Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
The Chinese human hepatocellular carcinoma cell SMMC7721 has been analysed by flow cytometry, Southern blotting and Western blotting. The results indicated that SMMC7721 cell is a hypoploid cell with a 0.81 DNA index, and that SMMC7721 cell has internal deletion in the 5'-end of its Rb gene and has no Rb gene product (Rb protein). The normal Rb cDNA has been inserted into a retrovirus vector DOL and introduced into SMMC7721 cells by electrporation transfection technique.About 75% of transfected SMMC7721 cells have been killed by Rb gene product. The remain 25% cells are alive as exogenous Rb gene has been mutationally inactivated. （Chin J Ocul Fundus Dis,1994,10:21-24）
Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)
Objective To review the advance in the experimental studies of microRNA(miRNA) and the relationship between miRNA and stem cells. Methods The related literature was reviewed, and the research findings of miRNA and stem cell were summarized. Results miRNA was noncoding small RNA (20-25 nt) involved in posttranscriptional change, that have been shown to regulate gene expressions. Ithas been reported that some kinds of miRNAs were likely important regulators forstem cells maintaining their state of selfrenewal，and play key roles in theirdifferentiation. Conclusion miRNA as regulation of gene expressions, can be served as a new way for stem cells research.
Objective To review the research progress of the treatment of osteosarcoma, and to thoroughly understand its current state of research and prospect so as to lay a sol id foundation for the cl inical treatment. Methods The cl inical and experimental research l iteratures about treatment of osteosarcoma were extensively reviewed and analyzed. Results The present treatment of osteosarcoma is still need to comprehensive therapy which combine chemotherapy and surgical treatment. There are some progresses in gene therapy and molecular targeting therapy which can improve survival rate. Furthermore, well-designed studies and cl inical trials are needed to evaluate the potential therapeutic impact before they are used in cl inical. Conclusion Advancement in chemotherapeutic regimens has improved survival and l imb-sparing surgery in the treatment of osteosarcoma, but the progress of gene therapy and molecular targeting therapy gives new hope for osteosarcoma patients.
Despite its low incidence, retinoblastoma and its rela ted gene (Rb gene) have attracted some of the most brilliant minds in medicine and biology fi elds over the past years. Great advances have been achieved in the tumoregenesis mechanism and clinical management of retinoblastoma recently. However as always , more questions arise from those results. In order to improve retinoblastoma re search in China, we need to strengthen the communication and cooperation with di ffe rent countries, different institutes and disciplines, and utilize the great reso urces of retinoblastoma patients in China.
Objective To explore the feasibility of recombinant adeno-associated virus (rAAV) as a vector for the gene therapy of liver cancer. Methods The rAAV/enhance green fluorescein protein (EGFP) recombinant was prepared by the routine method of two plasmids cotransfection.Results The experiment showed that one 10cm plate could produce 107-108 infection unit recombinant by the method of two plasmids cotransfection, and the transduction of HepG2 cell was increased with the increase of infection dosage of rAAV. About 100 multiplicity of infection (MOI) AAV vector could make all the tumor cell light. Conclusion Liver cancer cell can be efficiently transduced by rAAV, and AAV vector may be a valuable vector for the gene therapy of liver cancer.
ObjectiveTo explore the new gene therapy method for tumor, the recombinant Caspase3 gene (rcaspase3) eukaryotic expression plasmid was constructed by molecular biologic method. MethodsThe eukaryotic expression plasmid pcDNA3.1(+)/rCaspase3 was constructed by rearrangement of the large subunit and small subunit of Caspase3 and it was transfected into pancreatic carcinoma cells(PCⅡ). After being transfected, the expression of rCaspase3 mRNA in pancreatic carcinoma cells was detected by RTPCR and it’s apoptotic activity was detected by FCM. ResultsThe sequence of rCaspase3 showed that the recombinant molecules (rCaspase3) now had its’ small subunit preceding its’ large subunit. After pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/rCaspase3 by liposomes, a 894 bp strap was observed by RTPCR. No strap was found in control groups. A transparent hypodiploid karyotype peak was found by FCM.ConclusionThe plasmid of pcDNA3.1(+)/rCaspase3 has been constructed successfully. rCaspase3 has apoptotic activity and can be used as target gene in gene therapy for pancreatic carcinoma.