Development of FAP-Targeted Chimeric Antigen Receptor NK-92 Cells for Non-Small Cell Lung Cancer.

OBJECTIVES: Over the past two decades, great progress has been made in advancing the early detection and multimodal treatment of non-small cell lung cancer (NSCLC). However, overall cure rates and survival rates of NSCLC are still not satisfactory, and research into new therapies is needed. This study attempted to construct human Fibroblast Activation Protein-Chimeric Antigen Receptor Natural killer (NK)-92 cells (hFAP-CAR-NK-92 cells) and explore their potential therapeutic effects in NSCLC. METHODS: Immunohistochemistry analysis was carried out to examine fibroblast activation protein (FAP) and Gasdermin E (GSDME) expression in clinical specimens of lung adenocarcinoma and squamous cell carcinoma tissue. Then the engineered hFAP-CAR-NK-92 cells efficiency was determined in vitro with lactate dehydrogenase (LDH) cytotoxicity assay and the cell morphology of A549, H226, and cancer-related fibroblast (CAF) was observed by electron microscopy. After the co-culture of target cells and effect cells, flow cytometry was employed for examining the CD107a expression in the effect cells, and western blotting was conducted for the cleavage levels of Caspase 3 and GSDME proteins in the target cells. The safety and efficacy of hFAP-CAR-NK-92 cells adoptive transfer immunotherapy in a tumor-bearing mouse were evaluated. RESULTS: Clinical studies have shown FAP positivity in patients with NSCLC. Compared with A549 or H226 cells alone, FAP expression was notably raised in A549+CAF cells or H226+CAF cells in nude mice, respectively (p < 0.05). The killing efficiency of K562 cells was not significantly different between hFAP-CAR-NK-92 and NK-92 cells (p > 0.05). The hFAP-CAR-NK-92 cells presented a higher killing efficiency against the hFAP-target (A549-hFAP, H226-hFAP and CAF-hFAP) cells than the NK-92 cells (p < 0.05). The degranulation of CD107a and cleavage levels of GSDME and Caspase 3 protein in the hFAP-CAR-NK-92 group were higher than those in the NK-92 group (p < 0.05). The 300 nM Granzyme B also induced pyroptosis in hFAP- or GSDME-positive cells (p < 0.05). In vivo experiments revealed that hFAP-CAR-NK-92 cells inhibited tumor progression of hFAP-positive NSCLC (p < 0.05). CONCLUSIONS: In this study, we successfully constructed hFAP-CAR-NK-92 cells and confirmed that hFAP-CAR-NK-92 cells could target hFAP-positive NSCLC to inhibit the progression of NSCLC by activating the Caspase-3/GSDME pyroptosis pathway.

Closantel Suppresses Angiogenesis and Cancer Growth in Zebrafish Models.

Angiogenesis has emerged as an important therapeutic target in several major diseases, including cancer and age-related macular degeneration. The zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. In this study, we have taken advantage of the transgenic Tg (fli1a:EGFP) zebrafish line to screen the U.S. Drug Collection Library and identified 11 old drugs with antiangiogenic activity, including Closantel, an FDA-approved broad-spectrum salicylanilide antiparasitic drug for a variety of types of animals. Closantel was confirmed to have antiangiogenic activity in zebrafish with a half-inhibitory concentration (IC50) at 1.69 µM on the intersegmental vessels and 1.45 µM on the subintestinal vessels. Closantel also markedly suppressed cancer growth in zebrafish xenotransplanted with human lymphoma, cervical cancer, pancreatic cancer, and liver cancer cells, generally in a dose-dependent manner. These data reveal that Closantel has antiangiogenesis and anticancer effects and could be a potential drug candidate for animal and human cancer treatments. Further study is needed to clarify the mechanisms involved in the antiangiogenesis and anticancer effects of Closantel.

Sanguinarine inhibits Rac1b-rendered cell survival enhancement by promoting apoptosis and blocking proliferation.

AIM: Small GTPase Rac1 is a member of the Ras superfamily, which plays important roles in regulation of cytoskeleton reorganization, cell growth, proliferation, migration, etc. The aim of this study was to determine how a constitutively active Rac1b regulated cell proliferation and to investigate the effects of the Rac1b inhibitor sanguinarine. METHODS: Three HEK293T cell lines stably overexpressing GFP, Rac1-GFP or Rac1b-GFP were constructed by lentiviral infection. The cells were treated with sanguinarine (1 µmol/L) or its analogue berberine (1 µmol/L) for 4 d. Cell proliferation was evaluated by counting cell numbers and with a BrdU incorporation assay. The levels of cleaved PARP-89 (an apoptosis marker) and cyclin-D1 (a proliferative index) were measured using Western blotting. RESULTS: In 10% serum-containing media, overexpressing either Rac1 or Rac1b did not significantly change the cell proliferation. In the serum-starved media, however, the survival rate of Rac1b cells was significantly increased, whereas that of Rac1 cells was moderately increased. The level of cleaved PARP-89 was significantly increased in serum-starved Rac1 cells, but markedly reduced in serum-starved Rac1b cells. The level of cyclin-D1 was significantly increased in both serum-starved Rac1 and Rac1b cells. Treatment with sanguinarine, but not berberine, inhibited the proliferation of Rac1b cells, which was accompanied by significantly increased the level of PARP-89, and decreased both the level of cyclin-D1 and the percentage of BrdU positive cells. CONCLUSION: Rac1b enhances the cell proliferation under a growth-limiting condition via both anti-apoptotic and pro-proliferative mechanisms. Sanguinarine, as the specific inhibitor of Rac1b, is a potential therapeutic agent for malignant tumors with up-regulated Rac1b.

[Prokaryotic expression and characterization of two recombinant receptor-binding domain(RBD) proteins of human coronavirus NL63(HcoV-NL63)].

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.

Resveratrol improves learning and memory in normally aged mice through microRNA-CREB pathway.

Resveratrol (RSV) is a natural compound found in grapes and red wine. It has been well known for its beneficial effects as a dietary supplement in prevention of cardiovascular diseases and cancer. Recently, in vitro studies have reported the neuroprotective role of RSV in neurodegenerative process in Alzheimer's disease (AD). However, in vivo effects of RSV on the decline of brain function accompanying the aging process, especially those on cognitive loss, have not been not investigated. Here we report that, after intraventricular injection of RSV for one week in 8-9 month-old mice, the long-term memory formation and the LTP induction from hippocampus CA1 were improved. The RSV enhancement effects were blocked in SIRT1 mutant mice. Additional experiments suggest that RSV effects are likely to be mediated through reduced expressions of miR-134 and miR-124, which may in turn up-regulate CREB levels to subsequently promote BDNF synthesis. These findings demonstrate a role for RSV in cognition and a microRNA-CREB-BDNF mechanism by which RSV regulates these processes, demonstrating its value as a potential therapeutic target against CNS disorders in aging.

[Intravesical anchoring of streptavidin-tagged interleukin-4 fusion protein for immunotherapy of mouse superficial bladder cancer].

OBJECTIVE: To evaluate the antitumor efficacy of streptavidin-tagged interleukin-4 (IL-4-SA) bifunctional fusion protein in the immunotherapy of mouse model of superficial bladder cancer. METHODS: IL-4-SA fusion protein was prepared and its biological activity was determined. One day after MB49 cell implantation, 100 µl of 1 mg/ml NHS-PEO4-biotin was instilled into the bladder for 30 minutes, followed by intravesical instillation of 100 µl PBS, GFP-SA+IL-4 or IL-4-SA and incubation for 1 hour. The bladder irrigation was performed twice a week for three weeks. The CTL cytotoxicity and profile of CD8(+) tumor-infiltrating lymphocytes were analyzed. RESULTS: The IL-4-SA fusion protein was durably anchored to the biotinylated mucosal surface of bladder wall for up to 5 days.On day 80 after the implantation of MB49 cells, all of PBS-treated mice died, and 8 out of 10 mice in the GFP-SA-treated group died from tumor burden.In contrast, 5 out of 10 mice in the IL-4-SA-treated group were tumor-free. The MB49 tumor-specific cytotoxicity from mice in the IL-4-SA group was (11.3 ± 1.2)%, (22.7 ± 1.5)% and (31.0 ± 3.0)% at the effector to target ratios of 1:1, 25:1 and 50:1, respectively. But the corresponding cytotoxicity was (4.3 ± 0.6)%, (9.0 ± 1.0)% and (14.3 ± 1.5)% in the GFP-SA+IL-4 group, and (3.3 ± 0.6)%, (7.3 ± 0.6)%, (12.7 ± 2.1)% in the PBS group. The tumor-specific cytotoxicity in the SA-CD40L group was significantly higher than that in the control groups (P < 0.05). The infiltrating CD8(+) T cells in tumors in the IL-4-SA-treated group were increased compare with those in other groups. CONCLUSION: Intravesical anchoring of IL-4-SA elicites strong and long-lasting immunoprotection against superficial bladder cancer, and the novel immunotherapy may be an attractive therapeutic alternative in future.

[Norcantharidin inhibits DNA replication initiation protein Cdc6 in cancer cells].

OBJECTIVE: To explore the inhibitory effect of norcantharidin (NCTD) on the expression of DNA replication initiation protein Cdc6 in cancer cells. METHODS: MTT assay was performed to detect the inhibitory effect on different cancer cell lines, including HeLa, HepG2, Jurkat and Ramos cells. The effect of NCTD on Cdc6 protein level was detected by Western blotting, and BrdU incorporation assay was used to evaluate the DNA replication of the cells. RESULTS: NCTD significantly inhibited the proliferation of the cells and caused degradation of Cdc6 protein to result in the inhibition of the DNA replication of the cells shown by BrdU incorporation assay. CONCLUSION: NCTD can induce the degradation of Cdc6 in cancer cells to produce an anti-cancer effect.

[Expression, purification and bioactivity evaluation of streptavidin-tagged human interleukin-21 fusion protein].

OBJECTIVE: To obtain streptavidin-tagged human interleukin-21 (hIL21) fusion protein and evaluate its bioactivities. METHODS: hIL21-SA-pET21 and pET24a-SA- hIL21 plasmids were constructed and expressed in BL21(DE3) host bacteria. The hIL21-SA and SA- hIL21 fusion protein were purified through Ni-NTA affinity chromatography and refolded by dialysis. Flow cytometry was used to detect hIL21-SA and SA- hIL21 fusion protein on the biotinylated MB49 tumor cells. MTT assay was used to evaluate the effect of the fusion protein on the proliferation of human peripheral blood lymphocytes (PBLs) stimulated by Anti-CD3. RESULTS: The recombinant fusion proteins were highly expressed in BL21(DE3) at about 30% of the total bacterial proteins. The two fusion proteins exhibited bifunctional activities, i.e. both biotin-binding property and hIL21 activity and SA-mediated high-affinity binding to biotinylated cell surfaces (with anchoring modified rate of about 95.18% and 96.91%). CONCLUSION: We have successfully obtained bifunctional fusion protein hIL21-SA and SA- hIL21,which will provide a basis for further study of tumor biotherapy using the proteins.

A novel immunotherapy for superficial bladder cancer by the immobilization of streptavidin-tagged bioactive IL-2 on the biotinylated mucosal surface of the bladder wall.

BACKGROUND AND OBJECTIVE: Intravesical administration of Bacillus Calmette-Guerin (BCG) after transurethral resection is by far the most effective local therapy for superficial bladder cancer, the fifth most common cancer in the world. However, approximately one-third of patients fail to respond and most patients eventually relapse. In addition, there are pronounced side effects of BCG therapy, such as BCG sepsis and a high frequency of BCG-induced cystitis. This study established a novel immunotherapy through immobilization of streptavidin-tagged human IL-2 (SA-hIL-2) on the biotinylated mucosal surface of bladder wall. METHODS: A mouse orthotopic model of MB49 bladder cancer was established by perfusing MB49 cells into mouse bladders. The SA-hIL-2 fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall. Treatment began on day 1 after MB49 implantation, once every 3 days for 6 times. Immunohistochemical assay was performed to assess the persistence of SA-hIL-2 immobilized on the biotinylated mucosal surface of the bladder wall. The mice were monitored for tumor growth and survival. On day 60 after MB49 implantation, the SA-hIL-2-cured mice, which were found to have no hematuria or palpable tumors, were challenged with wild-type MB49 cells implanted into the pretreated bladder and monitored for survival. RESULTS: SA-hIL-2 could be immobilized efficiently and durably on the bladder mucosal surface as long as 7 days. On day 60 after MB49 implantation, 9 out of 20 SA-hIL-2-treated mice survived, but all mice in PBS control group died. More importantly, 5 out of 9 tumor-free mice in the SA-hIL-2 group were protected against a second intravesical wild-type MB49 tumor challenge. CONCLUSIONS: SA-hIL-2 fusion protein could significantly inhibit tumor growth and extend the survival time in the orthotopic model of MB49 bladder cancer.

[Immobilization of streptavidin-tagged bioactive hTNF-alpha on biotinylated mucosal surface of the bladder wall for treatment of superficial bladder cancer in mice].

OBJECTIVE: To investigate a novel immunotherapy through immobilization of streptavidin-tagged hTNF-alpha on the biotinylated mucosal surface of the bladder wall for bladder cancer treatment in mice. METHODS: A total of 120 female C57BL/6j mice were randomized into 5 equal groups, namely blank control, PBS, soluble hTNF-alpha, SA-GFP, and SA-hTNF-alpha treatment groups. Twenty-four hours after establishment of a mouse model of orthotopic superficial bladder cancer, SA-hTNF-alpha fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall, which was repeated every 4 days for a total of 6 sessions. Immunohistochemistry was performed to detect the retention time of SA-hTNF-alpha fusion protein in the biotinylated mouse bladder mucosa and the distribution of CD4(+) and CD8(+) lymphocytes in the mucosa and tumor tissues, with the tumor growth and mouse survival also observed. The cytotoxiciy of the tumor-specific lymphocytes was evaluated. The mice responding well to the treatment were re-challenged by MB49 and monitored for survival. RESULTS: SA-hTNF-alpha could be efficiently and stably immobilized on the bladder mucosal surface for as long as 7 days. On day 60 after MB49 implantation, 18 out of 22 SA- hTNF-alpha-treated mice survived, with 9 appearing tumor-free, but all the mice in PBS control group died. Five out of 9 tumor-free mice in SA-hTNF-alpha group showed resistance to a re-challenge with intravesical MB49. The numbers of CD4(+) and CD8(+) lymphocytes were significantly greater in SA-hTNF-alpha group than in the other groups (P<0.05). The cytotoxicity of the tumor-specific lymphocytes was significantly stronger in SA-hTNF-alpha group than in the other groups (P<0.05). CONCLUSION: SA-hTNF-alpha immobilized on the biotinylated mucosal surface of the bladder wall can significantly inhibit the tumor growth and promote the survival of the mice bearing orthotopic superficial bladder cancer.

[Preparation and bioactivity evaluation of SA-mIL15 fusing protein].

AIM: To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins. METHODS: pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate SA/mIL15 fusion proteins. SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography, and mIL15-SA fusion protein through anion exchange chromatography, followed by refolding. The efficiency of surface modification of the fusion proteins on the biotinylated RM-1 tumor cells was evaluated by a flow cytometer. MTT method was used to evaluate the proliferating effect of SA/mIL15 fusion proteins on mouse spleen lymphocytes stimulated by ConA. RESULTS: Both SA-mIL15 and mIL15-SA fusion proteins were highly expressed in Rosetta (DE3) at about 20% of total bacterial proteins. They exhibited the bi-functionality: proliferation-promoting activity of mIL15 on mouse spleen lymphocytes with the specific activity of 1x10(6); IU/mg for SA-mIL15 or 2 x 10(5); IU/mg for mIL15-SA, and SA-mediated high-affinity binding to the biotinylated surfaces of RM-1 tumor cells with about 95% surface modification efficiency. CONCLUSION: SA/mIL15 bi-functional fusion proteins were generated, which made feasible the development of mIL15-surface-modified cancer cell vaccine.

[A simple and efficient method for establishing a mouse model of orthotopic MB49 bladder cancer].

OBJECTIVE: To establish a simple and efficient method for establishing a mouse model of orthotopic superficial bladder cancer. METHODS: C57BL/6 mice were anesthetized with sodium pentobarbital and catheterized with modified IV catheter (24 G). The mice were intravesically pretreated with HCl and then with NaOH, and after washing the bladders with phosphate-buffered saline (PBS), 100 microl (1 x 10(7)) MB49 cells were infused and allowed to incubate in the bladder for 2 h followed intravesical mitomycin C (MMC) administration. The tumor formation rate, survival, gross hematuria, and bladder weight were determined as the outcome variables, and the pathology of the bladders was observed. RESULTS: Instillation of MB49 tumor cells resulted in a tumor formation rates of 100% in all the pretreated groups while 0% in the control group without pretreatment. MMC significantly reduced the bladder weight as compared to PBS. CONCLUSION: We have successfully established a stable, reproducible, and reliable orthotopic bladder cancer model in mice.

[Preparation and bioactivity evaluation of streptavidin-tagged human interleukin-15 fusion protein].

OBJECTIVE: To obtain streptavidin-tagged human interleukin-15 (SA/hIL15) fusion protein and evaluate its bioactivity. METHODS: pET24a-6His-SA-hIL-15 and pET32a-hIL-15-SA-6His plasmids were constructed and expressed in BL 21(DE3) host bacteria to generate the fusion protein. The recombinant fusion protein IL-15/SA was purified using Ni-NTA affinity chromatography and refolded, and the efficiency of surface modification of the fusion protein on biotinylated cells was examined by fluorescence-activated cell sorting. CCK-8 method was used to evaluate the effect of IL-15/SA fusion protein in inducing the proliferation of human peripheral-blood lymphocyte (PBL) cells stimulated by PHA. RESULTS: The recombinant SA-hIL-15 and hIL15-SA fusion proteins were highly expressed in BL21(DE3) at about 20% of the total bacterial proteins. The purified hIL15-SA fusion protein exhibited a bifunctionality by promoting the proliferation of PBL cells activated by PHA and high-affinity binding to biotinylated cell surface mediated by SA, with a cell surface modification efficiency exceeding 95%. SA-hIL-15 showed a 4-fold higher hIL15 bioactivity than hIL15-SA. CONCLUSION: SA/hIL-15 bifunctional fusion protein has been successfully obtained to facilitate the future development of hIL-15-surface-modified cancer cell vaccine.

[Expression, purification and refolding of streptavidin-tagged human tumor necrosis factor-alpha fusion protein].

OBJECTIVE: To study the purification, refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-alpha (SA-TNF-alpha) bi-functional fusion protein. METHODS: SA-TNF-alpha fusion protein was expressed in BL21(DE3) host bacteria, purified using Ni-NTA affinity chromatography and refolded by dilution and dialysis followed by identification using Western blotting. The effect of SA-TNF-alpha fusion protein against L929 cells was evaluated by MTT assay. Flow cytometry was used to analyze the surface modification of biotinylated MB49 tumor cells by SA-TNF-alpha fusion protein. RESULTS: Recombinant SA- TNF-alpha fusion protein was expressed in BL21(DE3) at about 30% of total bacterial protein, with a purity of about 95% after purification. The SA-TNF-alpha fusion protein existed as dimmers, tetramers and higher order structures after refolding. The fusion protein exhibited a bi-functionality by inhibiting L929 cells and SA-mediated high-affinity binding to biotinylated cell surfaces, with an anchor modification rate of above 90%. CONCLUSION: The dimmers, tetramers and higher order structures of the obtained SA-TNF-alpha fusion protein all exhibit a bi-functionality, and may serve as a potential candidate therapeutic agent for tumors.

[Artemin and GFRalpha3 expressions and their relevance to perineural invasiveness and metastasis of pancreatic carcinoma].

OBJECTIVE: To investigate the association of artemin and GFRalpha3 expressions with perineural invasion and metastasis of pancreatic carcinoma. METHODS: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression of artemin and GFRalpha3 in pancreatic carcinoma tissues, adjacent tissues and normal pancreas tissues, and the relevance of artemin and GFRalpha3 expressions to the perineural invasion and metastasis of pancreatic carcinoma were analyzed. RESULTS: The positivity rates of artemin and GFRalpha3 expressions were 72.09% and 67.44% in pancreatic carcinoma, respectively, significantly higher than those in the adjacent tissue (18.19% and 22.73%). The positivity rates of artemin and GFRalpha3 expressions were significantly higher in patients with perineural invasion than in those without perineural invasion (chi(2)=11.11 and 11.78, respectively, P<0.01). Significantly higher expression of artemin mRNA was noted in pancreatic carcinoma (0.741-/+0.014) than in the normal pancreas tissue (0.101-/+0.031, P<0.05), and patients with perineural invasion showed significantly higher positivity rates of artemin mRNA expression (0.843-/+0.012) than those without perineural invasion (0.512-/+0.017, P<0.05). CONCLUSION: Artemin and GFRalpha3 expressions may play an important role in perineural invasion of pancreatic carcinoma and can be used a useful indicators for evaluating the biological behavior of pancreatic carcinomas.

[Effect of vascular endothelial growth factor and tumor necrosis factor receptor for treatment of avascular necrosis of the femoral head in rabbits].

OBJECTIVE: To evaluate the therapeutic effect of vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor (TNFR) on avascular necrosis of the femoral head in rabbits. METHODS: Avascular necrosis of the femoral head was induced in 26 New Zealand white rabbits by injections of horse serum and prednisolone. The rabbits were then divided into VEGF/TNFR treatment group, VEGF treatment group, and untreated model group, with another 4 normal rabbits as the normal control group. In the two treatment groups, the therapeutic agents were injected percutaneously into the femoral head. Enzyme-linked immunosorbent assay was performed to determine the concentration of TNF-alpha in rabbit serum followed by pathological examination of the changes in the bone tissues, bone marrow hematopoietic tissue and the blood vessels in the femoral head. RESULTS: Compared with the model group, the rabbits with both VEGF and TNFR treatment showed decreased serum concentration of TNF-alpha with obvious new vessel formation, decreased empty bone lacunae in the femoral head and hematopoietic tissue proliferation in the bone marrow cavity. CONCLUSION: Percutaneous injection of VEGF and TNFR into the femoral head can significantly enhance bone tissue angiogenesis and ameliorate osteonecrosis in rabbits with experimental femoral head necrosis.

[Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro].

OBJECTIVE: To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization. METHODS: The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells. RESULTS: The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection. CONCLUSION: arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.

Refolding and purification of recombinant human VEGF-121 expressed as inclusion bodies in Escherichia coli.

Vascular endothelial growth factor 121 (VEGF(121)) was expressed as inclusion bodies by recombinant Escherichia coli. High concentrations of both biomass (46 g dry cell/L) and VEGF(121) inclusion bodies (4.5 g/L) were obtained by applying a high-cell-density culture. After the inclusion bodies were washed and dissolved, VEGF(121) was refolded at 0.2 mg/ml by ultrafiltration in refolding buffer with a yield of 81%. Renatured VEGF(121) was purified by anion chromatography and Sephacry S-100 chromatography with purity higher than 95% and final purification yield of 31%. The purified VEGF(121) could stimulate the proliferation of human umbilical vein endothelial cells as demonstrated by a biological activity assay.