Objective To construct lentiviral vector carrying rat′s calcitonin gene-related peptide(CGRP) gene for the following-up study on the function of CGRP .Methods CGRP gene segment was subcloned into shuttle plasmid ,become Puc57-CGRP .The pLenO-DCE-CGRP expression vector was be constructed by double digests .The pLenO-DCE-CGRP and 4 auxiliary packaging plas-mids were co-transfected into 293T cells .Cells were cultured for 48 hours .The supernant was collected and concentrated ,and then the viral titers were tested by multiple proportions dilution method and flow cytometer .The expression levels of CGRP were detec-ted in CGRP-modified 293T cells by Real-time PCR .Results The results of digestion and sequencing show that the pLenO-DCE-CGRP vector was constructed successfully .The titer of the lentiviral particles was 5 .1 × 108 TU /mL .Conclusion The high-titer lentvirus vector containing CGRP gene is constructed successfully ,which lay a foundation for transfecting mesenchymal stem cell (MSC) and studying the function of CGRP .

Objective To optimize the method of transcription activator‐like effector transcription factors (TALE‐TFs) con‐struction ,some improvement and adaption were made based on the traditional methods .Methods We first constructed the basic tandem fragments with different length ,including trimer ,tetramer ,pentamer and hexamer by Golden Gate cloning technique and PCR ,then the procedure with the highest efficacy was chosen to construct our TALE‐TFs .To determine the function of the TALE‐TFs ,the plasmid pminCMV with the specific binding sequence of TALE‐TFs was constructed by fragment substitution reaction (FSR) .The transcription activating function of TALE‐TFs was confirmed by the intensity of red fluorescence ,after TALE‐TFs , pEGFP‐N1 and pminCMV plasmid were co‐transfected into 293HEK cells .Results An optimized method for TALE‐TFs construc‐tion and functional assay was established .Conclusion This method can potentially be wildly used in fields that the expression of some constitutively expressed genes needs to be modified .

Objective To observe the effect of adenovirus- receptor activity modifying protein-1 (RAMP1) on nuclear factor(NF κB) and myocardial fibrosis in heart of rabbit with myocardial infarction. Methods Myocardial infarction (MI) models were developed in 54 rabbits and they were randomly divided into RAMP1 group,EGFP group and control group according to whether pAd2-EGFP-RAMP1,pAd2-EGFP or PBS was injected into infarction region and its border.At 7 d,14 d and 28 d after injection,Left ventricular function indices such as LVEF,LVSd,LVDd and LVFS were estimated by echocardiogram,the expression of NF-κB in myocardium was detected by Western blot,the plasma level of TNF-α was measured by ELISA,and fibrosis and collagen content was measured by Masson stain. Results At 7 d after adenovirus injection,the expression of RAMP1was significantly increased in RAMP1 group (67.33 ± 3.97)％ as compared to EGFP group(20.59 ±3.26) ％ and PBS group ( 23.80 ± 5.08) ％ ( P ＜ 0.05 ).The expression of NF κB was decreased in RAMP1 group ( 26.54 ± 5.13 ) ％ versus EGFP group (62.60 ± 6.18) ％ and PBS group (62.95 ±5.17)％ (P＜0.05).The plasma level of TNF-α was lower in RAMP1 group than in EGFP group and in PBS group at different time[7 d:( 136.74 ± 5.42) μg/L vs.( 196.97 ± 14.17) μg/L,(203.67 ±13.90)μg/L; 14 d:( 154.51 ± 13.61 )]μg/L vs.( 112.22±6.74 )μg/L,(160.46± 14.27)μg/L ;28 d;(51.10± 5.62)μg/L vs.( 95.55 ± 9.94 )μg/L,( 98.96 ± 12.68) μg/L,all P＜ 0.05].The collagen content was reduced in RAMP1 group as compared with EGFP group and PBS group at 14 d and 28 d [14 d:(7.10±0.98)％ vs.(19.52±2.32)％,(17.91±0.96)％ ;28 d:(17.04±2.44)vs,(34.10±5.59) ％,(33.98±4.33)％,all P＜0.01].At 28 d after infarction,the infarct size was decreased in RAMP1 group (26.54 ± 5.13) ％ compared with EGFP group (32.20 ± 3.73) ％ and control group (35.58±2.65) ％ (P＜0.01),and better heart function appeared in RAMP1 group. Conclusions The high-expression of RAMP1 could decrease the collagen deposition and fibrosis in the border of infarction and improve heart function through lower expression of NF-κB and decreasing the plasma level of TNF-α.

Objective To study the effect of the recombinant Lentivirus containing calcitonin gene related peptide (CGRP) gene on cells biological activity and differentiation of rat bone marrow stem cells(MSCs).Methods Rat MSCs were isolated and cultured by granulocytes adherent.MSCs were transfected with Lenti-EGFP CGRP(MSCsCGRP+/+ group),While MSCs were transfected with Lenti-EGFP as control group.Cell transfection rate was detected by flow cytometry,protein secretion in the above-mentioned MSCsCGRP+ + supernatant was detected using ELISA method.Cells surface markers weare detected by flow cytometry and immunohistochemistry.Trypan blue was used to examin the survive rate,β galactosidase staining was used to examin aging of MSCs transfection,and MTT was used to examine cell vitality.Results At first day after transfecting with Lenti-EGFP-CGRP,fluorescence was not observed by fluorescence microscope,but a small amount of CGRP protein was detected by ELISA in MSCsCGRP+/+ group,at 3 days and 4 days after transfecting with MSCs,strong fluorescence was observed by fluorescence microscope (the cell transfection rates were 77.87％ and 79.58％).The CGRP expression was significantly higher in MSCsCGRP+ + group than in control group [(19.53±0.50) pg/ml vs.(3.12±0.00) pg/ml,t=48.964,P＜0.01].At three days after transfection with MSCs,CD29 and CD90 expression were significantly higher,as compared with control group,CD31 expression was increased in MSCsCGRP+ /+ group.Seven days after transfection with MSCs,CD31 expression was significantly increased in MSCsCGRP+ + group,vWF expression was significantly increased in MSCsCGRP+ + group after MSCs were transfected with LentiEGFP CGRP for 14 days,but a SMA expression was decreased in MSCsCGRP+ +group.At 3 days and 7 days after transfection with Lenti-EGFP-CGRP,the proliferation,survive and aging showed no difference in MSCsCGRP+/+group and in control group (the proliferation of cell:t=0.253,0.290the survive of cell t=-0.307,0.690,all P＞0.05).At 14 days after transfection with Lenti-EGFP-CGRP,aging of cell were decreased in MSCsCGRP+ + group as compared with control group (t=2.446,P＜ 0.05).Conclusions After MSCs are transfected with Lenti EGFP-CGRP,biological characteristics of MSCs has no significant effects,there is still proliferation and differentiation activity.Cell secretion of CGRP can promote the endothelial cell differentiation,and inhibit the differentiation to smooth muscle cells.The CGRP modification of MSCs may play a role in the regulation of angiogenesis.

The olfactory bulb(OB)is the first relay station in the olfactory system and functions as a crucial hub.It can represent odor information precisely and accurately in an ever-changing environment.As the only output neurons in the OB,mitral/tufted cells encode information such as odor identity and concentration.Recently,the neural strategies and mechanisms underlying odor representation and encoding in the OB have been investigated extensively.Here we review the main progress on this topic.We first review the neurons and circuits involved in odor representation,including the different cell types in the OB and the neural circuits within and beyond the OB.We will then discuss how two different coding strategies—spatial coding and temporal coding—work in the rodent OB.Finally,we discuss potential future directions for this research topic.Overall,this review provides a comprehensive description of our current understanding of how odor information is represented and encoded by mitral/tufted cells in the OB.

ObjectiveTo study the effects of matrine derivative C4 on migration， invasion and apoptosis of non-small cell lung cancer cells by targeting heat shock protein 90. MethodMolecular docking and Western blot were used to detect the regulation of matrine derivative C4 （0， 1， 2， 4， 8 μmol·L^-1） on heat shock protein 90 （Hsp90）. methyl thiazolyl-tetrazolium （MTT） assay was conducted to observe the effects of C4 （0， 0.25， 0.5， 1， 2， 4， 8， 16 μmol·L^-1） on viability of A549 cells. The impacts of different concentrations of C4 （0， 2， 4， 8 μmol·L^-1） on the proliferation， migration， invasion and apoptosis of A549 cells were explored by clone formation assay， wound healing assay， Transwell assay， and flow cytometry， respectively. Western blot was employed to detect the effects of C4 （0， 1， 2， 4， 8 μmol·L^-1） on the protein expression of A549 protein kinase B （Akt）， phosphorylated protein kinase B （p-Akt）， epidermal growth factor receptor （EGFR）， phosphatidylinositol 3 kinase （PI3K）， p-PI3K， cysteine aspartate protease 9 （Caspase-9）， B-cell lymphoma 2 （Bcl-2）-associated X （Bax）， Bcl-2， and Bcl-2-associated agonist of cell death （Bad）. ResultMatrine derivative C4 could effectively interact with Hsp90 protein through a water-mediated hydrogen bond network with residues of asparagine-51 （ASN-51）， glycine-135 （GLY-135）， and phenylalanine-138 （PHE-138）， and the π-π stacking interaction with the phenyl ring of PHE-138 contributed to its affinity. In addition， compared with blank group， C4 down-regulated the protein expression of Hsp90 in A549 cells （P<0.05， P<0.01）， and reduced the cell viability （P<0.05， P<0.01）. The half-maximal inhibitory concentrations （IC₅₀） of C4 on A549 cells were （12.32±0.14）， （7.79±0.16） and （3.26±0.09） μmol·L^-1 at 24， 48 and 72 h， respectively. Compared with the conditions in the blank group， the clone formation ability of A549 cells in C4 （2， 4， 8 μmol·L^-1） groups was decreased （P<0.05， P<0.01） in a concentration-dependent manner， and C4 inhibited the migration and invasion of A549 cells （P<0.05， P<0.01）， with the most significant inhibition observed at 8 μmol·L^-1 （P<0.01）. Moreover， C4 induced apoptosis of A549 cells （P<0.01）， and the apoptosis rates at 0， 2， 4 and 8 μmol·L^-1 were （2.28±0.35）%， （4.97±0.36）%， （8.86±0.50）% and （20.67±0.99）%， respectively. It also up-regulated the protein expression of Caspase-9， Bax and Bad （P<0.05， P<0.01）， while down-regulated those of PI3K， p-PI3K， p-Akt， EGFR and Bcl-2 to varying degrees （P<0.05， P<0.01）， and the protein expression level of Akt did not change significantly. ConclusionMatrine derivative C4 played an anti-tumor role by inhibiting cell viability， proliferation， migration and invasion and promoting apoptosis response， which might be realized by inhibiting Hsp90/PI3K/Akt signaling pathway.

Objective: To investigate whether microRNA(miR)-214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase-1. Methods: H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37 ℃ with 5%CO₂, repeat passage was made after cell density reached about 80%, The 5^th to 8^th generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR-214 on pyroptosis and caspase-1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimic-negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR-214 mimic-negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti-pyroptosis effect of miR-214 was mediated by targeted inhibition on caspase-1, cells overexpressing caspase-1 were used in the rescue experiment. The cells overexpressing caspase-1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimics+hyperglycosis+recombinant adenovirus (Ad-caspase-1-EGFP) group with caspase-1 gene and EGFP green fluorescent protein expression (mimics+HG+Ad-caspase-1-EGFP group, H9c2 cells were transfected with caspase-1-green fluorescent protein-carrying adenovirus for 48 hours, followed by transfection of miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR-214 mimics+HG+Ad-EGFP empty virus group (mimics+HG+Ad-EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA-214 and caspase-1 in cells were detected by real-time quantitative PCR. The expression and localization of caspase-1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase-1, cleaved caspase-1, NLRP3 and ACS with β-actin as internal reference. The secretion of IL-1β and IL-18 in cell culture medium was detected by ELISA. The correlation between miR-214 and caspase-1 was detected by double luciferase reporter gene. Results: (1) The mRNA expression levels of miR-214 and caspase-1 in each group: the mRNA expressions of miR-214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR-214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase-1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase-1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase-1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL-1β and IL-18 in the cell culture medium of each group: the content of IL-1β and IL-18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL-1β and IL-18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR-214 and caspase-1: miR-214 specifically binds to caspase-1 3 ′UTR. Meanwhile, Western blot results showed that cleaved caspase-1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase-1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase-1 expression among groups (P>0.05). (6) The expression levels of procaspase-1, cleaved caspase-1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase-1 in each group (P>0.05). Cleaved caspase-1, ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase-1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+ Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL-1β and IL-18 in rescue experiment: the secretions of IL-1β and IL-18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). Conclusion miR-214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase-1.

OBJECTIVETo explore the influence of BCL6, MYC, P53 genes abnormalities can on the prognosis of diffuse large B-cell lymphoma (DLBCL), and to identify independent prognostic factors for DLBCL in order to facilitate clinical prognosis and selection of stratification treatment for the patients.

METHODSSixty five newly diagnosed DLBCL pathological specimens were collected from 2009 to 2012. Interphase fluorescence in situ hybridization technique (I-FISH) was used to detect the status of BCL6, MYC and P53 genes. Clinical factors were combined with immunohistochemical results for multiple-factor survival analysis.

RESULTSThe rates of BCL6 gene rearrangement, P53 gene deletion and MYC rearrangement were 21.5% (14/65), 35.4% (23/65) and 7.7% (5/65), respectively. BCL6 rearrangement group has obviously poorer overall survival (OS)(P< 0.05). COX proportional hazards model analysis showed that gender, BCL6 protein, BCL6 rearrangement, Ki67 index were prognosis factors independent of international prognostic index (IPI).

CONCLUSIONBCL6 can influence the prognosis of patients with DLBCL at gene and protein levels and both are independent prognostic factors for DLBCL.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; DNA-Binding Proteins ; genetics ; Female ; Gene Deletion ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Lymphoma, Large B-Cell, Diffuse ; diagnosis ; genetics ; mortality ; Male ; Middle Aged ; Mutation ; Prognosis ; Proto-Oncogene Proteins c-bcl-6 ; Proto-Oncogene Proteins c-myc ; genetics ; Survival Analysis ; Survival Rate ; Tumor Suppressor Protein p53 ; genetics ; Young Adult

Objective To investigate whether microRNA(miR)?214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase?1. Methods H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37℃with 5%CO2, repeat passage was made after cell density reached about 80%, The 5th to 8th generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR?214 on pyroptosis and caspase?1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR?214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR?214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR?214 mimic?negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR?214 mimic?negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti?pyroptosis effect of miR?214 was mediated by targeted inhibition on caspase?1, cells overexpressing caspase?1 were used in the rescue experiment. The cells overexpressing caspase?1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR?214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR?214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR?214 mimics+hyperglycosis+recombinant adenovirus (Ad?caspase?1?EGFP) group with caspase?1 gene and EGFP green fluorescent protein expression (mimics+HG + Ad?caspase?1?EGFP group, H9c2 cells were transfected with caspase?1?green fluorescent protein?carrying adenovirus for 48 hours, followed by transfection of miR?214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR?214 mimics+HG+Ad?EGFP empty virus group (mimics+HG+Ad?EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR?214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA?214 and caspase?1 in cells were detected by real?time quantitative PCR. The expression and localization of caspase?1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase?1, cleaved caspase?1, NLRP3 and ACS with β?actin as internal reference. The secretion of IL?1β and IL?18 in cell culture medium was detected by ELISA. The correlation between miR?214 and caspase?1 was detected by double luciferase reporter gene. Results (1) The mRNA expression levels of miR?214 and caspase?1 in each group: the mRNA expressions of miR?214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR?214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase?1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase?1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase?1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL?1β and IL?18 in the cell culture medium of each group: the content of IL?1β and IL?18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL?1β and IL?18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR?214 and caspase?1: miR?214 specifically binds to caspase?1 3 ＇UTR. Meanwhile, Western blot results showed that cleaved caspase?1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase?1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase?1 expression among groups (P>0.05). (6) The expression levels of procaspase?1, cleaved caspase?1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase?1 in each group (P>0.05). Cleaved caspase?1 , ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase?1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+Ad?caspase?1?EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL?1β and IL?18 in rescue experiment: the secretions of IL?1β and IL?18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad?caspase?1?EGFP group than in mimics+HG group (P<0.05). Conclusion miR?214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase?1.