Objective To investigate the susceptibilities of HCMV clinical strains isolated to ganciclovir from the patients after hematopoietic stem cell transplantation.Methods Eight HCMV clinical isolates were isolated from the blood or the urine of hematopoietic stem cell transplantation recipients who had been treated with GCV.Tissue cell infection median dose(TCID50) were calculated by Reed-Muench method.Drug susceptibility was determined by MTT assay.Results TCID50 values of eight HCMV clinical strains were 10-4.12/0.1ml,10-4.29/0.1ml,10-4.3/0.1ml,10-4.4/0.1ml 10-4.42/0.1ml,10-4.5/0.1ml,10-4.52/0.1ml and 10-4.62/0.1ml respectively.50％ inhibitory concentration(IC50) to GCV of eight HCMV clinical strains were 0.638,1.438,0.965,0.698,0.482,1.167,1.519,1.511 mg/L respectively.Conclusion Our results suggest that resistant HCMV strains are not prevalent in Guangzhou.Continuous monitoring of HCMV is needed to understand the antiviral resistance status of the virus in patients after hematopoietic stem cell transplantation and guide its clinical management.

Objective To explore the expression of cullin 4A (CUL4A) in cholangiocarcinoma tissues and its clinical significance.Methods Primary fresh cholangiocarcinoma tissues (n =35) and normal bile duct tissues (n =15) from patients who underwent curative surgery in Binzhou People's Hospital of Shandong Province from February 2011 to December 2013 were collected.The expressions of CUL4A mRNA were detected by quantitative real-time PCR (qRT-PCR).Then,cholangiocarcinoma tissues (n =72) and normal bile duct tissues (n =36) from patients who underwent curative surgery in Binzhou People's Hospital of Shandong Province from January 2008 to January 2014 were collected.The expressions of CUL4A protein were tested by immunohistochemistry.The relationships between the expression of CUL4A and the patients' clinicopathologic features and prognosis were analyzed.Results The expression of CUL4A mRNA in cholangiocarcinoma tissues was obviously higher than that in normal bile duct tissues (3.876 ±0.975 vs.1.216 ±0.265),and the difference was statistically significant (t =12.23,P ＜ 0.001).The positive rates of CUL4A protein in cholangiocarcinoma and normal bile duct tissues were 70.8％ (51/72) and 2.8％ (1/36) respectively,and the difference was statistically significant (x2 =44.524,P ＜ 0.001).The expression of CUL4A protein in cholangiocarcinoma was related to the differentiated degree (x2 =4.341,P =0.037),neural invasion (x2 =8.326,P =0.004),TNM stage (x2 =7.745,P =0.005),lymph node metastasis (x2 =3.869,P =0.049) and vascular invasion (x2 =5.555,P =0.018).Survival analysis results showed that the median survival time of CUL4A positive patients was significantly shorter than that of CUL4A negative patients (32.40 months vs.51.30 months),and the difference was statistically significant (x2 =6.561,P =0.011).Conclusion The expression of CUL4A in cholangiocarcinoma tissues is higher than that in normal bile tissues.CUL4A plays an important role in the process of tumor invasion and metastasis and indicates poor prognosis,which may become a potential target for the diagnosis and therapy of the cholangiocarcinoma.

OBJECTIVETo monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.

METHODSHCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.

RESULTSHCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.

CONCLUSIONSPhenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; isolation & purification ; Drug Resistance, Viral ; genetics ; Ganciclovir ; pharmacology ; Genes, Viral ; Genotype ; Hematopoietic Stem Cell Transplantation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics

Objective To improve the performance of artificial joint at biological fixed interface by frettingmechanical stimulation. Methods Taking the bonding sample of active rat bone tissues and inactive titanium bead coating as the research object, by using the self-developed fretting mechanical stimulation test device, the effects of mechanical stimulation with different tangential fretting amplitudes on growth promotion and micro-injury of bone tissues at fixed interface were explored, and the mechanism of growth promotion and micro-injury of bonetissues on the surface of titanium bead coating under fretting stimulation was revealed. Results The mechanical stimulation group with 40 μm fretting amplitude showed the maximum interface bonding force between bone tissues and titanium bead coating. The tangential fraction force-displacement curves changed from elliptic shape to linear shape, and the bonding interface was in adhesion area, which was difficult to become loose. The bone tissues had the most complete structure, and the number of tissue cells adhering and growing on the surface of titanium bead coating was the largest, as well as the distribution range was the widest. With the increase of fretting amplitude, the promoting effect of fretting stimulation on bone growth weakened, the bonding strength between bone tissues and titanium bead coating interface gradually decreased, the internal cavity area increased, the number and proliferation activity of tissue cells decreased to varying degrees as well. Conclusions Fretting stimulation with different amplitudes can promote the growth of bone tissue and cause micro-injury at the same time, and the optimal fretting amplitude is 40 μm.

Objective : To investigate the effect of lentivirus mediated silencing of NIPBL gene on osteogenic differentiation of mouse bone marrow mesenchymal stem cells ( BMSCs) . Methods : The third generation C57 Mouse Bone marrow mesenchymal stem cells were divided into experimental group , negative control group and blank control group. The lentiviral vector was transfected into mouse bone marrow mesenchymal stem cells , the transfection results were observed by inverted fluorescence microscope , and the expression of NIPBL gene was detected by real- time PCR. The cells of each group were cultured by osteogenic induction. The alkaline phosphatase activity was 2 and RUNX⁃2. Results : The expression of NIPBL mRNA decreased in the experimental group (P < 0. 05) . The activity of alkaline phosphatase in experimental group was lower than that in negative control group and blank control group (P < 0. 05) . The gene transcription and protein expression levels of OCN, BMP⁃2 and Runx⁃2 in experimental group were lower than those in negative control group and blank control group ( P < 0. 05 ) . Alizarin red staining results showed that the negative control group and blank control group had more red calcium nodules than the experimental group. Conclusion Lentivirus mediated silencing of NIPBL gene reduces the proliferation of mouse bone marrow mesenchymal stem cells , inhibits the expression of osteogenic differentiation related genes , and reduces the osteogenic differentiation ability.