OBJECTIVETo detect the methylenetetrahydrofolate reductase(MTHFR) gene C677T mutation with molecular beacon technique and assess the revant applicability.

METHODSA total of 228 samples were analyzed using molecular beacons which are oligonucleotide probes to become fluorescent upon hybridization. Wild-type molecular beacon and mutant beacon were designed to detect the genotypes of MTHFR gene.

RESULTSAnalysis of the 228 samples indicated that there were three genotypes including 41 homozygous mutants, 113 heterozygous individuals and 74 wild-type individuals. Every sample was identified clearly.

CONCLUSIONThe present method, a closed-tube PCR/hybridization assay, is a simple, high-throughput and fast procedure that is fully automated for detecting gene mutation.

DNA Mutational Analysis ; methods ; Fluorescent Dyes ; chemistry ; Genotype ; Humans ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Oligonucleotide Probes ; chemistry ; genetics ; Point Mutation ; Sensitivity and Specificity

Objective To investigate the expression of Toll-like receptor(TLR) 9 of circulating plasmacytoid dendritic cell (pDC) and analyze the frequency and interferon (IFN)-?production of circulating pDC during hepatitis B virus(HBV) infection.Methods Peripheral blood was collected from 69 HBV-infected patients,including 14 cases of asymptomatie HBV infection,30 cases of chro- nic hepatitis B(CHB),25 cases of HBV-related liver cirrhosis,and 21 healthy blood donors as con- trols.Flow cytometry was used to analyze the frequency of circulating pDC and the mean fluorescence intensity(MFI) of TLR9.Fresh peripheral blood mononuclear cells(PBMC) were stimulated by CpG ODN 2216 for 24 h in vitro.IFN-?in the supernatant was measured using enzyme-linked immunosorbent assay(ELISA) to analyze the frequency and IFN-?production of pDC during HBV in- fection.Data were analyzed with SPSS 13.0 for windows.Results Compared with healthy controls (62.6?10.7),the MFI of TLR9 of patients with asymptomatic HBV infection,those of CHB and HBV-related cirrhosis were significantly reduced (P

OBJECTIVETo modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference.

METHODSThe U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference.

RESULTSThe sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection.

CONCLUSIONThe siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Polymerase Chain Reaction ; RNA, Small Interfering ; biosynthesis ; genetics ; RNA, Small Nuclear ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of lymphadenectomy adjacent to inferior mesenteric artery root on the prognosis of rectal cancer.

METHODSClinicopathological data of 260 cases with rectal cancer undergone radical operation were analyzed retrospectively. The patients were divided into two groups. Group D(2): the lymph nodes adjacent to mesenteric artery root were not excised (n=188). Group D(3): the lymph nodes adjacent to mesenteric artery root were excised (n=72). Prognosis of two groups was compared during the follow-up period.

RESULTSIn group D(2), the 1-, 3-, 5-year total survival rates (TS) were 97.3%, 87.2% and 77.1%, and tumor-free survival rates (TFS) were 93.1%, 83.0% and 76.8% respectively. In group D(3 ), the 1-, 3-, 5-year total survival rates (TS) were 94.4%, 79.2% and 73.6%, and tumor-free survival rates (TFS) were 86.1%, 76.4% and 71.0% respectively. The differences of TS and TFS between two groups were not significant according to Kaplan-Meier analysis (P>0.05). Multivariate analysis revealed that the excision of lymph nodes adjacent to mesenteric artery root was not statistically correlated with the recurrence, metastasis and survival time after radical operation of rectal cancer.

CONCLUSIONExcision of lymph nodes adjacent to inferior mesenteric artery root has no significant impact on prognosis and it is unnecessary in the radical operation of rectal cancer.

Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Lymph Node Excision ; methods ; mortality ; Lymph Nodes ; surgery ; Lymphatic Metastasis ; Male ; Mesenteric Artery, Inferior ; surgery ; Middle Aged ; Prognosis ; Rectal Neoplasms ; mortality ; pathology ; surgery ; Survival Rate ; Treatment Outcome

Objetive: To investigate whether apoptosis of SGC7901 cells can be induced by the expression of the recombinant gene of anti-HER2 ScFv/tBid. Methods: The recombinant anti-HER2 ScFv/tBid gene was cloned into vector pCMV and the recombinant plasmid was transfected into SGC7901 cells. The gene expression was detected by RT-PCR and immunofluorescent staining. Cell counting was carried out to show the effect of the gene transfection on cell growth. At the same time, significant apoptotic peak was detected by flow cytometry in recombinant anti-HER2 ScFv/tBid gene transfected cells. Results: The fusion protein of anti-HER2 ScFv/tBid was observed in the cytoplasm of transfected SGC7901 cells. The transfected cells displayed typical cell growth inhibition and apoptosis. Conclusion: Fusion protein of anti-HER2 ScFv/tBid can induce apoptosis of SGC7901.

In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.
Animals ; Birds ; virology ; Influenza A Virus, H7N9 Subtype ; genetics ; isolation & purification ; physiology ; Influenza in Birds ; prevention & control ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Species Specificity ; Taq Polymerase ; metabolism ; Time Factors

BACKGROUND & OBJECTIVE: Dendritic cells (DCs) or DClike cells had been successfully induced in vitro from leukemia cells, which may provide a promising immunotherapeutic protocol for leukemia. This study was designed to investigate the efficiency of in vitro generation of dendritic cells from CD14+ acute myelomonocytic (M4) or monocytic (M5) leukemia cells and their ability of stimulating specific antileukemia T-cell response.METHODS: Bone marrow mononuclear cells (BMMNCs) were isolated from 5M4/M5 leukemia patients with high CD14 expression, and then divided into 3groups: adherent leukemia cells, nonadherent blasts, and total unfractioned blasts. CD14 expression of the 3 groups was evaluated by flow cytometry (FCM). When cultured with or without granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α) for 7-10 days, monocytic leukemia cell-derived dendritic cells (Mo-LDCs) were identified through morphologic observation and immunophenotype analysis using FCM. The immune function of Mo-LDCs was detected through allogeneic mixed lymphocyte reaction (Allo-MLR) and cytotoxicity assay of antileukemia cytotoxic T lymphocytes (CTLs). The leukemic origin of Mo-LDCs was confirmed by chromosomal karyotype analysis combined with the aberrant expression of myeloid antigens. RESULTS: The amount of CD14+ cells, which could differentiate into CD83+ mature DCs under induction of the cytokine combination, was higher in adherent leukemia cells than in nonadherent blasts and total unfractioned blasts. Regarding each 3 cell groups of the same patient or the unfractioned blasts of various patients, initial CD14 expression was positively related to the yield of CD83 + DCs after induction (r=0.967, P=0.007). Mo-LDCs exhibited typical morphology and phenotype as mature DCs, induced potent proliferation of homogeneous T cells in Allo-MLR, stimulated the expansion of leukemia-specific CTLs, and continued to possess the cytogenetic abnormalities of the original leukemia, as well as the aberrant expression of myeloid antigens. CONCLUSIONS: In M4/M5 subtype of AML, CD14+ cells could differentiate into immune-competent Mo-LDCs under the induction of the cytokine combination. CD14 expression level may predict the DCs differentiation ability of monocytic leukemia. MoLDCs, which possess the classical phenotype and function of DCs, as well as the abnormal leukemic antigens, may be useful for the immunotherapy of M4/M5 AML.

Objective To explore the application of integrated medical model on drainage tube orifice seepage of peritoneal cavity.Methods A total of 110 patients, who hospitalized from March to September 2013, were divided into control group received traditional method to cope with tube seepage of peritoneal cavity. While 108 patients during October 2013 to March 2014 underwent integrated medical model ( including multidisciplinary collaboration, suitable dressing, improvement of seepage, personal psychological consultation) as experimental group.Results The time of dressing change and the cost were (12.56 ±5.31) min/d and (40.45 ±5.67) Yuan/d intubated and decreased to (10.62 ±2.79) min/d and (15.56 ±5.62) Yuan/d extubated in the experimental group.All those data were lower than those in the control group with statistical difference (t=11.277,9.019,11.715, -19.100,respectively; P <0.05).The investigated data in the experimental group compared with those in the control group showed as bellows:the rates of satisfaction (100%vs 90%), complaint rate (0% vs 3.6%), and wound infection rate (7.4% vs 26.4%) with statistical difference (χ2 =11.374,5.546,13.896, respectively;P<0.05).Conclusions The integrated medical model is an effective and economic method to cope with drainage tube seepage of peritoneal cavity, benefits doctors, nurses and patients, and is worth of clinical promotion.

The automatic cleaning machine we have developed, adopts a SCM system in automatic cleaning. The machine has five functions: ultrasonic cleaning, cold or hot water spraying, drying and greasing. The clinical applications show that the machine with a good effectiveness is suitable for the cleaning of many surgical instruments. It also raises working efficiency, cuts down on the cost of repair and maintenance and reduces the injury and infection to nurses caused by manual cleaning, satisfying the needs of clinical applications.
Automation ; instrumentation ; Disinfection ; instrumentation ; Equipment Design ; Surgical Instruments ; standards ; Ultrasonics ; instrumentation

OBJECTIVETo establish an improved method for stereotactic location of the supraoptic nucleus in rats.

METHODSTwenty-four SD rats were randomly divided into experimental group (12 rats) and control group (12 rats) for oblique (20° to the left) stereotactic puncture (OSP group) and vertical stereotactic puncture (VSP group), respectively, both targeting the supraoptic nucleus (SON). The surgical data and postoperative (within 24) mortality of the rats were compared between the two groups.

RESULTSThe nucleus locating time was longer in OSP group than in VSP group (59.55∓3.64s vs 27.44∓2.18 s, P=0.000), and the postoperative mortality rate of the rats did not differ significantly between the groups (0 vs 44.4%, P=0.082). In OSP group, compared with VSP group, the procedure was associated with a lowered rupture rate of the superior sagittal sinus (11.1% vs 88.9%, P=0.003), a shortened hemostatic time after craniotomy (52.89∓24.05 s vs 157.445 ime a s, P=0.000) and after puncture (24.33 reas 45 s vs 133.89∓28.81 s, P=0.000), and also a shortened operation time (178.89 on tims vs 362.44 timees, P=0.000).

CONCLUSIONThe improved method for locating supraoptic nucleus in rats is convenient, stable and reproducible, and helps to avoid important blood vessels and specific nuclei according to the needs of different experiments and allows the operators to choose different surgical paths.