Antioxidative protective effect of icariin on the FeSO4/H 2O 2-damaged human sperm based on confocal raman micro-spectroscopy / 华中科技大学学报（医学）（英德文版）

Oxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from Herba Epimedii, on the human sperm. We prepared the FeSO4/H2O2-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman micro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect of icariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the "Raman fingerprint" of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H2O2. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive functions.

Antioxidative protective effect of icariin on the FeSO4/H 2O 2-damaged human sperm based on confocal raman micro-spectroscopy / 华中科技大学学报（医学）（英德文版）

Oxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from Herba Epimedii, on the human sperm. We prepared the FeSO4/H2O2-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman micro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect of icariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the "Raman fingerprint" of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H2O2. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive functions.

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>

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>

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>

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>

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>

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>

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>

Selective exclusion of hepatic outflow and inflow in hepatectomy for huge hepatic tumor / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the effects of selective hepatic vascular exclusion (SHVE) on prevention of serious hemorrhage and air embolism during hepatectomy and on the liver function after operation.</p><p><b>METHODS</b>From January 2004 to March 2007, 29 huge hepatic tumors were resected in our department. Both SHVE and Pringle maneuver were used to control the blood loss during hepatectomy. They were divided into two groups: SHVE group (15 cases) and Pringle group (14 cases). Data regarding the intraoperative and postoperative courses of the patients were analyzed.</p><p><b>RESULTS</b>There was no significant difference between the two groups regarding the age, sex, tumor size, cirrhosis, HbsAg positive rate and operating time (P > 0.05). Intraoperative blood loss was reduced significantly in the SHVE group (P < 0.05). The serum prealbumin levels on the postoperative day 1, 3 and 7 in SHVE group were significantly higher than those in the Pringle group (P < 0.05). The serum ALT value in SHVE group was significantly lower than that in the Pringle group on postoperative day 1, 3 and 7. The mean drainage volume in SHVE group was significantly less than that in the Pringle group on postoperative day 1 and 2. Liver failure occurred in two cases of the Pringle group, while no one in the SHVE group. Rupture of hepatic vein with massive blood loss occurred in 3 cases and air embolism in one case of the Pringle group, but did not occur in any case of the SHVE group.</p><p><b>CONCLUSION</b>When the selective exclusion of hepatic outflow and inflow is applied in hepatectomy, the resection rate of huge hepatic tumors and operative tolerance of hepatectomy are improved. It is a safe and rational operation type, and provides an optimal choice for hepatectomy.</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>

Study on Expression,Purification of GFP-SA Recombine Protein and Anchoring Carcinoma Cells / 中国生物工程杂志

The GFP(green fluorescence protein)-streptavidin(SA) bi-functional fusion protein was generated and characterized in order to demonstrate novel platform for efficiently and durably modifying the cell surface with SA-tagged bi-functional proteins.The GFP-SA/pET24d construct was generated and expressed in BL21(DE3) host bacteria at the high level.The recombinant protein GFP-SA was purified through the Ni-NTA affinity chromatography,and then refolded.After biotinylation B16 tumor cells were modified with GFP-SA bi-functional fusion protein and then subjected to fluorescent microscopy and FACS analysis.The effect of surface modification on the viability and growth of B16.F10 tumor cells was evaluated by MTT staining.The GFP-SA recombinant fusion protein was expressed in BL21(DE3) at about 20 % of total bacterial proteins.The GFP-SA bi-functional fusion protein exhibited the bi-functionality,i.e.,SA-mediated high-affinity binding to biotinylated cell surfaces and GFP-emitted green fluorescence.The cell surface modification with GFP-SA bi-functional fusion protein did not affect the viability and growth of the modified B16.F10 tumor cells significantly.The GFP-SA bi-functional fusion protein was obtained and could be displayed efficiently on the surface of the biotinylated B16.F10 tumor cells through the specific and tight interaction between streptavidin and biotin,thus can be used as good trace protein and experimental control in the development of surface-modified tumor vaccine.

Pathological changes of the blood vessels in rabbit femoral head with glucocorticoid-induced necrosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the pathological changes in the blood vessels in rabbit femoral head with glucocorticoid-induced necrosis and investigate the pathogenesis of glucocorticoid-induced osteonecrosis.</p><p><b>METHODS</b>Twenty New Zealand white rabbits were randomly divided into two groups, namely group A. which was injected with horse serum and prednisone and group B as the control group. Chinese ink was injected into the femoral cavity of the rabbits to observe the blood vessels in the femoral head under optical microscope and the femoral head was examined histopathologically.</p><p><b>RESULTS</b>Compared with the normal control group, the rabbits in group A had significantly decreased number of perfused vessels, which was featured by defective perfusion, osteocytie pyknosis or necrosis, increase of empty ostoocyte lacunae and fat cells, decrease of hematopoietic tissue, and blood vessel occlusion.</p><p><b>CONCLUSION</b>Vascular occlusion and vasculitis due to glucocorticoid treatment may cause avascular necrosis of the femoral head.</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.

Diagnosis and treatment of benign duodenal tumor / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the diagnosis and treatment of benign duodenal tumor.</p><p><b>METHODS</b>Clinical data of 14 patients with benign duodenal tumor confirmed pathologically or by operation from Oct.1988 to Oct.2001 were analyzed retrospectively.</p><p><b>RESULTS</b>Of 14 patients, 5 had Brunner's grand adenoma, 4 mesenchymoma, 2 leiomyoma, 2 hemangioma, 1 lipoma. Upper abdominal discomfort (64% ), gastrointestinal bleeding(50% ) and abdominal pain(20% ) were common manifestations. All cases received gastroscopy and only one case was diagnosed. Five cases received duodenoscope and the diagnosis was confirmed in 4 cases. Nine cases received hypotonic duodenography and lesions were found in 8 cases. Digital subtraction angiography was performed in 3 cases and detected all lesions. Computed tomographic scan and B-ultrasound were performed in 2 cases and only one case was diagnosed. Eleven cases (79% ) got definite diagnosis before operation. Tumor resection was performed in all patients. Perioperative death occurred in one patient. No recurrence occurred in 13 cases after following up from 2 to 11 years.</p><p><b>CONCLUSION</b>Upper abdominal discomfort and gastrointestinal bleeding are common features in patients with benign duodenal tumor. Duodenoscopy and hypotonic duodenography are good diagnostic approaches. Surgical tumor resection is the first choice of treatment.</p>