Transplantation of betatrophin-expressing adipose-derived mesenchymal stem cells induces ß-cell proliferation in diabetic mice.

Recent progress in regenerative medicine has suggested that mesenchymal stem cell (MSC)-based therapy is a novel potential cure for diabetes. Betatrophin is a newly identified hormone that can increase the production and expansion of insulin-secreting ß-cells when administered to mice. In this study, we evaluated the effect of betatrophin overexpression by human adipose-derived MSCs (ADMSCs) by in vitro experiments, as well as following their transplantation into a mice with streptozotocin (STZ)-induced diabetes. The overexpression of betatrophin did not affect the ADMSCs in terms of proliferation, differentiation and morphology. However, the co-culture of human islets with ADMSCs overexpressing betatrophin (ADMSCs-BET) induced islet proliferation, ß-cell specific transcription factor expression, and the islet production of insulin under the stimulation of glucose or KCl and Arg. In addition, ADMSCs-BET enhanced the anti-inflammatory and anti-apoptotic effects of the co-cultured islets compared with ADMSCs cultured alone. In mice with STZ-induced diabetes, the transplantation of ADMSCs-BET ameliorated the hyperglycemia and weight loss associated with STZ-induced diabetes; ADMSCs-BET also significantly enhanced the ratio of ß-cells per islet compared to the transplantation of ADMSCs alone. Thus, our study demonstrates a novel strategy for inducing ß-cell regeneration. ADMSCs-BET may replace insulin injections by increasing the number of endogenous insulin-producing cells in patients with diabetes. This combined strategy of ADMSC transplantation and gene therapy may prove to be a useful therapy for the treatment of diabetes.

Generation and characterization of immortalized rat retinal microglial cell lines.

Retinal microglia play an important role as resident immunocompetent and phagocytic cells in the event of injury and disease. Retinal microglia and microglia precursor transplantation show a rescue effect in ischemic retina and retinal degeneration. However, studies of retinal microglia have been hampered by the difficulty of obtaining sufficient numbers of microglia. One way to circumvent this difficulty is to establish permanent retinal microglia cell lines. In the present study, we report the generation of immortalized retinal microglia, T-MG cells, from postnatal day 3 rat retinal tissue using a lentiviral vector encoding SV40 large T antigen. The T-MG cells exhibited cell-type-specific antigens for monocyte/macrophage lineage cells, including CD11b (OX42), ED1 (OX6), and Iba1, and actively phagocytosed latex beads. In addition to primary retinal microglia, T-MG cells also have the ability to recruit into chemokines. Treatment of T-MG cells with lipopolysaccharide (LPS) led to increased levels of tumor necrosis factor-α, interleukin-1ß, and inducible nitric oxide synthase. Genome-wide microarray analysis showed a less than 1% difference in the genes between the T-MG cells and the control primary retinal microglia. The T-MG cells exhibited properties similar to those of the primary retinal microglia and should have considerable utility as an in vitro model for the study of retinal microglia in health and as a curative therapy and an in vivo model for the study of retinal microglia in disease.

[The cell-type-specific HSV-tk gene expression of suicide gene therapy system regulated by lens-specific promoter LEP503].

OBJECTIVE: To explore the specific expression of HSV-tk gene and killing effects on ocular leading cells of the enhanced specific HSV-tk/GCV gene therapy system regulated by lens-specific promoter LEP503. METHODS: Experimental research. The enhanced specific HSV-tk/GCV gene system of two vectors were constructed (Lenti-LEP503-HSVtk-Cre and Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk). The lentiviral vectors were produced by transient transduction of transfering vectors, packaging vectors and enveloping vector into 293T cells. Virus was collected with ultracentrifugation and resuspended with 1 ml phosphate buffered saline and stored at -80°C. The HLEC and RPEC, NIH3T3, 293T cells were transduced with the enhanced specific HSV-tk gene system. The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy, flow cytometry and RT-PCR. The killing effects of HLEC and RPEC at the concentration of 20 mg/L GCV were assayed and compared by flow cytometry and CCK-8 kit. Difference of RPE cell viability among groups was evaluated by analysis of variance (ANOVA). RESULTS: Expression efficiency of EGFP in RPEC group was 62.3%, 68.3% in NIH3T3 group, 75.8% in 293T group, whereas 17.5% in HLEC group. There was higher expression of HSV-tk at mRNA level in HLEC group than that in RPEC group. The relative intensity of HSV-tk mRNA in HLEC group transduced with the enhanced specific HSV-tk gene system was 4.01, whereas 0.29 in RPEC group. At the concentration of 20 mg/L GCV after 72 hours, the percentage of apoptosis detected by the flow cytometry in HLEC group transduced by the enhanced specific HSV-tk gene system was 76.51%, and 2.44% in RPEC group. There was no significant difference in the RPE cell viability among the enhanced specific HSV-tk gene combination-RPE group, normal-RPE group and negative-RPE control group at the concentration of 20 mg/L GCV after 72 hours (MD(1) = -0.047, P = 0.671; MD(2) = 0.027, P = 0.912). CONCLUSIONS: The enhanced specific HSV-tk gene system express HSV-tk selectively in HLEC. At the concentration of 20 mg/L GCV, it is effective against the proliferation of HLEC in vitro, but has less kill effect on RPEC.

ATRA enhances the bystander effect of suicide gene therapy driven by the specific promoter LEP 503 in human lens epithelial cells.

PURPOSE: To establish a novel, targeted lentivirus-mediated LEP503-HSV-tk/GCV suicide gene therapy system combined with all trans-retinoic acid (ATRA) for the inhibition of human lens epithelial cell (HLEC) proliferation and treatment of posterior capsular opacification (PCO) after cataract surgery; to estimate the enhancement of the bystander effect by ATRA; and to explore the role of Connexin43 (Cx43) mediated gap junctional intercellular communication (GJIC) in the bystander effect of the HSV-K/GCV system. METHODS: A Lenti-LEP503-HSV-tk-EGFP vector was generated by cloning the lens-specific promoter LEP503 (lens specific promoter 503) from genomic DNA of HLECs by PCR. The vector was then inserted into the promoter-less vector from lentivirus-based (CMV)-HSV-tk-EGFP. The expressional specificity of the LEP503 promoter was assessed by investigating the expression of EGFP (enhanced green fluorescent protein) and HSV-tk (herpes simplex virus thymidine kinase) mRNA, both driven by Lenti-LEP503-HSV-tk-EGFP vector, by fluorescence microscopy, RT-PCR, flow cytometry, and western blot assays in HLECs, human adult retinal pigment epithelium cells (RPECs), human adult skin fibroblast cells (ASFCs), and Hela cells. Morphological changes were observed by fluorescence microscopy and cell viability was determined using the Cell Counting kit-8 Cell Proliferation (CCK-8) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays after Lenti-LEP503-HSV-tk/GCV system combined with ATRA treatment on HLECs. Flow cytometry, DNA fragmentation, and western blot assays were employed to analyze the mechanisms of bystander effects. RESULTS: The promoter LEP503-mediated HSV-tk was specifically expressed in HLECs, and ATRA dose-dependently strengthened the bystander effect following LEP503-mediated HSV-tk/GCV gene therapy against lens cells by upregulating the expression of the gap junction protein Cx43. CONCLUSIONS: The Lenti-LEP503-HSV-tk/GCV suicide gene therapy system, combined with ATRA as an adjuvant, may be a feasible supplementary method for PCO treatment that targets residual lens cells.

CCR2 overexpression promotes the efficient recruitment of retinal microglia in vitro.

PURPOSE: Retinal microglia can be activated and recruited by chemokines and play a protective role in early retinal degeneration. CC-chemokine ligand 2 (CCL2) and its receptor, CC-chemokine receptor 2 (CCR2), have been implicated as key mediators for the trafficking and accumulation of microglial cells in lesioned tissue. The current study investigates whether the overexpression of CCR2 allows microglia to migrate toward CCL2 more efficiently. METHODS: Primary microglial cells were transduced with lentivirus carrying green fluorescent protein (GFP)-tagged CCR2 (CCR2-GFP). Overexpression of CCR2 was assessed by western blot analysis and fluorescence-assisted cell sorting. The chemotaxis of primary microglia transduced with lentivirus carrying CCR2-GFP was compared to either those transduced with GFP alone or those not transduced, using a chemotaxis chamber assay. RESULTS: Primary microglia showed a high transduction rate following lentivirus application and maintained normal microglial morphology and a significant overexpression of CCR2 protein. We found that CCL2-mediated chemotaxis is concentration and time dependent in microglia. The chemotactic response of microglia cells overexpressing CCR2-GFP was significantly increased compared to that of nontransduced and GFP-expressing microglia. CONCLUSIONS: These findings suggest that microglia can be efficiently transduced with CCR2-GFP lentiviral vectors and that the overexpression of CCR2 in retinal microglia promotes their chemotaxis in response to chemokines, suggesting that these cells may be promising targets for cell-based therapeutic manipulation in retinal disease.

[Preliminary research on the regeneration of injured rabbit vocal folds after the transplantation of human amniotic epithelial cells].

OBJECTIVE: To investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring. METHODS: hAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only ("injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively. RESULTS: hAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased. CONCLUSIONS: hAEC survived in the vocal folds lamina propria, and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.

[Expression and significance of telomerase activity of lens epithelial cells in posterior capsule opacification of rabbits].

OBJECTIVE: To explore the expression of telomerase activity of the lens epithelial cells in posterior capsule opacification of rabbits. METHODS: Clear corneal tunnel phacoemulsification was performed in both eyes of 11 New Zealand rabbits. After the model of posterior capsule opacification succeed in all eyes. Then, telomerase activity of the lens epithelial cells in the equator and posterior capsule opacification of 20 eyes was detected with TRAP-ELISA and TRAP-PAGE techniques. RESULTS: HepG2 cells were used as positive controls. Telomerase activity was detected in the lens epithelial cells in the equator capsule and posterior capsule opacification in rabbits, which showed several dim gradient stripped electrophoresis pattern. A450 - 690 value of telomerase activity in the equator capsule and posterior capsule opacification was 0.85 +/- 0.23 and 0.67 +/- 0.19, respectively, indicating statistically significant difference (t = 2.526, 0.021; P < 0.05). CONCLUSIONS: Telomerase activity exists in the lens epithelial cells of posterior capsule opacification in rabbits. The telomerase activity in this area is lower than that in the equator capsule.

[The effect of lentivirus-mediated suicide gene therapy on lens epithelial cells].

OBJECTIVE: To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death. METHODS: a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated. RESULT: the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect). CONCLUSION: GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.

[Construction of bicistronic lentiviral vectors carrying HSV-tk and EGFP gene].

OBJECTIVE: To construct CMV-mediated Lentiviral vectors coexpressing EGFP and HSV-tk gene in order to establish a novel lentiviral vector platform for the suicide gene therapy of eye disease. METHODS: The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. HSV-tk fragments from pcDNA3-HSV-tk were cloned into the site of lenti-internal ribosomal entry site (IRES)-EGFP to construct the bicistronic lenti-HSV-tk-EGFP vector. Human embryonic kidney 293T cells were co-transfected with the lentiviral vector (three plasmids) by calcium phosphate DNA precipitation. HLEC, HXO-Rb(44), SH-SY-5Y and Hela cells were transfected with viral production and the expression of EGFP was examined under fluorescent microscope after transfection. The expression of HSV-tk and EGFP was examined by RT-PCR. RESULTS: Lentivirus mediated stable integration and efficient expression of EGFP and TK genes in the cells tested. Coexpression of HSV-tk and EGFP in HLECs mediated by lentiviral vectors was confirmed by the result of RT-PCR. The transfection efficiency for HLECs was about 100% at MOI = 100, and kept the same level for at least 6 months. CONCLUSION: The bicistronic lentiviral vector platform carrying HSV-tk-EGFP is an efficient and stable gene transfer vector, it might be used for suicide genes therapy in the treatment some eye disease.

Effects of bicistronic lentiviral vector-mediated herpes simplex virus thymidine kinase/ganciclovir system on human lens epithelial cells.

Posterior capsule opacification (PCO) is the most common complication after phacoemulsification cataract surgery. Hyperplasia of the lens epithelial cell after phacoemulsification is thought to be an important feature contributing to PCO. In this study,we investigated the feasibility of killing the human lens epithelial cells (HLECs) by lentivirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene/ganciclovir (GCV) in HLECs and studied the bystander effect. HLECs were infected with lentiviral vectors coexpressing HSV-tk and enhanced green fluorescent protein (EGFP) or expressing EGFP alone and treated with ganciclovir. Infection efficiency was assessed by fluorescence microscopy, fluorescence-activated cell sorting, and reverse transcription PCR. The cytotoxicity of the HSV-tk/GCV suicide gene therapy system was assessed by DNA ladder and electron microscopy. The time effect and bystander effect of HLEC growth inhibition were evaluated with cell proliferation assay. Lentiviral vector-mediated stable integration and efficient expression of HSV-tk in HLECs, with infection efficiency exceeding 95% GCV at concentrations of 15 approximately 25 mug/ml, significantly induced apoptosis or necrosis of infected HLECs. GCV also killed normal cells mixed with HSV-tk infected cells. The bystander effect markedly increased the cytotoxicity of the HSV-tk/GCV system. Our results suggest that bicistronic lentiviral vectors can efficiently integrate several genes into HLECs and may be a gene therapy platform. Lentivirus-mediated suicide gene therapy might be a feasible treatment strategy to prevent capsule opacification.

A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis.

AIM: To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis. METHODS: OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibroblast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively. RESULTS: Weak A20 expression was found in MC3T3-E1 cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P< 0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P< 0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis. CONCLUSION: We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.