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 summarize the clinical experience in repair of deep burn and traumatic wounds with combined transplantation of different types of pedicled skin flaps in lower extremities.

METHODSTwo hundred and thirty-six patients with 271 deep wounds in lower extremities after burn or trauma were repaired with muscular skin flaps, local fascial flaps and island flaps with vascular pedicle (more than 20 types) in our department from Jan. 1998 to Sept. 2008.

RESULTSComplete necrosis of skin flaps occurred in 1 case, congestion and necrosis over the edge of skin flaps occurred in 3 cases, which were healed after grafting, and other skin flaps survived well with soft texture. Skin flaps were too bulky in 26 cases, among them 17 cases were thinned, and the appearance of other skin flaps were satisfactory. In 68 patients with functional region injury were recovered to certain extent without contracture.

CONCLUSIONSSkin flaps with pedicles, multiple transplantations if necessary, can repair deep wounds satisfactorily in lower extremities after deep burn or trauma injury.

Adolescent ; Adult ; Aged ; Burns ; surgery ; Buttocks ; surgery ; Child ; Child, Preschool ; Female ; Humans ; Lower Extremity ; injuries ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; Skin Transplantation ; Surgical Flaps ; Wound Healing ; Young Adult

OBJECTIVETo evaluate the clinical value of laparoscopy-assisted modified Soave procedure for Hirschsprung disease in adults.

METHODSTwenty-eight patients with a preoperative diagnosis of Hirschsprung disease underwent laparoscopy-assisted modified Soave procedure between March 2005 and December 2009. Clinical data were retrospectively analyzed.

RESULTSThere were no conversions to open surgery. The mean operative time was (165±12) minutes (range: 135-185 minutes). Estimated blood loss ranged from 50 to 250 ml, and no patients required intraoperative blood transfusion. Postoperative pathologic examination showed Hirschsprung diseases in 19 patients and Hirschsprung allied diseases in 9. Only two patients developed rectal cuff infection and three mild seepage. Other patients had no postoperative complications. The mean hospital stay was (17.5±1.0) days. No fecal incontinence or recurrent constipation occurred during follow-up.

CONCLUSIONLaparoscopy- assisted modified Soave procedure is safe and effective for Hirschsprung disease.

Adolescent ; Adult ; Female ; Follow-Up Studies ; Hirschsprung Disease ; surgery ; Humans ; Laparoscopy ; methods ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome ; Young Adult

The magnetic responsibility and antitumor effect of magnetic gemcitabine stealth nano-liposomes (MGSL) on breast cancer cell line MCF-7 in vitro and in vivo was evaluated. The magnetic response and targeting effect of MGSL in vivo were investigated. Morphological feature and ultrastructure changes of apoptosis of MCF-7 cells were observed. The effect of MGSL on proliferation inhibitory rate of MCF-7 cells was measured with MTT method. The FCM analysis was carried out to examine the cell cycle distribution and cell apoptotic rate. The antitumor effect on human breast cancer xenografts in nude mice was also studied. MGSL was able to converge at the targeting tissue under tridimensional magnetic field and the gemcitabine concentration around it increased, while the amount of gemcitabine in other organs decreased, such as in kidneys and heart. MCF-7 cell line was sensitive to MGSL and the cytotoxity was correlated with the loaded drug dose. The effect of MGSL on apoptosis of MCF-7 was obvious and the rate of apoptosis was 51.62%. The growth speed of tumor in the group of MGSL (+) significantly slowed down than that of other groups. MGSL prepared by reverse-phase evaporation method met with the demand of targeted delivery system, and it might be an effective antitumor agent.
Animals ; Antimetabolites, Antineoplastic ; administration & dosage ; pharmacokinetics ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; pharmacokinetics ; pharmacology ; Dose-Response Relationship, Drug ; Drug Delivery Systems ; Female ; Humans ; Liposomes ; chemistry ; Magnetics ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nanoparticles ; administration & dosage ; Neoplasm Transplantation ; Tissue Distribution ; Tumor Burden ; drug effects

Animals ; Female ; Liver ; blood supply ; Male ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; Sex Factors

OBJECTIVETo explore a new technique of reduction and internal fixation for tibia intercondylar eminence fractures under arthroscopy.

METHODSFrom June 2004 to February 2006, 9 patients with fresh tibia intercondylar eminence fracture (type II in 4 cases and type III in 5 cases) were treated with reduction and internal fixation using suture anchors (Depuy Mitek) under arthroscopy. All the patients, subject to regular post-operation functional exercise, were followed up for 6 to 22 months. The results were evaluated in the aspects of fracture reduction healing, knee joint relaxation and ROM, and functional restoration of overall limbs.

RESULTSIn the nine patients, the tibia intercondylar eminence fractures healed without displacement and nonunion. No knee joint relaxation or extension-restriction was found. Lysholm score indicated 93.8 +/- 2.3 at the 6th postoperative months.

CONCLUSIONThe reduction and internal fixation of tibia intercondylar eminence fractures under arthroscopy using suture anchors demonstrate a reliable and easy-to-use technique. Operation under arthroscopy helps diagnose and treat other complications inside knee joint, merely resulting in slight injury. In addition,early functional exercise contributes to rapid recovery of knee joint's functions.

Adult ; Arthroscopy ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Suture Anchors ; Tibia ; physiopathology ; surgery ; Tibial Fractures ; physiopathology ; surgery