Asialofetuin-hTERT-TK/GCV targeted gene therapy and its bystander effect on HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the targeted therapeutic effects of plasmid AF-pGL3-hTERT-TK on HepG2 cells.</p><p><b>METHODS</b>HepG2 cells were cultured and pGL3-hTERT-TK and AF-liposome were constructed. HepG2 and L02 cells were transfected with AF-pGL3-hTERT-TK. The growth, apoptosis of the cells and the bystander effects were studied using liquid scintillation analysis and tunnel and flow cytometry.</p><p><b>RESULTS</b>After the suicide gene was inserted into the downstream of hTERT, TK was effectively driven by the hTERT promoter, making the TK highly expressed in the HepG2 cells. The AF made the therapeutic gene enter the HepG2 cells more easily by recognizing and combining the ASGPR receptor protein on the HepG2 cell surfaces and induced their apoptosis and suicide with bystander effect. The apoptosis rate was 85%+/-3% in the HepG2 cells whereas in the normal L02 hepatic cells it was 16%+/-2%.</p><p><b>CONCLUSION</b>AF-pGL3-hTERT-TK can target and attack HepG2 cells and has almost no influence on normal L02 hepatic cells. AF-pGL3-hTERT-TK has a potential in the treatment of hepatocellular carcinomas.</p>

Transfection and anti-HBV effect mediated by the hepatocytes-targeting cationic liposomes co-modified with beta-sitosterol-beta-D-glucoside and Brij 35 / 药学学报

<p><b>AIM</b>To study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.</p><p><b>METHODS</b>Dipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.</p><p><b>RESULTS</b>The co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.</p><p><b>CONCLUSION</b>Co-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.</p>

Mechanism of the immunomodulatory activity of glycodelin.

Glycodelin, a progesterone regulated protein synthesized by the endometrium (GdA) has been well documented to inhibit the proliferation of activated T-cells and is an indispensable molecule in the maternal system for the establishment, maintenance and progression of pregnancy. Data from our laboratory have unequivocally shown that the immunosuppression by GdA is via induction of apoptosis in activated T cells. Another isoform of glycodein, GdS, from the male reproductive system, in spite of sharing an identical amino acid sequence as that of GdA has been shown not to harbour the immunosuppressive activity of GdA. As the only difference between the two proteins is glycosylation, we proposed to study the role of the sugars in imparting apoptotic activity to Gd. Using the recombinant baculovirus system, Gd lacking glycosylation was expressed and from the experimental observations we could conclude that the activity of Gd lies in the protein backbone. Recombinant Gd expressed in P. pastoris, and Chinese hamster ovary cells, like the GdS did not exhibit apoptotic activity. A close analyses of the glycans associated with the Gd molecules from various sources suggested that though the apoptogenic activity of Gd lies in the protein backbone, the glycans modulate the activity by masking (as in case of GdS and most recombinant Gd expressed in our laboratory) or unmasking (as in case of GdA and baculovirus expressed Gd), the functional region of the molecule.

Study on the hepatocytic cell targetability of liposomes / 药学学报

<p><b>AIM</b>To target for hepatocytic cell, liposomes was modified by special ligand.</p><p><b>METHODS</b>Sterically stabilized liposomes (SSL) was conjugated with asialofeticin (AF), the ligand of asialoglycoprotein receptor (ASGP-R) of hepatocyte. ASGP-R-BLM is the ASGP-R reconstructed on bilayer lipid membrane (BLM). The recognition reaction between AF-SSL and ASGP-R-BLM can be monitored by the varieties of membrane electrical parameters. The targetability of AF-SSL mediated to hepatocyte was detected by radioisotopic labeled in vitro and in vivo. The therapeutic effect of antihepatocarcinoma was observed also.</p><p><b>RESULTS</b>The lifetime of ASGP-R-BLM decreased with the added amount of AF-SSL. It was demonstrated that there was recognition reaction between AF-SSL and ASGP-R-BLM. The combination of AF-SSL with hepatocyte was significantly higher than that of SSL without AF-modified in vitro and in vivo. The survival time of rat for AF-SSL carriered ADM (adriamycin) group was much longer and the toxicities on heart, kidney and lung were lower than those SSL carried ADM group.</p><p><b>CONCLUSION</b>It is possible to actively target the cell with specific receptor by ligand modified liposomes. The result prvide scientific basis of hepatocyte targeted liposomes.</p>

Modulation of biochemical activity of hepatocytes in culture by matrix substratum.

The influence of extra cellular matrix on the biochemical activity of hepatocytes was studied by maintaining rat hepatocytes in primary culture in a serum free medium on different matrix protein substrata or biomatrices prepared from liver, aorta or mammary gland. There was significant difference in the individual protein synthesis and distribution by cells maintained on different substrata. Comparison of the kinetics of synthesis and secretion of albumin by cells maintained on different tissue biomatrix showed that those maintained on hepatic biomatrix synthesized more albumin and retained more of albumin synthetic capacity, when compared to those maintained on aortic and mammary gland biomatrix. Similarly, hepatocytes maintained on hepatic biomatrix synthesized significantly more apo B, the major apo protein of VLDL, than those maintained on heterologous tissue matrix. Induction of tyrosine aminotransferase by dexamethasone and the uptake of [14C]-amino isobutyric acid were found to be maximum in cells maintained on liver biomatrix than the heterologous biomatrix. But cells maintained on hepatic biomatrix incorporated less amounts of radioactivity into total cytoskeletal proteins as well as the individual proteins such as actin and the cytokeratins C8 and C18 while that by cells maintained on aortic biomatrix was significantly high. Quantitative analysis of the relative incorporation of radioactivity into individual cytoskeletal proteins and albumin in pulse labelling studies with cells maintained in culture on different matrix for different lengths of time revealed a reciprocal relationship between these two activities. These results indicate that the substrata with which the cells are in contact influence on a selective basis, the biochemical activity of hepatocytes in primary culture.