The goal of this work was to explore the prospect of standardized application of an in-vitro bioactivity assay for recombinant human follicle-stimulating hormone based on a reporter gene. The relative accuracy, intermediate precision, linearity and applicable range of the method were validated according to the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Three laboratories used this method to determine the in-vitro biological activities of six batches of drug product and three batches of drug substance manufactured by two different companies. The consistency of the potency determined by three laboratories, the intra-laboratory precision and inter-laboratory precision were analyzed. The method was optimized during the collaborative validation. The results of method validation meet the requirements of the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Aiming to resolve the problems found in the collaborative validation, the medium for cell seeding, the pre-diluted buffer solution of standard and sample, and the means of removing and discarding supernatant after stimulation were optimized. After optimization, there was no significant difference in the bioactivity among the different laboratories (P > 0.05), indicating statistical equivalency. Intra-laboratory and inter-laboratory precision were good and the geometric coefficient of variation (GCV%) were both less than 15%. In conclusion, the reporter gene assay has good intra-laboratory repeatability and inter-laboratory reproducibility and is suitable for analyzing recombinant human follicle-stimulating hormone drug product and drug substance by different manufacturers. It is expected to be used as a standardized method for the determination of the in-vitro bioactivity of such products.

OBJECTIVETo observe the effect of tetrandine on gene expression of collagen type I, collagen type III, transformation growth factor-beta1 and to investigate the inhibitory effect of tetrandine on the scar tissue hyperplasia in rabbits' ears.

METHODSAfter the scar model was formed on the rabbits' ears, the rabbits were divided into 4 groups to receive intro-lesion injection with saline, or prednisolone (Pre) or tetrandrine in low concentration (L-Tet, 1.0 mg/ml) or tetrandrine in high concentration (H-Tet, 7.5 mg/ml). The morphological changes of scar tissue were observed. The changes of fibroblasts quantity and collagen expression were observed with HE and Masson staining. Immunohistochemical study was used to observe the expression level of collagen type I and collagen type III and TGF-beta1. Collagen type I and collagen type III and TGF-beta1, and signal factor Smad 3 mRNA were detected with RT-PCR.

RESULTS(1) 24 days after injury, all the wounds healed completely with formation of red, tough and hypertrophic scar. HE and Masson staining showed significant increase of fibroblasts and collagen density with irregularly arrangement. (2) Compared with that in saline group, the scar in other groups became softer, lighter and thinner, especially in H-Tet group. (3) HE and Masson staining shows the scar in Tet and Pre groups contained less fibroblasts and lower collagen dentsity with comparatively regular arrangement than that in saline group (P < 0.01), especially in H-Tet group. (4) According to the immunohistochemical study, the expression of collage type I and III and TGF-beta was positive in all the groups, but the positive rate and the ratio of collagen density I to III decreased in the order of saline, L-Tet, H-Tet and Pre groups (P < 0.01). (5) PT-PCR detection results showed that the amplification bands brightness of collagen type I and III and TGF-beta1 and signal molecular Smad 3 mRNA in scar tissue were obviously different. Compared with that in saline group, the expression of collagen type I and III and TGF-beta1 and Smad 3 mRNA decreased in Tet and Pre groups (P < 0.01). H-Tet group showed the most obvious reduce in the expression of type I collagen and TGF-beta1 and Smad 3 mRNA. Conclusions Tetrandine can significantly suppress the expression of collagen type I and collagen type III and TGF-beta1 on hypertrophic scar of rabbit ears, and reduce signal factor Smad 3 mRNA' s expression. It may be one of the important mechanism for its inhibitory effect on scar hyperplasia.

Animals ; Benzylisoquinolines ; pharmacology ; Cicatrix, Hypertrophic ; drug therapy ; genetics ; pathology ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Ear ; Fibroblasts ; Gene Expression ; Male ; RNA, Messenger ; metabolism ; Rabbits ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

Objective To observe therapeutic effects of HuGanJieXian decoction on rats hepatic fibrosis induced by tetrachloride. Methods Rat models of hepatic fibrosis were constructed by intraperitoneal injection of tetrachloride.HuGanJieXian decoction composed of low, middle, and high dose curcumin were given to these rats respectively at the same time. Sho-saiko-to compound treatment group and Fufangbiejiarangan Tablets treatment group were made as positive control groups. After twelve weeks, all rats were executed. Serum samples were kept for measuring serum levels of PC-Ⅲ, LN, and HA. Left livers were extirpated for pathologic examination including H.E and Masson stainings. Grade of hepatic fibrosis were evaluated according to SSS system. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) of supematant centrifugated from hepatic tissue homogenate were detected. Results Serum levels of PC-Ⅲ, LN, and HA were depressed obviously in decoction groups compared with those of fibrotic group (P<0.05) , especially in the low-dose curcumin group.HuGanJieXian Decoction could increase the level of SOD and decrease the level of MDA (P<0.05) , especially in the low-dose curcumin group. Staining of H. E and Masson showed that degrees of hepatic fibrosis in decoction groups were improved obviously compared with that of the fibrotic group. Conclusion HuGanJieXian Decoction can improve rat hepatic fibrosis, the mechanism of this effect may be associated with protecting hepatic cell membrane and anti- peroxidative damage.

Objective To discuss the initial expression of serum procalcitonin (PCT) in patients with severe traumatic brain injury (TBI) and determine the potential value of PCT to predict the neurological outcome.Methods A retrospective analysis was made on patients admitted due to severe TBI (GCS≤8 points) from July 2011 to August 2012.Mortality and neurological outcome of the survivors were determined using Glasgow outcome scale (GOS) at 6 months after TBI.Results A total of 52 patients (39 males and 13 females),at median age of 38 years (range,15-65 years) were included in the study.Twenty-eight patients had good outcome (GOS of grade Ⅳ-Ⅴ),whereas 24 patients had poor outcome or died (GOS of grade Ⅰ-Ⅲ).Within 24 hours after TBI,serum PCT level was significantly higher in patients with bad outcome compared to those with good outcome (0.778 ng/ml:0.094 ng/ml,P ＜0.01).Enhanced PCT level presented a close correlation with the poor outcome (r =0.657,P ＜0.01).Area under the receiver operating characteristic curve (ROC) was 0.879 [95％ CI (0.757,1.000)].A cutoff value of 0.2 ng/ml had a sensitivity of 100％ and a specificity of 72.2％.Once the PCT level was superior to 4.7 ng/ml,none of the patients regained consciousness.Conclusion PCT is a simple and effective method for prediction of the outcome after severe TBI.

OBJECTIVETo explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells.

METHODSLuciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay.

RESULTSThe expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05).

CONCLUSIONmiR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Thyroid Hormones ; genetics ; metabolism ; Transfection

Objective To perform molecular cloning and sequencing, bioinformatics analysis,protein expression and function of extracellular signal regulated kinase (EgERK1) of Echinococcus granulosus in Xinjiang. Methods The specific primers of EgERK1 were designed and total RNA was extracted from Echinococcus granulosus in Xinjiang. EgERK1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and prokaryotic expression plasmid pET28a-EgERK1 was constructed and sequenced. The sequences were analyzed by DNA sequencing and bioinformatics technology. The recombinant EgERK1 protein was induced and expressed. The biological function was detected using sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot. Results The sequence of RT-PCR product was 1125 bp, encoding 374 amino acids with isoelectric point of 6.34.This gene was a new ERK-homologues gene indicated by BLAST, named EgERK1(EU701008).Homology comparisons indicated that the homology of EgERK1 and EmMPK1from Echinococcus multilocularis was 95.45%, and was 43.04%-61.88% to ERK from Caenorhabditis elegans, S. cerevisiae, D. melanogaster and human. Phylogenetic analysis showed that EgERK1 clustered with EmMPK1. Bioinformatics analysis predicted that EgERK1 contained a highly conserved T-X-Y motif and activation loop segment of ERK-like kinase.Western blot results showed the EgERK1 recombinant protein could reacted specifically with anti-human ERK monoclonal antibody. Conclusion A new EgERK1 gene of Echinococcus granulosus is successfully cloned and its recombinant protein could reacted specifically with ERK1/2 antibody, which provides the basis for further study of EgERK1 function in the host-parasite interaction.

OBJECTIVETo summarize the detailed failure mechanisms of revision hip arthroplasties and related risk factors.

METHODSFrom November 1988 to July 2008 revision of total hip arthroplasties was performed in 327 patients. The medical history, clinical and imaging material and operation records were investigated.

RESULTSRegarding revision as the end point of the study, the reasons for 327 revision arthroplasties were aseptic loosening in 226 hips (69.1%), infection in 52 hips (15.9%), periprosthetic fracture in 22 hips (6.7%), instability in 17 hips (5.2%), stem fracture in 5 hips (1.5%) and liner dissociation in 5 hips (1.5%).

CONCLUSIONSThe main failure mechanisms of primary hip arthroplasties are aseptic loosening and infection of implants, which could be attributed to improper selection of operation indications and implants and limitations to surgical philosophy and technique.

Adult ; Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Hip ; adverse effects ; Female ; Humans ; Male ; Middle Aged ; Periprosthetic Fractures ; Prosthesis Failure ; Reoperation ; Retrospective Studies ; Risk Factors ; Surgical Wound Infection ; Treatment Failure

OBJECTIVETo construct a mouse that specifically expresses Cre recombinase in hepatocyte.

METHODSA hepatocyte specific transgenic construct containing mouse albumin promoter, the Cre recombinase gene and the poly (A) of human growth factor gene was generated. The linearized constructs were introduced into the fertilized eggs by microinjection to obtain the transgenic mice. The transcriptional specificity of Cre recombinase was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression and function of Cre recombinase were detected by PCR and Southern Blot after crossing the Alb-Cre transgenic mice with the Smad4 conditional knockout mice.

RESULTSThe linearized constructs were microinjected into 837 fertilized eggs, and then the 797 effective eggs of microinjected eggs were implanted into the oviducts of 27 pseudo pregnant mice. In the 53 offspring, there were 6 mice carrying the transgene identified by polymerase chain reaction (PCR) and Southern Blot. Cre recombinase transcripts were detected in the livers and testis of the Alb-Cre transgenic mice using RT-PCR. The Cre recombinase was expressed in the livers of the double heterozygous for Alb-Cre and Smad4 floxed allele, and the exon 8 floxed by loxP site was deleted.

CONCLUSIONA hepatocyte-specific Cre transgenic mouse was generated successfully. The Cre recombinase expressed specifically in liver and could mediate the recombination between loxP sites in vivo.

Animals ; Female ; Hepatocytes ; metabolism ; Humans ; Integrases ; genetics ; Mice ; Mice, Transgenic ; Viral Proteins ; genetics

OBJECTIVETo investigate the value of spiral CT examination for diagnosis occult fracture of the patients with negative result of X-ray examination and with high suspicion of fractures,so as to reduce misdiagnosis.

METHODSFrom January 2007 to June 2010, 31 patients with ankle trauma performed spiral CT examination, including 18 males and 13 females, ranging in age from 21 to 67 years, with a mean of 35 years. The main symptoms of the patients included ankle pain, local swelling, obvious tenderness and activity limitation. All the patients had negative results of X-ray examination.

RESULTSThe spiral CT examination revealed 11 patients with fractures, involving a total of 17 points. Single fracture were found in 6 cases,and multiple fractures were found in 5 cases. Among single fractures, the lateral malleolus fracture was found in 1 case, talus fracture was found in 1 case, scaphoid fracture was found in 1 case, the fracture of the base of 5th metatarsal base was found in 1 case and calcaneal fractures were found in 2 cases. Within multiple fractures,internal and lateral malleolus fracture were found in 1 case; medial malleolus, calcaneus and talus fractures were found in 1 case; talus and scaphoid fractures were found in 1 case; the fractures of 1st and 2nd cuneiform bone were found in 1 case; the 2nd and 3rd metatarsal base fracture was found in 1 case.

CONCLUSIONFor the patients with negative X-ray examination and high suspicion of fractures,the spiral CT examination is needed, which could significantly improve the detection rate of occult fractures, and provide imaging basis for clinical treatment and judicial identify.

Adult ; Aged ; Ankle Injuries ; diagnostic imaging ; Female ; Foot Injuries ; diagnostic imaging ; Fractures, Closed ; diagnostic imaging ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Tomography, Spiral Computed ; methods

OBJECTIVETo measure and evaluate the personal noise exposure of cold rolling mill workers by using noise dosimeter.

METHODSAccording to job category and work type, all workers were divided into 11 groups. 3 to 5 day shift (8:00 to 16:00) workers from each group were selected as subjects for personal noise exposure measurement. SH-126 dosimeters were worn by each subject and collect noise data by a phone fix at collar. All subjects were asked to take notes about their working activities when they were wearing SH-126 dosimeters. Each worker's L(A)(eq) of 8 hours, geometric mean and range of each group were computed.

RESULTSThere were many noise sources in the workshop. Recorded data showed that noise exposure of cold rolling mill was unstable. The varieties of personal noise levels were quite large. Among 53 workers, the highest noise exposure level was 100.0 dB (A), the lowest was 81.2 dB (A); the highest work type was of the foreside welders [94.20 dB (A)], and the lowest was of the straight-cutters [89.02 dB (A)]; quality checkers had the biggest rang [16.3 dB (A)], and primary rolling workers had the lest [2.3 dB (A)].

CONCLUSIONNoise exposure of all the 11 groups were more than 85 dB (A). Noise protection of these workers should be improved. It suggested that measuring personal noise exposure individually with dosimeters might obtain the noise exposure level more integrally in the complicated environment.

Environmental Monitoring ; methods ; statistics & numerical data ; Humans ; Noise, Occupational ; prevention & control ; statistics & numerical data ; Occupational Exposure ; prevention & control ; statistics & numerical data