BACKGROUND:Chondromodulin-Ⅰ is expressed mainly in the cartilage, but it is little expressed in mesenchymal stem cells. Combined with the previous study of our group, we concluded that chondromodulin-Ⅰmaybe play an important role in inducing mesenchymal stem cells into chondrocytes accurately.
OBJECTIVE:To construct an expression plasmid stably carrying chondromodulin-Ⅰ to up-regulate the expression of chondromodulin-Ⅰ in bone marrow mesenchymal stem cells.
METHODS:Specific primers were designed in rat cartilage for chondromodulin-Ⅰ gene, then the pcDNA3.1 (+) plasmid expression vector was digested by enzyme and directional connected gene to construct pcDNA3.1(+)/ChM-Ⅰ expression vector. Bone marrow mesenchymal stem cells were obtained from rats using the method of density gradient centrifugation combined with adherent culture. Recombinant plasmid pcDNA3.1(+)/ChM-Ⅰ was transfected into rat bone marrow mesenchymal stem cells with liposome method, and G418 selection was used for stable screen of transfected cells. Reverse transcription-PCR and western blot were used to detect chondromodulin-Ⅰ expression in celllines.
RESULTS AND CONCLUSION:The positive clones were digested by enzyme and were identified and sequenced. The results showed that the reality length and sequence of chondromodulin-Ⅰ gene were consistent with the theoretical values, and reading frame was correct. Reverse transcription-PCR and western blot results showed that the expressions of chondromodulin-ⅠmRNA and protein were markedly up-regulated in bone marrow mesenchymal stem cells. Recombinant plasmid pcDNA3.1(+)/ChM-I was successful y constructed, and transfected into rat bone marrow mesenchymal stem cells. After G418 selection, expression of chondromodulin-Ⅰ was up-regulated stably in rat bone marrow mesenchymal stem cells.

Objective - To optimize the extraction and quantification methods for the determination of S phenylmercapturic acid - Methods (SPMA) in urine with performance liquid chromatography mass spectrometry. The urine was hydrolyzed with 50.0% sulfuric acid. The hydrolysate was purified by solid phase extraction column. Purified samples were separated by C18 chromatographic column and detected by tandem mass spectrometry. The isotope labeled SPMA was used as the internal Results - standard. The internal standard curve was used for quantification. The linear range of SPMA was 0.50 50.00 μg/L with the correlation coefficient of 0.999 8. The detection limit and the lower limit of quantification were 0.05 and 0.17 μg/L, - - - - respectively. The recovery rate was 97.0% 102.0%. The within run and between run relative standard deviation were 0.6% 1.0% - and 1.7% 6.5%, respectively. The mass concentration of urinary SPMA in the occupational benzene exposure group was - vs P higher than the non occupational benzene exposure group by this method (median: 2.81 0.28 μg/g creatinine, <0.05). Conclusion Compared to the national standard method, this optimized method of solid phase extraction and internal standard for quantification eliminates the matrix effect. This method is accurate and precise, and is suitable for the determination of SPMA acid in urine.

Objective To evaluate the short and mid-term effects of fixing comminuted posterior acetabular wall fractures with structural autologous iliac bone graft combined with mini screws.Methods From January 2010 to January 2014,29 patients with comminuted posterior acetabular wall fracture were treated by structural autologous iliac bone graf combined with mini screws.They were 21 males and 8 females,with a mean age of 44.2 years (range,from 22 to 58 years).The mean time form injury to operation was 7.8 days (range,from 1 to 25 days).The operations were performed through the Kocher-Langenbeck approach,with the patients in the lying position on the uninjured side.The fragments were reduced and fixed by mini screws and the ischemic ones were removed.Structural autologous iliac bone graft was used to reconstruct the posterior wall of acetabulum before a reconstruction plate was applied to compress and maintain it.The functional outcomes were evaluated by the modified Merle d'Aubigne and Postel clinical grading system at the last follow-ups.The radiographs were graded according to the Matta criteria.Results By the Matta criteria,10 cases achieved excellent reduction,16 good reduction,and 3 poor reduction,giving a good to excellent rate of 89.7％.Of this series,29 patients were followed up for 31.5 months on average (range,from 12 to 48 months).By the modified Merle d'Aubigne and Postel criteria,the functional recovery was rated as excellent in 16 cases,good in 9,fair in 3 and poor in one,giving a good to excellent rate of 86.2％.Two cases developed femoral head necrosis according to the magnetic resonance imaging 18 months postoperation.Three patients developed traumatic arthritis two years postoperation.Five patients developed heterotopic ossification postoperation,with no obvious clinical symptoms.Two patient with injury to the sciatic nerve recovered 4 months postoperation.Conclusions Structural autologous iliac graft combined with mini screws can reconstruct the integrity and stability of the fractured acetabular posterior wall,avoiding osteonecrosis of the acetabulum.This surgical technique is effective and safe in treatment of comminuted fracture of the acetabular posterior wall.

Objective To study the clinical and electrophysiological features of myasthenia gravis (MG)accompanied by myogenic lesion.Methods The data of the patients who were diagnosed MG accompanied by myogenic lesion from 1998 to 2006 were collected in EMG Room, Department of Neurology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and were retrospectively analyzed.Results In this group of 53 patients, myogenic lesion was found more often in patients with early-onset MG than those with late-onset(69.81% vs 30.20%), and among the early-onset patients the frequency of female was significantly higher than male(26 vs 11,X~2=5.281, P

OBJECTIVETo study the simple infection and super/co-infection of HAV-HEV, HGV in patients with viral hepatitis.

METHODSUsing EIA method to detect anti-HAV IgM, HBV serum markers, anti-HCV IgM, anti-HDV IgM, anti-HEV IgM, anti-HGV IgM in viral hepatitis patients with different clinical types.

RESULTSSeventy-three percent patients (154/210) had HBV infection markers, twenty-nine percent patients (61/210) had HAV infection marker, eight percent patients (17/210) had HCV, HDV infection markers, ten percent patients (21/210) had HEV infection and seven percent patients (15/210) had HGV infection. Only nine percent patients (20/210) had viral hepatitis serum markers negative. In all clinical types, sixty-one percent patients had only one type hepatitis virus infection, thirty-two percent patients had two types of hepatitis virus super/co-infection, six percent patients had three types of hepatitis virus super/co-infection. Super/co-infection often occurred in patients who had cirrhosis or hepatic failure.

CONCLUSIONHBV and HAV infection is very common in viral hepatitis patients, whereas HCV, HDV, HEV and HGV infection is relatively low; double super/co-infection of HAV-HEV, HGV frequently occurs in severe patients with viral hepatitis.

Antibodies, Viral ; blood ; China ; epidemiology ; Female ; GB virus C ; isolation & purification ; Hepatitis A ; epidemiology ; virology ; Hepatitis A virus ; isolation & purification ; Hepatitis E ; epidemiology ; virology ; Hepatitis E virus ; isolation & purification ; Hepatitis Viruses ; isolation & purification ; Hepatitis, Viral, Human ; epidemiology ; virology ; Humans ; Male ; Superinfection

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.

OBJECTIVETo investigate the expression of follistatin-like protein 1 (FSTL-1) in bone metastasis of prostate cancer (BMPC), the correlation of serum FSTL-1 with the chronic inflammatory factor interleukin-6 (IL-6) and bone morphogenetic protein 6 (BMP6) , and the clinical application value of serum FSTL-1 in BMPC.

METHODSUsing ELISA, we measured the expression levels of serum FSTL-1, IL-6, and BMP6 in 35 patients with BMPC and another 30 with benign prostatic hyperplasia (BPH) and performed correlation analysis on the data obtained.

RESULTSCompared with the BPH controls, the BMPC patients showed a significantly decreased expression of serum FSTL-1 ([34.45 ± 12.35] μg/L vs [20.23 ± 8.69] μg/L, P < 0.01) and increased levels of IL-6 ([11.21 ± 8.62] μg/L vs [23.56 ± 20.12] μg/L, P < 0.05) and BMP6 ([293.50 ± 39.72] μg/L vs [428.30 ± 178.40] μg/L, P < 0.05). There was a significant negative correlation between the level of serum FSTL-1 and those of IL-6 and BMP6 in the BMPC patients, with correlation coefficients of -0.971 and -0.972, respectively (P < 0.05).

CONCLUSIONThe expression of serum FSTL-1 decreases in patients with bone metastasis of prostate cancer, and it is correlated with the levels of inflammatory factor and cell transformation factor. This finding offers a novel biological marker for the development and progression of prostate cancer as well as a new biological target factor for its intervention.

Aged ; Biomarkers, Tumor ; blood ; Bone Morphogenetic Protein 6 ; blood ; Bone Neoplasms ; blood ; secondary ; Disease Progression ; Follistatin-Related Proteins ; blood ; Humans ; Interleukin-6 ; blood ; Male ; Prostatic Hyperplasia ; blood ; Prostatic Neoplasms ; blood ; pathology

OBJECTIVETo study the pathogenic effect of hepatitis G virus (HGV) infection on hepatic failure.

METHODSUsing the RT-PCR and EIA techniques to detect HGV RNA and anti-HGV in sera of hepatic failure patients and compare them with their liver function and mortality rates.

RESULTSThere was no significant difference about the positive rates of HGV among acute hepatic failure, subacute hepatic failure and chronic hepatic failure groups (X(2)=2.54, P>0.05). The level of ALT in HGV-positive group was slightly lower than that in HGV-negative group. The concentration of bilirubin and globulin was higher in HGV-positive group than HGV-negative group, and the concentration of albumin in HGV-positive group was significantly lower than that in HGV-negative group (t=2.59, P<0.05). The mortality rate in HGV-positive group was significantly lower than that in HGV-negative group (X(2)=4.68, 0.01

CONCLUSIONSThe virulence of HGV is mild, and the HGV infection does not aggravate hepatic failure.

Adult ; Female ; Flaviviridae Infections ; complications ; GB virus C ; pathogenicity ; Hepatitis, Viral, Human ; complications ; Humans ; Liver Failure ; etiology ; Male ; Middle Aged

OBJECTIVETo evaluate the frequency of microdeletions in the long arm of Y chromosome of idiopathic infertile males with azoospermia and oligospermia in Shaanxi province in China and to investigate the relevance of sperm count to Y microdeletion frequencies.

METHODSAccording to the sequence of sequence-tagged sits (STS) AZFa, AZFb, AZFc and SRY, 4 of the azoospermic factor regions on Y chromosome long-term supplied by GenBank, 5 sets of primers were synthesized. The Y microdeletions in AZF regions were screened by polymerase chain reaction (PCR) in 64 idiopathic cases of azoospermia and oligospermia and 20 men of known fertility.

RESULTSNo microdeletion was detected in the 20 normospermic subjects. Deletion of the AZFc/DAZ was detected in 11 individuals and one patient had both AZFb and AZFc deletion; no deletion of AZFa and SRY region was found. The frequency of Y microdeletions in the subgroups with different sperm count showed the highest value among azoospermic men (3 cases, 21.4%). The percentage progressively decreased with the deletion frequency (20.0%, 17.9% and 8.3%) in the subgroups with sperm counts of < 1 x 10(6)/ml, < (1-5) x 10(6)/ml and < (1 to approximately 10) x 10(6)/ml, respectively.

CONCLUSIONY chromosome microdeletions are specifically associated with severe spermatogenic failure. The rate of deletion involving AZF region of the Y-chromosome is higher in infertile men with azoospermia and oligospermia. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.

Adult ; China ; Chromosome Deletion ; Chromosomes, Human, Y ; Humans ; Male ; Oligospermia ; genetics ; Polymerase Chain Reaction ; Sequence Tagged Sites

OBJECTIVETo investigate performance of a biotinylated imaging probe 3a for targeted imaging of breast cancer cells.

METHODSUltraviolet absorption spectrum and fluorescence spectrum were employed to analyze the spectral characteristics of 3a. The fluorescence spectrums of 3a treated with different concentrations of glutathione (GSH) were obtained to determine the sensibility of 3a to GSH. Flow cytometry was used to determine the cellular uptake of 3a by MCF-7 cells, MDA-MB-231 cells and Hs 578Bst cells in the presence or absence of biotin, and the imaging performance of 3a in the 3 cell lines was assessed under an inverted fluorescent microscope. The toxicity of 3a to the cells was evaluated using MTT method.

RESULTS3a showed the strongest absorption peak at 510 nm, and its fluorescence emission signal was the strongest at 544 nm. As the concentration of GSH increased (0-6 mmol/L), 3a exhibited an increasing fluorescence signal at 544 nm. The cellular uptake of 3a was markedly higher in MDA-MB-231 cells and MCF-7 cells than in Hs 578Bst cells. The imaging studies showed that 3a had a good breast cancer cell-targeting property and produced clear images under fluorescent microscope. MTT assay demonstrated no obvious toxicity of 3a in Hs 578Bst cells even at the concentration of 20 µmol/L, but MCF-7 cells and MDA-MB-231 cells exposed to 2-20 µmol/L 3a showed a lowered cell viability.

CONCLUSION3a is capable of targeted imaging of breast cancer cells mediated by biotin. 3a at the concentration of 2-20 µmol/L has minimal cytotoxicity to normal breast cells but can lower the viability of breast cancer cells.