This study examined the effect of insulin on the expression of very low density lipoprotein receptor (VLDLR) subtypes of SGC7901 cells and discussed its biological implication. In vitro, moderately or poorly-differentiated human gastric adenocarcinoma cell line SGC7901 was incubated with insulin for different lengths of time, and then the expression of protein and RNA level in VLDLR subtypes were detected by Western blotting and real-time PCR, respectively. The results showed that, at certain time interval, insulin could down-regulate expression of type I VLDLR and up-regulate the expression of type II VLDLR in SGC7901 cells, at both protein and RNA level. We are led to conclude that insulin serves as a regulator in maintaining the balance between glucose and lipid metabolism in vivo, possibly through its effect on the differential expression of VLDLR subtypes.

This study examined the effect of insulin on the expression of very low density lipoprotein receptor (VLDLR) subtypes of SGC7901 cells and discussed its biological implication. In vitro,moderately or poorly-differentiated human gastric adenocarcinoma cell line SGC7901 was incubated with insulin for different lengths of time, and then the expression of protein and RNA level in VLDLR subtypes were detected by Western blotting and real-time PCR, respectively. The results showed that, at certain time interval, insulin could down-regulate expression of type Ⅰ VLDLR and up-regulate the expression of type Ⅱ VLDLR in SGC7901 cells, at both protein and RNA level.We are led to conclude that insulin serves as a regulator in maintaining the balance between glucose and lipid metabolism in vivo, possibly through its effect on the differential expression of VLDLR subtypes.

Very low density lipoprotein receptor (VLDLR) is thought to participate in the patho-genesis of atherosclerosis induced by VLDL and β-VLDL. The present study was undertaken to elu-cidate the effects of VLDL and β-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and β-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway in-volving VLDLR expression regulation. The results showed that VLDL and β-VLDL stimulated ERKI/2 activity in a PKC-dependent manner. VLDL or β-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERKI/2 or PKC. Our findings revealed that VLDL or β-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.

In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 x 10(9) cfu/microg was obtained. After amplification with helper phage, the titer of antibody library reached 5 x 10(12) cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

In order to obtain three isoforms of apolipoprotein E (apoE), the cDNA encoding apoE3 was obtained by RT-PCR from normal human liver tissue. Site-directed mutagenesis was used to obtain the cDNAs encoding apoE2 and apoE4 isoforms. The 3 cDNAs were subcloned into vector pGEM-3Z and verified by DNA sequencing. The expression recombinant which can express the target protein as a (His) 6-tagged fusion was constructed by subcloning apoE cDNA into vector pT7-PL. The purified proteins were gained by Ni-NTA column. The SDS-PAGE results revealed the 6 His fusion proteins (apoE2, apoE3 and apoE4) were correctly expressed and purified successfully.

In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E.coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 109 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 × 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

In the present study, we examined the regulation of the expression and function of AB CA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-Ⅰ for 24 h, RAW264.7 cells effluxed 37.65 % cholesterol loaded by acetyl LDL (ac-LDL), and 9.78 % cholesterol in ox-LDL group. The level of ABCA1 Mrna increased about three times either when cells were incubated with 100 μg/Ml ac-LDL or with 100μg/Ml ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.

Summary: To explore the functions of very low density lipoprotein receptor (VLDL-R) subtype II in lipoprotein metabolism and foam cells formation, the recombinant plasmid with the two subtypes cDNA was constructed respectively, the ldl-A7 cell lines were transfected and two cell lines expressing VLDL-R were obtained: one stably expressing the VLDLR with the O-linked sugar region (type I VLDLR) and the other without the O-linked sugar region (type II VLDLR). In the study on binding of VLDLR to their nuclein labeled natural ligands (VLDL and β-VLDL), it was found that surface binding of 125I-VLDL or 125I-β-VLDL of ldl-A7 cells transfected with type I VLDLR recombinant (ldl-A7-VRI) was more higher than that of ldl-A7 cells transfected with type II VLDLR recombinant (ldl-A7-VRII). After being incubated with VLDL for different time, the contents of triglyceride and total cholesterol in cells were mensurated, and the formation of foam cells and accumulation of lipid in cells was observed by oil-red O staining. The results showed that the contents of triglyceride and total cholesterol in ldl-A7-VR I were much higher than those in ldl-A7-VR II, and ldl-A7-VR I could transform into foam cells notably. It was suggested that type I VLDLR binds with relative higher affinity to VLDL and β-VLDL, and internalizes much more lipoprotein into cells. As a result, we can conclude that type I VLDLR plays a more important role in lipoprotein metabolism and foam cells formation than type II VLDLR.

Summary: Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the nuclide 131I was combined with Asor to form a kind of soluble nuclide-protein complex, which can be specifically endocytosed into hepatocyte by ASGP-R. After intravenous injection of the complex into experimental animals, the deposition of Asor in vivo and the targeting quality of hepatocyte was detected by ECT. This research testified the feasibility of targeting Asor complex delivery to hepatocyte mediated by ASGP-R in vivo, and provided foundation for the genetic diagnosis and gene therapy of hepatic cell-related diseases.

To explore the intracellular signal pathways for beta-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that beta-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of beta-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which beta-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by beta-VLDL in macrophages.
Cells, Cultured ; Lipoproteins, VLDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 3 ; metabolism ; physiology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Receptors, LDL ; biosynthesis ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; Transcription, Genetic ; Up-Regulation