Expression of subtypes of somatostatin receptors in hepatic stellate cells

AIM: To elucidate the mechanism by which somatostatin and its analogue exert the influence on liver fibrosis, and to investigate the mRNA expression of somatostatin receptors subtypes (SSTRs) and the distribution of somatostatin analogue octreotide in rat hepatic stellate cells (HSCs). METHODS: HSCs were isolated from Sprague Dawley (SD) rats by in situ perfusion and density gradient centrifugation. After several passages, the mRNA expression of 5 subtypes of SSTRs were assessed by reverse transcription-polymerase chain reaction (RT-PCR). HSCs were planted on coverslip and co-cultured with octreotide tagged by FITC. Then the distribution of FITC fluorescence was observed under laser scanning confocal microscope (LSCM) in 12-24 h. RESULTS: There were mRNA expression of SSTR2, SSTR3 and SSTR5 but not SSTR1 and SSTR4 in SD rat HSCs. The mRNA expression level of SSTR2 was significantly higher than that of other subtypes (P<0.01). FITC fluorescence of octreotide was clearly observed on the surface and in the cytoplasm, but not in the nuclei of HSCs under LSCM. CONCLUSION: The effect exerted by somatostatin and its analogues on HSCs may mainly depend on the expression of SSTR2, SSTR3 and SSTR5. Octreotide can perfectlycombine with HSCs, and thereby exerts its biological activity on regulating the characters of active HSCs. This provides a potential prevention and management against liver fibrosis.

A novel,rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+monocytes within 48 hours of in vitro culture

AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) and prostaglandin E2 (PGE2).One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocytederived dendritic cells within 48 h of in vitro culture (FastDC).METHODS: HCCLM3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/1 L-glutamine,100 μg/mL gentamicin, 1000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα(1000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE2(1 μg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/1 polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritornas. The FastDCs and novel dendritomas were immunostained with antiCD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS).Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy.RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas.CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.