Amyloid beta-Peptides ; toxicity ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Estradiol ; pharmacology ; PC12 Cells ; Peptide Fragments ; toxicity ; Rats

To investigate the inhibitory effects of siRNA expression vector on proto-oncogene Pokemon expression in SW480 cells and to provide experimental basis for further research about the biological function of Pokemon.siRNA expression vectors were constructed to express a short hairpin RNA against Pokemon.The recombinants were transfected into SW480 cells with liposome.Cellular morphology and transfection efficiency were observed.The expression of Pokemon were checked by real-time fluorescence quantitative PCR and Western blot.MTT assay was used to detect the effect of siRNA on the growth of SW480 cells and Fluorescence Activated Cell Sorter was applied to study cell apoptosis of SW480 cells.The transfection efficiency was about 36% and the cellular morphology changed greatly at 48h after transfection.siRNA expression vectors could specifically reduce the expression of Pokemon mRNA and protein in SW480 cells.Compared with negative control,the inhibition ratio of Pokemon mRNA expression was 34.2% and 67.7%,in 24th hour and 48th hour,the inhibition ratio of Pokemon protein was 48.3% and 73.6%,in 48th and 72th hour,respectively.Transfection with Pokemon siRNA expression vectors could inhibit cell growth and induce cell apoptosis of SW480 cells.siRNA expression vectors were successfully established that could effectively inhibit the expression of Pokemon in SW480 cells.Pokemon gene silencing could decrease growth and increase apoptosis of SW480 cells.

Animals ; Drugs, Chinese Herbal ; pharmacology ; PC12 Cells ; Patch-Clamp Techniques ; Potassium Channels, Voltage-Gated ; drug effects ; physiology ; Rats

Objective To investigate the effect of progressive neurological deterioration ( PND) of cerebral watershed infarction on early prognosis. Methods The consecutive patients with cerebral watershed infarction admitted in the Department of Neurology,Jinling Hospital,Nanjing University School of Medicine and their cerebral watershed infarctions confirmed by the imaging examination from March 2009 to March 2014 were enrolled. The clinical features, laboratory indicators and imaging features of internal watershed infarction,cortical-type watershed infarction,and mixed watershed infarction were identified and analyzed. The National Institutes of Health Stroke Scale was used to score neurological deficit. The modified Rankin scale ( mRS) was used to score the prognosis of patients. Single factor analysis was used to compare the differences between the groups. At the same time,the correlation between PND and poor prognosis of cerebral watershed infarction at day 90 was analyzed by multivariable Logistic regression analysis. Results A total of 89 patients with cerebral watershed infarction were enrolled,including 43 cortical-type watershed infarctions,36 internal watershed infarctions, and 10 mixed watershed infarctions. Single factor analysis indicated that the incidences of PND of internal watershed infarction and mixed watershed infarction were significantly higher than the cortical-type watershed infarction (36. 1% [n=13],50. 0% [n=5], and 16. 3% [n=7],respectively;P=0. 018). At day 90,28 patients had poor prognosis,and mRS was (3.4±1. 0) scores at day 90. There was significant difference in the types of infarction between the patients with poor prognosis and patients with good prognosis (P<0. 05). In patients with poor prognosis, most of them were internal watershed infarctions,accounting for 50. 0% (14/28),while in patients with good prognosis,most of them were cortical-type watershed infarctions(57. 4% [35/61]). The incidence of PND in patients with poor prognosis was significantly higher than that in patients with good prognosis (57.1% [16/28] vs. 14. 8% [9/61];P<0. 05). The result of multivariate Logistic regression analysis showed that after adjustment for confounding factor, PND was independently associated with the poor prognosis of cerebral watershed infarction at day 90 (OR 6. 969,95%CI 2. 451-19. 869;P<0. 01). Conclusion Compared with the cortical-type watershed infarction, the patients with internal watershed infarction is more prone to have PND, and PND is independently correlate with the poor prognosis at day 90.

Objective To evaluate the safety to perform 23G vitrectomy with scleral incisions at 5.0 mm behind the limbus.Methods This is a prospective uncontrolled case study.The data of 140 consecutive primary 23G vitrectomy patients (145 eyes) were enrolled in this study.There were 56 males (59 eyes) and 84 females (86 eyes),with the mean age of (56.34 ± 9.98) years and axial length of (23.99± 2.57) mm.There were 139 phakic eye and 6 aphakic eyes.All the eyes received 23G pars plana vitrectomy with scleral incisions at 5.0 mm behind the limbus.To measure the normal Chinese adult parameters of anteroposterior axis of the eyeball,lens thickness and scleral distances from the limbus to the plane passing through the lens posterior apex,the head CT scans of 105 patients without eye diseases in our hospital were studied during the same period of time for these vitrectomy surgeries.Pearson correlation analysis was used to analyze the relationship between age,anteroposterior axis of eyeball,lens thickness and scleral distances from the limbus to the plane passing through the lens posterior apex.Results Surgical related complications included retinal tears close to the scleral incision sites (3/145 eyes,2.1％) and lens injury (3/121 eyes,2.5％).No other surgical complications occurred,such as retinal hemorrhage,supra-choroidal expulsive hemorrhage and iatrogenic retinal detachment.Based on CT images,the average scleral distance from limbus to the plane passing through the lens posterior apex,anteroposterior axis of eyeball and lens thickness was (6.72± 0.81),(24.39± 0.97),(4.22 ± 0.47) mm,respectively.The results of Pearson correlation analysis showed that age and lens thickness had statistically significant correlation (r=0.328 9,P=0.000 6).Conclusion Primary 23G pars plana vitrectomy with incisions at 5.0 mm posterior to the limbus is safe.

In the present study, using fast cyclic voltammetry (FCV), atomic absorption/flame emission spectrophotometry and immunohistochemistry, we investigated the correlation between iron levels and degeneration of dopaminergic neurons in rat nigrostriatal system during the early 6-OHDA lesions in the medial forebrain bundle (MFB). The results showed that 1 d or 3 d after lesions in MFB, there was a 45% or 66% reduction, respectively, in the density of tyrosine hydroxylase (TH) immunoreactive cells in the substantia nigra (SN) of the lesioned side accompanied by an increase in iron staining intensity and iron concentration; while there was no change in dopamine (DA) release in the striatum (Str) of the lesioned side compared with the unlesioned side and the normal rats. There was no difference in the iron staining and concentration of SN and DA release of Str on the lesioned side between one-day group and three-days group. These results suggest that an iron level elevation in SN may be involved at the early stage of degeneration of DA neurons in SN. However, DA release in Str was unchanged due to the immense compensatory mechanism of DA system.
Animals ; Corpus Striatum ; metabolism ; pathology ; Dopamine ; metabolism ; Female ; Iron ; metabolism ; Medial Forebrain Bundle ; physiology ; Nerve Degeneration ; Neurons ; physiology ; Oxidopamine ; pharmacology ; Rats ; Rats, Wistar ; Substantia Nigra ; metabolism ; pathology ; Sympathectomy, Chemical

Objective To clone and express Dengue virus nonstructural protein 4A (NS4A) gene and express in eukaryotic cells.Then,to isolate and purify and isolate cellular proteins interacted with NS4A.Methods With specific primers,NS4A gene fragment tagged with FLAG and HA (FLAG-NS4A-HA) was amplified by PCR and cloned into an expression vector,pSG5 vector.Recombinant plasmid was transfected into A549 cells by LipofectAMINETM2000.Transient expression of FLAG-NS4A-HA was detected by Western blot.The NS4A interacting proteins were isolated and purified by tandem affinity purification (TAP) system using HA and FLAG antibodies,and then assayed by silver stained SDS-PAGE.Results Dengue virus NS4A gene tagged with FLAG and HA was successfully constructed into pSG5 vector and expressed in A.549 cells.Silver stained SDS-PAGE showed that the expressed NS4A and two potential interacting proteins that interact with NS4A were isolated after TAP purification and SDS-PAGE.Conclusion Cellular proteins that potentially interacted with Dengue virus NS4A were successfully purified and isolated,which provided a basis for further research.

To obtain the genome sequence of human bocavirus 2 (HBoV2), different regions of HBoV2 genome were amplified through PCR in fecal specimens which had been identified as single-positive for HBoV2 in 2010. A genome sequence of HBoV2 (HBoV2-NC, 5444 bp) was obtained after sequence assembly. The phylogenetic analysis showed that HBoV2-NC had the closest evolutionary relationship with HBoV2 Lanzhou strain. The predication of inverted terminal repeats of HBoV2-NC by DINAMelt showed that inverted terminal repeats were contained in HBoV2-NC 5' terminal, which had the typical stem-loop structure in other parvoviruses. Finally, some flanking sequences of HBoV2-NC were amplified by linker-PCR.
Base Sequence ; Gene Amplification ; Genome, Viral ; Human bocavirus ; chemistry ; classification ; genetics ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Parvoviridae Infections ; virology ; Phylogeny ; RNA, Viral ; chemistry ; genetics ; Terminal Repeat Sequences

In vivo fast cyclic voltammetry (FCV) was used to investigate dopamine (DA) release from amygdala (Amy) of female rats in different phases of estrus cycle, ovaricectomized (OVX) rats and male rats. Tyrosine hydroxylase (TH) immunohistochemistry was employed to measure the numbers of immunoreactive neurons in ventral tegmental area (VTA) of midbrain in the rats. We also observed the effect of intracerebroventricular injection of phytoestrogen-soy isoflavones on DA release from the Amy. The results are as follows: DA release from the Amy of proestrus female rats was apparently higher than that in estrus, metaestrus, diestrus female rats and OVX rats. Amy DA release and the numbers of the TH immunoreactive neurons in VTA showed a significant sex difference. DA release from Amy of female and OVX rats increased significantly within 5 min after i.c.v injection of soy isoflavones, which elicited no effects in male rats. The above-mentioned results suggest that endogenous estrogen may play an important role in regulating the activity of DA neurons in mid-limbic systems, and that soy isoflavones exert an estrogen-like effect on the dopaminergic systems in the Amy.
Amygdala ; metabolism ; physiology ; Animals ; Dopamine ; metabolism ; Estrogens ; physiology ; Female ; Isoflavones ; pharmacology ; Male ; Neurons ; physiology ; Ovariectomy ; Parkinson Disease ; metabolism ; Phytoestrogens ; Plant Preparations ; pharmacology ; Rats ; Rats, Wistar ; Soybeans ; chemistry ; Ventral Tegmental Area ; metabolism

Background Our previous study demonstrated that the aptamer S58 specifically targeted transforming growth factor-β receptor Ⅱ (TβRⅡ) and inhibited the transdifferentiation of human Tenon capsule fibroblasts (HTFs) mediated by transforming growth factor-β (TGF-β).Chitosan-nanoparticles (CS-NP) are good drug carriers,but the efficacy and safety of CS-NP/aptamer complexes deserve attention.Objective The aim of this study was to synthesize a novel CS-NP/aptamer complex called CS (S58)-NP and investigate its properties and applicability.Methods Human Tenon capsule tissue was obtained from patients during strabismus surgery,and HTFs were cultured and passaged using the explant culture method.The fourth to tenth generations of cells were used in the experiment.Different concentrations of CS-NP were used to prepare the CS(S58)-NP by the ionic cross-linking method with a surface charge rate (N/P) for S58 of 10,20,30 or 40.The particle size and Zeta potential were measured by the Zeta analyzer.The shape and distribution of CS (S58)-NP particles were examined under the scanning electron microscope.The binding of CS-NP with S58 and resistance of CS (S58)-NP to DNase Ⅰ were examined by agarose gel eletrophoresis.The release rate of S58 from CS (S58)-NP in PBS was quantitatively analyzed by a ultraviolet spectrophotometer.The cytotoxicity of CS(S58)-NP to HTFs was evaluated by detecting the production of lactate dehydrogenase (LDH).Results The Zeta analyzer showed that the particle size of CS (S58)-NP was 130-270 nm and its electric potential ranged from + 16 to +28 mV.The CS (S58)-NP particles appeared spherical with an even distribution under the scanning electron microscope.The mean encapsulation efficiency of CS(S58)-NP was 88.9％,89.3％,91.7％ or 90.5％,respectively,when the N/P was 10,20,30 or 40.After being encapsuled by CS-NP,S58 could resist the degradation from DNase I.Its total releasing level in PBS increased with the lapse of time,with a maximum releasing speed at 24 to 36 hours.The total releasing level reached 100％ at 96 hours.With increaseing concentrations of CS(S58)-NP,the relative releasing level of LDH in HTFs suspension gradually elevated with a significant difference among the groups (F =588.018,P =0.000),with the highest released LDH level at 50 nmol/L of CS(S58)-NP (12.853％ ±0.375％).Conclusions CS-NP provides a protective and slow-releasing effect on the S58 aptamer.CS (S58)-NP shows a good biocompatibility with HTFs with a low cytotoxicity at a concentration of ＜50 nmol/L.CS(S58)-NP could be used to inhibit TGF-β induced transdifferentiation of HTFs in the future.