0.05.[Conclusion]VDR TruⅠ and FokⅠpolymorphisms are not related to LDD in Han nationality.

OBJECTIVE: To determine the changes of the plasma metabolic phenotype in rats with chronic restraint stress (rats with syndrome of liver qi stagnation and spleen deficiency), so as to reveal the biological features of the syndrome of liver qi stagnation and spleen deficiency, and to examine potential application of metabonomic analysis in studies of syndromes in traditional Chinese medicine. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into four groups: group A, 7 d normal control group; group B, 21 d normal control group; group C, 7 d stress group; and group D, 21 d stress group, with 6 rats in each group. Chronic restraint was used to induce stress in rats. Blood was collected from the cardio-ventricle under anesthesia on the 8th (groups A and C) or 22nd day (groups B and D) and detected by using the Fourier variable superconducting nuclear magnetic resonance (NMR) spectrometer (Varian UnityInova 600 M). Free induction decay signals were transferred into one-dimensional NMR spectrogram via 32 k Fourier transformation. Segmental integral calculus (0.04 ppm per segment) was performed from 4.5-0.5 ppm (Carr-Purcell-Meiboom-Gill, CPMG) or 6.0-0 ppm (longitudinal eddy-delay, LED) as defaulted 1H spectra values by using the VNMR software. Data were saved as text or excel files after normalization and then used for pattern recognition analyses. All the data were analyzed by principal component analysis (PCA) using the SIMCA-P 10.0 software (Umetrics AB, Umea, Sweden). RESULTS: The PCA analysis of rat plasma (1)H NMR spectra revealed different metabolic spectra between stress and control groups, which were consistent with alterations of in vivo metabolisms in rats under stress stimuli. Compared with the normal control group, rats with repeated stress displayed significant changes in spectral peak shapes of acetate, lactate, tyrosine, low-density lipoprotein, and unknown compounds (3.44 ppm). These altered metabolites can be used as biomarkers of syndrome of liver qi stagnation and spleen deficiency for further studies. CONCLUSION: The (1)H NMR spectra of metabolites in the rat blood are differentially changed after chronic stress. Specific, characteristic metabolic products can be identified by analyses of metabonomics, which lead to interpretation of biological feature of Chinese medicine syndromes. Therefore, metabonomic analysis is an approach with good development prospects to studies of TCM syndromes.

A PCR method was used to amplify the sequence encoding the mature peptide of?-mannanase of Bacillus subtilis. The gene was inserted into the Pichia pastoris vector pPIC9K, downstream of?-factor signal peptide sequence. The resultant recombinant plasmid pPIC9K-MAN was lineared by BglII digestion and introduced into the host Pichia pastoris GS115 by PEG method. After screen, the recombinant P. pastoris strain MAN22 was obtained and fermented in large scale 5L fermenter. The recombinant mannanase activity could reach to 1102IU/ml. The properties of the recombinant mannanase were characterized.

Objective To evaluate clinical efficacy of multiple regimen combination in treatment of osteoporosis of perimenopausal or postmenopausal women. Methods From Jul. 2008 to Dec. 2009, 109 women with low bone mineral density (BMD) or osteoporosis treated in Department of Obstetrics and Gynecology, Affiliated Second Hospital, Wenzhou Medical College were enrolled randomly into 3 group,including 36 women in Group A managed by osteoform 1000 mg/d + alfacalcidol 0. 25 μg/bid orally, 40 women in group B managed by osteoform 1000 mg/d + alfacalcidol 0. 25 μg/bid + tibolone 1.25 mg/d orally and 33 women in group C managed by ostcoform 1000 mg/d + alfacalcidol 0. 25 μg/bid +bisphosphonates 70 mg/w orally. After 48 weeks BMD on lumbar 1 -4 (L1-4) and left femur were detected by X-ray. Bone alkaline phosphatase(BALP) ,cross linked clelopeptide of type Ⅰ collagen(CTX) and 25-hydroxychole calciferol [25 (OH) D3] was measured by enzyme linked immunosorbent assay (ELISA).Result Seven women (6. 4%, 7/109) were withdrawed form this study, including 2 cases losing follow up in group A, 3 cases stopping treatment in group B, 2 cases giving up treatment due to severe adverse effect (burning in upper abdomen) in group C. (1) Pain relieve: after 48 weeks treatment, women in 3 groups improved symptom of pain significantly, the rates of pain relieve were 85% (29/34)in group A, 92% (34/37) in group B and 94% (29/31) in group C. (2) BMD: BMD was improved significantly in women in 3 groups after treatment. BMD of L1-4 were (0.88±0.15) g/cm2 in group A,(0.89±0.18) g/cm2 in group B and (0.87±0.10) g/cm2 in group C before treatment, and converted to (0.90±0.01) g/cm2 in group A, (0.93±0.09) g/cm2 in group B and (0.91±0.11) g/cm2 in group C after treatment. BMD of left femur were (0.87±0.07) g/cm2 in group A, (0.87±0.07) g/cm2 in group B and (0.85±0. 12) g/cm2 in group C before treatment and converted to (0.90± 0.03) g/cm2 in group A, (0.91±0.08) g/cm2 in group B and (0.89 ±0.12) g/cm2 in group C after treatment. It was shown significantly different BMD between group B or C and group A (P < 0. 01), however, there was no significant different BMD between group B and C (P >0. 05). (3) Index of bone metabolism: BALP were (26±6) μg/L in group A, (26±9) μg/L in group B and (28±7) μg/L in group C before treatment and converted to (22±5) μg/L in group A, (20±9)μg/L in group B and (22±8)μg/L in group C after treatment, which showed statistical difference (P < 0.05). CTX were (0.85±0.20) ng/L in group A, (0.84±0.47) ng/L in group B, and (0. 88 ±0. 11) ng/L in group C before treatment and converted to (0. 81 ±0. 19) ng/L in group A, (0. 77±0.33) ng/L in group B, and (0.82 ±0. 14) ng/L in group C after treatment, which showed statistical difference (P < 0. 05). Conclusions Those 3 regimens combination could be used in treatment of osteoporosis by decreasing bone conversion, increasing bone density, decreasing bone absorption. Regimen A was only suitable for basic therapy,the other two regimens could provide better treatment.

Objective To investigate the expression and significance of JWA in Acute Myeloid Leukemia (AML). Methods Bone marrow mononuclear cell specimens were taken from 22 AML patients in newly diagnosis stage and complete remission stage respectively , and the JWA expression were detected at RNA and protein level. Results (1) JWA was expressed in all the samples at RNA and protein level. (2) At protein level, JWA expression was higher in the cells from newly diagnosed AML patients than in those from patients in complete remission stage (P < 0.05). (3)The complete remission rate of patients with higher expression level of JWA (87.5%) was similar to those with lower expression (83.3%). The complete remission rate of patients with higher expression level of JWA after the first course (31.25%) was lower than those with lower expression (66.7%). The early recurrence rate of patients with higher expression level of JWA (42.86%) was higher than those with lower expression (20%). Conclusion JWA may play an important role in the development and progression of AML. Increased expression of JWA may be one of the causes of refractory and relapsed AML.

Objective To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2)in the apoptosis,cell cycle and autophagy of starvation-induced colorectal cancer HCT116 p53 +/+ (p53 wild-type) cell line.Methods Six groups were included:(1) control group; (2) green fluorescent protein adenovirus (rAd-GFP) infection group; (3)ASPP2 adenovirus (rAd-ASPP2) infection group; (4)starvation group; (5)rAd-GFP + starvation group; (6) rAd-ASPP2 + starvation group.HCT116 cells were infected with ASPP2 adenovirus (rAd-ASPP2),resulting ASPP2 gene over-expression.The apoptosis,autophagy and cell cycle changes were induced by culturing with serum-free medium for 24 h.Apoptosis was evaluated by Calcein/PI uptaking test,and autophagy was observed by counting the red fluorescent protein autophagy plasmid CFP-Lc3 which was transfected into cytoplasm.Cell cycle was detected by flow cytometry.Statistical analysis was performed by one-way analysis of variance (ANOVA).Results Over-expressed ASPP2 was found to significantly promote starvation-induced HCT116 apoptosis and autophagy.The cell apoptosis rate in rAd-GFP + starvation group was 10.00％ ± 1.42％,and 18.44％ ±2.06％ in rAd-ASPP2 + starvation group(q =9.548,P =0.000).The cell autophagy rate in rAd-GFP+ starvation group and rAd-ASPP2 + starvation group was 35.00％ ± 5.34％ and 57.61％ ± 6.06％ respectively(q =7.657,P =0.000).Over-expressed ASPP2 accelerated HCT116 G2/M arrest under starvation,but resulted in both G0/G1 and G2/M arrest without starvation.Conclusion These results suggest that ASPP2 can promote starvation-induced HCT116 p53 +/+ cells apoptosis and autophagy,and affect the cell cycle.

Objective To investigate the effects of virtual reality prism adaptation on visuospatial neglect in stroke patients.Methods Thirty stroke patients with visuospatial neglect were studied.The subjects were divided into atreatment group and a control group.The subjects in the treatment group were treated with virtual reality prism adaptation and routine rehabilitation interventions for 2 weeks,while those in the control group were treated with routine rehabilitation interventions only.All the patients performed a battery of spatial attention tests including line bisection,letter cancellation,clock drawing and the Attention Network Test at the beginning and after 2 weeks of treatment.Results The virtual reality prism adaptation training had significant positive effects on all the measures of visuospatial neglect.Pair-wise comparisons confirmed significant differences between the treatment and control groups after 2 weeks of treatment with regard to all of the measures.Conclusions Virtual reality prism adaptation treatzment combined with routine rehabilitation can be more effective than conventional measures alone in improving the visuospatial performance of stroke survivors.

Objective To explore the role of brief ischemia pretreatment in the induction of renal ischemic tolerance,and investigate its effects on tubular cell necrosis,apoptosis and proliferation. Methods Male Sprague-Dawley rats were randomly divided into three groups,including sham-operated group (Sham),ischemia/reperfusion injured group subjected to theocclusion of both renal pedicles for 40 min followed by reperfusion(I/R),and preconditioned group with 20-min ischemia pretreatment induced 4 days before I/R(IPC).Histological changes were evaluated by PAS staining.The ultra-structure of tubular cells was observed by transmission electron microscopy(TEM).Apoptosis was confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL).The proliferation of tubular cells was evaluated with proliferating cell nuclear antigen(PCNA). Results Twenty-minites ischemia pretreatment offered both promising functional and histological protection against 40-min ischemia/reperfusion injury (P<0.01).The mortality rate wag reduced from 33%in I/R group to 0 in IPC group.The renopmtection offered by 20-min ischemia pretreatment was accompanied with reduced postischemic tubular cell apoptosis and necrosis (P<0.05), and increased cell proliferation (PCNA positive) (P< 0.01). Conclusions Brief and sublethal prior ischemia can render the kidney more tolerant to subsequent prolonged I/R injury. Its ability to tilt the balance of tubular cell fate toward survival, reducing postischemic cell death and enhancing cell proliferation, may play an important role in renal protection of ischemic preconditioning.

Objective:Semaphorin 4D (Sema4D) acts as a regulator for axon guidance in central nervous system development. However, new evidence indicates that Sema4D has a previously unrecognized function, namely, compensatory angiogenic factor. This study aimed to investigate the effect of Sema4D on tumor growth and vascularity of colorectal carcinoma (CRC) in nude mice. Meth-ods:Sema4D was knocked down in CRC cells by infecting the cells with lentiviruses coding for Sema4D shRNA. Two groups of cells, namely, those infected with control viruses and those infected with Sema4D shRNA viruses, were subjected to migration assay to test their ability induce endothelial cell migration. The two cell groups were subcutaneously injected into nude mice. Tumor growth was documented, and the tumors harvested from the mice were subjected to immunohistochemistry or immuno fl uorescence analyses. Re-sults:In vitro migration assay results indicated that media conditioned by HCT-116 cells infected with Sema4D shRNA lentiviruses in-duced low endothelial cell migration. The two groups of subcutaneously inoculated cells showed 100%tumorigenicity. However, tumor growth rates were significantly different between the two groups. Xenografts in which Sema4D was downregulated showed marked re-duction in tumor size and vascularity. Conclusion:Cancer cells may highly express Sema4D to trigger net neo-angiogenesis and gener-ate a tumor blood supply system. Thus, Sema4D could potentially be a target in anti-angiogenic therapy of CRC patients.

200 citrinin mutants were screened with the inhibition zone method from the transformants library of Monascus ruber M-7 by Agrobacterium-mediate DNA transfer, which contains more than 5,000 transformants.Then 53 mutants, whose citrinin contents ranged from 0.04?g/g to 154.57?g/g in the red fermented rice (RFR), were achieved by high performance liquid chromatography (HPLC).Color values of RFR prepared by these mutants were also detected.The results showed that there was a positive correlation between the citrinin content and the color value among the mutants.These results provide materials and research bases for ferrther studying the relationship between the production of citrinin and pigment of Monascus ruber at molecular level.