The Arabidopsis thaliana tonoplast Na+ /H+ antiporter gene, AtNHX1, was transferred into buckwheat by Agrobacterium-mediated method. Transgenic buckwheat plants were regenerated and selected on MS basal medium supplemented with 2.0mg/L 6-BA, 1.0mg/L KT, 0.lmg/L IAA, 50mg/L kanamycin and 500mg/L carbenicillin. 426 seedlings from 36 resistant calli originated from 864 explants (transformed about at 4.17 percentage) exhibited resistance to kanamycin. The transformants were confirmed by PCR, Southern blotting, RT-PCR and Northern blotting analysis. After stress treatment for 6 weeks with 200mmol/L NaCl, transgenic plants survived, while wild-type plants did not. After 3 days of stress treatment through different concentrations of NaCl, transgenic plants accumulated higher concentration of Na+ and proline than the control plants. However, the K+ concentration of transgenic plants declined in comparison with the control plants. Moreover, the rutin content of the roots, stems and leaves of transgenic buckwheat increased than those of the control plants. These results showed that it could be possible to improve the salt-tolerance of crops with genetic technology.
Adaptation, Physiological ; drug effects ; genetics ; physiology ; Arabidopsis Proteins ; genetics ; physiology ; Blotting, Northern ; Blotting, Southern ; Cation Transport Proteins ; genetics ; physiology ; Fagopyrum ; genetics ; metabolism ; physiology ; Plant Roots ; genetics ; metabolism ; physiology ; Plant Stems ; genetics ; metabolism ; physiology ; Plants, Genetically Modified ; genetics ; metabolism ; physiology ; Potassium ; metabolism ; Proline ; metabolism ; Regeneration ; Reverse Transcriptase Polymerase Chain Reaction ; Rutin ; metabolism ; Sodium ; metabolism ; Sodium Chloride ; pharmacology ; Sodium-Hydrogen Exchangers ; genetics ; physiology ; Transformation, Genetic

Objective To study the efficacy of late course accelerated fractionation(LCAF) radio- therapy in the treatment of nasopharyngeal carcinoma(NPC).The end-po s were local control,radiation-in- duced complications,factors influencing survival.Methods From December 1995 to April 1998,178 NPC patients were admitted for radiation treatment.The radiation beam used was ~(60)Co?or 6 MV X-ray.For the first two-thirds of the treatment,two daily fractions of 1.2 Gy were given to the primary lesion ,with an interval of≥6 hours,5 days per week to a total dose of 48 Gy/40 fractions,over a period of 4 weeks.For the last one third of the treatment,i.e.beginning from the 5th week,an accelerated hyperfractionation schedule was carried out.The dose per fraction was increased to 1.5 Gy,2 fractions per day with an interval of≥6 hours,the total dose for this part of the protocol was 30 Gy/20 fractions over 2 weeks.Thus the total dose was 78 Gy in 60 fractions in 6 weeks.Results All patients completed the treatment.Acute mucosi- tis:none in 2 patients,Grade 1 in 43,Grade 2 in 78,Grade 3 in 52,and Grade 4 in 3 patients.Local control rate:the 5-year nasopharyngeal local control rate was 87.7%,and the cervical lymph node local control rate was 85.7%.The 5-year distant metastasis rate was 26.1%,and 5-year survivals was 67.9%. Sixteen patients had radiation-induced cranial nerve palsy.Conclusions With this treatment schedule, patient's tolerance is good,local control and 5 year survivals are better than control groups of conventional fractionation and hyperfractionation radiotherapy.Radiation-related late complication does not increase.Ran- domized clinical trials are being carried out to further confirm the efficacy of LCAF for nasopharyngeal carci- noma.

Objective To investigate the effect of telmisartan on the expression of peroxisome proliferator activated receptor gamma(PPARγ)and its regulation in myocardial remodeling in spontaneously type 2 diabetic male Otsuka Long-Evans Tokushima Fatty(OLETF)rats fed with high-fat diet.Methods Twenty-eight four-week-old male OLETF rats were fed with high-fat diet. From 22-week of age, the pre-diabetic OLETF rats were randomly assigned to three groups:telmisartan-treated group[O-T group,5 mg/(kg·d),n=10],pioglitazone-treated group[O-P group,10 mg/(kg·d),n=8]and untreated group ( O-C group, equal volume of normal saline, n=10), continuously administration for 22-week. Twelve sex and age matched Long-Evans Tokushima Otsuka(LETO)rats were used as control(LETO group).At 22 and 48-week of age, the glucose tolerance of the rats was assessed by the oral glucose tolerance test(OGTT).At 48 weeks of age,five rats were randomly selected from each group,and clamp experiments were carried out.The glucose infusion rate from 60 min to 120 min(GIR60-120)was measured.All rats were sacrificed and the myocardial tissues were dissected.The ratio of heart weight to body weight(HW/BW)was calculated.Blood samples were collected,and serum PPARγ,tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and adiponectin were measured using ELISA and radioimmunoassay.The mRNA expressions of IL-6,PPARγ1 and PPARγ2 were measured by real-time PCR.The protein expression levels of PPARγ,adiponectin,IL-6 and NF-κB were determined by Western blot assay.The myocardial pathological changes were observed under light microscope (HE staining, Masson staining and PAS staining), and ultrastructural changes were observed under transmission electron microscope.Results At 22-week of age,neither type 2 diabetes mellitus(T2DM)nor IGT were found in three groups.At 48-week of age,T2DM was found in seven rats of O-C group,and IGT was found in two rats.T2DM was found in one rat of O-C group,and IGT was found in three rats.Neither T2DM nor IGT was found in the other two groups.GIR60-120and HW/BW were all significantly lower in the O-P group and O-T group than those of the O-C group at 48-week of age (P<0.05). Systolic blood pressure(SBP)and diastolic blood pressure(DBP)were significantly lower in O-T group than those in other three groups(P<0.05).Compared with the O-C group,the serum levels of PPARγ and adiponectin were significantly up-regulated,whereas the serum levels of IL-6 and TNF-α were down-regulated by telmisartan administration in O-T group (P<0.05).There were no significant differences in the above indexes between O-P and O-T groups.The results of real-time PCR and Westen blot assay showed that the mRNA expression of PPARγ1 was increased,and IL-6 expression decreased in O-T group compared with those of O-C group. The protein expressions of PPARγ and adiponectin were increased, and protein expressions of NF-κB and IL-6 were significantly decreased in O-P group and O-T group compared with those of O-C group(P<0.05).In the O-C group,the arrangernent of myocardial cells was irregular,myocardial fibers were swollen,a large amount of fibrotic tissue in the myocardial interstitium, and glycogen accumulation under light and electron microscope. Besides, myofibril breakage and perinuclear space expansion, myocardial mitochondria were apparently damaged or even dissolved. Compared with the O-C group, myocardial fibers arranged neatly, no obvious glycogen deposition and the ultrastructural changes of myocardium were obviously reduced in O-T group and O-P group. Conclusion Telmisartan can increase the expression level of PPARγ in the serum and myocardial tissue, reduce myocardial fibrosis and alleviate cardiac remodeling in the high-fat-diet OLETF rats.

OBJECTIVETo study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.

METHODSThe cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.

RESULTSThe plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.

CONCLUSIONThe antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.

Animals ; Central Nervous System ; embryology ; Cloning, Molecular ; Digoxigenin ; chemistry ; Gene Expression Regulation, Developmental ; Oligonucleotide Probes ; RNA Probes ; Uridine Triphosphate ; chemistry ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

OBJECTIVETo investigate the expression of miR-9 in B lymphocytes, B cell lymphoma and classical Hodgkin's lymphoma (cHL) cell lines and its significance.

METHODSCD19(+) B lymphocytes were sorted from normal lymph node by magnetic beads. Total cellular micro-RNA was extracted from cHL cell line L428, B cell lymphoma cell lines Ly1 and Ly10 (diffuse large B cell lymphoma), Raji cells (Burkitt's lymphoma) and CD19(+) B lymphocytes, respectively. These micro-RNAs were separately transformed into cDNA by reverse transcription. The expression levels of miR-9 were measured by fluorescence quantitative PCR. In situ hybridization was used to detect the expression of miR-9 in cell lines.

RESULTSThe expression of miR-9 was high in L428 cells (104.44 ± 1.61), and low in cell lines of B cell lymphoma (Ly1: 2.17 ± 0.38; Ly10: 1 ± 0.015; Raji: 2.65 ± 0.89), and extremely low in CD19(+) B lymphocytes (0.0026 ± 0.00040). Compared with that in the other cell lines, the expression of miR-9 in L428 cells was statistically significant (P < 0.05). miR-9 localized in the cytoplasm diffusely and strongly in L428, but scattered and slightly with some prominent distribution around the nuclear membranes in Ly1 and Ly10, and only weakly in Raji.

CONCLUSIONSmiR-9 highly expressed in cHL cell line and might be a molecular marker for diagnosis and treatment of cHL.

B-Lymphocytes ; metabolism ; Cell Line, Tumor ; Cell Lineage ; Hodgkin Disease ; metabolism ; pathology ; Humans ; Lymphoma, B-Cell ; metabolism ; pathology ; MicroRNAs ; metabolism

Objective To establish of a close loop blood information system forreal time monitoring in the entire process of clinical blood transfusion.Methods In accordance with files and laws in clinical blood transfusion and combining the work in blood transfusion department with the actual needs of clinical blood transfusion,the blood transfusion department in Shaanxi Provinical People's Hospital developed the close-loop blood information system with Skynet Software Co.Ltd.Results The close-loop blood information system was achieved informational management patternthat was detailed information from blood products arrival to departure and whole process of evaluation transfusion effect.The link of Hospital Information Manage System (HIS),Laboratory Information System (LIS) and blood information system was completed with the system.The system realized real-time and accurate data collection and monitoring.The system achieved a closed-loop management of the entire process of clinical blood transfusion.Conclusion By using theclose loop blood information system,the quality of medical services can be improved,the work productivity can be increased,and the clinical blood transfusion can be rationalized.

Objective This study aimed to comprehensively analyze and compare the clinicopathological features and prognosis of Chinese patients with human epidermal growth factor receptor 2(HER2)-low early breast cancer(BC)and HER2-IHC0 BC. Methods Patients diagnosed with HER2-negative BC(N=999)at our institution between January 2011 and December 2015 formed our study population.Clinicopathological characteristics,association between estrogen receptor(ER)expression and HER2-low,and evolution of HER2 immunohistochemical(IHC)score were assessed.Kaplan-Meier method and log-rank test were used to compare the long-term survival outcomes(5-year follow-up)between the HER2-IHC0 and HER2-low groups. Results HER2-low BC group tended to demonstrate high expression of ER and more progesterone receptor(PgR)positivity than HER2-IHC0 BC group(P<0.001).The rate of HER2-low status increased with increasing ER expression levels(Mantel-Haenszel χ2 test,P<0.001,Pearson's R=0.159,P<0.001).Survival analysis revealed a significantly longer overall survival(OS)in HER2-low BC group than in HER2-IHC0 group(P=0.007)in the whole cohort and the hormone receptor(HR)-negative group.There were no significant differences between the two groups in terms of disease-free survival(DFS).The discordance rate of HER2 IHC scores between primary and metastatic sites was 36.84%. Conclusion HER2-low BC may not be regarded as a unique BC group in this population-based study due to similar clinicopathological features and prognostic roles.

Aim To establish human U87-MG glioma model in nude mice brain and to observe the characteristics of the tumor growth. Methods Human U87-MG glioma cells were cultured in vitro. 5 μL of cell suspension containing 3.0 ×1010·L-1, 4.0×1010·L-1and 5.0×1010·L-1respectively was inocula-ted into the right caudate nucleus of 18 male nude mice brain un-der the guidance of stereotaxic apparatus, separately, whereas another 6 nude mice as the control group, were inoculated into the same volume of Hanks solution. The moving and survival state of rats with gliomas were observed. The examinations of the tumors formation, volumes, metastasis and histopathology were performed and the obtained brain samples were stained with HE and immunohistochemistry. Results All the tested rats of dif-ferent inoculation doses developed brain tumors without extracra-nial metastasis. The mean survival time of three groups was (46.50 ± 3.27) d,(38.50 ± 3.28) d and (30.67 ± 3.51) d,respectively. The tumors showed the similar morphological fea-tures and immunophenotype to human glioma. There was positive expression of GFAP and S-100 in the tumors. Conclusions The orthotopic implantation model of human U87-MG glioma, by in-oculating quantitative U87-MG cells stereotaxically into the brains of the nude mice, is successfully established with 100 yield of intracranial tumor and no extracranial growth extension. It resembles the histopathological and morphological features of human glioma,which can be used as a reliable animal model for the study of the tumorigenesis, pathogenesis, biological charac-teristics and therapy of glioma.

Aim To investigate the effect of homocys-teine(Hcy)on the permeability of pulmonary micro-vascular endothelial cells(PMVECs)and the role and mechanism of RASAL1.Methods CBS+/-mice were fed a high methionine diet(HMD)for 16 weeks to replicate an animal model of hyperhomocysteinemia(HHcy).HE staining was used to observe the changes in lung tissue structure.qRT-PCR was used to detect the levels of RASAL1 and DNMT1 mRNA in lung tis-sue.Western blot was used to detect the expression of RASAL1,DNMT1,ZO-1,and VE cadherin proteins.Methylation specific PCR was used to detect methyla-tion in the RASAL1 promoter region.PMVECs were transfected with Ad-RASAL1 to detect the expression of ZO-1 and VE cadherin.The si-DNMT1 interference fragment was transfected into PMVECs,and the ex-pression of the RASAL1 was detected by qRT-PCR and Western blot.Results Serum Hcy level of HMD mice was significantly raised,and HE staining showed severe structural disorder in lung tissue.The expres-sion of RASAL1,ZO-1,and VE cadherin was de-creased,while the expression of DNMT1 was in-creased.The degree of methylation in the RASAL1 promoter region was raised.The expression of ZO-1 and VE cadherin increased after PMVECs were trans-fected with Ad-RASAL1.After knocking down DN-MT1,RASAL1 expression was increased.Conclusion Hcy can increase the permeability of PMVECs,and its mechanism is related to the upregulation of RASAL1 methylation level.

Objective: To investigate the role of ClC-3 chloride channel in the proliferation of breast cancer cell line Mcf-7 treated with curcumin and its specific mechanism. Methods: MTT assay was used to detect the effect of chloride channel blocker (DIDS) and curcumin on Mcf-7 and human normal cell viability. Patch-clamp technique was used to determine the current density before and after drug treatment. Apoptosis assay by flow cytometry was performed for further examination of cell apoptosis. Results: Curcumin had toxicity on Mcf-7 and HUVEC cells and DIDS reduced the survival rate of Mcf-7 cells by inhibiting proliferation. Curcumin could activate the chloride ion current on MCF-7 cell membrane, which would be inhibited by DIDS. Finally, curcumin in low concentration combined with DIDS could significantly promote the MCF-7 cells apoptosis. Conclusions: Our results suggest that ClC-3 protein is involved in the regulation of curcumin induced proliferation inhibiting in breast cancer cells through inducing cell apoptosis. ClC-3 may be a potential target of tumor therapy.