Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

Hepatitis B virus envelope as a targeting gene transfer vector for hepatic cancer cells

Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.

SEQUENCE STUDY AND RETROVIRUS MEDIATED GENE TRANSFER IN MAMMALIAN CELL SYSTEMS OF HUMAN INTERLEUKIN 2 cDNA

Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification using a pair of synthesized primers. DNA sequence analysis demonstrated that the IL- 2 cDNA cloned from the Chinese tonsil donor is identical with the data reported so far, reflecting the high structural conservation of this gene. The human IL- 2 cDNA was inserted into XM6 to construct a recombinant retroviral expression vector XM6-IL2, having human IL-2 cDNA driven by the 5' LTR of MMLV. This vector was successfully passaged through ψ 2 and PA317 cells to yield high producer lines of ecotropic and amphotroplc infectious viruses. The murine myeloma cell line SP2/0 after being infected by retrovirus released from high titer PA317 constitutlvely secreted IL- 2 activity into the culture medium when assayed for T cell proliferative capacity. Activated human T cells are also exposed to the infectious retrovirus XM6- IL2. In sharp contrast to the parallel controls, the infected T cells with or without the furtheraddition of 500 units/ ml of exogenous IL- 2 proliferated and formed colonies of significant size under the selection pressure of G418. However, their growth in vitro could only be maintained for about 3 weeks. These facts demonstrated that gene transfer of human IL- 2 cDNA viaretrovirus is far from being sufficient to maintain the relatively long-termgrowth and clonal expansion of human T cell subpopulations in vitro.

RETROVIRUS-MEDIATED TNF-α GENE TRANSFER INTO TCA8113 CELLS

Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retroviral vector carring genes for both TNF-α and NeoR. After that, presence and expression of exoge-nous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate ofthe cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique,EL1SA technique, FACS technique, 60Co irradiation inactivation test, cryopreservation test, and Ames test, respec-tively. Results The presence of both TNF-a and NeoR gene and expression of TNF-α gene were demonstrated intransgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parentalcells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, allthe cells died by dl4 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h wasno different from that cryopreserved for 1 week after irradiation, the level of TNF-α secreted by the cryopreservedcells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cellshave no mutagenic activity. Conclusion TNF-α gene can be transduced into Tca8113 cells with retroviral vec-tor, and the cells can express TNF-α. Expression of HLA 1,11 antigens on Tca8113 cells can be increased by TNF-αgene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservationmethod for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.

Gene transfer and expression in rat anastomotic artery in vivo using adenoviral vector

Objective To observe the efficiency and time course of gene expression and the safety of adenoviral vector mediated gene transfer in vivo.Methods After soaking soluble stents in a high concentration of glucose solution containing Adv5-CMV (cytomegalovirus) (control group) or Adv5-CMV/LacZ (treatment group) for 30 minutes, the stents were inserted into the lumina of cut rat carotid arteries and end-to-end anastomoses of the cut carotid were performed with standard microvascular surgical techniques. On days 2, 7, 14, 28, 60 and 90 after gene transfer, anastomotic arteries of the two groups were observed. On days 7 and 14, the ascending aortas, hearts, brains, livers, lungs, spleens and kidneys of the treatment group were observed. All samples were analyzed for the presence of β-galactosidase activity and histochemical staining.Results β-galactosidase activity was not detected in the carotid arteries of the control group and organs not directly exposed to adenoviral vector of the treatment group. The amount of β-galactosidase activity (×10-3?U/g tissue) in the treatment group on the 2nd, 7th, 14th, 28th, 60th and 90th day after gene transfer was 3.87, 11.38, 9.8, 6.43, 3.18 and 2.43, respectively. Microscopic examination of sections from vessels of the control group and from the aortas, hearts, brains, livers, lungs, spleens or kidneys of the treatment group revealed no X-gal staining. Microscopic examination of carotid arteries of the treatment group revealed blue-staining in all anastomotic arteries and in all layers of the arterial wall observed on days 7 and 14 after gene transfer.Conclusion Adenoviral vector can effectively infect blood vessels in vivo. After adenoviral vector mediated direct gene transfer into anastomotic rat carotid arteries, recombinant gene expression began on day 2, peaked between days 7 and 14, prominently declined after day 28, and persisted at low levels more than three months. A recombinant gene could be delivered to a specific site by direct gene transfer in vivo by adenoviral vector infection.

Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer

Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing