BACKGROUND: Cryopreservation can decrease tissue and organ immunogenicity. The effects of cryopreservation on cell immunogenicity are disputed.OBJECTIVE: To investigate the effects of liquid nitrogen cryopreservation on osteoblast immunogenicity. DESIGN: Randomized,controlled ,paired-sample experiment. SETTING: This study was performed in the Laboratory Center, Qilu Hospital Affiliated to Shandong University between July 2003 and March 2004. MATERIALS: Four New Zealand rabbits of either gender were included for this study. 3H-TdR was provided by Nuclear Medicine Institute of Shandong University. METHODS: Osteoblasts were cultured from the tibial periosteum of New Zealand rabbits and then cryopreservated in the liquid nitrogen for 3 months and defrosted. Cryopreservated and thawn osteoblasts were set as cryopreserved group and freshly cultured osteoblasts were set as non-cyropreserved group. Major histocompatibility complex (MHC)-I positive rate was examined by flow cytometry assay prior to and after cryopreservation. Simultaneously, mixed lymphocyte-osteoblast cultures were established. Lymphocyte stimulation index was calculated after counting the flares using β liquid scintilloscope. MAIN OUTCOME MEASURES: MHI-I antigen positive rate and lymphocyte stimulation index prior to and after cryopreservation of osteoblasts. RESULTS: MHI-I antigen positive rate and lymphocyte stimulation index of osteoblasts was significantly higher in the non-cryopreserved group than in the cryopreserved group (P＜0.01). CONCLUSION: The immunogenicity of cryopreserved osteoblasts was significantly decreased. Liquid nitrogen cryopreservation is an ideal method to decrease the immunogenicity of osteoblasts.

Objective: To evaluate the effect of synthesized small interfering RNA targeting to HIF-lα on the adhesion and invasion of human tongue squamous cell carcinoma cell line (Tca8113). Methods; A double strand small interference RNA (siRNA) targeting HIF-1α (siRNAH1Fla) was transfected into cultured Tca8113 cells by lipofectamine2000. The expression of HIF-1α was investigated on mRNA level by real time-PCR and protein level by Western blot. The adhesion and invasion of Tca8113 cells to extracellular matrix (ECM) was also analyzed. Results: Exposure to hypoxia induced a prolonged elevation of HIF-lα protein and siRNAHIF.la reduced HIF-la synthesis as measured on mRNA level and protein level compared with the controls. No matter under normoxic or hy-poxic conditions, the adhesion potency of siRNAHIF-1α treated Tca8113 cells was markedly inhibited compared with controls(P<0.05 or P <0.01). So did the invasion potency (P<0.01). The adhesion and invasion potency of siRNAHIF.,a treated Tca8113 cells were inhibited more greatly under hypoxic condition than under normoxic condition ((36.4±2.7)% vs(26±2.35);(44.2±2.2)% vs (35±1.75), P<0.01)). Conclusion; siRNAH1F.lo can knockdown the expression of HIF-la and inhibit the cell adhesion and invasion to ECM in Tca8113 cells. HIF-la may play an established role in the regulation of Tca8113 cells invasion and metastasis. Interfering with HIF-1α pathways by siRNA strategy may provide a therapeutic target for human tongue squamous cell carcinomas.

Objective: To provide an efficeut protocol for constructing recombinant adenovirus, an in vitro ligation was used instead of homologous recombination. Methods: Gene BMP-2 was ligated into pShuttle 2 vector ( pShuttle 2-BMP-2 ) and then fragment containing BMP-2 gene and promoter pcmvie excised by PI-SCe Ⅰ and Ⅰ-Ceu Ⅰ endonuclease. The fragment was further combined with adenovirus vector (Adeno-X-BMP-2) , which was finally linearized with Pac Ⅰ and trans-fered to HEK293 to package adenovirus particles. Results: Both PCR assay and restiction analysis showed that the recombined rectors pShuttle2-BMP-2 and Adeno-X-BMP-2 contains the target BMP-2 gene. THe packaged adenovirus was also i-dentified by PCR assay with specific primers for BMP-2. Conclusions: The BMP-2 incorporated recombinant adenovirus was obtained and this laid a foundation for further study on BMP-2 mediated gene therapy. The in vitro ligation method de-scinbed here for constructing recombined adenovirus was more efficient than traditional homologous recombination.

OBJECTIVETo establish a novel nude mice model which can be visualized in real time and detected in a continuous and dynamic way for the development and metastasis of adenoid cystic carcinoma.

METHODSHuman adenoid cystic carcinoma cells, ACCM cell line, were infected with retroviral vector of pLEGFP-N1 and then screened for a single colony of ACCM-GFP cells. Cell proliferation and morphological analysis were conducted for ACCM and ACCM-GFP cells. Nude mice lingual carcinoma model was set up with ACCM-GFP cells injection and real time observation with fluorescence imaging on ACCM-GFP tumors was performed subsequently. Histological assay was analyzed for ACCM and ACCM-GFP tumors as well.

RESULTSACCM-GFP cells were able to express GFP stably in the long term. ACCM and ACCM-GFP cells showed no significant difference in cell proliferation and morphology, and no significant difference of histological characteristics in vivo could be found between ACCM and ACCM-GFP tumors. Tumor development could be monitored in real time with fluorescence imaging system in vivo.

CONCLUSIONGFP-expressing ACCM tumor model can be applied to detect and observe its development in the long term in a noninvasive, real time and dynamic way. It is also a kind of ideal in vivo mouse model for adenoid cystic carcinoma research.

Animals ; Carcinoma, Adenoid Cystic ; Cell Line, Tumor ; Cell Proliferation ; Disease Models, Animal ; Green Fluorescent Proteins ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude