Objective:To clone the yeast cytosine deaminase(YCD) gene and to elucidate the killing efficacy of yeast cytosine deaminase / 5 - fluorocytosine gene therapy system on gene- transfered cells. Methods:YCD gene was cloned and YCD expression retroviral vector was constructed,the vector was transfered into packaging cell line and high- titer virus was abtained. Then the tum origenic leukemia cell line K5 6 2 B was infected,selected and evaluated. Finally,the killing efficacy of 5 - FC on gene- transfered cell clone observed,and the 5 - FC to 5 - FU conversion of YCD- K5 6 2 B cell lysate was estim ated. Results:YCD gene was cloned and the sequence was confirm ed. A YCD expression retroviral vector,MSCV- YCD- IRES- EGFP was constructed. Transfering it into packaging cell line? NX- A ,high- titer (3.5? 10 6 CFU / ml) virus was obtained. The virus were used to infect tumorigenic leukem ia cell line,K5 6 2 B,and the infecting rate was quite high (30 % ) . A gene- transfered cell clone,YCD- K5 6 2 B,was selected,and YCD gene m RNA expression was detected in it.YCD- K5 6 2 B cell lysate demonstrated CD enzyme activity,and it could deaminase 5 - FC to 5 - FU .MTT assay showed 5 - FC could kill YCD- K5 6 2 B cell in 96 h even at lower concentration(15?mol/ L ) . Conclusion:YCD/ 5 - FC suicide gene therapy system has a significantkilling efficacy on gene- transfered cells. [

Objective To evaluate the appropriateness of hospitalization days at a tertiary hospital in 2014 by means of the Appropriateness Evaluation Protocol ( AEP ) , and to analyze the causes of inappropriate stays. Methods Medical records of inpatients admitted at a tertiary hospital in 2014 were randomly selected. AEP( US version) was used to evaluate the appropriateness of every hospitalization day, while the causes of inappropriate hospitalization day were also analyzed. Results A total of 1 641 days of stay from 148 medical records were reviewed, and 129 days of stay (7. 9%) were seen as inappropriate. Two major factors for inappropriate stays were waiting for surgery and waiting for test, roughly 89. 1% of the inappropriate hospitalization days. The proportion of inappropriate hospital stays reduced to 4. 8% after adjustment of two-day weekend. Inappropriate hospital stays mostly appeared during the second day to the eighth day after admission(93. 8%). Logistic analysis results showed that with concomitant symptoms, preoperative waiting days > 5 days, high level surgery, non-emergency admission were significantly associated with appropriateness of hospital stays (P<0. 05). Conclusions The rate of inappropriate stays will be reduced and the quality of medical services will be improved if comprehensive measures could be carried out according to the causes of inappropriate stays.

Objective To observe the ultrasonographic features of primary female genital system lymphoma and its histopathological characteristics.Methods Sonographic appearances and histopathological characteristics of 14 patients who were pathologically comfirmed as primary female genital system lymphoma were reviewed.Results Fourteen patients underwent ultrasonography and 15 lesions were found,6 cases of which were found in the ovary and cervix separately,3 cases in the uterine body.Among the lesions detected,10 of which respectively represents extremely low echo and homogeneous internal echo,all lymphomas had posterior enhancement.The margins were most frequently circumcribed (13/15),5numbers primary lymphomas of the ovary were mostly elliptical in shape,while 5 numbers of cervix tumors showed the shape of lobular,all of the tumors in the uterine body showed diffuse symmetrical enlargement without disruption of the endometria.Color Doppler imaging showed hypervascularity in most tumors(13/15).The pathological examination showed that all tumors were non-Hodgkin lymphoma diffuse B-cell type.Conclusions Primary female genital system lymphoma has some ultrasonic features,including extremely low echo,homogeneous echo and posterior enhancement.Final diagnosis depends on the histopathology.

ObjectiveTo investigate the value of strain ratio (SR) in the diagnosis of Hashimoto's thyroiditis.Methods 116 patients who had been diagnosed clinically as Hashimoto' s thyroiditis were enrolled in the study.Those patients were classified into four groups:the hyperthyroidism group,the euthyroidism group,the subclinical hypothyroidism group,and the clinical hypothyroidism group.50volunteers with normal thyroid functions were enrolled as a control group.SR of the thyroid and sternocleidomastoid muscle of same side was calculated.The correlation coefficient between the elasiticity SR and the serum thyroid stimulating hormone (TSH) was evaluated and their differences in the diagnosis of Hashimoto's thyroiditis were compared.ResultsThe Spearman's correlation coefficient between the SR and the serum TSH was 0.605,which was significant (P =0.000).The SR increased in an ascending order among the group with hyperthyroidism,the control group,the group with euthyroidism,the group with subclinical hypothyroidism,and the group with clinical hypothyroidism.ConclusionsElastography SR is valuable way for evaluating the progression of Hashimoto's thyroiditis.

Objective: To construct retroviral vector pLDAAOSN and observe the function of DAAO gene. Methods: With recombinant DNA technology, DAAO cDNA was cloned into retroviral vector (pLDAAOSN). The vector was transfected into ?XNA cells by CaPO4 method, and the DAAO cDNA was transferred by recombinant retroviral vector into leukemia cell line K562. The positive clones were obtained after G418 selection and termed K DAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in K DAAO. In order to observe the function of DAAO, K DAAO was treated with 50 mm/L D-Ala. Results: Results of plasmid pLDAAOSN digested with KpnⅠ and the sequence determination confirmed pLDAAOSN contains full-length DAAO cDNA. Infectious titer generated by the packaging cells was 5.2?10 6 CFU/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in K DAAO and expression of DAAO gene at mRNA level. Preliminary observation suggested that D-Ala could effectively kill K DAAO. Conclusion: Retroviral vector pLDAAOSN may be useful for futher study of gene therapy in cancer.

Objective: To investigate the effect of BSO on DAAO/D-Ala system killing K562e cells. Methods: KDFGC cell stably expressing DAAO was obtained by retrovirus transfection. The integration and expression of DAAO gene in KDFGC cells were identified by PCR and in situ hybridization. The killing effects of D-Ala on KDfGC cells treating with Immol/L BSO were observed. Results: PCR and in situ hybridization analysis confirmed integration of DAAO gene in positive clone and expression of it at mRNA level. The IC50 of KDFGC and KDFGC + BSO treating with D-Ala for 24 hours were 10.21 mmol/L and 7.92 mmol/L, respectively. The 95% confidence limits of them were different. Conclusion: BSO can enhance the killing effect of DAAO/D-Ala system on K652e cells.

Objective To retrospectively review and compare the clinical results of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from HLA- matched sibling donors mobilized with different regimens. Methods Seventy-one patients with hematological malignant diseases received allo-PBSCT from HLA-matched sibling donors in our department. Among them, 24 received allografts mobilized with G-CSF (group G), and the remaining (47 cases) were mobilized with G-CSF and GM-CSF (group G+ M). CD34+ subsets and T cell subsets in the allografts were analyzed, and the time of hematopoietic reconstitution and the incidence of graft versus host diseases (GVHD) were compared. The adverse effects on the donors after mobilization were also observed. Results The enough targeted CD34+ cells in all donors were harvested by 1-3 aphereses. Ninety-six h after mobilization, WBC counts of the donors were significantly higher in group G than in group G + M [(49. 6± 19. 5) 109/L vs (25.4 ± 10. 4) 109/L, P＜0. 05]. Analysis of the CD34+ subsets showed that the percentage of cells with the CD34+/CD38- phenotype was significantly higher in group G + M than in group G [(37. 7 ± 5. 7) % vs (31.4 ± 4. 5) %, P＜0. 05]. There was no significant difference in T cells and subsets of grafts. There was no significant difference in the number of total CD34+ cells and CD34+ CD38- cells, and infusion of T cells between two groups. The days required for the recovery of neutrophils and platelets was inversely correlated with the infused CD34+ and CD34+ /CD38- cell number. There was no significant difference in incidence of acute and chronic GVHD between two recipient groups. Seventeen cases and 10 eases among 71 eases died of relapses of primarydiseases, and complications of transplantation such as severe GVHD and infections respectively.Fourteen cases in group G (58.3 %) and 31 cases in group G+ M (66.0 %) survived. The most common adverse events in the donors were bone pain and fever, which mostly occurred 36 h after mobilization and could be relieved by non-steroidal anti-inflammatory drugs. Conclusion Two mobilization regimens showed equivalent clinical results. But the combined regimen of G-CSF and GM-CSF demonstrated a significantly greater mobilization of cells with the CD34+/CD38- phenotype.Meanwhile in allogeneic PBSCT, a greater number of total CD34+ cells and CD34+ CD38- cells infused may be associated with faster hematopoietic reconstitution of recipients.

OBJECTIVETo elucidate the killing activity of yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) gene therapy system on gene-transferred tumorigenic cell line K562B in vivo.

METHODK562B cell was infected with high titer virus and a gene transferred cell clone, YCD-K562B, was selected. Twelve male SCID mice of 4 week old were divided into 2 groups at random and both YCD-K562B and K562B cells were implanted to each mice. 5-FC or saline was given i. p for 10 days after tumor developed, and relative tumor volume was measured every 3 days. At the end of experiment, animals were sacrificed and the specimens were processed for histopathological examination.

RESULTSAt the end of experiment (21 days after tumor cell implantation), the relative tumor volume of the 4 groups were: YCD-K562B + 5-FC 2.922 +/- 0.581, YCD-K562B + saline 24.434 +/- 4.790, K562B + 5-FC 22.701 +/- 2.350 and K562B + saline 24.460 +/- 1.670; t-test analysis showed that 5-FC could kill cells (YCD-K562B) in vivo (P = 0.0001), but had no effect on the growth of gene-untransferred cells (K562B) (P = 0.096). In YCD-K562B + 5-FC group, relative tumor volume reduced in 3 approximately 6 days after treatment (the minimum was 0.681). Necrosis around artery could be found in the tumor of YCD-K562B + 5-FC group.

CONCLUSIONYCD/5-FC suicide gene therapy system has a significant in vivo killing activity to gene-transferred tumorigenic YCD-K562B cell.

Animals ; Cytosine Deaminase ; Flucytosine ; metabolism ; pharmacology ; Genetic Therapy ; methods ; Humans ; K562 Cells ; Male ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Neoplasms, Experimental ; genetics ; therapy ; Nucleoside Deaminases ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; Transfection ; Treatment Outcome ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the in vitro killing efficiency of D-amino acid oxidase (DAAO)/D-alanine (D-Ala) system on K562e cells.

METHODSK(DfGC) cell line stably expressing DAAO was obtained by retrovirus transfection technique. The integration and expression of DAAO gene were identified by PCR and in situ hybridization. The killing activities of D-Ala to DAAO(+) cells alone or the mixtures of DAAO(+) and DAAO(-) cells in different ratios were observed. H(2)O(2) production by K(DfGC) cell was measured by phenol red oxidation assay.

RESULTSPCR and in situ hybridization analysis confirmed the integration of DAAO gene in positive clone and its mRNA expression. There was no significant difference in cell proliferation between the two kinds of K562 cells. Ninety percent of K(DfGC) cells was killed by 12.5 mmol/L D-Ala after 24 hour-treatment and the H(2)O(2) levels were in accord with the killing activities of D-Ala. When K(DfGC) was mixed with K562e at different ratio, no significant bystander effect could be found after treating with 15.0 mmol/L D-Ala for 24 hours.

CONCLUSIONThe leukemia cell line K562e was sensitive to DAAO/D-Ala system and there was no significant bystander effects observed within this cells.

Alanine ; metabolism ; pharmacology ; Cell Survival ; drug effects ; D-Amino-Acid Oxidase ; genetics ; metabolism ; Gene Expression ; Humans ; Hydrogen Peroxide ; metabolism ; K562 Cells ; cytology ; drug effects ; metabolism ; Plasmids ; genetics ; RNA, Messenger ; genetics ; metabolism ; Transfection

Objective: To explore the prognostic value of CD34, CD2, CD56 expressions and FLT3-ITD mutation in adults with acute promyelocytic leukemia (APL) . Methods: The immuno-phenotypic and molecular characteristics of 137 adult patients with APL (from January 2010 to March 2016, in Henan Provincial People’s Hospital) were investigated. And the relationships between CD34, CD2, CD56 expressions, FLT3-ITD mutation and the outcomes of high WBC counts at onset, complete remission (CR) rate, early mortality, relapse rate (RR) , overall survival (OS) , disease free survival (DFS) were explored. Results: ①Among the 137 patients, the positive ratios of CD34, CD2, CD56 expressions and mutation rate of FLT3-ITD were 26.3%, 25.5%, 10.2% and 17.5%, respectively. The morbidities of positive CD34, CD2, CD56 expressions and FLT3-ITD mutation in the high-risk group were 43.2%, 47.7%, 18.2% and 27.3% respectively, while those in the low-/intermediate-risk groups were 18.3%, 15.1%, 6.5% and 12.9%, respectively (P<0.05) . ②At a median follow-up of 41 months, the total CR rate of the 137 adults APL patients was 96.9%, early mortality 6.6% and relapse rate 7.3% respectively. And RR of positive CD34 or CD2 expression patients was higher than negative CD34/CD2 expression ones (18.8% vs 3.3%, χ²=8.462, P=0.004; 16.1% vs 4.3%, χ²=4.382, P=0.028, respectively) . In addition, the early mortality of patients with positive CD56 expression or FLT3-ITD mutation was extremely higher than in negative ones (21.4% vs 4.9%, χ²=5.610, P=0.018; 16.7% vs 4.4%, χ²=4.833, P=0.028, respectively) . ③The whole OS and DFS were 88.3% and 84.7%, respectively. Wherein, OS and DFS in patients with CD34⁺, CD56⁺ or FLT3-ITD mutation were worse (P<0.05) . Conclusions Positive CD34, CD2, CD56 expression and FLT3-ITD mutation were latent poor prognostic factors in adults with APL.