Objective To explore an alternative strategy of ganciclovir-mediated cytotoxicity and study the mechanism of enhancing bystander effect of HSV-TK/GCV system by combination gene transfer of Rb gene and HSV-TK gene.Methods Recombinant eukaryotic expression plasmid pcDNA3.1-Rb harbour ing1.65kb of Rb gene was constructed.The pcDNA3.1-Rb and the plasmids tgCMV/HyTK car-ry ing HSV-TK gene were transfected respectively or co-transfected into the osteosarcoma cell line OS732by lipofection using DOTAP reagent.The mRNA expression of Rb gene and HSV-TK gene were detected by RT-PCR and Northern blot.Cell counting,cell cycle analysis and soft agar colony formation test were adopted to observe cell growth features.The expression of gap junction Connexin43gene was performed by RT -PCR and Western blot.The direct confirmation of gap junction intercellular commu ni cation was demonstrated by Lucifer yellow dye transfer.Cell sensitivity to GCV and″bystander effect″of HSV-TK/GCV system were measured by MTT assay.Results The eukaryotic expression plasmid pcD NA3.1-Rb was constructed successfully.The transfected cell lines OS732TK,OS732Rb and OS732RbTK were har-vested.mRNA expression of HSV-TK gene was demonstrated in OS732TK and OS732RbTK cells.Both exogenous and endogenous Rb gene expression could be detected in OS732Rb and OS732RbTK cells si-multa neously.Compared with parental cell OS732,OS732Rb and OS732RbTK cells changed their mor -phology and decreased the growth rate;the ability of soft agar colony formation reduced and the cell cycles were arrested at G 0 G 1 checkpoint.The Connexin43expression and gap junction in tercellular communica-tion en hanced in OS732Rb cell.GCV was of toxicity to only TK -positive cells,OS732TK and OS732RbTK,in a concentration dependent manner,the mixed coculture experiments of OS732and OS732Rb with the TK-negative cell,both showed sensitive to GCV,but the survival rate of OS732Rb cell was significantly lower than OS732cell under the same condition.Conclusion Coordinate ex pression of Rb andHSV-TK gene conferred a more po tent bystander effect by enhancing gap junction intercellular communica-tion and in hibiting prolifera tion.

Objective To evaluate the clinical curative effect and methods for recurring NPC with treatment of intracavitary radiotherapy after interventional chemotherapy. Methods 32 cases of recurring NPC were divided into two groups, each of which consisted of 16 cases. Group one was treated with intracavitary chemotherapy plus intracavitary radiotherapy. On the third day after interventional chemotherapy, the first intracavitary radiotherapy was performed (5 Gy) and the second treatment was done next day. Group two was only performed with intracavitary radiotherapy. The dose and interval were the same. The time of re examination was two weeks later. Results In the first group, clinical symptoms were improved, local swelling lymph nodes were shrunk, secretion on the nasopharyngeal surface was hardly left or disappeared. Tumours were shrunk and atrophy. On the intracavitary radiotherapy group, clinical symptoms changed a little, except the secretion on the mass surface was reduced. The white blood counts of the two groups showed no difference and there were no functional changes of liver, kidney and digestive system in the interventional group. Conclusions It is a new and effective therapy to treat recurring NPC with interventional chemotherapy plus intracavitary radiotherapy.

Bone morphogenetic protein-1(BMP-1) and its related molecules are members of metalloendoproteinase astacin family, including BMP-1, mTLD, mTLL-1 and mTLL-2. Even though all of them lack of the ability to induce bone or cartilage formation directly, they play key roles in numerable activities in ECM from embryo to adult, then affect the procedure and the result of osteogenesis and bone remodeling directly or indirectly. They are critical in maturation and deposition of some major collagen types, and in regulating the signaling of some growth factors in TGF-? superfamily by degradation of TGF-? inhibitor such as Chordin. The investigations about tissue distribution of BMP-1 and its related proteinases and also gene knock-out studies strongly indicate that they play key roles in osteogenesis and bone development.

Objective To evaluated the treatment results of late-course accelerated hyperfractionated radiotherapy combined with chemotherapy for advanced esophageal carcinoma. Methods 72 patients with advanced esophageal cancer were randomized into two groups. The late-course accelerated hyperfractionated radiotherapy combined with chemotherapy(LCAF+CT)group received the induction chemotherapy for two cycles, followed by conventional fractionation radiotherapy to a dose of 36 Gy, then changed into accelerated hyperfractionated radiotherapy to a total dose of 60 ~ 66 Gy. The conventional fractionation radiotherapy combined with chemotherapy(CF+CT)group received the chemotherapy that it was similar to LCAF+CT group, and used the conventional fractionation radiotherapy to a total dose of 60 ~ 66 Gy. Results The 1-, 3- and 5-year survival rates were 82.3 %, 51.6 %, 38.2 % in the LCAF+CT group, and 73.8 %, 31.5 %, 18.6 % in the CF+CT group. The differences of 3- and 5-year survival rates were statistically significant (P 0.05). Conclusion Late-coures accelerated hyperfractionated radiotherapy combined with chemotherapy might improve the treatment efficacy of advanced esophageal cancer and lengthen the suvival time. The toxic and side effects of LCAF+CT group were more severe than those of CF+CT group, but they were well tolerable.

AIM: To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1. METHODS: We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein. RESULTS: The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins. CONCLUSIONS: The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.

Objective The results and side effects of nasopharyngeal carcinoma treated by combined external radiotherapy and Californium -252 neutron after loading intracavitary radiotherapy. Methods From November 2005 to March 2007, 30 nasopharyngeal carcinoma patients with staged T1 and T2 by 1992 Fuzhou staging system, were treated by external beam radiotherapy combined with Californium-252 neutron after loading intraeavitary radiotherapy. Results All patients were followed up for 2 years. 2 cases recurred in the nasopharynx. 4 cases developed distant metastases. 2 cases developed trismus. No perforations in hand and soft palate occurred. Conclusion External beam radiotherapy combined with Californian-252 neutron after loading intracavitary radiotherapy for nasopharyngeal carcinoma is indicated for boosting the dose to the nasopharyngeal cavity; reducing the dose for external irradiation. It can improve local control rate of nasopharyngeal carcinoma and reduce occur rate for the trismus.

ObjectiveTo investigate the therapeutic effect of the frozen and fresh preparations of human umbilical cord mesenchymal stem cells （hUC-MSC） on a rat model of liver cirrhosis after transplantation via the portal vein or the caudal vein. MethodsA total of 70 specific pathogen-free healthy male Sprague-Dawley rats were randomly divided into normal group （13 rats fed with ordinary tap water and rat food） and liver cirrhosis model group （57 rats given subcutaneous multi-point injection of mixed carbon tetrachloride/olive oil solution）. At week 8， the growth of rats was observed for both groups， and 3 rats were selected from each group for histopathological examination to confirm the formation of liver cirrhosis. A total of 50 rats were selected from the liver cirrhosis model and were divided into model group， portal vein group+fresh cell preparation group， portal vein+frozen cell preparation group， caudal vein+fresh cell preparation group， and caudal vein+frozen cell preparation group using a random number table， with 10 rats in each group. Fresh or frozen hUC-MSC were transplanted via the portal vein or the caudal vein， and after 4 weeks of administration， the different groups were compared in terms of the changes in liver function parameters and liver fibrosis degree. Continuous data were expressed as mean±standard deviation， and the independent-samples t test was used for comparison between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsAt week 8 of modeling， the model group showed the formation of pseudolobules of different sizes in the liver and met the diagnostic criteria for liver cirrhosis， with significant increases in the levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBil）， and alkaline phosphatase （ALP） compared with the normal group （all P<0.001）， suggesting that the rat model of liver cirrhosis was established successfully. There were significant differences in the levels of ALT， AST， TBil， and ALP between the five groups （F=232.00， 177.10， 112.30， 121.70， all P<0.001）. Further comparison between two groups showed that the model group had significantly higher levels of ALT， AST， TBil， and ALP than the normal group （all P<0.01）， and the portal vein group+fresh cell preparation group， the portal vein+frozen cell preparation group， the caudal vein+fresh cell preparation group， and the caudal vein+frozen cell preparation group had significantly lower levels of ALT， AST， TBil， and ALP than the model group （all P<0.01）. ConclusionThere are significant improvements in liver function and liver fibrosis degree in a rat model of liver cirrhosis at week 4 after the transplantation of hUC-MSC， and frozen or fresh cell preparation and different transplantation approaches have no significant influence on treatment outcome.

Objective:To investigate the changes of T1 and T2 values in residual liver after major liver resection in rats and the relationship with pathologic indices related to liver regeneration.Methods:Seventy healthy male Sprague Dawley rats, SPF grade, aged 7-8 weeks, weighting 250-280 g, were divided into MR scan group ( n=14) and pathologic analysis group ( n=56). The MR scan group was further divided into partial hepatectomy group ( n=7) and the sham operation group ( n=7). MRI T 1 mapping and T 2 mapping were performed before surgery and on day 1, 2, 3, 5, 7, 14, 21 after surgery. T1 and T2 values of liver parenchyma were measured. In the pathologic analysis group, 7 rats were randomly included at each time point before and after surgery for pathologic examination, the diameter and proliferative activity (Ki-67 indices) of hepatocytes were assessed. The changes of imaging and pathologic indices were observed, and the correlations between MR parameters and liver volume and pathologic indices were analyzed. Results:Both T1 and T2 values in liver parenchyma were increased on day 1 after surgery and reached their maximum values on day 2 ( P=0.005 and P<0.001, compared with baseline), then were gradually decreased, and recovered to the preoperative level on day 14 and 21 ( P>0.05), respectively. T2 value was correlated with hepatocyte diameter, liver volume and Ki-67 indices better ( r=0.640, -0.764, 0.765, respectively, all P<0.001). T1 value was correlated with hepatocyte diameter, liver volume and Ki-67 indices ( r=0.472, -0.481 and 0.444, all P<0.001). Conclusion:The T1 and T2 values of rats liver remnant parenchyma showed regular changes, and were correlated with liver regeneration indices, which reflect the microscopic changes of rat liver remnant parenchyma, and are expected to be used for quantitative monitoring of liver remnant regeneration.