were type Ⅳ.The lateral approach was adopted for all the patients in the treatment in which locking compression plate fixation was used but no external fixation.Early and suitable rehabilitation was carried out postoperatively.The patients could bear part of the loading six weeks after the operation and all the loading twelve weeks after the operation.The results were validated using the Anerican Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot scale.Results The patients were followed up for 8-20 months (average,12 months).Bone union occurred in all cases with roentgenographic evidence during 8-12 weeks postoperation (average,10 weeks).None of the patients had such complications as nerve injury or osteomyelitis.Incision dehiscence occurred in one case in which a little part of titanium plate and screw was exposed,but the incision was healed by dressing 1 month later.Two patients had subtalar arthritis and suffered from the pain while walking.Of the two patients,the pain was relieved for one after the operation of subtalar joint fusion,and the other was lost to the follow-up after he or she refused further treatment for economic reasons.According to AOFAS foot score standard,the effects of 20 cases were excellent,17 were good,and 3 were fair.The excellent and good rate was 92.5％.Conclusion The application of locking compression plate is an effective and satisfied treatment of calcaneal fractures which is beneficial for healing and functional exercise.Moreover,the rate of soft tissue complication after operation is low.

The adhesion of leukocytes to vascular endothelium is crucial for the generation of inflammatory responses. The selectins and ?2-integrin (Mac-1) play a major role in the process. Recently, it was reported that RO-heparin can inhibit selectin-mediated leukocyte adhesion. The effect of RO-heparin on the Mac-1-mediated neutrophils adhesion were further tested. The results showed that RO-heparin could effectively inhibit neutrophils binding to ICAM-1, adhering to COS-7 cells expressing human ICAM-1, and adhering to human umbilical vein endothelial cells (HUVECs) under static and flow conditions. The findings suggest that the effect of RO-heparin on leukocyte adhesion is mainly due to its inhibition on the interaction between selectins or Mac-1 and their ligands and that RO-heparin might be useful in preventing inflammation diseases.

Objective To report 1 case with renal immunoglobulin light and heavy chain deposited disease (IgG-κ light chain and γ1 heavy chain).Methods The clinical manifestations,serum immunofixation electrophoresis,light and heavy chain abnormalities of blood and urine,bone marrow biopsy and renal biopsy data laboratory data were recorded and analyzed.Results A 63-year-old woman presented with massive proteinuria,microscopic hematuria,hypertension,anemia and serum IgG-κ light chain.Bone marrow aspirate revealed 4％ plasma cells.Renal biopsy revealed nodular glomerulopathy with congo red staining-negative.Immunofluorescence showed that κ light chain and IgG1 (γ1 heavy chain) were deposited along the glomerular basement membranes (GBM) and tubular basement membranes (TBM).Electron-microscopy revealed electrondense deposits that delineate the outer aspect of TBM and endothelial aspect of GBM.Conclusion The diagnosis of renal immunoglobulin light and heavy chain deposited disease (IgG-κ light chain and γ1 heavy chain) should be addressed combine with clinical and is pathology,especially with immune pathological examination.

Objective To explore the pancreatic kininogenase enteric-coated tablets in the treatment of diabetic retinopathy (DR) patients and its effect on the optic disc and macula retinal hemodynamics. Methods 86 cases (140 eyes) of DR patients were randomly divided into pancreatic kininogenase group and normal group with 43 cases in each group, two groups were treated with basic therapy, pancreatic kininogenase group were combined with pancreatic kininogenase treatment. The best corrected visual acuity and the clinical effect of optic disc and macula retinal hemodynamic changes were compared between two groups. Results After treatment, the best corrected visual acuity (BCVA) in pancreatic kininogenase group was greater than the normal group (P<0.05), the retinal neovascularization and fluorescein leakage area in pancreatic kininogenase group was less than the normal group (P<0.05). The disc vascular and macular retinal blood flow volume (VOL), blood flow velocity (FLW)values in pancreatic kininogenase group were larger than those of normal group, and the clinical curative effect of pancreatic kininogenase group was better than that of normal group (P<0.05). Conclusion Pancreatic kininogenase enteric-coated tablets in the treatment of DR patients can improve the optic disc and macular retinal hemodynamic parameters, improve visual acuity and reduce the retinal neovascularization and fluorescein leakage area, so as to improve the clinical treatment effect.

In recent years,many new microtubule-associated proteins are found or studied extensively in the treatment of breast cancer.End-binding protein 1 (EB1)has the promotional effects on cell proliferation and enhances the carcinogenesis of breast cancer,and also can affect the chemosensitivity of the treatment. MAP7 domain-containing protein 3 (Mdp3)expresses in breast epithelial cells and plays an important role on the growth and metastasis.Nucleolar and spindle-associated protein (NuSAP)gene is up-regulated in breast cancer and is considered as a biomarker for breast cancer.Tau expression level has a strong correlation with estrogen receptor (ER),progesterone receptor (PR)and human epidermal growth factor receptor-2 (HER-2), and it remains controversial in the results of the effect of paclitaxel chemotherapy and hormone treatment.

The adhesion of leukocytes to vascular endothelium is crucial for the generation of inflammatory responses. The selectinsand β2-integrin (Mac-1) play a major role in the process. Recently, it was reported that RO-heparin can inhibit selectin-mediated leukocyte adhesion. The effect of RO-heparin on the Mac-1-mediated neutrophils adhesion were further tested. The results showed that RO-heparin could effectively inhibit neutrophils binding to ICAM-1, adhering to COS-7 cells expressing human ICAM-1, and adheringto human umbilical vein endothelial cells (HUVECs) under static and flow conditions. The findings suggest that the effect of RO-heparin on leukocyte adhesion is mainly due to its inhibition on the interaction between selectins or Mac-1 and their ligands and that RO-heparin might be useful in preventing inflammation diseases.

Objective To prepare insulin thiolated hyaluronic acid nanoparticles (Ins-HA-Cys-NPs)and study its physicochemical properties. Methods The Ins-HA-Cys-NPs was prepared by ultrasonic emulsifying method,and the properties of nanoparticles including morphology,mean diameter,Zeta potential,entrapment efficiency and drug loading efficiency were studied,as well as the cryoprotectant selection.Results The prepared nanoparticles was round in appearance and the mean diameter was(178.5 ±0.8)nm,the polydispersity index was (0.214 ±0.013)and the Zeta potential was -(38.47 ±0.46 )mV,while the entrapment efficiency was (48.85 ±0.66 )%,drug loading efficiency was (4.79 ±0.13 )%;10%mannitol as cryoprotectant provided uniform and well dispersed suspension of nanoparticles with blue opalescence after redispersion.Conclusion The thiolated hyaluronic acid nanoparticles may be used as the carrier for oral drug delivery system of insulin,and it provides a basis for studies on rats in vivo.

Objective To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy(X-ALD).Methods The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells.Maternal contamination was evaluated by paternity test.PCRRFLP,sequencing and denaturing high performance liquid chromatography(DHPLC)were used to detect the ABCD1 gene of fetal genome.Results In the pedigree 1,the PCR product(799 bp)of the fetus 1 and her father(normal control)could be digested with BcnI. No P560L mutation,which was present in the index patient,was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing.In the pedigree 2,the PCR product(232 bp)of the fetus 2 and her father could not be digested with MaeI and no Q177X mutation,which was present in the propositus,was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing.In the pedigree 3,the PCR product(271 bp)was digested with AciI.the pattern of the fetus 3 and the propositus being the same,and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing.In the pedigree 4,the PCR product(269 bp)was analyzed with the DHPLC,and the pattern of elution peaks of the fetus 4 and her father was similar,but different from that of the propositus.No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing.Judging from the sex of the fetuses,fetuses 1 and 2 were normal homozygotes,fetus 3 was an ALD hemizygote,and fetus 4 was a normal hemizygote.Conclusion A new protocol for X-ALD prenatal molecular diagnosis is proposed,which would ensure the accuracy of prenatal diagnosis.