BACKGROUND:Chondromodulin-Ⅰ is expressed mainly in the cartilage, but it is little expressed in mesenchymal stem cells. Combined with the previous study of our group, we concluded that chondromodulin-Ⅰmaybe play an important role in inducing mesenchymal stem cells into chondrocytes accurately.
OBJECTIVE:To construct an expression plasmid stably carrying chondromodulin-Ⅰ to up-regulate the expression of chondromodulin-Ⅰ in bone marrow mesenchymal stem cells.
METHODS:Specific primers were designed in rat cartilage for chondromodulin-Ⅰ gene, then the pcDNA3.1 (+) plasmid expression vector was digested by enzyme and directional connected gene to construct pcDNA3.1(+)/ChM-Ⅰ expression vector. Bone marrow mesenchymal stem cells were obtained from rats using the method of density gradient centrifugation combined with adherent culture. Recombinant plasmid pcDNA3.1(+)/ChM-Ⅰ was transfected into rat bone marrow mesenchymal stem cells with liposome method, and G418 selection was used for stable screen of transfected cells. Reverse transcription-PCR and western blot were used to detect chondromodulin-Ⅰ expression in celllines.
RESULTS AND CONCLUSION:The positive clones were digested by enzyme and were identified and sequenced. The results showed that the reality length and sequence of chondromodulin-Ⅰ gene were consistent with the theoretical values, and reading frame was correct. Reverse transcription-PCR and western blot results showed that the expressions of chondromodulin-ⅠmRNA and protein were markedly up-regulated in bone marrow mesenchymal stem cells. Recombinant plasmid pcDNA3.1(+)/ChM-I was successful y constructed, and transfected into rat bone marrow mesenchymal stem cells. After G418 selection, expression of chondromodulin-Ⅰ was up-regulated stably in rat bone marrow mesenchymal stem cells.

Objective: To study the association between the AKAP12 promoter methylation and recurrence of hepatocellular carcinoma. Methods: A total of 142 primary liver cancer patients underwent surgery in department of Hepatobiliary surgery in Peking University Cancer Hospital from 2003 to 2009 were selected as subjects in the survey; with the inclusion criteria as hepatocellular carcinoma, no cancer cells were observed in the surgical margin(SM) samples. All patients had neither lymph nor distant metastasis at the time of surgery, and receiving complete follow-up data for at least 3 years. By the end of May 2014, a total of 75 patients had relapsed of whom 71 died and there were no lost. All samples were acquired from the frozen surgical tissues. Genomic DNA was extracted using phenol/chloroform method and performed bisulfite modification following with polymerase chain reaction (PCR). AKAP12 methylation in hepatoma and the corresponding SM samples from 142 patients was determined by denature high-performance liquid chromatography (DHPLC) and bisulfite clone sequencing. Kaplan-Meier and Cox proportion hazard regression model were used to identify the factors related to the survival time. Results: In 142 cases, 125 patients (88.0%) were male and 17 (12.0%) cases were female. The median age was 52.5 years, ranging from 34 years to 76 years. AKAP12 methylation-positive rate was significantly higher in hepatomas than SMs (54.9% vs. 10.2%, P<0.001). Patients with AKAP12 methylation-positive had less risk of the recurrence (HR=0.62, 95%CI: 0.39-0.99); with tumor diameter more than 5 cm (HR=1.53, 95%CI: 1.00-2.50),portal vein invasion(HR=4.53, 95% CI:2.69-7.64) increased the recurrence risk. Moreover, portal vein invasion had a higher risk of death (HR=2.98, 95% CI: 1.73-4.98). Conclusion There was significant association between AKAP12 DNA methylation and low risk of recurrence and long progression-free survival of hepatocellular carcinoma patients.

Objective To investigate the expression of Aspartate Beta-Hydroxylase (ASPH) in retroperitoneal liposarcoma (RL)and evaluate its clinical significances.Methods Relevant clinical data of 69 RL cases after surgical resection were collected.The expression of ASPH in tumor tissues was detected by immunohistochemistry.The CTL epitopes of ASPH protein HLA-A2 were predicted by SYFPEITHI and NetMHCpan software.Results The overall positive rate of ASPH expression for the whole group was 81％,that for well-differentiated liposarcoma was 73％,dedifferentiated liposarcoma was 87％ (P ＜ 0.05).ASPH expression was positively correlated with the postoperative recurrence free survival rate (P ＜ 0.05).Five HLA-A2 restricted CTL epitopes (9 peptides) were screened with the method of motif prediction.Conclusions ASPH expression is positively correlated with the degree of malignancy of RL,and the ASPH expression is an independent risk factor for postoperative recurrence free survival rate of RL.Moreover,ASPH was found to have 5 HLA-A2 restricted CTL epitopes,which are expected to be used for the immunotherapy of RL.