To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

Objective To study the effect of siRNA targeting survivin and tumor necrosis factor related apoptosis-inducing ligand on the proliferation and apoptosis of hepatocellular carcinoma ( HCC) cells. Methods siRNA eukaryotic expression vector was constructed and the effects of soluble tumor necrosis factor related apoptosis-inducing ligand ( sTRAIL) on HCC cells were observed. Results The recombinant plasmid Psilence ( + )-survivin was successfully constructed. Survivin mRNA expression inhibition ratio reached 73% by RT-PCR. Observed by MTT method sTRAIL failed to inhibit the survival rate of HepG2、HepG2/Silence( - ) cells at 12 h、24 h、48 h when compared to control groups. With the survivin gene being inhibited, the survival rate of HepG2/Silence( + ) cells(0. 518?0. 017) decreased in 12 h compared to control groups (0. 741?0. 005 ) and reached the lowest level in 48 h ( P

Background and purpose:Radical nephroureterectomy can be performed in a variety of ways, and each method has its advantages and disadvantages. It still remains controversial for choosing the surgical methods. In this study, we chose six surgical methods and investigated the safety and efficacy of different methods in treating upper urinary tract carcinoma.Methods:We retrospectively analyzed 135 patients with upper urinary tract transitional cell carcinoma who underwent operations in our hospital from Jan. 2002 to Oct. 2013, and compared the data of six different operations in-cluding operating time, volume of bleeding, time of bowel function recovery and incidence of bladder carcinomas.Results:The operations were successfully completed in groups A and B. Five cases in group C were transferred into group A be-cause of failing to pull the nub of the ureter. Two cases in group D were transferred into group A because of failing to pull the nub of the ureter. Three cases in group E were transferred into group D and 1 case was transferred into group A because of adhesion or bleeding. One case in group F was transferred into group A because of bleeding. There was no statistically significant difference in survival rates among six operations.Conclusion:Six operations are all safe and effective for the treatment of upper urinary tract carcinomas. Each method has its advantages and disadvantages. We should choose differ-ent methods according to particular cases.

Gynecology and obstetrics is a theoretical and practical subject. It is an important goal for the medical students to develop the clinical thinking ability and operating skills and apply them in the diagnosis and treatment of disease. To overcome the limited teaching resources, the rare clinical skills opportunities caused by doctor-patient relationship tension, virtual patient (VP) combined with clinical teaching was applied in clinical teaching, which can reproduce the real, and bring the students to the role of the clinician , enrich the content of the obstetrics and Gynecology clinical teaching. Along with the reform the teaching faculty with high quality was established, their clinical teaching experiences and innovative thinking were improved significantly. The results were evaluated by means of clinical comprehensive ability test. The present study aimed to establish virtual patients of OBGYN (virtual patient, VP) learning to promote learning of basic knowledge, clinic skills, and thinking ability.

OBJECTIVE: To evaluate the inhibiting effects of the root of Mallotus apelta (Lour.) Muell.-Arg. on duck hepatitis B virus (D-HBV) in vivo. METHODS: Forty nestling ducks with congenitally infection of D-HBV detected by PCR were randomly divided into five groups: untreated group, lamivudine-treated group, and high-, medium- and low-dose root of Mallotus apelta-treated groups. The ducks in the lamivudine-treated group were fed lamivudine with a dose of 50 mg/kg once. Ducks in the three-dose Mallotus apelta-treated groups were treated with different doses of decoction of this herbal medicine for 21 days respectively. The serum content of D-HBV DNA was determined by quantitative real-time PCR technique before and 7 days after the treatment, and on the 7th, 14th and 21st day of the treatment. Liver biopsy was also executed before and after the treatment to observe the histopathological changes. RESULTS: Lamivudine showed a rapid inhibiting effect on D-HBV DNA, but this effect didn't last long, and the serum level of D-HBV DNA increased again after treatment. The serum level of D-HBV DNA dropped markedly in the high- and medium-dose Mallotus apelta-treated groups on the 14th and 21st day. Low-dose Mallotus apelta revealed no obvious inhibiting effect on D-HBV. After treatment, the inhibiting effect in the root of Mallotus apelta-treated group continued as compared with that in the untreated group. The histopathological changes of liver tissues showed that the inflammation in the high-dose root of Mallotus apelta-treated group was weakened as compared with that in the lamivudine-treated group. CONCLUSION: The root of Mallotus apelta has therapeutic effect on D-HBV. It can restrain the duplication of D-HBV in vivo. Although this effect is weaker than that of lamivudine, it continues longer. Thus this herbal medicine is an effective, safe and economical drug for hepatitis B.

BACKGROUND: Deletion of glial cell-derived neurotrophic factor and endothelin receptor type B gene will induce abnormal development of enteric nervous system. Neural stem cell transplantation can repair nervous system from anatomy and function,and be considered as a vector of gene transfection.OBJECTIVE: To transfect recombinant adenovirus carrying glial cell-derived neurotrophic factor and endothelin receptor type B gene into mouse neural stem cells, and to observe expression of target gene.DESIGN: A cell-gene study.MATERIALS: New-born Kunming mice were provided by the Animal Center of Tongji Medical College, Huazhong University of Science and Technology, China. jetPEI reagent was purchased from PolyPlus Co, France. The pAdTrack-CMV-GE with green fluorescent protein (GFP) was gifted by Doctor Sun Nianfeng and Zhang Jinghui in our laboratory.METHODS: Neonatal mouse brain tissues were sterilely obtained to prepare monoplast suspension. Adenovirus expressing glial cell-derived neurotrophic factor and endothelin receptor type B gene with GFP was dissolved in NaCI to prepare JetPEI/DNA complex. Subcultured neural stem cells in DMEM/F12 were regulated to 5×10~8/L, and 400 μL cell suspension and 100 μL JetPEI/DNA complex were seeded on a 24-well plate at 37 ℃ in 5% CO_2 incubator. Neural stem cells were harvested at 24, 48 and 72 hours following transfection.MAIN OUTCOME MEASURES: The efficiency of transfection was detected using fluorescence microscope and flow cytometry.Target gene expression in neural stem cells was determined using RT-PCR.RESULTS: Bright green fluorescence of the transfected cells could be observed under fluorescence microscope after 24 hours of transfection. The positive rate of GFP was 15.36%, 24.67%, 25.73% at 24, 48 and 72 hours following transfection respectively.Neural stem cells expressed glial cell-derived neurotrophic factor and endothelin receptor type B gene at various time points.Strap brightness was low at 24 hours, and exogenous gene expression was great at 72 hours.CONCLUSION: The target genes were successfully transfected into neural stem cells by using jetPEI reagent. Moreover, glial cell-derived neurotrophic factor and endothelin receptor type B gene effectively transcribed and expressed in target cells.

Objective To evaluate the antitumor effect of SEB-scFv fusion Drotein on gastric cancer. Methods Typical changes of morphologic features and super-microstructure were observed when SEB-scFv fusion protein was used in SGC7901 cens;and the effects of SEB-scFv and its concentration on the cell growth were examined by methyltetrazolium(MTT)assay;DNA ladder and flow cytometry were employed respectively to detect the inhibition phenomenon or apoptosis.We produced a subcutaneous gastric tumor model in baby SD rats by implanting SGC7901 cells.The SEB and SEB-scFv were injeeted to the vena caudalis in the trial groups,and normal saline to the control group.The weight of the tumor and the survival were recorded after treatment. Results Cell inhibitory rate was increased along with increased concentration of SEB-scFv fusion protein.Electron microscopy revealed that the cell presented typical changes of apoptosis.FCM indicated that the apoptotic rate of SGC7901 cell lines significantly increased with increasing dose of SEB-scFv fusion protein,and agarose gel electrophoresis appeared marked DNA ladder.The average weight of tumor in the SEB-scFv group was lower than that in control groups(P＜0.05).The tumor inhibition rate was 61.3%,and the mean survival period of rats in SEB-scFv group was longer than that of other group(P＜0.05)with a survival prolongation rate of 54.6%. Conclusion The resuhs indicate that SEB-scFv fusion protein has an obvious antitumor effect on gastric cancer.

Objective To evaluate the feasibility,safety and short-term outcome of laparoscopic-assisted surgery for colorectal cancer.Methods From August 2001 to November 2004,laparoscopic resection of colorectal carcinoma were performed in 112 cases,including right hemicolectomy(n=23),left himicolectomy(n=7),radical resection of sigmoid cancer(n=15),Dixon procedure(n=49),and Miles procedure(n=18).Results One hundred and five patients underwent laparoscopic resection successfully,7 cases were converted to open surgery because of hemorrhage,obesity or adhesion with adjacent organ,6 of which were left colon or rectal cancer.The mean operating time was(161.2?48.6)min,and the mean operative blood loss was 78.5 mL.There were 8 cases occurred postoperative complications,and no mortality during perioperative period.The length of upper and lower segment of resection for colonic cancer was (14.5?3.2)cm and(11.0?2.6)cm respectively.The length of upper and lower segment of resection for rectal cancer was(15.3?2.7)cm and(2.8?1.6)cm,respectively.The mean number of lymph nodes dissected was(8.2?4.6),and lymph node metastases were found in 49 cases.One hundred and seven cases(95.5%) were followed up for 8-44 months,of which,7 cases had local recurrence and 6 cases had distant metastases.No case of trocar port tumor implantation was observed.Conclusions Laparoscopic surgery for colorectal cancer is feasible and safe,can result in the same outcome as open radical surgery,and has the advantages of mini-invasive procedure.

Objective To establish the mathematical models of stature estimation for Sichuan Han female with measurement of lumbar vertebrae by X-ray to provide essential data for forensic anthropology re-search. Methods The samples, 206 Sichuan Han females, were divided into three groups including group A, B and C according to the ages. Group A(206 samples) consisted of all ages, group B(116 samples) were 20-45 years old and 90 samples over 45 years old were group C. All the samples were examined lumbar vertebrae through C Rtechnology, including the parameters of five centrums (L1-L5) as anterior border, posterior border and central heights (x1-x15), total central height of lumbar spine (x16), and the real height of every sample.The linear regression analysis was produced using the parameters to establish the mathematical models of stature estimation. Sixty-two trained subjects were tested to verify the accuracy of the mathematical models. Results The established mathematical models by hypothesis test of linear regression equation model were statistically significant (P<0.05).The standard errors of the equation were 2.982-5.004 cm, while correlation coefficients were 0.370-0.779 and multiple correlation coefficients were 0.533-0.834.The return tests of the highest correlation coefficient and multiple correlation coefficient of each group showed that the highest accuracy of the multiple regression equation, y=100.33+1.489 x3-0.548 x6+0.772 x9+0.058 x12+0.645 x15, in group Awere 80.6% (±1SE ) and 100% (±2SE ). Conclusion The established mathematical models in this study could be applied for the stature estimation for Sichuan Han females.

AIM: To construct prokaryotic expression vector of human angiogenesis inhibitor arresten gene and express recombinant arresten in Escherichia coli. METHODS: Human arresten gene was amplified from recombinant plasmid pGEM-Arr with polymerase chain reaction (PCR), and then cloned into prokaryotic expression vector pRSET by means of recombinant gene technology. The recombinant plasmid pRSET-Arr was transformed into E.coli BL21(DE3), and recombinant arresten was expressed in the bacteria under induction of IPTG. The expressed products were detected by SDS-PAGE analysis. RESULTS: Restriction analysis indicated that the arresten gene was successfully inserted into the expression vector, and DNA sequencing verified that the reading frame of the recombinant vector was correct. Recombinant arresten was successfully expressed in Escherichia coli; its molecular weight was about 26 kD and its amount was approximately 30% of total bacterial proteins.CONCLUSION: The successful construction of prokaryotic expression vector containing human arresten gene and the effective expression of recombinant arresten in Escherichia coli laid the foundation for further study on its biological functions.