Differentiation of bone mesenchymal stem cells into hepatocyte-like cells induced by liver tissue homogenate.

This study investigated the efficacy and feasibility of inducing the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro using Sprague Dawley rats, as a model of hepatocyte generation for cell transplantation. BMSCs were isolated and grown using the adherent method and exposed to 5 or 10% liver tissue homogenate, before being collected for analysis after 0, 7, 14, and 21 days. Immunofluorescence and western blotting were employed to detect the liver-specific markers a-fetoprotein (AFP) and albumin (ALB). Supernatant urea content was also measured to verify that differentiation had been induced. After 7 days in the presence of 10% liver tissue homogenate, BMSCs demonstrated hepatocyte-like morphological characteristics, and with prolonged culture time, liver-specific markers were gradually produced at levels indicating cell maturation. AFP expression peaked at 14 days then began to decrease, while both urea and ALB levels increased with induction time. Overall, marker expression in the 5% homogenate group was less than or equal to the 10% group at each time point. Thus, in a rat model, liver tissue homogenate obtained from partial hepatectomy can induce the differentiation of BMSCs into hepatocyte-like cells. This method is simple, feasible, and has remarkable real-world application potential.

Immune function of nonparenchymal liver cells.

The liver is the human body largest digestive and metabolic organ, and a very important immune organ. This paper discusses the location and morphology of hepatic sinusoidal endothelial cells, dendritic cells, hepatic stellate cells, and Kupffer cells in the liver and their role in regulating immune functions. Therefore, here we provide a preliminary understanding of the immune regulatory function of liver cells, and information on the occurrence and treatment of liver diseases.

NDC80 promotes proliferation and metastasis of colon cancer cells.

Chromosome instability is a common feature of tumor cells, and may be an important mechanism in tumor formation. Nuclear division cycle 80 (NDC80) is closely associated with the stability of chromosomes. Therefore, we investigated the relationship between NDC80 and development of colon cancer using a range of methods. Western blotting and immunohistochemistry were employed to determine the expression of this protein in different colon cells and tissues, cell proliferation was measured with an MTT assay, levels of proliferating cell nuclear antigen were examined by immunofluorescence, and cell migration was observed using wound healing tests. Our results showed that the expression of NDC80 in colon cancer cells (CACO2, HCT8, HCT116, and SW480) and tissues (from 20 patients) was higher than that in controls. Moreover, cell proliferation and migration rates were elevated in cells transfected with NDC80 compared to control groups. In summary, NDC80 promotes the proliferation and metastasis of colon cancer cells, and may constitute a new target for gene therapy in treating this disease. Combined with clinicopathological grading, measurement of positive NDC80 expression may be helpful in diagnosing and estimating the prognosis of colon cancer patients.

Cloning and transformation analysis of isoflavone synthase gene into Minshan Trifolium pratense.

The aim of this study was to clone the isoflavone synthase (IFS) gene and establish the recombinant Minshan Trifolium pratense. The IFS gene was cloned from the callus of Minshan T. pratense using reverse transcription-polymerase chain reaction. The plant expression vector pRI101-AN-IFS was constructed and introduced into Agrobacterium tumefaciens strain LBA4404, and then screened under cephalosporin. IFS expression was detected by reverse transcription-polymerase chain reaction. The IFS gene was cloned successfully. Sequence analysis indicated that IFS gene had high homology with similar genes from other plants. The IFS-overexpressing callus was obtained by introducing the LBA4404-harboring IFS-pRI101-AN-IFS vector into T. pratense calluses.

Construction and identification of pIRES2-LIF-NT-3 bicistronic eukaryotic expression vector.

We used a simple and efficient method to construct a bicistronic eukaryotic expression vector pIRES2-LIF-NT-3. The leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3) genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The LIF cDNA fragment was inserted into the multiple cloning sites of a vector containing internal ribosome entry site and enhanced green fluorescent protein (EGFP) (pIRES2-EGFP) to generate the bicistronic eukaryotic expression plasmid pIRES2-LIF-EGFP. Next, the NT-3 cDNA fragment was cloned into pIRES2-LIF-EGFP in place of EGFP to create the plasmid pIRES2-LIF-NT-3. pIRES2-LIF-NT-3 was transfected into HEK293 cells and reverse transcription-polymerase chain reaction and Western blotting were used to test the co-expression of double genes. LIF and NT-3 genes were cloned and the DNA was sequenced. Sequencing analysis revealed that LIF and NT-3 were consistent with the sequence recorded in GenBank. Restriction analysis indicated that the LIF and NT-3 genes were inserted correctly into the expression vector pIRES2-EGFP. Following transfection of pIRES2-LIF-NT-3 into HEK293 cells, the double gene was expressed at the mRNA and protein levels. The LIF and NT-3 coexpression plasmid is a novel expression system that will enable further study of the functions of the LIF and NT-3 genes.

Construction and identification of pIRES2-VEGF165-NT-3 bicistronic eukaryotic expression vector.

We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-VEGF165-NT-3. The neurotrophin-3 (NT-3) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NT-3 cDNA fragment was cloned into the pIRES2-VEGF165-EGFP vector in place of enhanced green fluorescent protein (EGFP) to create the plasmid pIRES2-VEGF165-NT-3. Next, pIRES2-VEGF165-NT-3 was transfected into HEK293 cells, and reverse transcription-polymerase chain reaction and Western blotting were used to test co-expression of the double genes. The vascular endothelial growth factor 165 (VEGF165) and NT-3 genes were cloned; DNA sequencing analysis demonstrated that the VEGF165 and NT-3 sequences were the same as those recorded in GenBank. Restriction analysis indicated that the VEGF165 and NT-3 genes were correctly inserted into the expression vector pIRES2-EGFP. The double gene was expressed at both the mRNA and protein levels. The VEGF165 and NT-3 co-expression plasmid was successfully constructed, providing a novel expression system for further study of the functions of the VEGF165 and NT-3 genes.

Construction and identification of pIRES2-NGF-VEGF165 bicistronic eukaryotic expression vector.

We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-NGF-VEGF165. The nerve growth factor (NGF) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NGF cDNA fragment was inserted into the multiple cloning sites of the pIRES2-EGFP vector to generate the bicistronic eukaryotic expression plasmid pIRES2-NGF-EGFP. The vascular endothelial growth factor 165 (VEGF165) gene was obtained from the pIRES2-VEGF165-EGFP plasmid by polymerase chain reaction. Next, the VEGF165 cDNA fragment was cloned into pIRES2-NGF-EGFP in place of enhanced green fluorescent protein creating the plasmid pIRES2-NGF-VEGF165. pIRES2-NGF-VEGF165 was transfected into HEK293 cells and reverse transcription-polymerase chain reaction and Western blot analysis were used to test the co-expression of double genes. The NGF and VEGF165 genes were cloned and the DNA was sequenced, which revealed that NGF and VEGF165 were consistent with the sequence recorded in GenBank. Restriction analysis showed that the NGF and VEGF165 genes were inserted into the expression vector pIRES2-EGFP. Transfection of pIRES2-NGF-VEGF165 into HEK293 cells resulted in expression of the double gene at the mRNA and protein levels. The NGF and VEGF165 coexpression plasmid provides a novel expression system, enabling further study of the functions of the NGF and VEGF165 genes.

Induction of new ADAM related proteins from treated human Chang-liver cells.

Chang-liver cells is a cell line generated from human liver tissue, which is often used in scientific research. ADAMs are a family of proteins that consist of multi-domains, possess multi-functions and play a central role in normal or abnormal physiological conditions, such as regeneration and tumorigenesis. To investigate the expression and functional alteration of the ADAMs or ADAM related proteins in Chang-liver cells, this cell line was treated with heat stress, modified Hanks solution containing ATP or other buffers. Our results showed that the treatment with Hanks solution containing ATP induces Chang-liver cells to express new ADAM related proteins. To analyze these new ADAM related proteins, a cDNA expression library was constructed for the treated Chang-liver cells. A series of positive clones were obtained through immunoscreening with an ADAMs common antibody. A new ADAM related protein possessing alkaline protease activity was confirmed in these clones.