Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 ³ °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 ⁶ °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.
Freezing ; Vitrification ; Cryopreservation/methods* ; Trehalose ; Gold ; Rewarming ; Metal Nanoparticles ; Cryoprotective Agents ; Lasers

[Objective]This study was conducted to examine the effects of dexmedetomidine on the proliferation and angiogenesis of MHCC97H and SMCC7721 human hepatocellular carcinoma(HCC)cell lines cultured in hypoxia condition in vitro,and investigated the possible mechanism involved.[Methods]MHCC97H and SMCC7721 human HCC cell lines under hypoxia culture condition were treated with presence or absence of dexmedetomidine(100 μmol/L). Cell viability,colony formation,vasculogenic mimicry(VM) formation were assessed. The effects of dexmedetomidine on α-2A adrenergic receptor(α2A),hypoxia induced factor-1a(HIF-1a),and vascular endothelial growth factor(VEGF)protein expression were evaluated with Western blot analysis.[Results]Cell proliferation assay and colony formation assay indicated that hypoxia obviously promoted the proliferation of MHCC 97H and SMCC7721 cells(CoCl2 group vs corresponding control group,the proliferation rate of MHCC97H and SMCC7721:Day 3,142.2%and 133.8%;Day 4,134.7%and 131.0%;Day 5,133.5%and 136.2%;all P<0.05),and VM formation assay suggested that hypoxia increased angiogenesis of MHCC97H and SMCC7721 cells. Whereas dexmedetomidine significantly inhibited the proliferation(Dex+CoCl2 group vs CoCl2 group,the proliferation rate of MHCC97H and SMCC7721:Day 3,55.7%vs 60.7%;Day 4,46.9%vs 58.1%;Day 5,46.4%vs 57.0%,all P<0.05)and angiogenesis of MHCC97H,SMCC7721 cells induced by hypoxia. Dexmedetomidine may exert these functions by activating α-2A adrenergic receptor,causing an decrease in HIF-1a and VEGF protein,while hypoxia activated HIF-1a and VEGF protein to promote the growth and angiogenesis of cells.[Conclusion]The findings provide evidence that hypoxia could promote the proliferation and angiogenesis of MHCC97H and SMCC7721 cells,while dexmedetomidine might inhibit these effects by down-regulating HIF-1a and VEGF protein expression through activatingα-2A adrenergic receptor.

Objective To compare the efficacy difference of Green Forsythiae Fructus and Ripe Forsythiae Fructusby using Lianqiao powder and Yinqiao powder in the classic formula; To provide experimental evidence for the guidance of one for dual-use of the Forsythiae Fructus. Methods The guinea pig sore model was made with Staphylococcus aureus. 40 guinea pigs were randomly divided into blank group, model group, Lianqiao powder Green Forsythiae Fructus group and Lianqiao powder Ripe Forsythiae Fructus group. The blank group and the model group were fed with normal saline, while Lianqiao powder Green Forsythiae Fructus group and Lianqiao powder Ripe Forsythiae Fructus group were treated with 1.2 g raw medicine/kg liquid Lianqiao powder Green Forsythiae Fructus and Lianqiao powder Ripe Forsythiae Fructus for 7 d. The symptom score, blood and pathological changes of guinea pig soreness were observed. The model of acute lung injury was induced by 10% LPS aerosol inhalation. 40 rats were randomly divided into blank group, model group, Yinqiao powder Green Forsythiae Fructus group and Yinqiao powder Ripe Forsythiae Fructus group. The blank group and the model group were fed with normal saline, while Yinqiao powder Green Forsythiae Fructus group and Yinqiao powder Ripe Forsythiae Fructus group were treated with 4 g raw medicine/kg liquid Yinqiao powder Green Forsythiae Fructus and Yinqiao powder Ripe Forsythiae Fructus for 10 d. The levels of IL-6, TNF-α and IL-1β in the lung lavage fluid were detected. Results The effect of Lianqiao powder Green Forsythiae Fructus on the wound healing of guinea pig sore wound was faster than that of Lianqiao powder Ripe Forsythiae Fructus, but there was no significant difference between Lianqiao powder Green Forsythiae Fructus group and Lianqiao powder Ripe Forsythiae Fructus group in inhibiting the secretion and pathological changes of guinea pig sore wound. The levels of IL-6, TNF-α and IL-1β in Yinqiao powder Green Forsythiae Fructus group was lower than that in Yinqiao powder Green Forsythiae Fructus group, without statistical significance. Conclusion It is verified that there is efficacy differences between Green Forsythiae Fructus and Ripe Forsythiae Fructus in the different Chinese herbal compound.

Objective: To understand the serotypes, genotypes and transmission source of dengue viruses(DENV) isolated in Yunnan from 2013 to 2015. Methods: Viral RNA was extracted from serum samples of dengue fever(DF) cases at the acute stage in Yunnan, then the gene fragments of envelope protein(E) region were amplified by RT-PCR. The homology and phylogenetic analysis was made on the nucleotide and deduced amino acid sequences by bioinformatics softwares including Clustal X, DNAStar and MEGA5. Results: Viral nucleic acid detection and sequencing indicated that 40 E genes of DENV were obtained. The serotypes and genotypes of DENV were revealed by homology and phylogenetic analysis based on E genes of DENV. Fifteen virus strains belonged to DENV serotype 1(DENV-1), of these, 14(11 from Ruili, 1 from Lincang and 2 from Kunming) were genotype I(G-I), 1 from Kunming was G-V. Twenty-two virus strains belonged to DENV serotype 2(DENV-2), of these, 10 from Ruili were G-I and 12 from Xishuangbanna were G-IV. Two virus strains belonged to DENV serotype 3(DENV-3) and G-II. One virus strain belonged to DENV serotype 4(DENV-4) and G-I. All detected DENV genotypes were mainly predominant in Southeast Asia. All the 40 Yunnan DENV strains shared high homology with the DENV strains in Southeast Asia countries. Conclusions Four serotypes and multiple genotypes of DENV had been co-circulating in Yunnan from 2013 to 2015. The DENV transmitted from Southeast Asia countries was the main cause of DF in Yunnan. It is necessary to strengthen the surveillance and management on the imported cases of DF in Yunnan.

Objective To assess the value of noninvasive MR imaging biomarkers in evaluating the early efficacy of anti?angiogenesis drugs. Methods Subcutaneous colon cancer xenograft models in thirty nude mice were established. The mice were randomly divided into three groups (n=10 for each): avastin injection group (dose 10 mg/kg), fluorouracil group (dose 150 mg/kg), physiological saline group (dose 20 mg/kg). Dynamic contrast?enhanced (DCE?MRI) and multiple b value diffusion weighted imaging (muti?b?value DWI) were acquired before or 1 h, 24 h and 48 h after the treatment. The parameters of contrast transfer coefficient (Ktrans), reflux constant (Kep), plasma volume fraction (Vp), extravascular extracellular volume fraction (Ve) and various apparent diffusion coefficients (ADCs) (ADC10b, ADChigh and ADCperf) were measured. Forty eight hours after the treatment, the mice were sacrificed following MRI. Aimmunohistochemical examination determined microvessel density (MVD) and proliferating cell nuclear antigen (PCNA) score. A repeated measures analysis of variance was used to compare the difference between the quantitative parameters among the three groups. A multivariate variance analysis was performed to compare the difference between the parameters at the same time point among the three groups. The correlation between MRI quantitative parameters with MVD and PCNA score were analyzed by Pearson and Spearman correlation analysis respectively. Factor analysis method summarized MRI quantitative parameters. Results One hour after the treatment, the parameters of Ktrans, Kep, ADC10b, ADChigh and ADCperf value immediately changed, they were(0.009 ± 0.005)/s,(0.042 ± 0.031)/s,(0.043 ± 0.002)× 10?3 mm2/s,(0.031 ± 0.005)× 10?3 mm2/s,(0.089 ± 0.006)× 10?3 mm2/s, Ktrans, Kep, ADC10b and ADChigh values all had significant differences in the three groups (F=42.058, 25.979, 9.870 and 8.511, respectively, all P<0.05). There were also statistical difference in the change trend of the above parameters among the three groups (F=22.108, 7.280, 65.698 and 19.900, respectively, all P<0.05). The change trend of ADCperf showed significant difference among the three groups (F=38.780, P<0.01). Ktrans, Kep and ADCperf positively correlated with the MVD count and PCNA score (r values were 0.421 to 0.811, both P<0.01), while ADC10b showed a negative correlation (r=-0.656 and-0.560, both P<0.01), ADChigh had negative correlation with the PCNA score (r=-0.568, P<0.05). Ktrans, Vp, Kep and ADCperf were classified as tumor microcirculation factor, whereas ADC10b and ADChigh were normalized for cell metabolism factor through the factor analysis. Conclusions Combination of DCE?MRI and muti?b?value DWI can reflect the early changes of drug therapy from the aspects of tumor microcirculation and cell metabolism. Ktrans, Kep, ADCperf, ADC10b and ADChigh can be taken as noninvasive imaging biomarkers to quantify the early efficacy of anti?angiogenesis drugs.

Objective To study the CT findings of struma ovarii(SO)and improve the understanding of SO imaging features. Methods CT images of 6 cases were retrospectively reviewed.CT plain scan was performed in 6 patients;CT enhancement scan was performed in 2 patients.Results All tumors were unilateral.On non-enhanced CT,the lesions presented as well-defined irregu-lar cystic-solid masses.The cystic portions presented as well-defined,multiple,various size,and there were entire cystic walls with smooth inner wall.Four tumors showed high attenuation lesions in the cyst portion of the mass on precontrast scans.The solid por-tions showed irregular tissue density,and were often distributed in the cysts.The tumors showed stippled calcification in solid por-tions and/or cystic wall in 4 cases.One tumor accompanied a great of ascites liquid.After contrast administration,the cystic por-tions showed no enhancement,and the cystic walls and the solid portions showed mild enhancement.Conclusion CT findings of SO have certain characteristics such as a cystic-solid and well-defined mass with calcification,high attenuation lesion on plain CT,and marked solid part enhancement on contrast CT.

ObjectiveTo observe the effect of Astragalus membranaous on angiotensinⅡ (AngⅡ)-induced transform-ing growth factor β1 (TGF-β1) production of cardiac ifbroblasts.Methods Cardiac ifbroblasts were culturedin vitro. Cells were allocated into 3 groups: control group, Astragalus membranaous groups (50, 100, 200 mg/ml), Ang II group (10-7 mol/L) and AngⅡ/Astragalus membranaous groups (50, 100, 200 mg/ml). The proliferation of each group was tested by methyl thiazolyl tetrazolium method. TGF-β1 was measured by ELISA.Results The proliferation of cardiac ifbroblasts had signiifcant difference between each groups (F=71.84,P=0.000). The proliferation of cardiac ifbroblasts with Ang II stimulation was higher than that of cells without Ang II stimulation (P<0.05). Astragalus membranaous inhibited Ang II-induced cardiac ifbroblasts proliferation dose dependently (P<0.05). The TGF-β1 production had signiifcant difference between each groups (F=786.81,P=0.000). The TGF-β1 production in AngII/astragalus membranaous groups was lower than that in Ang II group (P<0.05). The TGF-β1 production in Ang II group was the highest, and had signiifcant difference as compared to other groups (P<0.05). Astragalus membranaous inhibited Ang II-induced TGF-β1 production dose dependently (P<0.05).Conclusions Ang II can stimulate the proliferation of cardiac ifbroblasts, and promote the TGF-β1 production. Astragalus membranaous can inhibit the proliferation of Ang II-induced cardiac ifbroblasts, and reduce the TGF-β1 production of cardiac ifbroblasts.

Objective To investigate the expressions of Wnt2 and β-catenin in Doxorubicin (DOX)-induced myocardial injury and to explore their roles in myocardial cell apoptosis.Methods Cardiomyoblast cells were damaged by different concentrations of DOX(1 mg/L,2 mg/L,3 mg/L,4 mg/L) for 72 h.The effect of different concentrations of DOX on cardiomyocyte growth curve was detected according to the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazolium bromide(MTT) assay.DOX(1 mg/L) was used to induce the model of cardiomyoblast cell injury.Cardiomyocytes were divided into 4 groups:group A:DOX-injured cardiomyocytes for 12 h ;group B:DOX-injured cardiomyocytes for 24 h ; group C:DOX-injured cardiomyocytes for 48 h; group D:normal cardiomyocytes.The expressions of Wnt2,β-catenin and p53 were observed by Western blot and reverse transcription polymerase chain reaction(RT-PCR) at the time point of 12 h,24 h and 48 h.Results DOX significantly inhibited cardiomyocyte proliferation in a dose dependent fashion.The protein and mRNA expressions of Wnt2 increased in the DOX-induced myocardial injury group compared with the group D,with statistical significance (F =224.115,P ＜ 0.05) ;The expressions of β-catenin,p53 were significantly increased compared with the group D,and the higher expression appeared with the time extending(F =188.145,231.927,all P ＜ 0.05).Significantly positive correlation between Wnt2 and β-catenin expression was observed(r =0.940,P ＜ 0.05).Conclusions These findings suggest that Wnt2/β-catenin signaling pathway may play important roles in the cardiovascular disease and be useful for exploring the molecular mechanism of myocardial injury..

Objective To explore the feasibility and value of dynamic contrast-enhanced (DCE-MRI) parameters in assessing early effects of anti-angiogenesis medicine in targeted therapy for tumors.Methods Twenty BALB/C-nu nude mice were injected subcutaneously with human colon cancer cells HT-29 to the right hind leg.The nude mice were evenly divided into the experimental group and control group with 10 mice in each group.The mice of experimental group were intraperitoneally injected with bevacizumab,and the control group were injected with the same volume of saline.DCE-MRI was performed before medication and one hour,24 h and 48 h after medication.The Ktrans,Kep,Ve and initial area under enhancement curve (iAUC) of DCE-MRI were analyzed.The animals were sacrificed 48 hours after medication.Microvessel density (MVD) of the tumors was detected by immunohistochemistry.One way analysis of variance was performed to analyze parameters of DCE-MRI.The Pearson correlation coefficient was used to analyze the correlation between parameters of DCE-MRI and MVD.Results Under DCE-MRI,the edge of subcutaneous colon cancer xenografts was obviously gradually enhanced,pseudo color indicated high perfusion,the strength degree of the central region was low and which meant low perfusion.The differences in Kep of different time point of experimental group were statistically significant (F=3.752,P=0.016) ; there as no significant difference in other parameters of DCE MRI (all P＞0.05).There was no significant difference in Ktrans and Kep before medication and one hour after medication (all P＞0.05).There were significant difference in Ktrans and Kep 24 hour and 48 hour after medication between experimental group (24 hour∶ (0.095 ± 0.039) min-1 and (0.297 ± 0.141) min-1,48 hour∶ (0.090±0.033) min 1 and (0.314±0.148) min-1) and control group (24 hour∶ (0.150±0.074) nin-1 and (0.494±0.126) min-1,48 hour∶ (0.171±0.045) min-1 and (0.441± 0.092) min-1) (F24h =4.824 and 11.386,F48h =22.605 and 5.455,all P＜0.05).There was no significant difference in Ve and iAUC between two groups at different time points (all P＜0.05).MVD of experimental group was lower than that of control group.Ktrans and Kep were positively correlated with MVD (r=0.745 and 0.400,both P＜0.05).Conclusion Ktrans and Kep parameters of DCE-MRI may be used in monitoring the earlier effects of anti-angiogenesis medicine in targeted therapy for colon cancer.

Zebrafish is a relatively new and booming vertebrate animal model.Over the past three decades, ze-brafish has been applied in various aspects of life science, as well as health sciences, environmental studies and aquacul-ture research.To meet the requirement for different research purposes, large amounts of zebrafish resources, including mu-tant and transgenic lines, have been developed with different techniques.All of these resources need well and careful col-lection and maintenance, therefore several zebrafish resource facilities have been built worldwide.As one of them, the Chi-na Zebrafish Resource Center (CZRC, http://zfish.cn) was founded in 2012.This review is trying to introduce the devel-opment and maintenance of zebrafish scientific resources, and the updated progress of CZRC.