This study investigated the changes of CD4(+) CD25(+) regulatory T cells (Tregs) in peripheral blood of patients with hepatocellular carcinoma before and after transcatheter arterial chemoembolization (TACE). The proportion of CD4(+) CD25(+) Tregs among CD4(+) T lymphocytes in peripheral blood of 33 patients with hepatocellular carcinoma was determined by flow cytometry before, 1 week and 1 month after TACE. And 25 healthy volunteers served as control. One month after TACE, the patients were divided into two groups: 22 in group A, who were in stable condition or getting better; and 10 in group B, who were deteriorating. One patient died and was excluded. The results showed that the percentage of CD4(+)CD25(+) Tregs among CD4(+) T lymphocytes did not significantly change in the 33 patients 1 week after TACE as compared with that before TACE, however, the difference was significant (P<0.01) between the patients with hepatocellular carcinoma and the healthy subjects. The percentage of CD4(+) CD25(+) Tregs among CD4(+) T lymphocytes in group A 1 month after TACE was decreased significantly in comparison with that before and 1 week after TACE (P<0.01), whereas, that in group B was increased significantly 1 month after TACE (P<0.01). It was concluded that patients with hepatocellular carcinoma had a higher proportion of CD4(+)CD25(+) Tregs in peripheral blood. TACE did not significantly affect the level of CD4(+) CD25(+) Tregs within short time (such as 1 week). The proportion of CD4(+)CD25(+) Tregs in peripheral blood 1 month after TACE was related to the prognosis of hepatocellular carcinoma.
Antineoplastic Agents/administration & dosage ; Carcinoma, Hepatocellular/*immunology ; Carcinoma, Hepatocellular/therapy ; Chemoembolization, Therapeutic/*methods ; Liver Neoplasms/*immunology ; Liver Neoplasms/*therapy ; T-Lymphocytes, Regulatory/*immunology

OBJECTIVETo study the MRI manifestation of testicular tumor and the value of MRI in the diagnosis of the disease.

METHODSWe retrospectively analyzed 23 cases of pathologically confirmed testicular tumor, and observed the morphological characteristics, signals and surrounding conditions of the tumor using plain and enhanced MRI scanning.

RESULTSOf the 23 cases, seminoma was identified in 7, mixed germinoma in 3, teratoma in 3, endodermal sinus tumor in 2, epidermoid in 1, Leydig cell tumor in 1, leucoma in 1, nonspecific inflammatory mass in 3, and tuberculosis in 2. MRI revealed the precise locations and specific characteristics of

CONCLUSIONBased on MRI findings and clinical manifestation, most testicular tumors can be diagnosed correctly.

Adolescent ; Adult ; Carcinoma, Embryonal ; diagnosis ; Child ; Child, Preschool ; Endodermal Sinus Tumor ; diagnosis ; Germinoma ; Humans ; Infant ; Leydig Cell Tumor ; diagnosis ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Retrospective Studies ; Seminoma ; diagnosis ; Teratoma ; diagnosis ; Testicular Neoplasms ; diagnosis ; Young Adult

OBJECTIVETo develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.

METHODSThe double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.

RESULT AND CONCLUSIONDNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.

Base Sequence ; Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Inverted Repeat Sequences ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Restriction Mapping ; methods ; Sequence Analysis, DNA ; Time Factors

OBJECTIVETo establish transgenic mouse models of familial amyotrophic lateral sclerosis (FALS) and identify the genotype of the first filial generation.

METHODSSix male B6SJL SOD1G93A/+ hemizygote mice were mated with 6 female B6SJLF1/J+/+ mice to produce the filial generation. The genomic DNA was extracted from the tail vein blood of the first filial generation mice and PCR was performed to amplify the hmSOD1 gene fragment. The genotype of the mice was determined by electrophoresis, and the PCR product was purified for further gene sequence analysis and detection of mutation loci.

RESULTSFifty-three progeny mice were born and the survival rate before ALS onset was 98% (52/53), and among the survived mice, the positivity rate for hmSOD1 gene was 44.2% (23/52). Electrophoresis result showed that the PCR product of 236 bp was consistent with the hmSOD1 gene fragment, and the sequence of the PCR product was identical with hmSOD1 gene sequence of G93A mutant.

CONCLUSIONTransgenic mouse models of ALS can be established in the first filial generation of male B6SJL SOD1G93A/+ mice mated with female B6SJLF1/J+/+. PCR technique can precisely identify the genotype of the filial generation.

Amyotrophic Lateral Sclerosis ; enzymology ; genetics ; pathology ; Animals ; Animals, Newborn ; Base Sequence ; Breeding ; Disease Models, Animal ; Electrophoresis, Agar Gel ; Female ; Genotype ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Mice, Transgenic ; Point Mutation ; Sequence Analysis, DNA ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1

OBJECTIVETo review the clinical manifestations, imaging, tumor markers, treatment methods, pathology results and clinical curative effects of pineal region tumors and to evaluate the characteristics and intervention strategies for those tumors.

METHODSThe clinicopathological data of 132 patients with pineal region tumor treated in our department between January 2000 and May 2008 were retrospectively studied.

RESULTSA moderate predominance in males was presented. The clinical manifestations of the disease included increased intracranial pressure and ocular movement impairment. There were some features but no regularity and specific appearance on imaging including CT and MRI. 88.6% of patients associated with hydrocephalus. A high serum level of alpha-fetoprotein (AFP) was presented in 14 cases and high HCG in 9 cases. Eighteen cases received direct radiation therapy and 7 had radiotherapy post biopsy. 107 cases were treated surgically and 63 cases received postoperative adjuvant treatment. 114 cases had pathology results including 56 germ cell tumors. The patients were followed up for 12 approximately 132 months. Recurrence developed in 23 cases and 12 cases died. The 5-year survival rate was 89.3%.

CONCLUSIONPineal region tumors are often associated with hydrocephalus and this makes preoperative diagnosis difficult. Imaging examination may help diagnosis but less specific. Germ cell tumors may diagnosed by some tumor markers. Radiation therapy is the choice of treatment for pure germinomas. Other types of pineal region tumors should receive surgical treatment. Postoperative adjuvant treatment based on pathology can provide a good prognosis in pineal region tumor.

Adolescent ; Adult ; Aged ; Brain Neoplasms ; blood ; diagnosis ; therapy ; Child ; Child, Preschool ; Chorionic Gonadotropin ; blood ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Humans ; Hydrocephalus ; etiology ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Pineal Gland ; pathology ; surgery ; Pinealoma ; blood ; diagnosis ; therapy ; Retrospective Studies ; Sex Factors ; Survival Rate ; Tomography, X-Ray Computed ; Young Adult ; alpha-Fetoproteins ; metabolism

OBJECTIVETo look into the character of the expression of collagen type II and transforming growth factor beta1 (TGF-beta1), basic fibroblast growth factor (bFGF) in the apical articular process cartilages of adolescent idiopathic scoliosis (AIS) and congenital scoliosis (CS) patients.

METHODSThe articular processes of 22 AIS and 18 CS were collected. The techniques of HE staining, immunohistochemistry and in situ hybridization were adopted in this research. By comparing the apical processes with the end processes, the convex processes with the concave processes, the AIS processes with CS processes, the pathological changes of the articular process cartilages of these patients and the distribution of collagen type II and TGF-beta1, bFGF in them were studied. The images of immunohistochemistry and in situ hybridization were input into the image analysis system and were analyzed semi-quantitatively. The SAS software (8.01) was adopted, and P < 0.05 was defined as the significant level.

RESULTSThe expression of collagen type II and TGF-beta1, bFGF in AIS was similar to CS: the concave sides of apexes were higher than the convex sides. The comparisons had statistical significance. There was no statistical significance between upper and lower end vertebrae in convex and concave sides, between convex and concave sides in upper and lower end vertebrae. The apical vertebrae were significantly higher than the ipsilateral sides of upper or lower end vertebrae for collagen type II. There was no statistical difference of the expression at the concave, convex, upper, lower end vertebrae between AIS and CS.

CONCLUSIONSThe cartilages of the apical processes show some signs of regression and hypoplasia in scoliosis. The concave side is more severe than the convex side. Increase of collagen type II and TGF-beta1, bFGF in the concave sides of apical processes in scoliosis may be the results of reconstruction of extracellular matrix and the compensation reactions which are caused by abnormal biomechanical forces such as compressive stresses. Compressive stress on the concave sides has more influences on the expression of collagen type II than tensile stress on the convex sides.

Adolescent ; Cartilage, Articular ; metabolism ; pathology ; Child ; Collagen Type II ; metabolism ; Fibroblast Growth Factor 2 ; metabolism ; Humans ; Scoliosis ; metabolism ; pathology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.

METHODSBased on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.

RESULTSIn the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).

CONCLUSIONThe dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.

Animals ; Cell Line ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; Luciferases ; genetics ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

BACKGROUNDLow-density lipoprotein (LDL) receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line (HepG2) cells under physiological and inflammatory conditions.

METHODSIntracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum (ER) to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies.

RESULTSLDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1 beta (IL-1 beta) further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1 beta caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium.

CONCLUSIONSIL-1 beta disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.

Cell Line, Tumor ; Cholesterol ; analysis ; Endoplasmic Reticulum ; metabolism ; Feedback, Physiological ; Humans ; Interleukin-1beta ; pharmacology ; Intracellular Signaling Peptides and Proteins ; analysis ; genetics ; Membrane Proteins ; analysis ; genetics ; Protein Transport ; RNA, Messenger ; analysis ; Receptors, LDL ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sterol Regulatory Element Binding Protein 2 ; analysis ; genetics

OBJECTIVETo observe the characteristics of gene expression of type X collagen in the cartilage of end-plate and the fibrous annulus in the intervertebral disc of idiopathic scoliosis (IS) patients.

METHODInvestigating the expression of type X collagen in the peak disc and the lower end disc of 21 IS patients, the peak disc of 16 congenital scoliosis (CS) and the lumbar disc of 3 normal people (according with the principle of medical ethnics) by reverse transcript polymerase chain reaction.

RESULTSThe expression of type X collagen in the concave side of IS peak disc was higher than the convex side (P < 0.05). There was no significant difference of gene expression of type X collagen between the convex side and the concave side of the lower end disc (P > 0.05). The gene expression of type X collagen in the IS peak disc was higher than those of lower end disc (P < 0.05). For the CS peak discs, the expression of type X collagen of the concave side was higher than the convex side (P < 0.05).

CONCLUSIONThe expression of type X collagen of the IS peak disc increases, and the expression of type X collagen of the concave side is higher than the convex side. These changes may be secondary.

Adolescent ; Child ; Collagen Type X ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Intervertebral Disc ; metabolism ; Male ; Scoliosis ; genetics ; metabolism

OBJECTIVETo observe early clinical efficacy of general spine system (GSS) in spondylolisthesis combined with lumbar canal stenosis, lumbar decompression, reduction and bone graft.

METHODSSixteen patients with degenerative lumbar spondylolisthesis combined with lumbar canal stenosis, 10 male, 6 female, average age 58.5 years (range 42 - 72 years) underwent lumbar decompression, bone graft and internal fixation using GSS. Preoperatively 10 patients had degree I spondylolisthesis and 6 patients had degree II spondylolisthesis. Clinical efficacy, reduction effectiveness and complications were recorded.

RESULTSThe 16 patients in this group were followed up postoperatively for an average of 21.2 months (18 - 24 months). At latest follow-up after surgery, preoperative clinical symptoms had disappeared completely in 15 of 16 patients, and low back pain relief was seen in 15 patients. Average duration of surgery was 170 min (120 - 270 min), and average blood loss was 375 ml (100 - 800 ml). X-ray results showed complete reduction for all spondylolisthesis patients, and results remained good in follow-up. Dura mater tearing, pedical fracture, nerve injury and other surgical complication did not occur. Screw breakage, screw loosening and instrument loosening at the screw-rod juncture were not observed after surgery or in follow-up.

CONCLUSIONGSS provides good reduction for spondylolisthesis, and shows good early clinical efficacy.

Adult ; Aged ; Bone Transplantation ; Decompression, Surgical ; Female ; Follow-Up Studies ; Humans ; Internal Fixators ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Spinal Fusion ; instrumentation ; methods ; Spinal Stenosis ; etiology ; Spondylolisthesis ; surgery ; Treatment Outcome