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.

Gas Chromatography-Mass Spectrometry ; methods ; Humans ; Malathion ; metabolism ; urine

Objective·Giving the ovariectomized (OVX) mice exogenous estrogen replace therapy (ERT) so as to explore the inflammatory mechanism of estrogen in the pain modulation of OVX mice.Methods·Twenty-four 12-week-old female C57BL/6J mice were randomized into three groups:sham group (S),ovariectomy+placebo group (P),ovariectomy+estrogen group (E).The ERT began from one week after OVX and last for four weeks.Paw withdraw threshold (PWT) and paw withdraw latency (PWL) were measured at one day before OVX,and three days,one week,two weeks,three weeks,four weeks and five weeks after OVX.Concentrations of inflammatory cytokines in serum were measured at three days and five weeks postoperatively.Five weeks after operation,the lumbar intumescentia of spinal cord was taken and the expression of p38 was analyzed by Western blotting.Results·All the OVX mice's PWTs or PWLs were decreased after operation compared with non-operation mice;PWTs and PWLs were significantly different from group S separately from one week and three days after OVX (P＜0.01).The inflammatory cytokines of OVX mice were significantly higher than group S (both P＜0.01) at three days after OVX.Western blotting results showed that the p38 of group P was significantly higher than that of group S after five weeks postoperatively (P＜0.01).By receiving ERT,both PWTs and PWLs of group E increased slightly and were significantly different from group S (P＜0.01) at four and five weeks after OVX.At five weeks after OVX,inflammatory cytokines of group E were significantly lower than group P (P＜0.01) but still much higher than group S (P＜0.01).Meanwhile the content of p38 in group E was less than that in group P (P＜0.01).The p38 in group E was still more than group S,however it was no longer significantly different from group S (P＞0.05).Conclusion·Estrogen may reduce concentrations of inflammatory cytokines through the p38 MAPK pathway and plays an important role in the pain modulation of OVX mice.

Aging ; Animals ; Brain ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Male ; Neurotransmitter Agents ; metabolism ; Rats ; Rats, Sprague-Dawley

Adult ; Aged ; Anthracosis ; complications ; Humans ; Male ; Middle Aged ; Pulmonary Emphysema ; diagnosis ; etiology ; Respiratory Function Tests ; Tomography, X-Ray Computed

Testicular germ cell tumor (TGCT) is a most common testicular malignancy with an increasing incidence, and its pathogenesis and mechanisms are not yet clear. The next generation sequencing has become the main tool to uncover the underlying mechanisms of TGCT. The differential gene expressions, gene mutation, predisposing gene-dominated signaling pathways, and changes of the relevant genes in the sex chromosome are largely involved in the occurrence and development of TGCT. Studies on the genomics of TGCT contribute a lot to identifying the pivotal pathogenic genes and paving a theoretical ground for the early screening and targeted therapy of TGCT. This paper summarizes the advances in the studies of the genomics of TGCT so as to reveal thetmechanisms of the disease at the genetic level.
Genomics ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Neoplasms, Germ Cell and Embryonal ; genetics ; Testicular Neoplasms ; genetics

OBJECTIVETo observe preliminary efficacy of decompressive unilateral improved transforaminal lumbar interbody fusion (TLIF) for the treatment of lumbar degenerative diseases.

METHODSFrom August 2009 to December 2011, 28 patients with lumbar degenerative diseases were treated by decompressive unilateral improved TLIF,including 16 males and 12 females with an average of 61 (aged 46 to 71) years old,the courses of disease ranged from 6 months to 6 years. Among them, 20 cases suffered from lumbar spinal stenosis, 8 cases were lumbar disc herniation. Decompressive range included single segment in 24 cases,and double segments in 4 cases; 15 cases were performed operation on the left side, 13 cases on the right side. JOA lower back pain scoring system (29 points) were applied for evaluate preoperative and postoperative symptoms, physical signs and sphincteral functions;Visual analogue scale (VAS) were used to evaluate preoperative and postoperative low back pain.

RESULTSAll patients were followed up 6 to 28 (mean 14) months. Postoperative JOA score and VAS score were 17.9 +/- 2.2, 2.8 +/- 0.7 respectively,and preoperative JOA score and VAS score were 8.5 +/- 1.7, 8.6 +/- 1.2, respectively. There were significant meaning in JOA and VAS scores before and after operation (P < 0.05). Twenty-eight patients were all obtained intervertebral synostosis.

CONCLUSIONDecompressive unilateral improved TLIF for treatment of unilateral radicular lumbar spinal stenosis and lumbar disc herniation,which has advantages of minimally invasive,curative effects,decrease medical costs,is worthy spreading in clinical.

Adult ; Aged ; Decompression, Surgical ; Female ; Humans ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Spinal Diseases ; surgery ; Spinal Fusion ; Treatment Outcome

Definite elements is the first step of systemic analysis. Quality of thesis was determined by a combination of three factors in non-substantial dimension: ability,attitude and system. They affect the quality of undergraduate thesis in health management through different mechanisms respectively,such as selection and externalization of ability,power,adaptation,adjustment and defense of attitude,guide and regulation of system.

Objective To study the better way of avoidance of hypothalamus injury in surgical management of craniopharyngioma.Methods 40 cases with craniopharyngioma accepted surgery treatment were analyzed respectively.Results The pterional approach was performed in 30 cases, interfornical approach in 8 cases,subfrontal approach in 2 cases.Total removal of tumor was achieved in 35 cases,subtotal removal in 5 cases.One died from extradural hematoma after operation. No one died from hypothalamus injury.Conclusion Some way was key to avoid hypothalamus injury and to achieve a better surgical result, such as proper approach, separating strictly along tumor, identification of pituitary stalk and hypothalamus structure,preservation of perforating arteries to hypothalamus.

Objective To investigate the effects of total flavonoids of Herba Epimedii(HEF)on the metabolism of typeⅠcollagen and the expression of cathepsin K in the bone of ovariectomized(OVX)rats. Methods Fifty-four female SD rats were allocated into 6 groups;OVX group,sham operation group,OVX rats followed by three doses of HEF(40,80 and 160 mg?kg~(-1)?d~(-1))and nilestriol(0.1 mg?kg~(-1)?d~(-1))for 12 weeks respectively.Bone mineral density(BMD)of whole body was determined by dual-energy X-ray absoptiometry.The level of cross-linked N-telopeptide of typeⅠcollagen(NTx)in the urine were determined by ELISA.The amounts of typeⅠcollagen protein and cathepsin K protein in bone tissue were detected by immunohistochemical method and Western blotting.Results Compared with OVX group,the total BMD values in the HEF treated groups were increased(all P＜0.05),and the expression levels of typeⅠcollagen in three HEF treated groups rose significantly in a dose-dependent manner after 12 week,and simultaneously,both the expression of cathepsin K in bone and the level of NTx/Cr were reduced markedly(P＜0.05),being most significant(P＜0.01)in the group treated with the highest dose of HEF(160 mg?kg~(-1)?d~(-1)).Conclusion HEF seems to be able to elevate BMD and improve bone quality of rats via promoting synthesis and inhibiting proteolysis and absorption of typeⅠcollagen in the bone.