1.Two-step Tandem Chromatography Purification of Anti-human CD80 Monoclonal Antibody 4E5 from Mouse Ascites
Hong-Bing MA ; Yu-Hua QIU ; Ran TAO ; Wen-Xiang LI ; Ying XU ; Xue-Guang ZHANG ;
China Biotechnology 2006;0(08):-
A two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0,50 mmol/L Tris-HCl and satisfactory results were obtained using a 0~0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.
2.Dose escalation of cisplatin with 5-fluorouracil in concurrent chemoradiotherapy for esophageal carcinoma
Qiang LIN ; Xian-Shu GAO ; Xue-Ying QIAO ; Zhi-Guo ZHOU ; Jun ZHANG ; Xiang-Ran YANG ; Xin WAN ;
Chinese Journal of Radiation Oncology 1992;0(04):-
Objective To define the maximum-tolerated dnse(MTD)and observe the side effect of escalating cisplatin with 5-fluorouracil in concurrent chemoradiotherapy for esophageal carcinoma in Chinese,with toxicity studied.Methods Previously untreated fifteen Chinese patients suffering from esophageal carcinoma received conventional fractionafiun radiotherapy,with 5 daily fractions of 2.0 Gy per week.The total radiation dose was 60 Gy.Concurrent chemotherapy dose escalation was given by the relatively safe and kidney-sparing modified Fibonacci sequence.The starting dose was cisplatin 37.5 mg/m~2 D1 and 5-fluorouracil 500 mg/m~2 D1-5, respectively.This regimen was repeated 4 times every 28 days.Escalation dose was eisplatin 7.5mg/m~2 and 5- fluorouracil 100mg/m~2.Every cohort contained at least 3 patients.If no dose-limiting toxicity(DLT)was observed, the next dose level was opened for entry.These courses were repeated until DLT appeared.MTD was declared as one dose level below which DLT appeared.Results DLT was defined as grade 3 radiation-induced esophngitis at the level of cisplatin 60 mg/m~2,5-fluorouracil 700 mg/m~2.MTD was defined as eisplafin 52.5 mg,/m~,5- fluorouracil 700 mg/m~2.The major side effect were radiation-induced esophagitis,leucopenia,nausea,vomiting and anorexia.Conclusion Maximun tolerated dose of cisplatin with 5-fluorouracil in concurrent chemoradiotherapy in the Chinese people with esophageal carcinoma were eisplatin 52.5 mg/m~2 D1,5-fluorouracil 700 mg/m~2 D1-5,repeated 4 times every 28 days.
3.Investigation of Coptis chinensis on jaundice of glucose-6-phosphate dehydrogenase (G6PD) deficient neonates from Guigang, Guangxi province.
Xiu-Lan LIN ; Na LIN ; Chun-Fang LIU ; Yuan LIU ; Zhi-Ran LIANG ; Rong WAN ; Xiang-Ying KONG
China Journal of Chinese Materia Medica 2007;32(23):2543-2546
OBJECTIVETo investigate the effect of Coptis chinensis on jaundice of G6PD deficient neonates.
METHOD122 G6PD deficient neonates with jaundice who were in People' s Hospital of Guigang of Guangxi province from January 1999 to October 2004 were divided into two groups: C. chinensis group (62 neonates with C. chinensis administration before jaundice' s appearance) and none C. chinensis group (60 neonates without C. chinensis administration before jaundice' s appearance). The initial time, duration of jaundice, hemoglobin and serum bilirubin level and the incidence of kernicterus were analyzed between the two groups.
RESULTThe initial time of jaundice is significantly later and the duration of jaundice is markedly shorter in the neonates with C. chinensis than that without C. chinensis. Simultaneously, the level of hemoglobin is significantly increased, and there is a low tendency of serum total bilirubin and direct bilirubin level in C. chinensis group as compared to that in none C. chinensis group. Moreover, there is no kernicterus in C. chinensis group and no difference in the treating result out of hospital between the two groups.
CONCLUSIONOur results do not support the view that C. chinensis could aggravate jaundice of G6PD deficient neonates.
Bilirubin ; blood ; China ; Coptis ; chemistry ; Female ; Glucosephosphate Dehydrogenase Deficiency ; blood ; chemically induced ; complications ; Hemoglobins ; metabolism ; Humans ; Infant, Newborn ; Jaundice, Neonatal ; blood ; chemically induced ; complications ; Kernicterus ; blood ; chemically induced ; complications ; Male ; Plant Preparations ; adverse effects ; Plants, Medicinal ; chemistry ; Retrospective Studies ; Time Factors
4.Experimental research on bletilla carrying exogenous basic fibroblast growth factor that promotes wound healing
Xiao WANG ; Jing ZHAO ; ying Meng DU ; juan Hai SUN ; wei Xiang GAO ; Ran WU
Chinese Journal of Tissue Engineering Research 2017;21(34):5481-5486
BACKGROUND: Bletilla bletilla striata gelatin (BSG) is found to remarkably promote the growth of granulation tissue and capillary vessels, as well as the expression of vascular endothelial growth factor in the wound tissue in rabbits with full-thickness skin defect of the back. Basic fibroblast growth factor (bFGF) remarkably promotes the growth of collagen fibers and the growth and dilation of capillary vessels in the wound tissue in rabbits with full-thickness skin defect of the back. However, BSG is easy to decompose under normal temperature, affecting fulfillment of its functions. OBJECTIVE: To explore the effect of BSG carrying exogenous bFGF on wound healing. METHODS: Forty healthy rabbits were used to make animal models of full-thickness back skin defects, and then randomly divided into four groups, namely, group BSG+bFGF, group bFGF, group BSG and group saline. Rats in each group were subjected to the corresponding treatment once a day until the wound was completely healed. Wound healing time was recorded. Wound healing rate was detected at 3 and 10 days after modeling. Real-time PCR and western blot assay were used to detect the expression of vascular endothelial growth factor, α-smooth muscle actin and type I collagen at mRNA and protein levels at 7 days after modeling. RESULTS AND CONCLUSION: The wound healing time in the BSG+bFGF group was shortened by 4.5, 3.0 and 2.8 days as compared with the normal saline group, BSG group and bFGF group, respectively (P < 0.05). The wound healing rates in the BSG+bFGF group were also higher than those in the other groups at 3 and 10 days after modeling (P< 0.05). Findings from both PCR and western blot assay showed higher expression of vascular endothelial growth factor and α-smooth muscle actin and lower expression of type I collagen in the BSG+bFGF group than the other three groups at 7 days after modeling (P < 0.05). To conclude, BSG carrying exogenous bFGF can promote wound healing, and the underlying mechanism may be to promote vascular endothelial growth factor and inhibit type I collagen.
5.Influence of the reductase deficient Escherichia coli on the solubility of recombinant proteins produced in it.
Sheng XIONG ; Mei-Ying ZHANG ; Chui-Wen QIAN ; Yan-Chao RAN ; Yi-Fei WANG ; Xiang-Rong REN ; Kuan-Yuan SU ; Zhou-Yao YU
Chinese Journal of Biotechnology 2003;19(6):686-691
The cytoplasm of E. coli is a reducing environment where cysteines do not engage in disulfide bonds. Any disulfide bonds that do appear are rapidly reduced through the action of disulfide reducing enzymes such as thioredoxin and glutaredoxin. To study the influence of E. coli cytoplasm on the solubility of recombinant proteins produced in it, bovine fibroblast growth factor (BbFGF), with single disulfide bond, and anti-HBsAg single-chain Fv (HBscFv), with two disulfide bonds, were selected as the pattern molecules of simple protein and complex protein, respectively. pJN98-BbFGF, a BbFGF expressing plasmid based on the vector pET3c, was constructed and transformed into normal host BL21(DE3) and a reductase deficient strain, E. coli Origami(DE3). At the same time, pQE-HBscFv, a HBscFv expressing plasmid was constructed and transformed into M15 [pREP4] and Origami(DE3). The recombinant BbFGF and HBscFv were produced in 2 types of bacteria and their solubilities and bioactivities were determined, respectively. It was found that the majority of BbFGF had formed inclusion body in the cytoplasm of BL21 (DE3) and all of them turned into soluble protein in Origami(DE3). It was also found the productivity of BbFGF in Origami (DE3) was 5% - 10% of the total protein and the value was 15% - 23% in BL21(DE3). BbFGFs produced in 2 recombinant bacteria were purified by cation exchange and heparin affinity chromatography. MTT assay revealed that the bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGFs from different bacteria was 1.6ng/mL and 2.2ng/mL, respectively. As far as HBscFvs, both of them formed inclusion body in the cytoplasm of M15 [pQE-HBscFv] and Origami [pQE-HBscFv]. The inclusion body was solubilized in 6mol/L GuHCl, purified with a His-Trap column and then refolded by dialysis step-by-step against buffers containing downtrend concentration of GuHCl. Indirect ELISA was applied to determine the HBsAg binding activity of HBscFvs. It was found there was no obvious difference between the bioactivity of refolded HBscFvs produced from 2 recombinant bacteria. On the other hand, the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15 [pQE-HBscFv] displayed without any bioactivity. The soluble HBsFv in the cytoplasm of Origami [pQE-HBscFv] was purified by cation exchange and immobilized metal affinity chromatography (IMAC) and the yield was 1 - 2mg/L. Those results suggested that modification of the redox environment of E. coli cytoplasm greatly improved the solubility of recombinant disulfide-bonded proteins produced in it. In the next step, we had like to co-express of molecular chaperones or refoldase to raise the yield of soluble recombinant proteins, as well as optimizing the culture condition of the "oxidizing" E. coli.
Animals
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Antibodies
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genetics
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immunology
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metabolism
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Cattle
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli
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enzymology
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genetics
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metabolism
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Escherichia coli Proteins
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genetics
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Fibroblast Growth Factors
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genetics
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metabolism
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Genetic Vectors
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genetics
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Hepatitis B Surface Antigens
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immunology
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Inclusion Bodies
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chemistry
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metabolism
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Oxidoreductases
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genetics
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Plasmids
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genetics
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Protein Engineering
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Recombinant Proteins
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chemistry
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genetics
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metabolism
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Solubility
6.Efficacy of glycyrrhizin combined with cyclosporine in the treatment of non-severe aplastic anemia.
Cui-ai REN ; Yan-xiang LI ; Jing-ying CUI ; Zhi-xin SHENG ; Xue-hong RAN ; Bao-hong WANG ; Mao-hong ZHANG
Chinese Medical Journal 2013;126(11):2083-2086
BACKGROUNDCyclosporine A (CsA) has been widely used in the treatment of aplastic anemia (AA), but the application of CsA was limited in patients who had liver diseases or abnormal liver function due to its liver toxicity. Glycyrrhizin has long been used in China in the treatment of various liver diseases to lower transaminases. In this study, we observed the efficacy and safety of glycyrrhizic acid combined with CsA in the treatment of newly diagnosed patients with non-severe AA (NSAA).
METHODSA total number of 76 patients with newly diagnosed NSAA were enrolled into the study at our hospital between July 2005 and June 2010. The patients were divided randomly into two groups: the glycyrrhizin-treatment group (group A) and the control group (group B) with 38 patients in each group. All patients received 3 - 5 mg×kg(-1)×d(-1) CsA for at least 4 months and were treated either with or without glycyrrhizin for 4 months.
RESULTSsixty-eight patients were eligible for evaluation. In the control group, 9.09% patients (n = 3) achieved a complete response while 51.52% (n = 17) attained a partial response. The overall response rate was 60.61% (n = 20). The remaining 13 patients (39.39%) did not have any response. In the glycyrrhizin-treatment group, complete response rate was 20% (n = 7) and partial response rate was 62.86% (n = 22). The overall response rate was 82.86% (n = 29) and the non-response rate was 17.14% (n = 6). Response rate was significantly increased with the addition of glycyrrhizin to CsA compared with CsA alone (P < 0.05).
CONCLUSIONThe combination of glycyrrhizin and cyclosporine regimen was an effective treatment for NSAA in terms of improvement of response rate, reduction in CsA-related liver injury, and attenuation of severity of nausea and other adverse events in the treatment of patients with NSAA.
Adolescent ; Adult ; Aged ; Anemia, Aplastic ; drug therapy ; immunology ; Cyclosporine ; administration & dosage ; adverse effects ; Drug Therapy, Combination ; Female ; Glycyrrhizic Acid ; administration & dosage ; adverse effects ; Humans ; Interferon-gamma ; blood ; Interleukin-2 ; blood ; Male ; Middle Aged
7.Quantitation of HTLV-I proviral load using real-time quantitative PCR with Taqman MGB probe.
Jin-Zhen XIE ; Chang-Rong CHEN ; Jun ZHANG ; Hong-Ying NI ; Sheng-Xiang GE ; Juan-Juan ZHOU ; Shan-Hai OU ; Xiu-Juan ZHENG ; Peng RAN ; Bin PEI
Chinese Journal of Virology 2009;25(5):339-343
A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.
Gene Products, gag
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genetics
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Gene Products, pol
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genetics
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Human T-lymphotropic virus 1
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genetics
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isolation & purification
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Humans
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Molecular Probes
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Polymerase Chain Reaction
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methods
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Viral Proteins
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genetics
8.Association of single nucleotide polymorphism of MnSOD gene with carcinogenesis and development of esophageal squamous cell carcinoma.
Yun-jie CHENG ; Ya-di WANG ; Qing LIU ; Zhi-ming DONG ; Feng-peng WU ; Xiang-ran YANG ; Xue-ying QIAO ; Jun ZHANG
Chinese Journal of Oncology 2009;31(11):831-835
OBJECTIVETo investigate the association of single nucleotide polymorphism (SNP) of manganese superoxide dismutase (MnSOD) gene with carcinogenesis and progression of esophageal squamous cell carcinoma.
METHODSThe MnSOD9 T-->C SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 103 patients with esophageal squamous cell carcinoma and 195 healthy controls.
RESULTSA significant difference was observed in the MnSOD allelotype distribution among esophageal squamous cell carcinomas and healthy controls (chi(2) = 4.645, P < 0.05). Individuals with the 9 C allele had a significantly higher risk to develop esophageal squamous cell carcinoma compared with those with the TT allele. The frequency of C allelotype among patients with lesions of different lengths (= 5 cm and > 5 cm) was 16.3% and 36.7%, respectively. A significant difference was observed in the MnSOD allelotype distribution between patients with lesions of different lengths (chi(2) = 5.147, P < 0.05). No significant association of the MnSOD polymorphism at 9 T-->C with the tumor site, maximal length and clinical staging was found in esophageal squamous cell carcinoma.
CONCLUSIONSingle nucleotide polymorphism (SNP) of MnSOD gene may be correlated with the susceptibility and disease progression of esophageal squamous cell carcinoma, and may become a tumor marker for prediction of this cancer.
Aged ; Carcinoma, Squamous Cell ; genetics ; pathology ; Case-Control Studies ; Esophageal Neoplasms ; genetics ; pathology ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Superoxide Dismutase ; genetics ; Tumor Burden
9.Protective effects of Ginkgo biloba extract on presbycusis in the rat model via autophagy pathway
Qing-Ling WANG ; Meng-Xian ZHANG ; Ying-Dong ZHOU ; Hao-Ran KANG ; Xiang-Dong GUO ; Qing-Lin WANG
Chinese Traditional Patent Medicine 2024;46(1):65-71
AIM To investigate the effects of Ginkgo biloba extract on hearing function,cochlear morphology and autophagy-related protein expression in a rat model of presbycusis.METHODS Forty-five rats were randomly divided into the control group,the model group and the low,medium and high dose G.biloba extract groups(10,20 and 30 mg/kg),with 9 rats in each group.The rat model of presbycusis was established by intraperitoneal injection of 500 mg/kg D-galactose(D-gal).Eight weeks after the corresponding administration,the rats had their changes of hearing threshold detected by the auditory brainstem evoked potential(ABR);their morphological changes of cochlear hair cells,stria vascularis(SV)and spiral ganglion cells observed by HE staining;their number of hair cells inside and outside the cochlea detected by immunofluorescence staining;their ultrastructure changes of cochlear hair cells observed by transmission electron microscopy;and their expression of autophagy-related proteins in cochlea tissue detected by Western blot.RESULTS Compared with the control group,the model group displayed increased ABR threshold(P<0.01);more severely damaged inner and outer hair cells,spiral ganglion cells and SV,decreased SV thickness and numbers of spiral ganglion cells,inner and outer hair cells and autophagosomes(P<0.01);decreased protein expressions of Beclin1 and LC3 Ⅱ and ratio of LC3 Ⅱ/LC3 Ⅰ in cochlear tissue(P<0.01),and higher P62 protein expression(P<0.01).Compared with the model group,the medium and high dose G.biloba extract groups shared decreased ABR thresholds(P<0.01);improved morphology of inner and outer hair cells and SV in the cochlea,normalized,morphology of spiral ganglion cells,and increased SV thickness and the numbers of spiral ganglion cells,inner and outer hair cells and autophagosomes(P<0.05,P<0.01);increased protein expressions of Beclin1 and LC3 Ⅱ and the ratio of LC3 Ⅱ/LC3 Ⅰ in the cochlea(P<0.01),and decreased P62 protein expression(P<0.01).CONCLUSION The protective effects G.biloba extract on hearing function and cochlear cells in the rat model of presbycusis may be associated with the up-regulated expression of Beclin1 and LC3 Ⅱ proteins and down-regulated P62 protein expression in cochlear tissues.
10.The feeder layer of human embryonic fibroblasts supports the growth of human spermatogonial stem cells.
Yu-Bin WANG ; Bin CHEN ; Ying-Chao WANG ; Zhi-Ling ZHANG ; Hong-Xiang WANG ; Yong-Ning LU ; Zu-Qiong XIANG ; Kai HU ; Yi-Ke YANG ; Yin-Fa HAN ; Zheng WANG ; Yi-Xin WANG ; Yi-Ran HUANG
National Journal of Andrology 2008;14(12):1063-1068
OBJECTIVETo investigate the methods and conditions for the isolation, purification and culture of human spermatogonial stem cells (SSCs) on the feeder layer cells of human embryonic fibroblasts (hEFs).
METHODSSSCs isolated and purified from normal human fetal testicular tissues by sequential two-step enzyme digestion and Percoll uncontinuous density gradient centrifugation were cultured on the feeder layer cells of hEFs isolated from 5-9 weeks old human embryos. The surface markers SSEA-1 and OCT4 of the SSCs were detected by immunohistochemistry; the alkaline phosphatase (AKP) activity of the SSC clones measured; and the expressions of the SSC-related genes determined by RT-PCR.
RESULTSSSCs survived, proliferated and formed colonies on the feeder layers, and the colonies were highly positive for SSEA-1 and OCT4, with strong AKP activity and high expressions of the SSC-related genes.
CONCLUSIONThe feeder layer of hEFs supports the growth of human spermatogonial stem cells.
Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; Humans ; Male ; Spermatogonia ; cytology ; Stem Cells ; cytology