Evaluation of Xpert MTB/RIF for the Diagnosis of Extrapulmonary Tuberculosis in China / 生物医学与环境科学（英文）

We evaluate the performance of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis (EPTB) in China. The performance of Xpert was evaluated compared to the composite reference standard (CRS), drug susceptibility testing (DST), and imaging examination. The overall sensitivity and specificity of Xpert were 64.1% (195/304) and 100% (24/24), respectively, using CRS as the gold standard. The sensitivity was significantly higher than that of culture for pus (P<0.05). The proportion of EPTB-positive cases diagnosed by imaging was two times more than that diagnosed using Xpert; however, 6 out of 19 cases may have been overdiagnosed by imaging. Compared to phenotypic DST, the sensitivity and specificity of Xpert were 80% (12/15) and 100% (75/75), respectively. Considering its high sensitivity and specificity, Xpert MTB/RIF may be used as a rapid initial test for EPTB diagnosis, and may also support a quicker decision on the treatment regimen. The combination of imaging and Xpert testing could provide high efficiency and accurate diagnosis of suspected EPTB.

Prokaryotic expression and characterization of two recombinant receptor-binding domain(RBD) proteins of human coronavirus NL63(HcoV-NL63) / 病毒学报

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.

Expression and purification of four single-stranded DNA-binding proteins and their binding on HCV RNA / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Express and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.</p><p><b>METHODS</b>The expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.</p><p><b>RESULTS</b>Suitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.</p><p><b>CONCLUSIONS</b>We have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.</p>

Circadian clock gene Rev-erb-α deficiency prolongs female estrous cyclicity / 生理学报

The circadian clock has been linked to female reproductive physiology and endocrine in mammals. Epidemiological studies of female shift workers have shown increased rates of abnormal reproduction and adverse pregnancy. But little is known how the circadian rhythms affect reproduction. The aim of the present study was to investigate the influences of circadian rhythms on estrous cycle in female mice using clock gene Rev-erb-α knock out (Rev-erb-α(-/-)) mice. To test the fertility of Rev-erb-α(-/-) mice, litter sizes were counted after mating with C57BL/6J male mice. HE staining was used to observe the change of follicle development. The number of embryos of Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice was compared 1.5 d after mating with C57BL/6J male mice. Then Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice were housed to adult, and daily vaginal lavage with 0.9% saline was used to monitor estrous cycle for at least 30 days. Quantity of various cells was counted on specified smears views after staining. We observed estrous cycles of Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice using line plots and periodic spectrograms. The results showed that the Rev-erb-α(-/-) female mice were infertility, and the number of embryos of Rev-erb-α(-/-) females was less than that of Rev-erb-α(+/+) females. However, the follicle development of Rev-erb-α(-/-) female mice was normal. The estrous cycle of Rev-erb-α(-/-) female mice was 3.22 days longer than that of Rev-erb-α(+/+) female mice. The results suggest that loss of Rev-erb-α prolongs estrous cycle, which is probably one of the reasons for female mice infertility, and circadian rhythm is important for mammalian estrous cycle.

Intravesical anchoring of streptavidin-tagged interleukin-4 fusion protein for immunotherapy of mouse superficial bladder cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Optimizing expression of the capsid protein VP2 from human Bocavirus and establish it's seroepidemiology assying methord / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain sufficient recombinant VP2 protein of human Bocavirus and establish it's seroepidemiology assying metbord. METHORD: Tbe capsid protein VP2 DNA genes of HBoV1 and 2 were optimized in accordance with tbe usage of the favorite codons in K coil so as to enhance its protein expression in prokaryotic expressing system. The protein was purified by Ni-NTA column, and its antigenicity was determined by Western Blot. Then establish ELISA to detect the specific anti-VP2 IgG antibodies against HBoV1 and 2 in healthy children aged 3-6 years in Nanjing, China.</p><p><b>RESULTS</b>The recombinant protein 6 x His-VP2 was produced in a larger quantity at 25 degrees C induced by IPTG (1 mmol/L) over night and purified by Ni-NTA column. Seropositive rates of HBoV1 and 2 were 62.2% and 55.5% and their mixed seropositivity was 37%.</p><p><b>CONCLUSION</b>The optimizing expression of the capsid protein VP2 from human Bocavirus constructed successfully and get a high yield under certain conditions. The established ELISA could be used to further analyze seroepidemiology of HBoV in China.</p>

Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).</p><p><b>METHODS</b>The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.</p><p><b>RESULTS</b>CFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.</p><p><b>CONCLUSION</b>The reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.</p>

Expression and purification of different segments from HCoV-NL63 nucleocapsid protein and their application in detection of antibodies / 病毒学报

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.

Preparation and application of a novel HCV diagnostic antigen fused to streptavidin / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare streptavidin-tagged hepatitis C virus (HCV) fusion protein and explore its application for the detection of antibody against HCV infection.</p><p><b>METHODS</b>A recombinant plasmid pET-11d-C44P-SA was constructed, which coding a novel HCV diagnostic antigens (C44P) and streptavidin (SA) fusion protein, and the fusion protein was generated with BL21 (DE3) E Coli and identified by Western Blot analysis. Then the fusion protein was purified through the Ni-NTA affinity chromatography and over 90% purity has been achieved. Anti-HCV ELISAs were developed when the fusion protein was used in the biotin-pre-coated microplate or ordinary microplate, and then the sensitivity and specificity of the ELISA were evaluated with confirmed human sera panels.</p><p><b>RESULTS</b>The fusion protein was expressed in high yields and purified successfully, the ELISA detection of anti-HCV with human sera panel indicated that its sensitivity and specificity is higher when SA-tagged HCV antigen (C44P-SA) coated in biotin-pre-coated microplate, compared to C44P or C44P-SA coated in ordinary microplate.</p><p><b>CONCLUSION</b>The sensitivity and specificity of anti-HCV ELISA can be improved when a novel HCV diagnostic antigen fused to SA combined with the biotin- pre-coated microplate. This study laid a foundation for improving the performance of HCV diagnostics.</p>

Norcantharidin inhibits DNA replication initiation protein Cdc6 in cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the inhibitory effect of norcantharidin (NCTD) on the expression of DNA replication initiation protein Cdc6 in cancer cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>NCTD can induce the degradation of Cdc6 in cancer cells to produce an anti-cancer effect.</p>

Immobilization of streptavidin-tagged bioactive hTNF-alpha on biotinylated mucosal surface of the bladder wall for treatment of superficial bladder cancer in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Expression, purification and bioactivity evaluation of streptavidin-tagged human interleukin-21 fusion protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain streptavidin-tagged human interleukin-21 (hIL21) fusion protein and evaluate its bioactivities.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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%).</p><p><b>CONCLUSION</b>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.</p>

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 / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>SA-hIL-2 fusion protein could significantly inhibit tumor growth and extend the survival time in the orthotopic model of MB49 bladder cancer.</p>

Preparation and bioactivity evaluation of streptavidin-tagged human interleukin-15 fusion protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain streptavidin-tagged human interleukin-15 (SA/hIL15) fusion protein and evaluate its bioactivity.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>SA/hIL-15 bifunctional fusion protein has been successfully obtained to facilitate the future development of hIL-15-surface-modified cancer cell vaccine.</p>

Expression, purification and refolding of streptavidin-tagged human tumor necrosis factor-alpha fusion protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the purification, refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-alpha (SA-TNF-alpha) bi-functional fusion protein.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>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.</p>

Artemin and GFRalpha3 expressions and their relevance to perineural invasiveness and metastasis of pancreatic carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the association of artemin and GFRalpha3 expressions with perineural invasion and metastasis of pancreatic carcinoma.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

A simple and efficient method for establishing a mouse model of orthotopic MB49 bladder cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a simple and efficient method for establishing a mouse model of orthotopic superficial bladder cancer.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>We have successfully established a stable, reproducible, and reliable orthotopic bladder cancer model in mice.</p>

Effect of vascular endothelial growth factor and tumor necrosis factor receptor for treatment of avascular necrosis of the femoral head in rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Refolding and purification of recombinant human VEGF-121 expressed as inclusion bodies in Escherichia coli / 南方医科大学学报

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.