With the rapidly development of the biotechnology industry,large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications.Bacterial cells are most often used for the production of recombinant proteins.However,recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive.To circumvent this problem,several eukaryotic expression systems have also been developed over the years,ranging from yeast to mammalian cell-based technologies.For many mammalian proteins,especially those secreted and modified posttranslationally,a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells,i.e.,mammalian cells.Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins.Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supernatants containing the protein of interest.As it is allowed for easy and efficient purification,many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted.The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells.The noncatalytic C-terminal hemopexin-like domain of MMP-2,PEX,can block angiogenesis and tumor growth in vivo.Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties,as well as for their industrial applications.For this purpose,the rat growth hormone,mouse IgG? chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells,and their secretion efficiencies were compared by Western blotting and ELISA.Western-blotting of PEX protein from culture media,resulted in detection of proteins with the predicted molecular mass,which indicate that all of the signal sequences could direct PEX secretion successfully.The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency.Thus,expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly.To examine whether the observed difference in secretion yields is promoted at the transcriptional level,a RT-PCR analysis was performed at 6 h after transfection.The presence of mRNA transcripts of PEX was observed in all the DNA constructs.Moreover,semiquantitative reverse transcription(RT-PCR)results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection.Though there was no difference in the expression levels of PEX at an early time point after transfection,the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells.Hence,the described difference in exported yields is probably promoted at the secretion level,rather than at the transcriptional level.Chick chorioallantoic membrane(CAM)bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis.The MMP-9's signal peptide is used for the first time as leader sequence for secretion of foreign proteins.The results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.

Animals ; Animals, Newborn ; Hyperoxia ; physiopathology ; Lung ; drug effects ; pathology ; Oxygen ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tretinoin ; pharmacology ; therapeutic use

AIM: To investigate the therapeutic effect of endothelial progenitor cell-conditioned medium (EPC-CM) on the lung structure of neonatal rat exposed to hyperoxia, and to explore the mechanisms.METHODS: Bone marrow-derived endothelial progenitor cells (EPCs) were collected from new born Sprague-Dawley (SD) rats and the EPCs were identified.The conditioned medium from the passage 3 EPCs was collected.Newborn SD rats (n=40) were randomly divided into 4 groups.The rats in room air group were exposed to the room air (21% O2) for 21 d.The rats in hyperoxia group were exposed to hyperoxia (85% O2) for 21 d.The rats in endothelial cell basal medium (EBM) group were exposed to hyperoxia for 21 d, and received 100 μL EBM on postnatal day 14 (P14) in a single intratracheal (IT) injection.The rats in EPC-CM group were exposed to hyperoxia for 21 d, and received 100 μL EPC-CM on P14 in a singlie IT injection.The rats were sacrified on the 21st day.The left lungs were excised, placed in 4% paraformaldehyde, serially dehydrated in ethanol and embedded by paraffin.Serial sectioning of the paraffin-embedded left lung tissues was prepared for 5 μm thickness, and stained with hematoxylin and eosin.The pulmonary radical alveolar count (RAC) and alveolar mean linear intercept (MLI) were then calculated.The microvascular density was determined by FVIII immunostaining.The mRNA expression of KGF, VEGF, SP-A and SP-C in the right lung tissues was detected by real-time fluorescence quantitative PCR.RESULTS: The cultured cells had typical EPC morphological characteristics, and had the abilities to bind to FITC-UEA-1 and uptake DiI-ac-LDL.The body weight of the rats on day 21, RAC, MLI and microvascular density were significantly lower in hyperoxia group and EBM group than those in room air group (P<0.05).The EPC-CM group had significantly higher RAC and microvascular density than those in hyperoxia group and EBM group (P<0.05), but the body weight and MLI had no significant difference.The mRNA expression levels of KGF, VEGF, SP-A and SP-C in hyperoxia group and EBM group were significantly lower than those in room air group (P<0.05).The mRNA expression levels of KGF, VEGF, SP-A and SP-C in EPC-CM group were significantly higher than those in hyperoxia group and EBM group (P<0.05).CONCLUSION: EPC-CM promotes the lung alveolarization and microvascular formation in neonatal rats exposed to hyperoxia.These benefits may be correlated with the increased KGF and VEGF mRNA expression.

Background The main mechanism of diabetic retinopathy (DR) is the formation of retinal neovascularization. Vascular endothelial growth factors (VEGF) is primarily factor for DR. Objective The goal of this study was to investigate the effects of angelicae sinensis decoction for supplementing blood ( ASD), a kind of traditional prescription of Chinese herbal medicine, on vascular endothelial growth factor (VEGF) level in vitreous and serum of STZ-induced type 2 diabetes mellitus. Methods Thirty-four SPF male SD rats were fed using high fat diet for 8 weeks and then the streptozocin of 25 mg/kg was intraperitoneally injected to create the type 2 diabetic models. ASD (3.6 g/kg) and 10 mg/kg Gli of 5 mi were administered intragastrically from the 4th day of high fat die though 8 weeks respectively. Other 10 matched rats were used as normal control group. The body weight, blood glucose level ,triglycerides (TG) and high density lipoprotein (HDL) were detected in 4 and 8 weeks after usage of drug. The vitreous and serum samples were obtained in the 8 th weeks for the VEGF detection by enzyme-linked immunosorbent assay (ELISA). This experiment comlied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The body weight and HDL in DM group were significantly lower than those of normal control group in 4 or 8 weeks ( P＜0. 01 ), but in ASD and Gli groups ,the body weight and HDL were higher than those of the DM group (P＜0. 05 ). the blood glucose level and TG were elevated in DM group compared with normal control group (P＜0. 01 ), but those in ASD group and Gli group were decreased in comparison with DM group ( P＜0.05 ). EIISA revealed that no significant differences were found in VEGF levels between normal control group and ASD group or Gli group (P＞0. 05 ), but in DM group, the serum VEGF level (147.65±4. 55 ng/L) was reduced and vitreous VEGF level (673.45± 100. 85 ng/L) was raised,showing the considerable differences (P＜ 0.05 ). No any correlation was found between serum VEGF level and vitreous VEGF level (r=0.314,P＞0. 05). Conclusion ASD can decrease the VEGF level in vitreous,implying that ASD postpone the occurrence of diabetic retinopathy in DM rat.

AIM:To establish the murine model of oxygen induced retinopathy ( OIR) and to evaluate the inhibition of retinal neovascularization by erythropoietin ( EPO) blockade.
METHODS: Neonates of C57BL/6 mouse ( P7 ) were exposed to 75%±2% oxygen for 5d and return to normal air environment when 12d ( P12 ) to establish oxygen-induced retinal neovascularization model. The neonates were divided into groups, injected with 0. 5μL solution containing 25ng ( group A), 50ng ( group B), 250ng ( group C) of soluble erythropoietin receptor ( EPO-R) or PBS (group D) at P12, P14 and P16 in the right eye. On P17, the litters were sacrificed and their right eyes were enucleated and fixed with 4% paraformaldehyde, made pathological section. The number of breakthrough internal limiting membrane neovascular nuclei was counted with pathological retinal morphology, understanding theproliferative degree of retinal neovascularization.
RESULTS: The pathological sections showed the neovascular cell nuclei which penetrating the inner limiting membrane in intravitreal EPO receptor injection group was reduced statistically than that in PBS injection group, the difference was statistically significant ( P<0.01). And, neovascular nuclei count differences in the
various concentrations of EPO receptor group was statistically significant (P<0. 01). With the EPO receptor concentration increase, neovascular endothelial cells broken through the internal limiting membrane was reduced.
CONCLUSION: Intravitreal injection of soluble EPO receptor can block EPO and improve neovascularization. The new method is expected to become new treatment of ocular neovascular diseases.

Objective To investigate the effect of acupuncture plus astragalus polysaccharide on the expression of Bcl-2 protein in pancreatic islet b cells in db/db mice. Method C57BL/Ksj-db/db mice as an animal model of spontaneous type 2 diabetes were selected for this experiment. Five-week-old db/db mice were randomized into model, acupuncture, medication and acupuncture+medication groups. Meanwhile, db/m mice were selected as a normal group. The acupuncture group received acupuncture at points Housanli (equivalent to Zusanli, ST36), Neiting(ST44) and Yishu(Extra) and the medication group, an oral gavage of astragalus polysaccharide (1400 mg/kg). Both groups were treated once daily, for 12 consecutive weeks. After the end of experiment, blood glucose, insulin and resistin were measured, and the expression of Bcl-2 protein in islet b cells was determined by immunohistochemical method. Result Blood glucose, insulin and resistin levels were significantly lower in the acupuncture+medication, acupuncture and medication groups than in the model group. They were significantly lower in the acupuncture+medication group than in the acupuncture and medication groups and significantly lower in the acupuncture group than in the medication group. The expression of Bcl-2 protein in islet b cells was higher in the medication, acupuncture and acupuncture+medication groups than in the model group; there was a statistically significant difference (P<0.01). The expression of Bcl-2 protein was higher in the acupuncture+medication groups than in the medication group (P<0.01) and it was basically the same in the medication group as in the acupuncture group; there was no statistically significant difference (P>0.05). Conclusion Acupuncture plus astragalus polysaccharide can significantly reduce blood glucose and serum insulin and resistin levels and increase the expression of Bcl-2 protein in islet b cells to effectively inhibit apoptosis in islet b cells in db/db mice. Its effect is better than that of acupuncture alone or medication.

Mitochondrial oxidative stress has been recognized as a preliminary and critical factor that aggravates the pathological cascade of Alzheimer's disease, which induces the production of β-amyloid protein, upregulates the expression of phosphorylated tau protein and triggers oxidative damage to lipids, proteins and mitochondrial deoxyribonucleic acid. Central neurons are more vulnerable to oxidative stress than non-neuronal cells due to their high oxygen demand, abundant unsaturated fatty acids and antioxidant enzymes deficiency. On this account, this review introduces the causes of mitochondrial oxidative stress, and analyzes the important role of mitochondrial oxidative stress in the pathogenesis of Alzheimer's disease. Meanwhile, the review focuses on the design and intervention strategies of drug delivery systems targeting mitochondrial oxidative stress in neurons, aiming to provide new ideas for the prevention and treatment of Alzheimer's disease.

In order to study the accumulation of Fritillaria thunbergii cultivar, peimine content in Xiaye, Kuanye, Duozi and Xiaosanzi bulbs of different sizes and parts was determined by HPLC-ELSE. The results indicated that the peimine content varied significantly with the cultivar type, the size and part of bulb. The distribution laws of peimine were as follow: Xiaosanzi > Duozi > Xiaye > Kuanye, small-size bulb > big-size bulb, core bud > scale. The peimine yield per plant in Duozi was the highest.
Cevanes ; analysis ; Chromatography, High Pressure Liquid ; Fritillaria ; chemistry ; growth & development

Objective To study the deleterious effect of prolonged hyperoxia exposure on preterm rat lungs. Methods On the 2nd postnatal day, preterm SD rats were randomly assigned to the air group (I) and hyperoxia group (II, exposed to 85% O2). After 3, 7 and 14 days of exposure, the contents of total protein (TP), hydroxyproline (HYP) and malondialdehyde (MDA), total cell counts and differentiation in bronchoalveolar lavage fluid (BALF), ratio of lung wet weight/dry weight (W/D), and lung collagen content were examined. After 3, 7, 14 and 21 days of exposure, lung histopathology and radial alveolar counts (RAC) were performed. Results On day 3 of hyperoxia exposure, only the MDA content increased in Group II (P<0.05). On day 7 and 14, TP, HYP, total cell counts, the percentage of neutrophils in BALF and lung W/D also significantly increased (P<0.05 or 0.01). The differences of lung collagen contents between the two groups were not significant (P>0.05). Hyperoxia exposure resulted in subacute alveolitis and inhibition of lung development on day 7, 14 and 21. RAC was similar between the two groups on day 3 (4.9±0.7 vs 5.0±0.8), but different on day 7 (5.9±0.9 vs 7.1±0.9; P<0.05. On day 14 and 21, RAC decreased more obviously in Group II compared with that in Group I (7.0±0.8 vs 9.9±0.6, 7.3±0.9 vs 10.5±0.8; P<0.01). Conclusions Prolonged exposure to 85% O2 may result in subacute inflammatory lung injury and inhibition of lung development in preterm rats.

The purpose of this study was to isolate and purify polysaccharide from Gynura divaricata and analyze its monosaccharide composition. A water-soluble crude polysaccharide was obtained by hot water extraction, ethanol precipitation and deproteinization after degreasing. The crude polysaccharide then purified with DEAE-Sepharose Fast Flow column chromatography and dialysis. The monosaccharide composition and structure were analyzed by HPLC, UV spectrophotometer and 1H-NMR. The results showed that the purity and molecular weight of GDPS-2 and GDPS-3 were 87.3%, 2.03 x 10(4) Da and 90.9%, 4.29 x 10(4) Da, respectively. The UV spectrophotometer and 1H-NMR data suggested that glycosidic bond of GDPS-2 and GDPS-3 were a type. Both GDPs-2 and GDPs-3 were homogeneous polysaccharides, and GDPs-2 was mainly composed of glucuronic acid and xylose at a molar ratio of 1.1:0.63. GDPs-3 was mainly composed of rhamnose, glucuronic acid, galactose, xylose and galacturonic acid at a molar ratio of 0.32:6.0:0.21:1.75:4.3.
Asteraceae ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Magnetic Resonance Spectroscopy ; Molecular Weight ; Polysaccharides ; chemistry ; isolation & purification