The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated. FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology, PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine 2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels of mFasL protein on N1H3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3 cells membrane was investigated by the inducing apoptosis of Fas⁺ Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately co-cultured with Yac-1 cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants from twelve resistant clones was 8. 5 × 10⁵ colony-forming-unit (CFU)/ml. The NIH3T3 cells transfected by above supernatants had a higher level of mFasL (53. 81 ±6.9 %), and significantly induced the apoptosis of Fas⁺ Yac-1 cells (56.78±4. 5 %), as both were cocultured for 5 h at 1:1 ratio, whereas it is 7. 08±3. 4 % in control group (P<0.01). Supernatant from PLFIN-PA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.

To investigate the effect of calcitonin gene-related peptide (CGRP) on bone resorption mediated by interleukin-1β(IL-1β)in vitro, the osteoclasts isolated from the long bones of newborn SD rats were co-cultured with osteoblasts on ivory slices placed in 24-well plates. 24 h later, conditioned media containing CGRP and/or IL-1β were added to the wells respectively, and continued culturing for 48 h. After the cells were stripped off by ultrasonication, the ivory slices were stained in toludine blue. The number and the total area of resorption lacunae on each slice were measured by computer imaging analysis system. Our results showed that IL-1β significantly stimulated bone resorption, but CGRP inhibited the effect mediated by IL-1β in a dose-dependent manner, It is suggested that CGRP may inhibit osteoclastic bone resorption through two ways: One is that CGRP functions directly on osteoclasts to block their activation; the other is that CGRP regulates the release of cytokines by osteoblasts and indirectly affects the function of osteoclasts.