Effect of CXCR4 pretreated with ultrasound-exposed microbubbles on accelerating homing of bone marrow mesenchymal stem cells to ischemic myocardium in AMI rats.

OBJECTIVE: To investigate the role and potential mechanism of CXCR4 in promoting targeted homing of bone marrow mesenchymal stem cells (BMSCs) with ultrasound-exposed microbubbles (UM) pretreatment. METHODS: Third generation BMSCs were divided into four groups control group, ultrasound (US) group, UM group and ultrasound-exposed microbubbles plus catalase group. RT-PCR and western blot were performed to determine the levels of CXCR4 mRNA transcription and protein expression, respectively. Third generation BMSCs were labeled with Fluo-α/AM and divided into three groups: control group, US group and UM group, and fluorescence intensities in the cells were observed immediately, 5 min and 15 min after intervention under fluorescence microscope. The calcium iron levels in the cells were analyzed. BMSCs were divided into five group: group A without calcium in the medium, group B, group C, group D and group E containing calcium chloride with concentration of l mol, 2 mol, 4 mol, anti-calcium-sensing receptor antibody, respectively. RT-PCR and western blot were performed to determine the levels of CXCR4 mRNA transcription and proteins expression of the third generation BMSCs of each group, respectively. RESULTS: The levels of CXCR4 mRNA transcription and protein expression between US group and control group had no statistically significant difference (P > 0.05) shown by RT-PCR and western blot; the transcription level in the UM group was significantly higher than that in US group and control group (P < 0.05); and in the ultrasound-exposed microbubbles plus catalase group, the transcription level was much lower than that in UM group. Fluorescence intensify in the cells of US group had no significant difference compared with that in the cells of the control group (P > 0.05), which in the cells of UM group was significantly higher than that in the cells of both US group and control group (P < 0.05). Compared to group A, expressions of CXCR4 of group B to D were significantly increased in concentration-dependent manner showed by RT-PCR and western blot (P < 0.05). Compared to group C, expressions of CXCR4 of group E were significantly decreased (P < 0.05). CONCLUSIONS: UM can promote the influx of calcium in BMSCs and increase mRNA transcription and protein expression of CXCR4. The latter may partly be caused by influx of calcium.

[Effects of heat shock protein gp96 on function of macrophages from mouse].

BACKGROUND & OBJECTIVE: Heat shock protein gp96 (HSPgp96) plays an important role in antigen presenting and specific antitumor immunotherapy, but its effects on macrophages need further investigation. This study was to investigate the effects of HSPgp96 derived from H22 tumor cells on mouse peritoneal elicited macrophages (PEMphi) by detecting some factors that are related to the function of the macrophages. METHODS: After macrophages were stimulated by HSPgp96, dynamic changes of intracellular free calcium (Ca2+), nitric oxide (NO) and reactive oxygen species (ROS) in the macrophages were measured by the laser scanning confocal microscopy (LSCM) with sensitive fluorescent dye Fluo-3/AM, DAF-FM-DA, and H2DCF-DA. The color immunofluorescence assay was used to observe the expression intensity and distribution of MHC II and CD86 in the macrophages. RESULTS: The production of Ca2+, NO and ROS increased rapidly in the macrophages after stimulation of HSPgp96. Their concentration peaks appeared at 30 s, 80 s, and 580 s after stimulation with the increase scopes of (161.06+/-70.99)%, (77.58+/-31.17)%, and (647.28+/-185.70)%, respectively. The positive rates of MHC II and CD86 were significantly higher in 60 mg/L HSPgp96-stimulated macrophages than in control macrophages [(61.8+/-5.1)% vs. (40.9+/-3.5)%, P<0.01; (54.9+/-2.0)% vs.(23.1+/-3.1)%, P<0.01]. CONCLUSION: The in vitro stimulation of HSPgp96 may enhance the production of Ca2+, NO and ROS in mouse PEMphi, and up-regulate the expression of MHC II and CD86.

[Induction of anti-tumor immunity by HSP gp96-peptide complexes].

AIM: To immunize the mice with gp96-peptide complexes extracted and purified from different kinds of malignant tumor cells and to observe anti-tumor immunity induced by the complexes. METHODS: HSP gp96-peptide complexes were extracted and purified from different kinds of malignant tumor cells. Mice were immunized subcutaneously with the complexes from different sources at different doses. Then the mice were challenged with 2 x 10(5) tumor cells on the seventh day after the last immunization. Tumor incidence, weight and histology were observed and compared with those of control group. At the meantime, cytotoxicity activity and nitric oxide production of mouse peritoneal macrophages were detected in vitro after being stimulated with gp96-peptide complexes. RESULTS: The SDS-PAGE and Western blot showed that gp96-peptide complexes were purified successfully. The anti-tumor immunity was correlated with the dose of the complex and the immunized mice could resist the challenge of homogeneous tumor cells. NO secreted from the peritoneal macrophages stimulated with the complex was significantly higher than that of control group and the tumoricidal activity of the macrophages in vitro was markedly promoted by the complex. CONCLUSION: Mice immunized with appropriate dose of gp96-peptide complexes from H22 tumor cells can resist the challenge of syngeneic tumor cells. NO secreted by macrophages stimulated with the complex might play a key role in the anti-tumor immunity.

[Expression of PTEN, Cx43, and VEGF in hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Previous studies showed that expression of phosphatase and tensin homology deleted on chromosome ten (PTEN), gap junction protein connexin 43 (Cx43) and vascular endothelial growth factor (VEGF) may play an important role in tumor occurrence and progression. This study was designed to investigate the relationship among the protein expression of PTEN, Cx43, and VEGF in carcinogenesis and progression of hepatocellular carcinoma. METHODS: The expression of PTEN, Cx43 and VEGF were determined in 47 cases of HCC by streptavidin peroxidase immunohistochemistry. RESULTS: In the hepatocellular carcinoma, the total positive rates of PTEN, Cx43 and VEGF were 76.4%, 42.6%, and 70.2%, respectively. With progression of tumor course, PTEN and Cx43 positive rates decreased (P< 0.05), but the VEGF positive rate significantly increased (P=0.001). In comparison with the group of intrahepatic vascular embolism, PTEN positive rate increased apparently in the group without intrahepatic vascular embolism (P=0.006). Cx43 positive rate was lower in the group with cirrhosis background than that in the group without cirrhosis background (Chi(2)=4.713 5, P=0.03). No significant difference of PTEN positive rate was observed in tumor size, differentiation degree in cancerous tissue and cirrhosis background, Cx43 positive rate in tumor size, differentiation degree in cancerous tissue and intrahepatic vascular embolism, VEGF positive rate in tumor size, differentiation degree in cancerous tissue, cirrhosis background and intrahepatic vascular embolism. Correlation analysis revealed that PTEN positive rate was positively correlated with Cx43 positive rate (Cramer's V=0.394 9, P=0.023), negatively correlated with VEGF positive rate (Cramer's V=0.393 7,P=0.024), but no significant association was observed between Cx43 positive rate and VEGF positive rate (Cramer's V=0.307 2, P=0.064). CONCLUSION: The aberrant expression of PTEN, Cx43, and VEGF may play a role in occurrence, progression, and intrahepatic metastasis of the hepatocellular carcinoma. Determining the expression of PTEN, Cx43, and VEGF may contribute to synthetically evaluating the biological behavior of hepatocellular carcinoma.