Experimental investigation of HGF inhibiting glial scar in vitro.

To study the inhibitory effect of Hepatocyte growth factor (HGF) on the responsive hyperplasia of damaged astrocytes in vitro. We prepared damaged model of astrocytes to simulate the responsive hyperplasia of damaged astrocytes in vivo by culturing astrocytes in vitro; After the first day of Ad-HGF transfection, astrocytes were scratched, then after the first, the third, and the fifth day of scratch, we detect the expression amount of astrocytes specific glial fibrillary acidic protein (GFAP) and the ratio of S-phase cells with flow cytometry, both of which can reflect the proliferation status of damaged astrocytes; After HGF was added in scratched astrocytes, the activity of SPK and MAPK (P42/44) were detected by autoradiography and immunoblotting test; After adding different concentrations of HGF protein in astrocytes cultured in different serum concentrations and adding diverse concentrations of HGF protein, SPK and SPK inhibitor DMS in scratched astrocytes, we detect cell proliferation with 3H-TDR incorporation. The first day after Ad-HGF transfected astrocytes were scratched, the amount of GFAP secreted by astrocytes were decreased significantly (P < 0.05), and the cells in S phase were declined obviously. HGF has bidirectional regulation on SPK of scratched astrocytes: increases the SPK activity when HGF in low dose, while inhibits when in high dose. In addition, DMS can block the signal passage; HGF had no effects on MAPK (P42/44) of damaged astrocytes cells. In conclusion, after the transfection of Ad-HGF, it can inhibit the responsive hyperplasia of damaged astrocytes by the means of blocking SPK passage.

[Relationship of motor deficits and imaging features in metastatic epidural spinal cord compression].

OBJECTIVE: To explore the relationship of motor deficits of the lower extremities with the imaging features of malignant spinal cord compression (MESCCs). METHODS: From July 2006 through December 2008, 56 successive MESCC patients were treated at our department. All were evaluated by magnetic resonance imaging and computed tomography and were scored according to motor deficits Frankel grading on admission. Imaging assessment factors of main involved vertebrae were level of vertebral metastatic location, epidural space involvement, vertebral body involvement, lamina involvement, posterior protrusion of posterior wall, pedicle involvement, continuity of main involved vertebrae, fracture of anterior column, fracture of posterior wall, location in upper thoracic spine and/or cervicothoracic junction. RESULTS: Occurrence was the same between paralytic state of MESCCs and epidural space involvement of imaging features. Multiple regression equation showed that paralytic state had a linear regression relationship with imaging factors of lamina involvement (X1), posterior protrusion of posterior wall (X2), location in upper thoracic spine and/or cervicothoracic junction (X7) of main involved vertebrae. The optimal regression equation of paralytic state (Y) and imaging feature (X) was Y = -0.009 +0.639X, + 0.149X, +0.282X. Lamina involvement of main involved vertebrae has a greatest influence upon paralytic state of MESCC patients. CONCLUSIONS: Imaging factors of lamina involvement, posterior protrusion of posterior wall, location in upper thoracic spine and/or cervicothoracic junction of main involved vertebrae can predict the paralytic state of MESCC patients. MESCC with lamina involvement is more easily encroached on epidural space.

[Intraarterial neoadjuvant chemotherapy for extremity osteosarcoma].

OBJECTIVE: To summarize the experiences in intraarterial neoadjuvant chemotherapy for extremity osteosarcoma. METHODS: Between January 2002 and December 2007, 111 patients with stage IIB extremity osteosarcoma received preoperative intraarterial chemotherapy after placing chemotherapy pump subcutaneously, en bloc resection and postoperative adjuvant chemotherapy. There were 63 males and 48 females with an average age of 18 years old (range: 14 - 39). The time from symptom onset to hospitalization varied from several days to 6 months. The induction chemotherapy regimen included epirubicin [50 - 70 mg/m(2) by 4-hour intraarterial infusion/day for 3 days] and epirubicin plus adriamycin [100 - 120 mg/m(2) by 2-hour intraarterial infusion/day for 3 days] repetitively every 2 - 3 weeks. Among which 24 cases only received 2 cycles of induction chemotherapy was assigned into the nonstandard chemotherapy group and 87 cases receiving 3 - 6 cycles of induction chemotherapy the standard chemotherapy group. The number of preoperative chemotherapeutical cycles of standard chemotherapy group depended on the clinical and radiographic evaluation of chemotherapy efficacy. RESULTS: The median follow-up time was 28 (8 - 48) months. The rate of extremity preservation surgery was 89.53% (77/86) in the standard chemotherapy group and 37.5% (9/24) in the nonstandard chemotherapy group. Kaplan-Meier survival analysis showed that the 3-year overall survival rate and disease-free survival rate of all 111 cases were 68.3% and 65.9% respectively. There were significant differences in overall survival rate (38.9%, 80.0%, P = 0.000), disease-free survival rate (30.1%, 79.5%, P = 0.000), distant metastatic rate (66.67%, 16.09%, P = 0.0000) and local recurrence rate (58.33%, 13.79%, P = 0.0000) between two groups. CONCLUSION: Standard intraarterial neo-adjuvant chemotherapy is more effective than nonstandard intraarterial induction chemotherapy to treat stage IIB extremity osteosarcoma.

[Secretion expression in Pichia pastoris and immunogenicity comparison of two forms of HBsAg/GM-CSF fusion proteins].

AIM: To express two fusion forms of hepatitis B virus surface antigen (HBsAg-s and s-HBsAg) in the Pichia pastoris expression system, and compare immunogenicity of the two fusion proteins. METHODS: Was fused GM-CSF to 5' or 3' terminal of HBsAg by inserting the gene fragment of connecting peptide (Gly(4)Ser)(3) to linker gene of GM-CSF and HBsAg. The two fusion proteins were expressed by secreting type expression plasmid pPIC9K in the Pichia pastoris, then the expressed products were detected by SDS-PAGE, Western blot and purified by DEAE-Sepharose Fast Flow ion exchange columns. Mice were inoculated with the two purified HBsAg/GM-CSF fusion proteins and HBsAg respectively in each, and the levels of anti-HBsAg in mice sera were tested by ELISA. RESULTS: Two HBsAg/GM-CSF fusion proteins were successfully expressed in the form of secretion in Pichia pastoris strain GS115, and exhibited specific reaction with both anti-HBsAg and anti-GM-CSF antibodies in Western blot. ELISA results showed after the inoculation the levels of anti-HBsAg induced by the two HBsAg/GM-CSF fusion proteins was higher than by HBsAg alone (P<0.05). Furthermore, the effect by fusing GM-CSF to C terminal of HBsAg was better than by fusing GM-CSF to N terminal of HBsAg. CONCLUSION: The immunogenicity of HBsAg could be enhanced by fusing GM-CSF.

Enhancement of DNA vaccine-induced immune responses by a 72-bp element from SV40 enhancer.

BACKGROUND: Although DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed. METHODS: Vector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice. RESULTS: It was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells. gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner. CONCLUSIONS: The 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.