In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, ¹H NMR, and ¹³C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.

OBJECTIVETo discuss the relationship between estradiol and the mitogenic activated protein kinase signal transduction pathway and the expression of the MAPK in the MCF-7 breast cancer cell-line.

METHODSEpithelial growth factor (EGF) and different concentration of estradiol to induce the expression of phosphospecific ERK1/2 (pERK1/2) in MCF-7 cell line was used and the expression of pERK1/2 with western-blotting was detected. Then antiestrogen ICI 182780 and MAPK inhibitor PD98059 to inhibit the expression of pERK1/2 was used. The cell cycle of MCF-7 was detected by FACS.

RESULTSEGF could significantly induce the expression of pERK1/2. Estradiol could also induce the expression of pERK1/2, but the intensity was less than the induction of EGF. The percentage of cells in the G(2)/M cell cycle after estradiol induction increased (18.38%) compared to the control group (10.52%) (P < 0.05).

CONCLUSIONSMAPK is an important regulatory signal in breast cancer. Its measurement in breast cancer tissues provides information about the degree of activation of various growth factor pathways. This molecule may also provide a molecular target for compounds designed to block cell proliferation.

Breast Neoplasms ; enzymology ; pathology ; physiopathology ; Cell Proliferation ; drug effects ; Estradiol ; pharmacology ; Female ; Humans ; MAP Kinase Signaling System ; drug effects ; physiology ; Mitogen-Activated Protein Kinases ; drug effects ; metabolism ; Receptors, Estrogen ; metabolism ; Signal Transduction ; Tumor Cells, Cultured

OBJECTIVETo investigate the ability of antisense RNA eukaryotic expression plasmid pcDNA3.0/survivin targeting survivin gene to inhibit survivin expression and enhance the sensitivity to taxotere in multidrug resistant colon carcinoma cell line LOVO/Adr.

METHODSThe antisense RNA eukaryotic plasmid pcDNA3.0/survivin was transfected into LOVO/Adr cells by lipofectamine. The expression of survivin mRNA was measured using RT-PCR. After treated with taxotere, MTT assay and flow cytometry were used to evaluate the proliferation inhibition and apoptosis of LOVO/Adr cells.

RESULTSThe expression of survivin mRNA in LOVO/Adr cells transfected with pcDNA3.0/survivin was down-regulated in a time- dependent manner. The inhibitory rate of taxotere (0.5 micromol/L) was (37.3 +/- 2.9)% in pcDNA3.0/survivin transfected cells, significantly higher than (21.9 +/- 2.3)% and (21.1 +/- 1.9)% in pcDNA3.0 transfected and untransfected control cells respectively (P< 0.01). The apoptosis rate of taxotere was (28.7 +/- 1.7)% in pcDNA3.0/survivin transfected cells,significantly higher than (13.4 +/- 1.6)% and (14.3 +/- 1.8)% in pcDNA3.0 transfected and untransfected cells respectively.

CONCLUSIONThe antisense RNA eukaryotic expression plasmid pcDNA3.0/survivin could down-regulate the expression of survivin gene and enhance the chemosensitivity of LOVO/Adr cells to taxotere, which may provide a novel therapy for colon carcinoma.

Apoptosis ; drug effects ; Cell Line, Tumor ; Colonic Neoplasms ; drug therapy ; Drug Resistance, Neoplasm ; Drug Screening Assays, Antitumor ; Humans ; Microtubule-Associated Proteins ; genetics ; pharmacology ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics ; Taxoids ; pharmacology ; therapeutic use ; Transfection

Thrombotic microangiopathy (TMA) is a lethal transplantation-associated complication which exactly likes acute intestinal graft-versus-host disease (GVHD) in the clinical manifestation. 373 consecutive patients with hematological diseases received family HLA matched or mismatched HCT from May, 2002 to July, 2004. To analyse the clinical and pathological characteristics of TMA, 30 patients who suffered from severe diarrhea and received colonoscopic examination and gut biopsy were retrospectively analyzed. The results indicated that 7 patients originally diagnosed as gut GVHD showed the pathological evidence of enteric TMA. The incidence of TMA was 7 out of 30 specimen (23.3%). Pathological evidence of enteric TMA shown microvascular disorder characterized by thrombus in the capillary without infiltration of lymphocytes and perivascular hemorrhages in the mucosa, swelling and focal denudation of epithelial cells. All patients with TMA were associated with cytomegalovirus (CMV) antigenemia/disease. Among these patients, 4 cases, who only showed TMA without the evidence of gut GVHD pathologically, displayed treatment-resistant bloody diarrhea, renal failure, veno-occlusive disease, hemorrhagic cystitis, hemolytic anemia as well as thrombocytopenia. But the other 3 cases, with co-existence of both TMA and GVHD pathological characteristics had better treatment response. Survival analysis indicated that 3 patients with TMA-GVHD survived for 461 to 536 days but three out of four TMA patients died from VOD with liver failure as well as multiple organ failure during 101 to 254 days after HCT. In conclusion, to better diagnose those patients with severe and refractory diarrhea following HCT, pathological examination may indicate crux evidence to identify intestinal TMA from gut GVHD. Furthermore, this primary report has first evidenced that TMA and TMA-GVHD are two pathologically well-recognized subtypes with the difference between the pathological characteristics, treatment response and clinical outcomes.
Graft vs Host Disease ; etiology ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Intestinal Diseases ; diagnosis ; etiology ; pathology ; Purpura, Thrombotic Thrombocytopenic ; diagnosis ; etiology ; pathology ; Reference Standards ; Retrospective Studies ; Thrombosis ; diagnosis ; etiology ; pathology

OBJECTIVETo investigate the effects of exogenous wild PTEN gene stably transfection on growth of breast cancer cells in vitro.

METHODSAt first, a recombinant eukaryotic expression plasmid pcDNA3.1-PTEN was constructed. Human breast cancer cell line MDA468 was transfected with pcDNA3.1-PTEN or mock transfected plasmid pcDNA3.1(-) with lipofectamine. RT-PCR, immunohistochemical staining and Western blot were used to determine target gene expression. Cell viability was tested by MTT assay. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI.

RESULTSThe PTEN stably transfected cells demonstrated the integration of the exogenous target gene and corresponding mRNA and protein over-expression. There was a significant decline in cell viability of pcDNA3.1-PTEN transfected MDA468 cells in comparison with the mock-transfected ones (P < 0.01). The PTEN-trasfected MDA468 cells also showed an increase in the rate of apoptosis, compared with parental and mock-trasfected cells (P < 0.001).

CONCLUSIONStable expression of exogenous PTEN can suppress the malignant phenotypes of the human breast cancer cell line MDA468.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Eukaryotic Cells ; metabolism ; Humans ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; Phenotype ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the differences about RNA interference (RNAi) technique which focuses on single or multiple sites to suppress colon cancer LoVo cell line's epidermal growth factor receptor (EGFR) mRNA and protein expression, induce cell apoptosis and enhance 5-fluorouracil (5-FU) sensitivity.

METHODSThe human colon cancer LoVo cells were transfected by liposome with pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 expressive vectors which were established by p Genesil-1 plasmid and EGFR short hairpin RNA (shRNA) synthesized in vitro, then were selected for 4 weeks by using G418. Five groups were selected for the study: Group 1: the normal cultured LoVo cells; Group 2: the negative control plasmid HK; Group 3: pU6-EGFR-shRNA-1 plasmid vector; Group 4: pU6-EGFR-shRNA-2 plasmid vector; Group 5: pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2, half for each. The mRNA and protein expression were assessed using Real Time PCR and Western blot, the cell apoptosis was determined via flow cytometry, and the suppressive rate and IC(50) to LoVo cells by 5-FU of different concentrations and time points were carried out by using Cell Counting Kit-8 (CCK-8).

RESULTSExpression plasmids encoding shRNA were successfully established and transfected into the LoVo cells. In group 3, 4 and 5, the mRNA expression was decreased by (80.2 +/- 3.4)%, (81.3 +/- 2.8)% and (90.6 +/- 2.8)%, respectively, and protein expression was decreased by (74.1 +/- 4.0)%, (73.4 +/- 2.3)% and (90.4 +/- 3.3)%, respectively; meanwhile, cell apoptosis increased by (10.4 +/- 0.5)%, (10.1 +/- 0.4)% and (14.2 +/- 0.5)%, respectively. The IC(50) of 5-FU and cell suppressive rate analysis demonstrated that there were significant differences among group 5, groups 3 and 4, and groups 1 and 2, but there were no significant difference between group 1 and group 2, as well as group 3 and group 4.

CONCLUSIONSBoth pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 were capable of suppressing EGFR expression of LoVo cells, and therefore promoting apoptosis and increasing the cell toxicity of 5-FU. The targeting double combined sites RNAi technique was significantly better than single site interference. The new therapeutic modalities in the treatment of human colon cancer are suggested by this study.

Apoptosis ; genetics ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Combined Modality Therapy ; Fluorouracil ; pharmacology ; Genetic Therapy ; Humans ; RNA ; genetics ; RNA Interference ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection

OBJECTIVETo summarize the reoperation experiences in treatment of massive rebleeding after subtotal gastrectomy for bleeding gastroduodenal ulcer.

METHODSFrom 1980 to 2002, clinical data of 26 cases with massive rebleeding after subtotal gastrectomy for bleeding gastrorenal ulcer were analyzed retrospectively.

RESULTSPreoperative gastroscopy was performed in 6 cases, intraoperative gastroscopy in 11, and preoperative superselective angiography in 2 cases. Eleven cases with left ulcer or post- bulb ulcer bleeding underwent resection of the left ulcer or longitudinal incision of the duodenal descending part and direct hemostasis. Thirteen cases with anastomotic stoma bleeding underwent local suture hemostasis or resection of the stoma plus Billroth II or Roux- en- Y gastrojejunostomy. Two cases with gastric bleeding received reexcision of the stomach remnant. Twenty- four cases (92.3% ) were cured and 2 cases (7.7% ) died of gastric bleeding.

CONCLUSIONPreoperative superselective angiography and intraoperative gastroscopy are beneficial to clarify the bleeding position and causes for massive rebleeding after gastrectomy. It is very important to select proper operative method to prevent postoperative rebleeding.

Adult ; Aged ; Aged, 80 and over ; Angiography ; Female ; Gastrectomy ; adverse effects ; Gastrointestinal Hemorrhage ; surgery ; Humans ; Male ; Middle Aged ; Peptic Ulcer ; surgery ; Postoperative Hemorrhage ; surgery ; Retrospective Studies

OBJECTIVEA randomized trial of breast self-examination (BSE) Program was carried out to evaluate whether the intensive BSE could reduce the number of deaths among women from breast cancer.

METHODSThis study was a randomized controlled trial (RCT). A total of 266 064 women (age of 30 to 64 years) associated with 519 textile factories in Shanghai had been randomly assigned to a BSE group (132 979 women) or a control group (133 085 women) since 1989. Initial instruction in BSE group would include demonstration of proper palpation techniques and was followed by 2 reinforcement sessions during the subsequent 4 years including video shows, BSE instruction sessions and BSE practice under medical supervision. These activities were continued for 5 years. Attendance at all events was recorded. The cohort was followed through July 2000 for development of breast diseases, and the breast cancer cases were followed through 2001 for vital status. Data analysis methods used would include Kaplan-Meier plots, log-rank test and Cox modeling.

RESULTSAmong women under instruction, 864 breast cancers detected and 133 breast cancer deaths occurred while 896 breast cancers were detected and 130 deaths recorded in the control group. The tumor size (P = 0.07), TNM stage (P = 0.39) and cumulative breast cancer mortality rate (P = 0.72) were not significantly different between the 2 groups. However, more and smaller fibroadenomas were detected in the instruction group than in the control group (P< 0.01).

CONCLUSIONIntensive instruction in BSE did not seem to have reduced the mortality rate of breast cancer, but more and smaller benign breast lumps could be detected.

Adult ; Breast Diseases ; diagnosis ; Breast Neoplasms ; diagnosis ; mortality ; Breast Self-Examination ; China ; Female ; Humans ; Middle Aged ; Patient Education as Topic

OBJECTIVETo analyze the clinical features and treatment protocols of otogenic intracranial complications in Peking Union Medical College Hospital.

METHODSRetrospective study of 14 patients (10 males and 4 females, aged between 12 - 62 years, mean age 32.1 years) hospitalized from 1982 - 2006. Twelve cases were otitis media (OM) with cholesteatoma, the other 2 cases were non-cholesteatomatous OM. All the otogenic intracranial complications located at the same sides as otologic disorders. Brain abscess was the most common type of otogenic complications and Proteus was the most common microorganism detected. Suppurative ear discharge, headache, high fever and nausea with vomiting were the most common clinical manifestations with very high incidences. All the patients received combined protocols of mastoid surgeries and antibiotics treatment.

RESULTSAll the 14 patients recovered clinically. For patients discharged before 1987, there were 4 patients followed up for 22.5 - 24.4 years with a mean time of 23.8 without recurrence, 1 patient died of cardiovascular disease 19.2 years later after discharge, 4 patients lost follow-up. For the 5 patients discharged after 1997, brain abscess recurred in one patient with pseudo-recovery after 24 days and he fully recovered after re-hospitalization and treatment. All the five patients were followed up for 1.5 years to 10.6 years with a mean time of 6.5 years without recurrence.

CONCLUSIONSYoungsters and males seemed to be more vulnerable. Brain abscess was the most common intracranial complication and Proteus was the most common pyogenic microorganism. Combination of mastoid surgery and antibiotics were essential for effectively controlling the intracranial complications and improving the recovery. CT and MRI were essential for correct diagnosis bedtimes and MRI seemed to have a better performance.

Adolescent ; Adult ; Brain Abscess ; etiology ; Child ; Ear Diseases ; complications ; Female ; Humans ; Male ; Middle Aged ; Otitis Media ; complications ; Retrospective Studies ; Young Adult

Adult ; Arthroscopy ; methods ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Shoulder Joint ; diagnostic imaging ; pathology ; surgery ; Tomography, X-Ray Computed ; Treatment Outcome