OBJECTIVETo compare different expression profiles of all known chemokine receptors in human hepatocellular carcinoma (HCC) cell lines with different metastasis potentials.

METHODSEighteen pairs of chemokine receptor primers were designed using Premier software. Expression profiles of the 18 chemokine receptors on four HCC cell lines of lower to higher potentials of metastasis (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6) were analyzed by RT-PCR. Expression of CXCR4 was detected by RT-PCR.

RESULTSExpression profiles of chemokine receptors on four HCC cell lines with different metastatic potentials had significant differences (P < 0.01), in which CCR10, CXCR4 and CXCR6 expressions decreased gradually as the metastatic potential of the cell lines increased. The expressions of CCR3, CCR4, CCR10, CCR12 and XCR1 on HCCLM6 were significantly reduced compared with SMMC-7721 (P < 0.01), whereas the expressions of CXCR1 (P = 0.006) and CXCR5 (P = 0.003) exceeded that of SMMC-7721. Except for CXCR2, CXCR6 and XCR1, most of chemokine receptors on MHCC97-H were expressed differently compared with MHCC97-L (P < 0.05), in which expressions of CCR1 (P = 0.002), CCR2 (P = 0.004) and CCR5 (P = 0.046) exceeded MHCC97-L. CXCR4 was detected only on the positive controls and SMMC-7721 when the template of total RNA was reduced one-half in RT-PCR.

CONCLUSIONChemokine receptors are expressed very differently at mRNA level on HCC cell lines with different metastatic potentials. The different profiles of chemokine receptors in tumor microenvironment and the function of CXCR4 in HCC should be further studied. Our findings have important implications in understanding the relationship between chemokine receptors and the metastatic potential of HCC.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics ; Receptors, Chemokine ; metabolism

OBJECTIVETo evaluate the influence of Chinese drugs for cooling blood and dissolving stasis (CBDS) in different concentrations on morphology of krypton laser induced choroidal neovascularization (CNV) in brown Norway (BN) rats.

METHODSForty-eight rats received laser irradiation (659 nm) on fundus of one eye (power 360 mW, spot diameter 50 microm, time 0.05 s). They were divided into four groups equally: the control group (A) treated with normal saline, and the three CBDS groups treated respectively with high (B, 5.0 g/kg), median (C, 2.5 g/kg) and low (D, 1.25 g/kg) dosage of CBDS, twice every day via gastric perfusion for 21 successive days. Fundus fluorescein angiography (FFA) on 4 selected rats in each group was performed at the 7th, 14th and 21st day after photocoagulation, and histopathologic examination using light microscope with immuno-histochemical stain was conducted on them as well.

RESULTSFFA showed that CNV was firstly appeared on day 7 after photocoagulation, in Group A, it expanded gradually and reached the peak on day 21, but showed no significant expansion in the three CBDS groups. The fluorescein leakage in Group C (52343.13 +/- 12973.92 dots) and D (66252.78 +/- 20659.71 dots) was significantly less than that in Group A (91457.19 +/- 29309.11 dots) and B (95973.40 +/- 53950.43 dots) on day 21, all showing statistical significance (P<0.05). The variation of CNV in thickness showed that in Group A it increased gradually from day 7 and reached the peak on day 21 (55.3383 +/- 8.5036 microm); but in the CBDS groups, the peak was reached on day 14, then became thinned gradually, on day 21, it was 40.0913 +/- 13.3448 microm in Group B, 38.8473 +/- 7.9862 microm in Group C and 38.9372 +/- 5.1728 microm in Group D, all thinner than that in Group A significantly (P<0.05).

CONCLUSIONCDBS can effectively suppress the krypton laser induced CNV proliferation and prevent the CNV leakage in BN rats.

Animals ; Choroidal Neovascularization ; drug therapy ; pathology ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Fluorescein Angiography ; Male ; Rats ; Rats, Inbred BN

A new method for establishing ES cell lines from 129/ter. C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM) for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.
Alkaline Phosphatase ; metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; Cell Differentiation ; Cell Division ; Cell Line ; Embryo, Mammalian ; cytology ; Female ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Rats ; Stem Cells ; physiology

OBJECTIVETo introduce a technique pertaining to S2 iliosacral screw insertion.

METHODSThe screw pathway was first measured on the preoperative pelvic CT scan or the standard sacral lateral radiograph to make sure the existence of the "safe zone" in the S2 segment for screw insertion. Under general anesthesia, patients were positioned supine or prone, depending on the injury pattern of pelvic ring or associated injuries requiring concomitant operation. The operation field was routinely sterilized using iodine and subsequent alcohol solution and draped. The tip of a guide wire was inserted through a stab wound to the posterior outer iliac table, manipulated in the "safe zone" being enclosed by the anterior aspect of the S2 nerve root tunnel, the anterior aspect of the sacral vertebrae, and the inferior aspect of the S1 foramen under the guidance of the standard sacral lateral fluoroscopy, and then the tip was hammered one to two millimeters into the iliac cortex. The guide wire progressed along the trajectory between the inferior aspect of the S1 foramen and the superior aspect of the S2 foramen on the pelvic outlet fluoroscopic view, and then along the posterior to the anterior aspect of the S2 sacral vertebrae and alae on the pelvic inlet fluoroscopic view with a predetermined length. At that moment, in order to ensure the safety, another standard sacral lateral view was imaged to detect the guide wire's tip which should locate posterior to the anterior aspect of the sacral vertebrae and anterior to the anterior aspect of the S2 nerve root tunnel. Subsequently, the depth was measured, the trajectory was drilled and tapped, and the screw was inserted. Following the removal of the guide wire, the wound was irrigated and sutured.

RESULTSUtilizing this insertion technique, there were 30 S2 iliosacral screws in total being placed to stabilize the injured and unstable posterior pelvic ring in 27 patients. Each S2 screw was accompanied by an ipsilateral S1 screw. The S2 screw location was completely intraosseous in all patients, which was verified by postoperative pelvic outlet and inlet radiographs and CT scans. The insertion accuracy was 100 percent in the present series.

CONCLUSIONThe S2 iliosacral screw insertion technique is safe and reproducible to guide the placement of the S2 screw, enhancing the stability for the compromised posterior pelvic ring.

Adult ; Bone Screws ; Female ; Fractures, Bone ; surgery ; Humans ; Ilium ; injuries ; surgery ; Male ; Sacrum ; injuries ; surgery

Objective To investigate the effect of collagen scaffold loaded with collagen-binding domain neurotrophin-3 (CBD-NT3) on the extension of cellular processes of dorsal root ganglions (DRGs), and explore the significance of this kind of combinatorial strategies in the spinal cord injury repair. Methods The tail tendons of SD neonatal rats were performed the removal of cellular components to prepare the collagen scaffold; HE staining was employed to evaluate whether the cells were completely removed from the collagen scaffold. The collagen scaffold was loaded with CBD-NT3,and then, they were co-cultured with primary DRGs for 1, 3 and 5 d, respectively. NT3 and PBS were also co-cultured with primary DRGs for 1, 3 and 5 d, respectively, as controls. Cells on the scaffold were stained by fluorescein diacetate (FDA) for morphology observation and the lengths and angles of the processes in each group were also quantitatively analyzed. Scanning electron microcopy (SEM) was employed to observe the topography of scaffold and the ultrastructure of DRGs 3 d after the co-culture.Results HE staining indicated that the cellular components in the scaffold were removed completely.The length of processes elongation in CBD-NT3 treatment group was significantly longer than that in the controls 3 d after the co-culture (P＜0.05). The 95% confidence interval of the angle between the line which the process emerged from the cell soma to the growing tip of the process and the long axis of fiber was 18.8-20.7 degrees. The results of SEM showed that cells could rely on the topography of the scaffold to anchor and grow. Conclusion The combinatorial strategies of collagen scaffold with CBD-NT3 can play a double function for oriented guiding and inducing extension of cellular processes effectively,which may provide a better therapeutic approach for spinal cord injury repair.

Objective ] To study the molecular characteristics and antibiotic resistance of Salmonella diarrhea cases in sentinel hospitals through active surveillance system conducted by public health laboratory. [ Methods] Two sentinel hospitals were chosen for collection of stool specimens from food-borne infectious diarrhea cases and for Salmonella separation and detection immediately following serotyping and antimicrobial susceptible test ( AST ) on those isolates .Moreover , pulsed field gel electrophoresis ( PFGE) was used for the genetic homology analysis . [ Results] A total of 2 579 diarrhea specimens were collected and analyzed from 2010 to 2012, with 185 Salmonella isolates, covering 23 different serotypes (annual positive rates were 9.1%, 6.8%, 5.1%, with an average 7.2%).Salmonella enterica serovar Enteritidis(S.Enteritidis) and Typhimurium(S.Typhimurium) were the most common serotypes, of which 68.9% cases were seen in those aged 21 to 60 and 21.4% cases in those over 60 years old. 27.7%-96.9%S.Enteritidis and 2.6%-63.2% S.Typhimurium(P all <0.05) proved resistant to Nalidixic acid, Sultisoxazole, Streptomycin, Sulfamethoxydiazine, Gentamicinand Tetracycline. PFGE analysis on 22 S.Enteritidis strains showed 11 different clusters , while 20 S.Typhimurium strians showed 6. [Conclusion] S.Enteritidis and S.Typhimurium are the most common Salmonella serotypes, and molecular typing indicates the existence of clustering and sporadic outbreaks caused by dominance clones . We should be alert to early warnings on potential outbreaks of multiple-drug-resistant ( MDR ) S.Enteritidis.Active surveillance system based on public health laboratory should play an important role in the control of food-borne infectious diseases .

OBJECTIVETo compare the detection sensitivity of epidermal growth factor receptor (EGFR) mutations between allele specific oligonucleotide PCR (ASO-PCR) and bi-loop probe and specific primer quantitative PCR (BPSP-qPCR).

METHODSA total of 96 non-small cell lung cancer specimens were selected from West China Hospital from September 2009 to December 2010. ASO-PCR was developed to detect the presence of classical EGFR mutations. A total 39 available specimens were also tested by BPSP-qPCR.

RESULTSEGFR mutation detection rate was 30.2% (26/96) by ASO-PCR. The mutation rate was higher in female than in male patients [45.5% (20/44) vs. 17.3% (9/52), P = 0.003], non-smokers than smokers [44.1% (26/59) vs. 8.1% (3/37), P < 0.001] and adenocarcinomas than other subtypes of lung cancer [37.0% (27/73) vs. 8.7% (2/23), P = 0.01]. Among mutation negative cases by ASO-PCR, BPSP-qPCR increased the rate of detection of 19-del and L858R mutation by 10.3% (4/39) in adenocarcinomas and non-smoking subset. Overall, the mutation detection rate of BPSP-qPCR was higher than that of ASO-PCR [66.7% (26/39) vs. 41.0% (16/39), P = 0.02].

CONCLUSIONBPSP-qPCR has a better detection sensitivity than that of ASO-PCR.

Adenocarcinoma ; genetics ; Carcinoma, Non-Small-Cell Lung ; genetics ; DNA Mutational Analysis ; Female ; Genes, erbB-1 ; Humans ; Lung Neoplasms ; genetics ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; methods ; Receptor, Epidermal Growth Factor ; genetics ; Sensitivity and Specificity ; Sex Factors ; Smoking

OBJECTIVEEstablishing cDNA microarray, in order to study functional genomics of Salvia miltiorrhiza.

METHODTotal RNA samples were prepared from S. miltiorrhiza roots using a modified CTAB method. mRNA was isolated by Quichprep Micro mRNA Purification Kit from Pharmacia. Then cDNA was synthesized and cloned into the EcoRI-XhoI sites of the ZAP Express vector using a cDNA synthesis kit, and the ligation mixture was packaged using a ZAP-cDNA Gigapack Gold III cloning kit (Stratagene). The single phage was isolated for PCR amplification, Aliquots of the PCR reactions were analyzed in a 1.5% agarose gel to verify the quality of PCR. The remaining cDNA was purified by Multiscreen filter plates (Millipore) and aliquots were analyzed by agarose gel again to verify the quality of purification. Clones passed verification was resuspended in 15 microL 50% DMSO for arraying. An actin gene from S. miltiorrhiza was used for positive control. PloyA and 50% DMSO was used for negative controls.

RESULTBacterial colonies containing cNDAs of S. miltiorrhiza were inserted with average insert size of 0. 5 kb to 2. 5 kb. Total 4 354 genes were singled out from the first 8 736 PCR product and used for cDNA microarray manufacture. Single color fluorescence hybridization showed that all positive controls had signals while negative controls had no signals.

CONCLUSIONIt was the first cDNA microarray about traditional Chinese herbs especially for geoherbs. It could be a powerful tool for studying functional genomics of S. miltiorrhiza.

Gene Expression Profiling ; Gene Expression Regulation, Plant ; Genomics ; methods ; Oligonucleotide Array Sequence Analysis ; methods ; Plant Roots ; genetics ; Plants, Medicinal ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Salvia miltiorrhiza ; genetics

OBJECTIVETo explore in-vivo targeted imaging techniques for liver cancer detection using quantum dots (QDs) labeled probes in a nude mouse model of human hepatocellular carcinoma.

METHODSMercaptoacetic acid (MAA) modified QDs were linked to mouse-anti-human alpha-fetoprotein (AFP) monoclonal antibody to form water soluble QD-AFP-Ab probes, which were validated by spectra analyses and transmission electron microscope. The probes were firstly used to detect AFP antigen in human hepatocellular carcinoma cell line HCCLM6 in-vitro by one-step immunofluorescence method. In-vivo tumor xenografts and lung metastases models were then established by inoculation of HCCLM6 cells subcutaneously and into the tail vein of nude mice, respectively. QD-AFP-Ab probes were injected into the tail vein of the tumor bearing mice for live animal fluorescence imaging. Spectra of tumor and normal tissue were analyzed under illumination of Ti: sapphire laser. Serum levels of alanine amino transferase, aspartate amino transferase, blood urea nitrogen and creatinine were determined by conventional biochemical analysis. The liver, spleen, lungs, kidneys, heart and brain of the experimental nude mice were investigated for nonspecific uptake of the probes by confocal microscope.

RESULTSThe QD-AFP-Ab probes had broad excitation spectra and high fluorescence intensity. They could specifically and efficiently recognize AFP antigen in hepatocellular carcinoma cells. Tumor targeting imaging using these probes were successful without any acute toxicity to the experimental animals. Spectra analysis showed that the probes per field were lower in the centre than the periphery of the tumor. Non-specific uptake of QD-AFP-Ab probes occurred mainly in the liver, spleen and lungs.

CONCLUSIONSQD-AFP-Ab probes have good optical properties and biocompatibility for in-vivo targeted imaging of hepatocellular carcinoma. Such approach promises to be highly desirable for molecular targeted research of liver cancer.

Animals ; Antibodies, Monoclonal ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Diagnostic Imaging ; methods ; Fluorescent Antibody Technique ; methods ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Lung Neoplasms ; metabolism ; secondary ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microscopy, Fluorescence ; Molecular Probes ; metabolism ; pharmacokinetics ; toxicity ; Neoplasm Transplantation ; Quantum Dots ; Tissue Distribution ; alpha-Fetoproteins ; immunology ; metabolism

OBJECTIVETo investigate the effectiveness of reconstruction of immunological functions of T cells on the degree of metastases of mouse hepatocarcinoma and the mechanisms of their functioning.

METHODSThe T cell model of immunological functions in Balb/c nu/nu mice was established and the effectiveness of the model was evaluated. The mice were divided into 4 groups. The immunological functions of T cells in experiment groups of Balb/c nu/nu mice were reconstructed. Metastases of the cancer in lymph nodes in each group were examined histologically. The formation time and growth rate of the tumors were calculated. The expression of MHCI and II of the tumor cell line and the difference of expression of immune associated gene were detected by Th1-Th2-Th3 gene array.

RESULTSThe ratio of CD3, CD4, CD8 and CD4/CD8 in the reconstructed group was higher than that in the control group. The average formation time was 7.7+/-0.6 days in Balb/c nu/nu mice and 11.5+/-1.3 days in Balb/c mice. The extent of metastases of the experiment group was lower than that of the control group (P < 0.05). The expression of MHCI of the high metastasis cell line was lower than that of the low metastasis cell line (P < 0.05). The expressions of Th1/Th2 associated genes in lymphocytes of high metastasis mice were lower than those of the low metastasis mice.

CONCLUSIONReconstruction of the immunological function of T cells can influence the metastasis of mouse hepatocarcinoma. The alteration of MHC molecule and low expression of Th1/Th2 correlated genes in lymphocytes may be a factor influencing the metastasis of liver cancer.

Animals ; CD4-CD8 Ratio ; Carcinoma, Hepatocellular ; immunology ; pathology ; Liver Neoplasms ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; T-Lymphocytes ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Tumor Cells, Cultured