[Transplantation of atrial natriuretic peptide-expressing fibroblasts reduces blood pressure and increases urine volume in spontaneously hypertensive rats].

To investigate the potential of gene therapy for the treatment of chronic diseases such as hypertension, chronic heart failure, and chronic renal failure, we established the neonatal rat fibroblast line engineered to secrete the mutant human atrial natriuretic peptide (mhANP), and then transplanted the cell line into young spontaneously hypertensive rats (SHR) subcutaneously. We found that a single transplantation of the cell line caused an obvious rise in the concentration of mhANP in serum 7 d after transplantation ((135 +/- 8) vs (106 +/- 7) pg/mL, P < 0.01). The animals' blood pressure in test group was always remarkably lower than that of empty vector group within 42 d after transplantation, even though the blood pressure in all groups was constantly increasing in the process of ontogeny ((175 +/- 10) mm Hg vs (189 +/- 12) mm Hg, P < 0.05). A maximal blood pressure reduction of 33 mm Hg ((157 +/- 9) mm Hg vs (124 +/- 112) mm Hg, P < 0.01) was observed 14 d post cell transplantation. There was a marked increase in urine volume in test group from second week after treatment beginning ((5.9 +/- 0.7) mL/6 h vs (4.3 +/- 0.8) mL/6 h, P < 0.01) and the effect lasted 14 d ((6.1 +/- 1.1) mL/6 h vs (4.0 +/- 0.8) mL/6 h, P < 0.01), however the statistical difference in concentration of K+ and Na+ in serum and urine was not observed. The results suggested that subcutaneous implantation of fibroblasts-expressing mhANP significantly reduced blood pressure in young SHR during the period of ontogeny and efficiently improved their renal function and the somatic gene transfer of mhANP may have potential value in the treatment of human chronic diseases such as hypertension, chronic heart failure, and chronic renal failure.

[T-bet gene modified dendritic cells abrogate and reverse the airway inflammation in allergic asthma: experiment with mice].

OBJECTIVE: To explore the feasibility of the therapeutic strategy to use T-bet gene modified dendritic cells (DCs) to reverse the course of asthma. METHODS: (1) Mature DCs were derived from mononuclear cells obtained from femur of BALB/c mouse and divided into 3 groups, T-bet group transfected with recombinant adenovirus Ad-T-bet containing T-bet gene, LacZ group transfected with recombinant adenovirus Ad-LacZ containing LacZ gene, and control group. Seven days later ELISA was used to detect the interferon (IFN)-gamma level in the culture fluid. (2) Airway inflammation abrogating trial. Twenty-four BALB/c mice were sensitized with intraperitoneal injection of ovalbumin (OVA) (on day 1 and 15) to establish asthma models, and then randomly divided into 3 equal groups: T-bet group injected intravenously with T-bet-modified DCs on day 27, LacZ group injected with LacZ-modified DCs, and model control group without intravenous injection. Two days later the model mice began to undergo challenge by inhalation of OVA twice (on day 29 - 31). Eight mice were used as control group treated with PBS. On day 37 all mice were killed, ELISA was used to detect the blood interleukin (IL)-4 and IFN-gamma levels, and microscopy was conducted to observe the airway inflammation. (3) Airway inflammation reversing trial. Another 24 model mice were divided into 3 equal groups as well: re-challenged T-bet group injected intravenously with T-bet-modified DCs on day 27 and 42, re-challenged LacZ group injected intravenously with LacZ-modified DCs on day 27 and 42, and model control group. Since the day 45 OVA inhalation was given once a day for successive 3 days. On day 49 these mice were all killed to undergo the tests as mentioned above. RESULTS: The IFN-gamma level in the culture fluid of the T-bet gene modified DCs was (15.24 +/- 4.75) ng/ml, significantly higher than that of the LacZ gene modified DCs and control DCs [(3.08 +/- 0.61) and (2.35 +/- 0.41) ng/ml respectively, both P < 0.01]. The IFN-gamma in mice blood plasma of T-bet groups in abrogating and reversing trial were (130.2 +/- 10.5) and (145.7 +/- 16.7) pg/ml respectively, both significantly higher than those of the abrogating and reversing trial normal control groups [(25.0 +/- 6.5) and (24.6 +/- 5.9) pg/ml respectively], asthmatic model control groups [(20.7 +/- 4.5) and (16.5 +/- 7.0) pg/ml respectively] and LacZ groups [(17.6 +/- 7.0) and (24.2 +/- 9.0) pg/ml respectively] (all P < 0.01). However, the IL-4 levels in mice blood plasma of T-bet groups were both significantly lower than those of asthmatic model control groups and LacZ groups (all P < 0.01). The airway inflammation of T-bet groups were remarkable milder than those of the model control groups and LacZ groups. CONCLUSION: The asthma management strategy based on T-bet gene modified DCs is feasible with the plausible mechanism that the T-bet gene modified DCs regulate the T cells differentiation and polarization on the antigen presenting level.

[A strategy for targeting gene therapy against cancer mediated by epidermal growth factor receptor].

OBJECTIVE: To establish a protocol for the targeting gene therapy against cancer with rich epidermal growth factor receptor (EGFR). METHODS: A recombinant pcDNA3.1-PE III mut was constructed and combined with a non-viral vector, a fusion protein histone H1, epidermal growth factor C-loop previously expressed by us, to be a protein-DNA complex in vitro. Using the complex to treat BT-325 and Hela cancer cells with EGFR and JK cells without EGFR. The killing rates of the cells was calculated after 48 h of incubation at 37 degrees C. RESULTS: To BT-325 and Hela cells, the killing rates were 46.03% and 48.12% respectively. To JK cells, the complex had no killing function. CONCLUSION: The protocol for targeting gene therapy against cancer with EGFR has been established successfully.

[Construction of a non-viral vector H1s-EGFc and a preliminary study on its function].

OBJECTIVE: To construct a non-viral vector for targeting cancer gene therapy. METHODS: The coding sequence of H1s-EGFc was inserted into the expression vectors of Pichia pastoris, and the fusion protein was expressed in secretary way. H1s-EGFc was purified by anion exchange chromatography and size exclusion chromatography. H1s-EGFc fusion protein and "killing gene" expression recombinant pKG plasmid DNA were dissolved in serum-free RPMI-1640 culture to produce H1s-EGFc/pKG complex. HeLa cells, an epidermal growth factor receptor (EGFR) highly expressing cell line, and Jurkat cells, an EGFR non-expressing cell line, were cultured and transfected with H1s-EGFc/pKG complex of different concentrations. Trypan blue staining was used to calculate the number of live cells and the killing rate of H1s-EGFc/pKG. RESULTS: H1s-EGFc fusion protein was constructed and expressed with a purity of over 90%. When the concentrations of H1s-EGFc/pKG complex were 3 microg/ml, 6 microg/ml, and 9 microg/ml respectively the killing rates were 30.6%, 36.2%, and 58.1% respectively. CONCLUSION: The fusion protein H1s-EGFc binds functional gene efficiently and targets it into specific cells. It can be used as non-viral vector in target cancer gene therapy.

Overexpression and purification of recombinant atrial natriuretic peptide using hybrid fusion protein REF-ANP in Escherichia coli.

Atrial natriuretic peptide (ANP), a small peptide consisting of 28 amino acids, has been applied in clinical treatment for heart failure, but it can encounter proteolytic degradation during its expression in host cells. Therefore, it is usually reported that ANP was expressed as a part of fusion protein. The aim of our study was to use an overexpression system to express the fusion protein REF-ANP and to optimize a purification method. First, Escherichia coli DH5alpha was transformed with constructed expression vector containing two tandem copies of ref-anp gene and the fusion protein REF-ANP was overexpressed in shaking flask culture. Subsequently, the inclusion bodies were purified with reverse phase chromatography and pooled fractions were lyophilized. After this step, REF-ANP can be solubilized under native conditions without urea. After cleavage reaction, the sample was subjected to size exclusion chromatography and then rANP was polished with reverse phase chromatography. The final purity of rANP was more than 98% and the recovery of rANP per liter of shaking flask culture was more than 3mg. Such methods as mass spectrometry, capillary isoelectrofocusing analysis, and N-terminal amino acid sequence were used to identify rANP. The capillary isoelectrofocusing analysis showed that the pI of ANP was about pH 9.7. In this study, an efficient refolding and purification process should make scaling-up procedures easier and more successful than earlier reports. Moreover, it is possible that the refolding and purification method along with the overexpression system described in this article may offer new ideas on optimizing expression and purification of other kinds of short peptides.

[Hypotensive effect of encapsulated genetically engineered fibroblasts expressing mutant atrial natriuretic peptide in hypertensive rats].

OBJECTIVE: To study the inhibitive effect of subcutaneous implantation of capsule filled with fibroblasts engineered to secrete the mutant human atrial natriuretic peptide (mhANP) on blood pressure in young spontaneously hypertensive rats (SHR) and to explore the feasibility of gene therapy for the treatment of hypertension. METHODS: A recombinant retroviral vector pLHY24 bearing mhANP cDNA was constructed. Primary fibroblasts derived from the skin of new born SHR were cultured and transfected with the vector pLHY24 to establish a genetically modified fibroblast line or transfected with the blank vector pLNCX. Then the two kinds of cell culture were put into specially made capsules with microholes. The capsules filled with the genetically modified allogenic fibroblasts and those with blank vector were implanted into the dorsal subcutaneous tissues of two groups of 10 young SHR respectively. The plasma ANP, blood pressure, urine volume, potassium and sodium concentrations in urine, and body weight were determined every week for 7 weeks. RESULTS: After delivery of retroviral vector bearing mhANP gene into the packaging cell PA317 and the primary fibroblasts, immunoreactive mhANP were detected in the cell culture medium at the concentration of (5.84 +/- 0.07) and (13.37 +/- 2.36) ng.10(-6) cells.24 h(-1) respectively. One week after implantation of the genetically modified allogenic fibroblasts the plasma level of mhANP was 131 pg/ml +/- 8 pg/ml, significantly higher than that in control group (104 pg/ml +/- 7 pg/ml, t = 8.62, P < 0.001). Although the blood pressure increased along with aging after the gene transfer, an obvious delay of blood pressure increase was seen significantly lower in test group [from (129 +/- 9) to (169 +/- 9) mm Hg] than that in the control group [from (145 +/- 10) to (181 +/- 9) mm Hg, P < 0.05 or 0.01]. A maximal blood pressure reduction of 28 mm Hg in young SHR was observed 7 days after transplantation as compared with controls. In addition, there was an obvious increase in urine volume of test group 2 weeks after transplantation and the effect lasted for more than 2 weeks. However, there were no statistical differences in body weight and the concentrations of K(+) and Na(+) in urine. CONCLUSION: Subcutaneous implantation of the encapsulated genetically modified fibroblasts engineered to secrete mutant ANP causes a lowering effect of blood pressure in young SHR.

High-level expression of foreign genes via multiple joined operons and a new concept regarding the restricted constant of total amount of plasmid DNA per Escherichia coli cell.

OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell. METHODS: Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR). RESULTS: No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P < 0.01). Further calculation showed that the total amount of plasmid DNA per cell was not significantly different in each series (P > 0.05) and restricted to some extent. CONCLUSIONS: Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.

[Expression and purification of gene transfer vehicle mediated by epidermal growth factor receptor].

OBJECTIVE: To create a gene transfer vehicle for targeting gene therapy of cancer with epidermal growth factor receptor overexpressing. METHODS: Encoding sequences of the first domain of histone gene (H1) and EGF C-loop (EGFc) were obtained by PCR amplification. These two DNA fragments were ligated by EcoR I site, and cloned and sequenced. E. coli expression vector of the fusion gene was then constructed. The fusion protein H1EGFc was purified by specific band isolation from SDS-PAGE. RESULTS: The molecular weight of purified protein was consistent with the designed request. Its purity reached 94.02%. CONCLUSION: A fusion protein H1EGFc was expressed and purified.

[Site-directed mutagenesis of atrial natriuretic peptide gene and effect of the mutations on its diuretic activity in nephrotic rats].

OBJECTIVE: To prolong the half-life and enhance the biological activity of the human atrial natriuretic peptide (hANP), a peptide hormone, which is synthesized and released mainly by cardiac atrial myocytes and possesses potent natriuretic, diretic, and vasorelaxant properties. METHODS: The site-directed mutation technique based on polymerase chain reaction was performed to get the mutant of the human ANP gene (mhANP), and the retroviral expression vector, pLHY24, in which mhANP gene is under the transcriptional control of the human cytomegalovirus promoter, was constructed. The naked plasmid DNA of pLHY24 and positive control vector, pLHY19, in which the wild-type hNAP gene is in the same conditions as mhANP gene in pLHY24, and negative control vector, pLNCX without purpose gene, at a dose of 5 mg/kg body weight was injected intramuscularly into the rats with experimental renal disorder induced with adriamycin (ADR), respectively. RESULTS: DNA sequencing result proved that the respected mhANP gene with the point mutations of TTC(131)/Phe-->TCC/Ser and ATG(135)/Met-->ATA/Ile has been obtained. In comparison with negative control group (87 +/- 7.1 pg/ml), a single intramuscular injection of expression vector harboring mhANP or hANP gene resulted in an obvious increase in plasma level of mhANP (107 +/- 7.8 pg/ml, t = 4.65, P < 0.01) or hANP (113 +/- 8.6 pg/ml, t = 5.71, P < 0.01) 5 days after injection. A significant elevation in the ratio of urine volume to body weight was occurred after both of mhANP gene and hANP gene delivery as compared with negative control and the effect lasted for more than 15 days. The diuretic activity of mhANP gene delivery was 1.6-, 2.0-, and 1.9-fold higher than that of hANP gene 5, 10, and 15 days after gene transfer, respectively. However, there were no statistical differences in the concentrations of K(+) and Na(+) in urine. CONCLUSIONS: Both of mhANP and hANP gene delivery into the rats with experimental nephropathy could improve their directic function obviously and the diuretic activity of the former is stronger than that of the latter significantly.

[Comparative analysis of diuretic activities of human ANP gene injected intramuscularly and intravenously in adriamycin-induced nephrotic rats].

In order to explore the feasibility of gene therapy strategy based on the human atrial natriuretic peptide (hANP) gene delivery for the treatment of nephropathy and compare the diuretic activities of the hANP gene injected intramuscularly(i.m.) and intravenously(i.v.), the naked retroviral vector DNA harboring the hANP cDNA under the control of retroviral 5' long terminal repeat at a dose of 5 mg/kg body weight was injected i.m. or i.v. into the nephrotic model rats induced with adriamycin(ADR) injected i.v. at a dose of 7.5 mg/kg body weight. A single injection of the hANP gene resulted in a marked elevation in plasma level of hANP 5 days after gene delivery and a significant increase in the ratio of urine volume to body weight and the diuretic effect continued for more than 15 days. In addition, there was a significant rise in the body weight of treatment groups as compared with that of negative control group and no difference in the concentrations of electrolytes in urine between groups. There was no significant differences in total effects resulted from the two routes of gene delivery and the way of gene delivery through the skeletal muscle is simpler and easier. These results suggest that somatic gene delivery of the hANP gene could enhance the renal functions in nephrotic rats significantly and would be a potential strategy for the treatment of renal disorders.