BACKGROUND: Amniotic fluid cells have been widely used in antenatal diagnosis for gene mutation-related diseases. However,there are few reports concerning isolation, culture, surface character identification, differentiation and application perspective of fetal mesenchymal stem cells (MSCs) derived from amniotic fluid.OBJECTIVE: To investigate the differentiation of fetal mesenchymal stem cells (MSCs) derived from second-trimester amniotic fluid into osteoblasts in vitro.DESIGN, TIME AND SETTING: The cytological/n v/tro study was conducted at the Experimental Center of Xinhua Hospital from August 2005 to May 2006.MATERIALS: Ten amniotic fluid samples were obtained from pregnant women (18-22 weeks after conception) or aborted women,The informed consents were obtained from pregnant women.METHODS: Fetal MSCs were separated mechanically from amniocyte culture system and expanded in medium in vitro. At passage 3, fetal MSCs were induced in 100 nmol/L dexamethasone, 10 mmol/L β-glycerophosphoric acid and 50 mg/L vitamin C for 14 days.MAIN OUTCOME MEASURES: Collagen Ⅰ and alkaline phosphatase expression was detected by immunohistochemistry.Collagen Ⅰ protein expression was determined by Western Blot analysis. Calcium tuberoses were measured by Von Kossa staining. The cytoskeletal protein was detected by laser confocal microscopy.RESULTS: The isolated fetal MSCs were uniformly positive for CD44 and HLA-ABC, negative for CD34, CD45 and HLA-DR. After being induced with osteogenic medium for 14 days, 91% cells were positive for alkaline phosphatase, and 87% cells for collagen Ⅰ.Cells expressed collagen Ⅰ protein. Number of calcium tuberoses was increased and became big over time. Cytoplasm microfilament presented green fluorescence and the microfilament surrounding cells formed dense bundle.CONCLUSION: Fetal MSCs derived from amniotic fluid could be induced into osteeblasta and displayed a typical osteoblastic morphology and biological characteristic.

Objective To establish a TaqMan probe-based real-time fluorescent quantitative PCR ( real-time PCR) for the quantitative and rapid detection of viral reservoir in peripheral blood mononuclear cells (PBMCs) isolated from rhesus monkeys with simian immunodeficiency virus (SIV) infection and to evaluate its preliminary application. Methods A pair of primers and one TaqMan probe were designed ac-cording to the conserved sequence of SIVmac239 strain for real-time PCR amplification. A length of 2 090 bp of nucleotide fragment was digested from the plasmid p239SpSp5 containing 5′-end long segments of SIV-mac239 strain by restriction enzymes EcoRⅠand SpeⅠ. The standards used for quantitative detection of SIV DNA in peripheral blood samples were prepared by a 10-fold serial dilution and used for graphing the stand-ard curve. The numbers of SIV DNA ( copies per 106 PBMCs) in rhesus monkeys during acute and chronic phases of SIVmac239 infection were determined and the virological characteristics of SIV DNA at different phages of infection were analyzed. Results A linear positive correlation between cycle threshold ( Ct) val-ues and concentrations (10 copies/μl to 109 copies/μl) of the standards was found. High levels of SIV DNA were monitored in SIV-infected monkeys 14 to 22 days after acute infection. The levels of SIV DNA in the acute phase of infection were about 1 to 2 logs higher than those in the chronic phase of infection. The num-bers of SIV DNA ( copies per 106 PBMCs) were 1 log lower than the SIV viral load in peripheral blood of the same monkey. The ratios of SIV DNA load to SIV RNA load ( DNA/RNA) in chronic phase of infection were higher than those in the acute phase. Conclusion The established TaqMan probe-based real-time fluorescent quantitative PCR was a highly sensitive and specific assay for the detection of SIV DNA with an advantage of wide linear range. It could be used for the quantitative evaluation of latent reservoirs of SIV.

Objective To analyze the association of postnatal weight gain proportion of very low birth weight (BW) preterm babies and the onset of severe retinopathy of prematurity,and investigate the optimal cut-off points and predictive ability of postnatal weight gain (WG) proportion for the onset of retinopathy of prematurity (ROP).Methods A retrospective cohort study.257 preterm infants underwent screening whose weight was less than 1500 g were enrolled in this study.Risk factors include BW,gestational age (GA),history of oxygen inhalation,need for blood transfusions,Apgar score in 1 to 10 minutes,embryo number,delivery mode,in vitro fertilization infants,and WG proportion within 6 weeks after birth and other systemic diseases were recorded.Their correlation with severe ROP is analyzed.Clinical outcomes were divided into severe ROP group (patients who suffered from ROP and required treatment) and mild and no ROP group (patients who suffered from ROP but do not require treatment and-patient without ROP).The severe ROP group included 18 patients and mild and no ROP group included 239 patients.Multiple logistic regression and receiver operating characteristic (ROC) curve were used to determine if the WG proportion was independently related to severe ROP development and if it was capable of predicting severe ROP.This study determines the predict value by comparing the area under the ROC curve (AUC) of independent risk factors.Results GA (t=-4.835,P＜0.001),BW(t=-5.192,P＜0.001),history of oxygen inhalation (x2=6.001,P=0.009),proportion of infants who had oxygen inhalation for more than 10 days(x2 =10.019,P=0.002),postnatal WG proportion at 1 week(t =-3.663,P＜ 0.001),postnatal WG proportion at 2 weeks(t=-3.425,P=0.001) had significant difference between two groups.Multiple logistic regression analysis revealed that GA (β =-0.858,P =0.008),BW (β =-0.005,P =0.010),postnatal WG proportion at 2 weeks (β=-8.745,P =0.035) were correlated to severe ROP significantly.And their area under the ROC were 0.836[95％ confidence interval (CI):0.752-0.920],0.826 (95％CI:0.947-0.903),0.744 (95％CI:0.598-0.891) respectively.The optimal cut-off points of GA,BW,and postnatal WG proportion at 2 weeks were 28.41 weeks,1241.96 g,12.80％ respectively.Conclusion Low WG proportion at 2 weeks of very low BW preterm babies is an important and independent risk factor for severe ROP and has certain predictive value of the onset of severe ROP.

Objective To investigate the diagnosis and treatment of pseudomyxoma peritonei (PMP) and provide a reference for diagnosis and treatment of PMP.Methods The clinical features,laboratory examinations,treatment and outcomes of 8 PMP misdiagnosed cases were analyzed with recent relevant reference.Results Ultrasonography,CT,peritoneal cytological examination,tumor markers results are helpful for diagnosis of PMP.PMP will be confirmed and classified by pathological examination after operation.Complete cytoreductive surgery (CRS)or major debulking surgery (MDS) of the tumor combined intraperitoneal chemotherapy and systemic chemotherapy,eight patients in seven cases survived 4-71 month range,one patient died of respiratory failure of pulmonary infection after the third operation.Conclusions Ultrasonography,CT,peritoneal cytological examination,tumor markers tests help avoid misdiagnosis of PMP before operations.Intraoperative findings follow after PMP,CRS or MDS should be executed in the operation or the next time.Intraperitoneal chemotherapy and conventional chemotherapy can improve survival in patients with PMP and prolong their survival time.

OBJECTIVE: To construct a system of therapeutic effect evaluation for patients with primary liver cancer according to the theory of syndrome differentiation in traditional Chinese medicine (TCM), and to examine its reliability. METHODS: Analytic hierarchy process and 100 mm surveyor's rod method were applied to obtain bottom layer and top level syndromes, which were used to construct the method of therapeutic effect evaluation, and its reliability was verified in clinical practice by comparing with some evaluation criteria in Western medicine, such as cancer severity scale; Karnofsky performance scale; Child-Pugh classification, cancer staging classification, and quality of life scale, etc. RESULTS: A system of therapeutic effect evaluation was constructed, and it could reflect the progress of tumor, changes of hepatic function and constitution. The evaluation scores acquired from the system were highly associated with the quality of life of the patients. CONCLUSION: The system of therapeutic effect evaluation can reflect the severity of disease and the characteristics of TCM treatment.

Although gene therapy was regarded as a promising approach for glioma treatment, its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems. Mesenchymal stem cells(MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy. Therefore, in this study, we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus. We firstly compared the infectivity of type 3, type 5, and type 35 fiber-modified adenoviruses in MSCs. We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo. Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus. MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups. In conclusion, MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma, making it a potential therapeutic strategy for treating malignant glioma.
Adenoviridae ; Animals ; Brain Neoplasms ; pathology ; therapy ; Cell Line, Tumor ; Genetic Vectors ; Glioma ; pathology ; therapy ; Humans ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oncolytic Virotherapy ; Random Allocation ; Virus Replication ; Xenograft Model Antitumor Assays

OBJECTIVETo explore a method for ectopic prefabrication of mandible with vascular pedicle.

METHODSCancellous bone blocks harvested from the dog ribs were packaged with mandible-shaped titanium mesh scaffold and implanted into latissimus dorsi of dog with thoracodorsal artery and vein through the scaffold. After 12 weeks, bone formation and vascularization were evaluated by gross inspection, histological examination and immunohistochemistry.

RESULTSVascularized mandible with thoracodorsal artery and vein were formed and histological staining and immunohistochemisty confirmed new bone formation and vascularization.

CONCLUSIONSThere is feasibility for ectopic prefabrication of vascularized mandible graft using cancellous ribs, which provides a new method for mandibular defect reconstruction. Experimental study on ectopic prefabrication of vascularized mandible graft with autogenous ribs.

Animals ; Bone Transplantation ; methods ; Dogs ; Female ; Male ; Mandible ; surgery ; Osteogenesis ; Reconstructive Surgical Procedures ; methods ; Ribs ; surgery ; Tissue Engineering ; methods ; Transplantation, Autologous

Objective To map the specific gene responsible for primary erythromelalgia and identify gene mutations in a Chinese family and one sporadic patient with primary erythromelalgia. Methods Geno-mic DNA was extracted from peripheral lymphocytes of the family members of the pedigree and the sporadic patient. Scanning the genes on chromosome 2q that had been identified was performed by using 6 microsatellite markers for the family members with primary erythromelalgia. Then linkage analysis and haplotype analysis were conducted. All exons of SCN9A gene were analyzed by PCR-DNA sequencing. The mutation identification was also confirmed by restriction fragment length polymorphism(RFLP). Results A maximum 2-point LOD score of 2.11 was found at a recombination fraction (? = 0.00) with markers D2S2370 and D2S2330. Recombination events were detected by markers D2S1353 and D2S2345 in this family by the haplotype analysis. There were two missense heterozygous point mutations in the 15th exon of SCN9A gene both in the family(T2573A) and the sporadic patient(T2543C), leading to the substitution of the amino acid leucine to histidine(L858H) and isoleucine to threonine(I848T), respectively. The above mutations were not found in 400 normal alleles. Conclusion It is proved that primary erythromelalgia is caused by mutations in SCN9A gene.

OBJECTIVETo investigate the expression and clinical significance of matrix metalloproteinase-9 and its complex in the urine of the patient with breast cancer.

METHODSUsing substract gel electrophoresis and western-blot analysis, expressions of MMP-9 and MMP-9/NGAL complex in breast cancer (n = 97), breast benign (n = 41) and normal (n = 60) were observed.

RESULTSThere MMP-9 and MMP-9/NGAL complex expressions were 76.29% and 64.95% in breast cancer, 46.34% and 43.90% in breast benign, and 23.33% in normal respectively. The MMP-9 and MMP-9/NGAL complex expressions were higher in breast cancer than those in breast benign and in normal (chi(2) = 7.456, P < 0.01). MMP-9 and MMP-9/NGAL complex expressions in urine of breast cancer had not any relationship with tumor size, TNM stage, patient age, menopause status as well as ER status, but was correlated to lymphatic node status (chi(2) = 5.206, P < 0.05).

CONCLUSIONSMMP-9 and MMP-9/NGAL complex expressions in urine are significant in estimating lymphatic node metastasis in breast cancer and a valuable early prognostic factors and screening in breast cancer.

Acute-Phase Proteins ; Blotting, Western ; Breast Neoplasms ; pathology ; urine ; Carrier Proteins ; urine ; Female ; Humans ; Lipocalin-2 ; Lipocalins ; Lymphatic Metastasis ; Matrix Metalloproteinase 9 ; urine ; Oncogene Proteins ; Proto-Oncogene Proteins

Objective To evaluate the risks of vaccination operation in order to provide scientific basis for policy makings of vaccination operation.Methods The risk matrix and analytic hierarchy process approach were used to evaluate the risks of vaccination operation,including the possibility,severity,risk weight,and risk level.Results Seven items for the first level and thirty one items for the second level vaccination risk factors were determined.In the first level,E level risk (Extremely serious risk ) was vaccination implementation risk,and H level risk (high risk ) was the risks of pre -notification and health education and risks of vaccine and cold chain management.Vaccination implementation risk accounted the largest weight (weight coefficient=38.95%).In second level,E level risk was three inspection and seven verification before vaccination,and H level risk were personnel professional quality,personnel responsibility,pre -inspection,informed before vaccination, vaccination route, site and dose, vaccination operation, post vaccination notification and retention,and three inspection and seven verification before vaccination,accounted the largest weight (weight coefficient=10.14%).The weight of the logical consistency of the test results were satisfactory (consistency ratio<0.1 ).Conclusion The integrated application of risk matrix and analytic hierarchy process in risk assessment of vaccination operation helps to further regulate vaccination services and has application and promotion value.