ObjectiveTo establish separate and appraisal methods of murine bone marrow mesenchymal stem cells (MSCs) and optimize the suitable conditions inducting MSCs directional differentiating into adipocytes in vitro.MethodsThe differential adherence to plastic was employed to separate MSCs. CFU-f and successive CFU-f cultures were employed to characterize the potent of proliferation and self-renewal of MSCs. The different adipogenic medium was used as induction for the differentiation of MSCs into adipocytes. The differentiated cells were identified by oil red O immunohistochemistry stain.ResultsThe purified MSCs showed the morphology of fibroblasts. It was found that the number of CFU-f formation depended on the planted number of MSCs. It showed a good relationship. Small type colony of CFU-f had little potent to re-clone, but almost 90% big type colony of CFU-f had the potent to regenerate CFU-f. The MSCs could directionally differentiated into adipocytes induced by different adipogenic medium. But more than 96% MSCs differentiated into mature adipocytes when induced by combined with dexamethasone (DM), 1-methy-3-isobutylxanthine (IBMX), insulin (IS) and indomethacin (ID).ConclusionThe purified MSCs can be harvested by method of differential adherence to plastic, and these MSCs have the potent of proliferation and self-renewal. Moreover, more than 96% MSCs can differentiate into mature adipocytes when induced by combined with DM, IBMX, IS and ID.

Objective To assess the effective length of jejunal graft when the 3~(rd) intestinal artery is u- tilized as vascular pedicle and afford a reliable theoretic base for clinical esophageal reconstruction.Methods In 32 formalin preserved and 21 fresh cadaver specimens,the diameter of 1st to 5th intestinal arteries and diameter of arterial arches are measured with linear calibre.Measure the length of jejunum that can be harves- ted as graft when the arches are extended.In the 21 fresh specimens,the 1st,2nd,4th and 5th intestinal ar- teries are ligated,acetic ester stained with red dye were injected into the lumen of 3rd intestinal artery via catheter.Extent of distribution of the arteries to the jejunum was observed.And then red ABS solution was in- jected into the 3rd intestinal artery to make into cast specimen.The blood supply distribution of jejunum through 3rd intestinal artery-arterial arch and communicating system were observed again.Results The di- ameter of the 3rd intestinal artery was the largest among the 1st to 5th intestinal arteries.The length of jejunum vascularized by 3rd intestinal artery can be as long as (142.2?62.3) (69.0～206.60cm) in acetic ester in- filtrated specimens.While in ABS east specimen,the average available extent of donor jejunum was(30.8?7.3) (23.0～37.3cm).Conclusion As observed by this applied anatomy study,the jejunum graft vascu- larized by 3rd intestinal artery alone has sufficient length to meet the need of esophageal reeonstrution.

OBJECTIVETo analyze the relationship of K-ras mutation with the development of liver metastasis in colorectal cancer patients and the survival outcomes.

METHODSFrom 2003 to 2008, 300 patients who underwent colorectal cancer surgery in the Department of General Surgery of Zhongshan Hospital, Fudan University were assigned to different groups, according to the diagnosis and follow-up results. The mutation of exon 2 of K-ras was detected in primary paraffin-embedded lesions by PCR and Pyrosequencing. The association of gene mutation with the development of liver metastasis and its prognosis was studied.

RESULTSAmong 300 cases, the mutations of exon 2 were present in 120 cases(40%). The G13D mutation was more common in metachronous metastasis group than that in synchronous group(17.0% vs. 8.0%, P=0.041). Multivariable regression analysis showed that G13D mutation was an independent risk factor(HR=1.108, 95%CI:1.032-5.062, P=0.048) for metachronous metastasis. Patients with mutated K-ras had a poorer overall survival compared to those without mutated K-ras for patients without liver metastasis(median overall, 65 vs. 72 months, P=0.039), and for patients who received metastasis resection(median disease-free survival 18 vs. 24 months, P=0.048). Multivariable analysis showed that K-ras mutation was an independent risk factors of overall survival(HR=1.561, 95%CI:1.022-6.422, P=0.045) in patients without liver metastasis.

CONCLUSIONDetection of K-ras mutation may predict the development of liver metastasis and prognosis.

Aged ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Genes, ras ; genetics ; Humans ; Liver Neoplasms ; genetics ; secondary ; Male ; Mutation ; Prognosis

Objective Data on the leukapheresis from 26 pediatric patients with hematologic or solid malignancies was retrospectively evaluated to screen predictive factors affecting the efficacy of peripheral blood stem cell(PBSC) collection from donors,as well as hematopoietic recovery in recipients.Methods We present our experience with 49 apheresis from 26 granulocyte-colory Stimulating factor mobilized donors and analyzed the correlations between the mobilization,the leukocyte count in the donor peripheral blood and the MNC and CD_(34)~+ cell yields in collecting products and the neutrophil and platelet recovery of recipients.Results The process of mobilization and apheresis were well tolerated by our pediatric donors.The median numbers for harvested MNCs and CD_(34)~+ cells were 4.5?10~8/kg and 1.9?10~6/kg of recipient body weight,respectively.Mobilizing dose positively affected the number of mononuclear ceus(MNC) but not CD_(34)~+ cells in the apheresis products.The CD_(34)~+ cell number in the apheresis product was influenced significantly by donor circulating MNC on the day of harvest and correlated with recipient′s engraftment after PBSC was reinfused.Conclusions The MNC yield was stable and met with the demand for autologous stem cell transplantation while the CD_(34)~+ cell number varies obviously from each donor.Since a rapid engraftment was associated with a high number of CD_(34)~+ cells collected,which was in turn predicted by the level of the pre-apheresis CD_(34)~+ cells in the peripheral blood of donors,it is necessary to monitor the donors′ CD_(34)~+ cell during mobilization to determine the optimal time for apheresis.J Appl Clin Pediatr,2006,21(3):148-150

Objective: To establish a method for isolation and purification of fetal membrane derived adherent cells (FMDACs) , and investigate their biological characteristics. Method: FMDACs were isolated with trypsin inducing and cultured in vitro. FMDACs were induced to differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique were used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: FMDACs were successfully isolated and expanded in vitro. They had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion; FMDACs have the possibility of multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs is stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.

OBJECTIVETo determine the optimal thread pitch for an experimental cylinder implant in Ansys Work-bench Design Xplorer environment.

METHODSFinite element models of segment jaw bone with a V-shaped thread implant were created. The thread pitch (P) was set from 0.5 mm to 1.6 mm. The maximum Equivalent stresses (EQV stresses) in jaw bone and in implant were evaluated.

RESULTSUnder axial load, the amplification of maximum EQV stresses in cortical bone, cancellous bone and implant were 7.1%, 123.4% and 28.7% respectively. Under bucco-lingual load, the amplification of maximum EQV stresses in cortical bone, cancellous bone and implant were 2.8%, 28.8% and 14.9% respectively. When P exceeded 0.8 mm, the response curve curvature of maximum EQV stresses in jaw bone and in implant to P was ranged from -1 to 1.

CONCLUSIONStresses in cancellous bone are more sensitive to thread pitch than in cortical bone. Stresses in jaw bone under axial load are easier affected by thread pitch than under bucco-lingual load. Thread pitch plays a greater role in protecting dental implant under axial load than under bucco-lingual load. Thread pitch exceed 0.8 mm should be the optimal design in a cylinder implant, but oversized pitch should be avoided too.

Biomechanical Phenomena ; Computer Simulation ; Dental Implants ; Dental Prosthesis Design ; Finite Element Analysis ; Mandible ; Stress, Mechanical

OBJECTIVETo construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.

METHODSThe recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.

RESULTSRestrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.

CONCLUSIONThe minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.

Animals ; COS Cells ; Cercopithecus aethiops ; Dystrophin ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Models, Genetic ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVEThe combination of oxaliplatin (L-OHP), 5-fluorouracil (5-Fu) and folinic acid (FA), being one of the effective regimens for advanced gastric cancer, is used in form of chronomodulated chemotherapy for advanced gastric cancer to investigate its efficacy and safety.

METHODSTwenty-six patients received a 4-day chronomodulated infusion of L-OHP, 5-Fu and FA. L-OHP (25 mg.m(-2).d(-1)) infused from 10:00 am to 22:00 pm, and followed by 5-Fu (600 mg.m(-2).d(-1)) and FA (300 mg.m(-2).d(-1)) from 22:00 pm to 10:00 am for 4 days using a multichannel programmable pump, every 2 weeks as an cycle for at least 2 cycles.

RESULTSTwenty-six patients with previously untreated advanced gastric cancer were eligible. Two complete and 13 partial remissions were observed with an overall response rate of 57.7%. Stable disease was observed in 6 patients (23.1%) and progressive disease in five (19.2%). Four of these patients underwent surgery. The median remission time was 3.5 months and time to tumor progression (TTP) was 4.5 months. The median overall survival time was 8 months. A total of 80 cycles were given without any grade 4 toxicity observed, but grade 3 thrombocytopenia (1.3%) and mucositis (1.3%) in one patient, two grade 3 neutropenia (2.5%) and nausea/vomiting (2.5%) in 2.

CONCLUSIONChronomodulated intravenous chemotherapy of oxaliplatin, 5-fluorouracil and folinic acid is effective and safe in the treatment of advanced gastric cancer.

Adenocarcinoma ; drug therapy ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Chronotherapy ; Drug Administration Schedule ; Female ; Fluorouracil ; administration & dosage ; Humans ; Leucovorin ; administration & dosage ; Male ; Middle Aged ; Organoplatinum Compounds ; administration & dosage ; Stomach Neoplasms ; drug therapy ; Treatment Outcome

OBJECTIVETo identify the pathogenic gene for a non-syndromic hearing loss family.

METHODSMutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons of SLC26A4 (solute carrier family 26, member 4) gene.

RESULTSCompound heterozygous mutations N392Y and S448X were detected in the proband of the family, heterozygous mutation S448X was detected in the father, heterozygous mutation N392Y was detected in the mother.

CONCLUSIONThe proband's hearing loss resulted from the compound heterozygous mutations N392Y and S448X for SLC26A4 gene.

Adult ; Base Sequence ; DNA Mutational Analysis ; Deafness ; diagnostic imaging ; genetics ; pathology ; Family Health ; Female ; Humans ; Male ; Membrane Transport Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Tomography, X-Ray Computed

OBJECTIVETo study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation.

METHODSPolymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation.

RESULTSBy DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before. Mismatch primer amplification and restriction fragment length polymorphism analysis suggested that this mutation was not detected in the normal control.

CONCLUSIONThe mutation 1633-26(C-->A) may be the disease-causing mutation in this patient with multiple exostoses.

DNA Mutational Analysis ; Exostoses, Multiple Hereditary ; genetics ; Female ; Humans ; Mutation ; N-Acetylglucosaminyltransferases ; genetics ; Young Adult