OBJECTIVETo observe analgesic effect of electroacupuncture ( EA) on rats with chronic inflammatory pain and its regulatory mechanism on ispilateral dorsal root ganglion (DRG) and spinal dorsal horn (SDH) Mas-related G protein-coupled C receptor (MrgprC).

METHODSTotally 40 healthy male SD rats were divided into 4 groups according to random number table, i.e., the normal (N) group, the model (M) group, the acupuncture (Acu) group, the EA group, 10 rats in each group. The model of chronic inflammatory pain was established by subcutaneous injecting 0. 1 mL complete Freund's adjuvant (CFA) into right hind paw. Paw withdrawal thresholds (PWTs) were measured before modeling, at day 1, 3, 5, 7, and after CFA injection, respectively. Expression levels of MrgprC in ispilateral DRG and SDH were detected by Western blot. The content of bovine adrenal medulla 22 (BAM22) in SDH was detected by immunohistochemical assay.

RESULTSCompared with N group at each time point, PWTs significantly decreased in M group (P <0. 01). Compared with M group, PWTs significantly increased at day 5 of EA and after EA in EA group (P < 0.05, P < 0.01). Compared with Acu group at each time point, post-EA PWTs significantly increased in the EA group (P < 0.05). Compared with N group, expression of MrgprC in ispilateral DRG and ratio of BAM22 positive cells in ispilateral SDH increased in M group (P < 0.01). Compared with M group, expression of MrgprC in ispilateral DRG and ratio of BAM22 positive cells in ispilateral SDH increased in the EA group (P < 0.05).

CONCLUSIONEA had favorable analgesic effect on chronic inflammatory pain induced by CFA, and its mechanism might be possibly associated with up-regulating MrgprC expression in ispilateral DRG and BAM22 content in ispilateral SDH.

Analgesia ; Animals ; Electroacupuncture ; Enkephalins ; metabolism ; Freund's Adjuvant ; Ganglia, Spinal ; drug effects ; Inflammation ; chemically induced ; drug therapy ; Male ; Pain Management ; methods ; Peptide Fragments ; metabolism ; Posterior Horn Cells ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore whether the mesenchymal stem cells (MSCs) of rats can be induced into hepatocytes and the condition of differentiation in vitro.

METHODSMesenchymal stem cells were collected from the femora of SD rats by density gradient centrifugation and identified by flow cytometric analysis and alkaline phosphatase (AKP) staining. MSCs were divided into 4 groups to induce differentiation with the different concentration of hepatocyte growth factor (HGF) in culture medium. The concentration of each group was group A 0 ng/ml, group B 10 ng/ml, group C 20 ng/ml and group D 40 ng/ml, respectively. The morphological changes of MSCs were observed by phase-contrast microscope. On day 1, 3, 7, 14, 21 and 28, mRNA of albumin, AFP and CK18 of MSCs of each group were examined by reverse transcription polymerase chain reaction (RT-PCR), and the expressions of them were also detected with immunohistochemistry technique.

RESULTSMesenchymal stem cells collected from the femora of SD rats expressed antigens of CD29, CD44 and CD90, but not CD34 and CD45. AKP staining was negative for all of MSCs. On day 7, AFP mRNA of MSCs in group C and D could be detected by RT-PCR, and increased on day 14, and then directed on day 21. Albumin and CK18 mRNA of MSCs in group C and D could also be detected from day 14 to day 28 by RT-PCR. On the contrary, mRNA of AFP, CK18 and albumin was not detected in group A and B of culture. Immunocytochemical analysis for CK18, albumin and AFP showed positive staining reaction for AFP on day 7, for CK18 and albumin on day 14 in group C and D, and negative staining reaction both in group A and B of culture.

CONCLUSIONMSCs of adult rats cultured in high concentration of HGF can differentiate into hepatocytes.

Animals ; Cell Differentiation ; drug effects ; Dose-Response Relationship, Drug ; Female ; Hepatocyte Growth Factor ; administration & dosage ; pharmacology ; Hepatocytes ; cytology ; In Vitro Techniques ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore whether rat mesenchymal stem cells (MSCs) can be induced to develop into hepatocytes and the role of the microenvironment of hepatocytes growth in inducing MSCs differentiating into hepatocytes in vitro.

METHODSMesenchymal stem cells were collected from the aspirates from femurs of SD rats by density gradient centrifugation and identified by flow cytometric analysis and alkaline phosphatase (ALP) staining. Rat hepatocytes were isolated by the modified two-step method described by Seglen. Two 6-well culture plates were piled up after the chambers' bottoms of the upper plate was removed. Then the upper and lower chambers were separated by a semi-permeable membrane. MSCs and hepatocytes of rats were plated separately in the upper and lower chambers of the two 6-well culture plates for co-culturing. MSCs cultured alone without co-culturing with hepatocytes served as controls. On days 1, 3, 7, 14, 21 and 28, mRNA of cytokeratin 18 (CK-18), alpha-fetoprotein (AFP) and albumin were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), and immunocytochemistry staining of CK-18 AFP and albumin were also examined.

RESULTSThe shapes of MSCs co-cultured with hepatocytes changed and their sizes and numbers increased in the course of the culturing. When MSCs were co-cultured with hepatocytes for 2 weeks, colonies composed of polygonal cells resembling mature hepatocytes were found. In the controls, shapes of cells also changed and their sizes and numbers increased, but colonies composed of polygonal cells resembling mature hepatocytes were not found. Of the MSCs co-cultured with hepatocytes, on day 7, the mRNA of AFP was detected by RT-PCR, and it increased on day 14, and then decreased on day 21. mRNA of albumin and CK-18 were detected by RT-PCR from day 14 to day 28 in the co-cultured cells, but mRNA of AFP and CK-18 and albumin were not detected in the controls in the course of the culturing. Immunocytochemical analysis for CK-18, albumin, and AFP, showed positive staining reaction for AFP on day 7, for CK-18 and AFP on day 14 in the co-cultured cells but not in the controls.

CONCLUSIONSRat MSCs co-cultured with hepatocytes can differentiate into hepatocytes.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Hepatocytes ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo assess the clinical value of digitalized minimally invasive technique in the treatment of the hepatolithiasis.

METHODSThe 64-slice spiral CT data were acquired from 4 hepatolithiasis patients for three-dimensional reconstruction and simulation operation using abdominal medical image-3D visualization system (MI-3DVS). Three-dimensional reconstruction was performed for the liver, hepatic arteries, hepatic veins, portal veins, intrahepatic bile ducts and calculi. Based on the size and position of the calculi and the distribution of the dilated or stenotic biliary ducts, several simulation operations such as partial hepatectomy and hepaticojejunstomy were performed. With guidance by the findings in the simulation operation, the actual minimally invasive operation was performed.

RESULTSThe three-dimensional models of the liver, hepatic arteries, hepatic veins, portal veins, intrahepatic bile ducts and calculi were reconstructed successfully, which clearly visualized the site and the number of calculi and the condition of the involved intrahepatic bile ducts. Guided by the three-dimensional models and the simulation operations, partial hepatectomy and hepaticojejunstomy were performed and the calculi were removed completely in all the 4 cases with maximum preservation of the residual liver volume.

CONCLUSIONThree-dimensional reconstruction and simulation operation allows digital minimally invasive treatment of hepatolithiasis, which can be a new approach to hepatobiliary surgery.

Adult ; Aged ; Bile Ducts, Extrahepatic ; diagnostic imaging ; surgery ; Bile Ducts, Intrahepatic ; diagnostic imaging ; surgery ; Computer Simulation ; Female ; Gallstones ; diagnostic imaging ; surgery ; Hepatectomy ; methods ; Humans ; Image Processing, Computer-Assisted ; Imaging, Three-Dimensional ; methods ; Middle Aged ; Minimally Invasive Surgical Procedures ; methods ; Tomography, Spiral Computed

Objective To analyze the failure ratio and the causes of the inoculation failure of hepatitis B virus(HBV)-vaccine in children and relevant the remedial measures. Methods One thousand three hundred and sixty cases treated in Suzhou Wuzhong people′s hospital during Jan.2007 to Jul.2008 were chosen,of whom 286 children from 1-5 years old to be anti-HBs negative or anti-HBs titre to be 0-10 IU/L were screened,and specific failure reasons for the vaccination were analyzed,also the timely treatment measures were taken.Then 286 children were divided into 5 groups randomly.Apart from one group was set up as blank control,the other 4 groups were arranged to accept different immunization methods with 0,1,2 month schedule,group A simply got revaccinated with HB vaccine(10 ?g) 3 times;group B revaccinated with double dosage of HB vaccine(20 ?g) 3 times;group C besides being revaccinated 3 times,the immune regulatory agent was jointly used;group D revaccinated 3 times with genetically engineered CHO hepatitis B vaccine. Results The ratio of failure of HBV-vaccine was 21.03%,what caused failure of hepatitis B vaccine included immunologic inadequacy 218(76.22%),repeated respiratory infection 192 cases(67.13%),abuse hormone 140 cases(48.95%),zinc deficiency 129 cases(45.10%),anaemia 108 cases(37.76%),passive smoking 80 cases(27.97%),the mother being chronic parenchymatous nephritis or HBV carrier 63 cases(22.03%),premature 54 cases(18.88%),adiposity 38 cases(13.29%),dystrophy 29 cases(10.14%).There were 4 methods of revaccination,the positive rate for group A,B,C,D were 90.00%,96.47%,99.08%,95.83%,respectively.Group C had the highest positive rate,compared with the other 3 groups,which were statistically significant(P a

OBJECTIVETo investigate the effect of cationic liposome-mediated RNA interference (RNAi) in silencing epidermal growth factor (EGF) receptor (EGFR) gene in breast cancer cells in vivo.

METHODSA small interfering RNA (siRNA) targeting EGFR gene was constructed and transfected into human breast cancer cell in vitro via cationic liposome. The transfected cells were inoculated into nude mice, and the tumor growth inhibition rate was calculated. The tumors were then removed for immunohistochemistry and Western blotting to examine the expression of EGFR protein. Quantitative RT-PCR was used to detect the mRNA expression of the EGF receptor gene, and enzyme-linked immunosorbent assay (ELISA) performed to assess the EGF level in both the serum and tumor extraction.

RESULTSIn athymic nude mice, MDA-MB-231 cells had obviously lower tumor formation rate than ZR-75 cells (30.00% and 88.89%). Transfection of the cells with EGFR siRNA significantly inhibited tumor formation capacity of the cells in vivo as compared with the cells transfected with empty vector or irrelevant siRNA. The results of ELISA demonstrated that in mice bearing the tumors grown from EGFR siRNA-transfected cells, the EGF levels in the serum and tumor extraction were lowered by 16.77% and 12.59%, respectively. Real-time RT-PCR showed that EGFR siRNA transfection caused a specific downregulation of EGFR mRNA expression by 21.05% in the tumor.

CONCLUSIONChemically synthesized 21-nucleotide siRNA duplexes can be effectively delivered via lipofectamine 2000 into breast cancer cells in vivo to induce a longer-lasting gene silencing effect than in vitro transfection. RNAi of EGFR gene may indicate a promising approach for management of lung cancers, especially those nodular ones with easy access.

Animals ; Breast Neoplasms ; genetics ; pathology ; therapy ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Expression Regulation, Neoplastic ; Mammary Neoplasms, Experimental ; genetics ; pathology ; therapy ; Mice ; Mice, Nude ; RNA Interference ; RNA, Small Interfering ; genetics ; Random Allocation ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effect of Leonurus Heterophyllus Sweet, (LHS) on tissue factor (TF) transcription and expression induced by thrombin in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were incubated with different concentrations of LHS and the TF mRNA expression was detected by reverse transcript-polymerase chain reaction (RT-PCR).

RESULTSLHS treatment of HUVECs at different concentrations and for different times resulted in significant differences in TF expression (Plt;0.01). The transcription of TF in LHS-treated cells was significantly different from that of the blank control group (Plt;0.01).

CONCLUSIONLHS can decrease the expression of TF and intervene with TF transcription in HUVECs in vitro.

Cells, Cultured ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Gene Expression ; genetics ; Humans ; Leonurus ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; Thromboplastin ; genetics ; Time Factors ; Umbilical Veins ; cytology

OBJECTIVETo study human hepatocarcinoma stem cell markers.

METHODSTumor tissue samples were obtained from 8 patients with hepatic cellular cancer undergoing surgical tumor resection and the tumor cells were cultured with primary tissue culture in vitro. The cell subpopulations with each marker were isolated from the tumor cells by immunopanning using oval cell markers (CD34, c-kit, Thy-1, CK7, CK19, CK14). The cells of each phenotype were injected into nude mice to measure their ability of tumor formation and the tumor mass was weighed one month after implantation. The tumorigenic subpopulations of the cells were injected into the nude mice again in 1/4 or 1/10 of their original densities to further analyze their ability of tumor formation.

RESULTSAmong all the cell subpopulations, those positive for CD34, c-kit and CK7, respectively, showed marked difference from the negative cells in their ability of proliferation and tumor formation. The CD34(-), c-kit(+) and CK7(+) subpopulations of the cells exhibited strong capacity of tumor formation even in only 1/4 or 1/10 of their original density.

CONCLUSIONSThere are heterogeneous subpopulations within human hepatocarcinoma with observable difference in tumor formation ability. The strong tumor formation ability of CD34(-), c-kit(+) and CK7(+) subpopulation of the cells suggests that CD34(-), c-kit(+) and CK7(+) represent part of the surface markers of hepatocarcinoma stem cells.

Antigens, CD34 ; analysis ; Biomarkers, Tumor ; analysis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Humans ; Keratin-7 ; analysis ; Liver Neoplasms ; metabolism ; pathology ; Neoplastic Stem Cells ; metabolism ; Proto-Oncogene Proteins c-kit ; analysis ; Tumor Cells, Cultured

OBJECTIVETo investigate the clinical effect of the minimally invasive surgical treatment with per-pancreat region for sever acute pancreatitis (SAP).

METHODSFify-four cases of SAP were divided into two groups, patients of group A (n = 28) were given minimally invasive surgical treatments (step 1: under local anesthesia, patients were put the home-made double cannula in the abdominal around the region of pancreas.step 2:patients with biliary stone were performed by laparoscopical operations). Patients of group B (n = 26) were treatment by open operations including biliary decompression, gastrostomy, jejunostomy, removing necrotic pancreatic organizations and puting the double cannula around the region of pancreas. Through double cannula around the pancreas area, all patient's cavity were persistently douched using 0.5% 5-FU saline solution.

RESULTSWashed after one week, two groups patient's drainage fluid amylase concentration were decreased significantly (t = 2.68, P = 0.013; t = 2.41, P = 0.028), patient's white cell count, body temperature, heart rate of Groups A were also decreased significantly (t = 2.32, P = 0.035; t = 2.39, P = 0.021; t = 2.38, P = 0.023). Compared with group B, the mortality, the incidence of complications, hospitalization time and total cost of treatment of group A patients were significantly lower than that of group B (chi(2) = 8.62, P = 0.001; chi(2) = 6.35, P = 0.014; t = 2.22, P = 0.034; t = 2.67, P = 0.010), but the cure rate was significantly higher than that of group B (chi(2) = 3.89, P = 0.045).

CONCLUSIONSMinimally invasive surgical treatment of per-pancreatic region for SAP can not only remove the causes, but also fully drainage and timely block the pathological vicious cycle of SAP. What is more, it is simple, minimally invasive and have few complications and significant effect.

Drainage ; Humans ; Laparoscopy ; Minimally Invasive Surgical Procedures ; Pancreas ; Pancreatitis ; therapy

OBJECTIVETo investigate the differential expression of genes encoding multidrug resistance-associated protein 2 (MRP2), MRP3 and MRP5 in human normal liver cell line L-02, hepatocarcinoma cell line BEL and its adriamycin-resistant counterpart BEL/ADM cell line.

METHODSThe total RNA was extracted from L-02, BEL and BEL/ADM cells, respectively, and the mRNAs were reversely transcribed into cDNA. The expression levels of MRP2, MRP3, MRP5 mRNAs were detected according to the standard curve using real-time fluorescence quantitative PCR.

RESULTSThe expression of MRP2 mRNA in BEL/ADM cell line was significantly higher than that in L-02 and BEL cells (P=0.000), and the latter two cells showed similar expression levels of MRP2 mRNA (P=0.468). The expressions of MRP3 and MRP5 mRNA showed statistically significant difference between the 3 cell lines (P=0.000).

CONCLUSIONMRP2 might play a role in the intrinsic drug resistance, and MRP3 and MRP5 are related to the acquired drug resistance of hepatocellular carcinoma.

Carcinoma, Hepatocellular ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction