Objective:To investigate the effects of Anhydroicaritin (AHI) , an isopentenylated flavo-noid compound, on proliferation, migration and apoptosis of human hepatocarcinoma cell line MHCC-97H.Methods:Human hepatocarcinoma cell line MHCC-97H and human normal liver cell line L02 were cultured in vitro. MHCC-97H cells were treated with 0, 20, 40, 80, 120, 160, 200 μg/ml of AHI respectively and L02 cells were treated with 0, 25, 50, 100, 150, 200, 400, 500 μg/ml of AHI respectively. CCK-8 and clone formation assay were used to detect cell proliferation. Scratch test was used to explore cell migration ability. Hoechst33342 assay and flow cytometer were used to detect cell apoptosis. The expressions of apoptosis-related proteins were detected by Western blotting. Results:The cell viabilities of MHCC-97H cells treated with 0, 20, 40, 80, 120, 160, 200 μg/ml of AHI for 24 h were (100.00±0.00) %, (97.41±2.10) %, (96.58±3.23) %, (87.72±4.85) %, (78.33±3.76) %, (56.97±2.61) % and (15.25±2.51) % respectively, and there was a statistically significant difference ( F=429.20, P<0.001) . There were statistically significant differences between 0 μg/ml and 80, 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . The cell viabilities of L02 cells treated with 0, 25, 50, 100, 150, 200, 400, 500 μg/ml of AHI for 24 h were (100.00±0.00) %, (96.82±3.79) %, (95.36±3.43) %, (90.79±5.75) %, (77.67±5.66) %, (63.98±5.22) %, (34.22±4.01) % and (33.84±4.41) % respectively, and there was a statistically significant difference ( F=233.20, P<0.001) . There were statistically significant differences between 0 μg/ml and 100, 150, 200, 400, 500 μg/ml of AHI treatment (all P<0.05) . The 24 h half maximal inhibitory concentration (IC 50) value of AHI treated L02 cells was (300.20±17.10) μg/ml, which was significantly higher than that of MHCC-97H cells [ (158.60±5.50) μg/ml], and there was a statistically significant difference ( t=13.65, P<0.001) . The cell clone numbers of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml of AHI for 24 h were 1 993.00±46.29, 1 355.00±54.84, 998.33±21.03 and 218.33±35.95 respectively, and there was a statistically significant difference ( F=954.80, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . The healing rates of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml of AHI for 24 h were (51.68±1.93) %, (16.04±0.73) %, (8.88±0.31) % and (-6.94±0.46) % respectively, and there was a statistically significant difference ( F=1 616.00, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . Hoechst33342 experiment showed that MHCC-97H cells treated with 0 μg/ml AHI showed uniform dark blue with a complete nuclear state under inverted microscope. Compared with 0 μg/ml AHI treated cells, cells in the 120, 160, 200 μg/ml AHI treatment groups wrinkled and broken, and nuclei were also morphologically abnormal, with some nuclei stained bright blue, and the situation became more obvious with increasing dose. The apoptosis rates of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml AHI for 24 h were (10.51±0.56) %, (42.23±0.87) %, (61.92±0.52) % and (72.05±0.74) % respectively, and there was a statistically significant difference ( F=4 677.00, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . There were statistically significant differences among the different expression levels of Bax, Cleaved Caspase-3/Caspase-3, Cleaved Caspase-9/Caspase-9, and Bcl-2 proteins in MHCC-97H cells of 0, 120, 160, and 200 μg/ml of AHI treatment ( F=30.43, P<0.001; F=212.80, P<0.001; F=475.30, P<0.001; F=10.75, P=0.004) . The Bax protein expression of 160 and 200 μg/ml was significantly increased than that of 0 μg/ml AHI (both P<0.001) . The Cleaved Caspase-3/Caspase-3, Cleaved Caspase-9/Caspase-9 protein expressions of 120, 160 and 200 μg/ml were significantly increased than those of 0 μg/ml AHI (all P<0.001) . The Bcl-2 protein expression of 120, 160, 200 μg/ml was significantly decreased compared with that of 0 μg/ml AHI (all P<0.05) . Conclusion:AHI can inhibit the proliferation and migration of hepatocellular carcinoma cell line MHCC-97H, and promote its apoptosis.

The brain pericyte is a unique and indispensable part of the blood-brain barrier (BBB), and contributes to several pathological processes in traumatic brain injury (TBI). However, the cellular and molecular mechanisms by which pericytes are regulated in the damaged brain are largely unknown. Here, we show that the formation of neutrophil extracellular traps (NETs) induces the appearance of CD11b⁺ pericytes after TBI. These CD11b⁺ pericyte subsets are characterized by increased permeability and pro-inflammatory profiles compared to CD11b^- pericytes. Moreover, histones from NETs by Dectin-1 facilitate CD11b induction in brain pericytes in PKC-c-Jun dependent manner, resulting in neuroinflammation and BBB dysfunction after TBI. These data indicate that neutrophil-NET-pericyte and histone-Dectin-1-CD11b are possible mechanisms for the activation and dysfunction of pericytes. Targeting NETs formation and Dectin-1 are promising means of treating TBI.
Blood-Brain Barrier/metabolism* ; Brain/pathology* ; Brain Injuries, Traumatic/metabolism* ; Extracellular Traps/metabolism* ; Histones ; Humans ; Lectins, C-Type ; Pericytes/pathology*

Objective To investigate the expression of Aire,Foxp3,AchR and other immune factors in human thymoma tissue and plasma and explore their role in myasthenia gravis with thymoma.Methods T lymphocyte subsets,immunoglobulin and other immune factors in plasma were compared,and the Expression of Aire,Foxp3 and AchR were examined in thymoma by reverse transcriptional polymerase chain reaction and immunohistochemical staining,and the results were analyzed by SPSS statistics software.Results The ratio of CD4 + to CD8 + T lymphocyte was much higher in plasma,while the expressions of Aire,Foxp3 and AchR at mRNA and protein level were much lower in thymoma patients with myasthenia gravis,and related to Ossermann subtype,WHO subgroup and Masaoka stage.The differences were statistically significant (P ＜ 0.05).Conclusion The ratio of CD4 + to CD8 + T lymphocyte and the abnormal expressions of Aire and Foxp3could used as an indicator of immune state in thymoma patient with myasthenia gravis and play an important role in the development of thymoma with myasthenia gravis,but the mechanism is indefinite.

Background and purpose: AMP-activated protein kinase (AMPK) plays an important role in the regulation of cell metabolism and energy balance and is associated with cell proliferation, survival and multiple signaling pathways. Recent reports found that AMPK is involved in tumor suppression and drug resistance. The aim of this study was to explore the effect of AMPK on the anti-tumor effect of adriamycin and underlying mechanism in breast cancer MCF-7/adr cells. Methods:The anti-proliferative effects of adriamycin was detected by methyl thiazolyl tetrazolium (MTT) assay in MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKαcells. The cell morphology in each group was stained with the lfuorescent dye Hoechst 33528, and the effects on apoptosis induction were examined by lfow cytometry (FCM). The intracellular concentration of adriamycin was detected by lfuorescence assay. The resis-tance-and apoptosis-related proteins were analyzed by Western blot. Results:The growth of breast cancer MCF-7/adr cells was inhibited by adriamycin in a dose-and time-dependent manner. The IC50 values at 24 and 48 h were (36.8±2.1) and (28.8±1.3) μg/mL, respectively. AMPKαover-expression enhanced the cytotoxic effect of adriamycin in MCF-7/adr-AMPKαcells in a dose-and time-dependent manner. Its IC50 values at 24 and 48 h were (16.0±0.7) and (4.2±0.2) μg/mL, respectively. Fluorescent morphological assay showed that AMPKαoverexpression contributed to adriamycin induced apoptosis in MCF-7/adr-AMPKαcells. After treatment with 1.0 μg/mL adriamycin for 48 h, the apoptosis rates of MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKα cells were (12.0±1.4)%, (12.7±1.6)% and (32.0±4.2)%, respectively, indicating that overexpression of AMPKα enhanced the adriamycin-induced apoptosis in MCF-7/adr cells. Fluorescence microplate assay showed that over expression of AMPKαsigniifcantly increased the intracellular accumulation of adriamycin, in a concentration dependent manner. Western blot analysis showed that, compared with MCF-7/adr and MCF-7/adr-vector cells, the expressions of Bax, caspase-3 and cleaved PARP proteins were increased. Meanwhile, Bcl-2 and P-gp protein expressions were decreased in MCF-7/adr-AMPKαcells. Furthermore, the release of cytochrome c from mitochondria into the cytosol was also observed in MCF-7/adr-AMPKαcells. Conclusion:AMP-Kαoverexpression can enhance the chemosensitivity of breast cancer MCF-7/adr cells to adriamycin through inhibiting the drug effux transporter and regulating the expression of apoptosis-related proteins.

Background Retinal ganglion cell (RGCs) death following ischaemic insult is the major cause of a number of vision-threatening diseases.Recent studies confirmed that micro RNA (miR-30b) can alleviate hypoxy-induced cardiac injury.However,whether miR-30b can protect RGCs against oxygen-glucose deprivation damage is still not ellucidated.Objective The aim of this study was to investigate the protective effect of miR-30b on RGCs damage caused by oxygen-glucose deprivation.Methods The retinas were isolated from the eyeballs of eight SD rats aged postnatal 24 hours and RGCs were primarily cultured.The cells were divided into the recombinant adeno-associated virus (rAVV) control group,rAAV-miR-30b mimic group and AAV-miR-30b inhibitor group.Then the cells were transfected using rAVV-miR plasmid,rAAV-miR-30b mimic plasmid and AAV-miR-30b inhibitor plasmid,respectively for 6 days with the RGCs ∶ AAV as 1 ∶ 10 000.The cells were cultured with low glucose medium in hypoxygen incubator (5％ CO2,17％ N2,3％ O2) or 5％ CO2 incubator respectively for 24 hours.Cell viability was detected by cell counting kit-8 assay.The expression of Tubulin Ⅲ,a neuron specific marker,was detected by immunofluorescence technology to evaluate the survival of RGCs.The apoptosis and necrosis of the cells were assessed by Hoechst/PI double staining.Results The RGCs grew well with round shape and 1 3 processes 7 days after cultured in the normal cells.However,the RGCs were diminished and the cell process disrupted in the oxygen-glucose deprivation group.The relative vability of the cells was 3.310-±0.162 in the rAAV-miR-30b mimic group,which was significantly higher than 0.949±0.141 in the rAAV-miR-30b inhibitor group and 0.900±0.181 in the rAAV-miR control group(t=10.508,10.296,both at P＜0.001).It was positively expressed in survival RGCs,with the red fluorescence.The number of Tubulin Ⅲ+ cells was (13.800± 1.924)/field in the rAAV-miR-30b mimic group,showing a significant increase in comparison with (0.600±0.548)/field in the rAAV-miR-30b inhibitor group and (0.800± 1.304)/field in the rAAV-miR control group (t =15.141,14.912,both at P ＜ 0.001).Significant differences were found in the apoptosis rate and necrosis rate among the rAAV-miR-30b mimic group,rAAV-miR control group and PBS group (F=10.851,P=0.002;F=6.378,P=0.013),and the apoptosis rate and necrosis rate in the rAAV-miR-30b mimic group were considerably lower than those in the rAAV-miR control group and PBS group (all at P＜0.05).Conclusions The oxygen-glucose deprivation models can be established in RGCs by hypooxygic and low-glucose cultivation.rAAV encoding miR-30b mimics transfection can protect RGCs against oxygen-glucose deprivation damage.

Objective To establish and identify the induced pluripotent stem cell(iPSC) line reprogrammed from human umbilical cord mesenchymal cells(HuMSCs).Methods HuMSCs were cultured by adhesion method,and OCT4,SOX2,KLF4,c-Myc,NANOG,LIN-28 were transfected into HuMSCs with lentiviral victor to reprogramme HuMSCs into iPSC.Morphological observation,pluripotency genes (SOX2,TDGF1,THY-1,OCT4,REX1 and TERF1) expression,alkaline phosphatase detection,karyotype analysis,embryonic stem cells (ESC) specific proteins (NANOG,OCT4,SSEA-4,TRA-1-81) immunofluorescence staining,differentiated into teratomas in vivo(inject the iPSC into SCID mice) and embryniod bodies in vitro were performed to exam the pluripotency of the iPSC.Results Four days after being infected by lentivirus,the HuMSCs became round-shape; 10 days after infection,some embryonic stem(ESC)-like colonies appeared.Fourteen days after infection,picked up the regularly shaped colonies and cultured several passages.About 1.25％ HuMSCs were reprogrammed into iPSC.The iPSC presented clone-like growth like ESC.All the cells were positive to alkaline phosphatase staining and expressed the pluripotency genes.The iPSC also expressed the ESC specific proteins,and karyotype analysis showed normal chromosome caryotype (46,XY).Furthermore,the iPSC could form embryoid bodies in vitro,expressed alpha fetoprotein(AFP),smooth muscle actin(SMA) and β-tubulin.The iPSC could alsoform teratomas in vivo.Conclusion OCT4,SOX2,KLF4,c-Myc,NANOG,LIN-28 can reprogram HuMSCs into iPSC efficiently.

Objective To investigate the effect of the multimodal analgesia on postoperative pain after renal transplantation and the cytokines .Methods 40 cases of allogaft renal transplantation due to chronic renal failure were randomly divided into two groups (n=20) .The group D received the multimodal analgesia :preemptive analgesia plus patient controlled epidural analgesia(PCEA) and the group C(control) received analgesic drugs by intermittent intramuscular injection .The visual analogue scale(VAS) scores , the Ramsay sedation scores ,HR ,MAP and SPO2 at postoperative 2 ,6 ,12 ,24 ,48 h were recorded .Blood interleukin-2(IL-2) ,in-terleukin-6(IL-6) and interleukin-10(IL-10) levels were measured before anesthesia ,at the end of operation and postoperative 6 , 24 ,48 h .Results Postoperative MAP and SPO2 had no obvious change in the two groups ,no statistical differences in the various time points existed between the two groups (P>0 .05) .HR was significantly increased at 6 ,24 h after operation in the group C , which had statistical difference compared with that at the same time points in the group D (P<0 .05) .The VAS scores at postoper-ative 6 ,12 ,24 h in the group D were significantly lower than those in the group C ,the difference showed statistical significance (P<0 .05) .The sedation scores at various time points had no statistical difference between the two groups (P>0 .05) .The levels of IL-2 and IL-10 at postoperative 6 ,24 ,48 h in the two groups were significantly higher than those before anesthesia and at the end of operation (P<0 .05) .The levels of IL-2 and IL-6 at postoperative 6 ,24 ,48 h in the group D were significantly lower than those in the group C(P<0 .05) .Conclusion Multimodal analgesia can reach the effective analgesic effect ,down-regulate the pro-inflam-matory cytokines and up-regulate anti-inflammatory cytokines for maintaining postaperative serum cytokines balance .

BACKGROUND:Multimodal analgesia provides sufficient analgesia in renal recipients and appears to be associated with the recovery of renal function after transplantation. OBJECTIVE:To investigate the effect of multimodal analgesia with dezocine on postoperative immunity after renal transplantation, and discuss the appropriate analgesic drugs and methods for patients with renal transplantation. METHODS:Forty patients undergoing renal transplantation were randomly divided into two groups. They al received general anesthesia combined with epidural blockage. Control group received intramuscular injection of analgesic drugs when needed, while dezocine group received multimodal analgesia:preemptive anaIgesia with dezocine+patient-control ed epidural analgesia. The heart rate, mean arterial pressure, and saturation of blood oxygen were detected before anesthesia, 12, 24, 48 hours after transplantation. T lymphocyte subsets, interleukin-2, interleukin-6 and interleukin-10 levels in venous blood were measured before anesthesia, 12, 24, 48 hours after transplantation. RESULTS AND CONCLUSION:Compared with before anesthesia, the CD4+, CD8+cellsubset counts, CD4+/CD8+ratio, the levels of interleukin-2 and interleukin-6 were decreased significantly (P<0.05), and the levels of interleukin were significantly increased after transplantation in the control group (P<0.05). The postoperative CD4+cellsubset counts, the levels of interleukin-2 and interleukin-6 were significantly lower at 12 hours after transplantation than that before anesthesia (P<0.05), then recovered to normal levels at 24 hours in dezocine group. The postoperative CD8+cellsubset counts, CD8+and CD4+/CD8+ratio were not changed before and after transplantation in the dezocine group. The levels of interleukin-10 in the dezocine group were significantly increased at 48 hours after transplantation compared with before anesthesia (P<0.05), which was stil lower than that in control group (P<0.05). Multimodal analgesia with dezocine can effectively protect the immune system, promote short-term turnover of renal function, and prolong graft survival for patients with renal transplantation.

Objective To investigate the effect of adenosine A2A receptor on pituitary-adrenal axis response in acute phase of moderate craniocerebral trauma.Methods Eighteen adenosine A2A receptor knock-out mice in a C57BL/6 background and another eighteen their wild-type littermates were divided into normal control group and craniocerebral trauma for 4 hours group,and craniocerebral trauma for 24 hours group according to random number table,with siμ mice per group.Plasma levels of adrenocorticotropic-hormone (ACTH) and corticosterone at hours 4 and 24 postinjury were determined using ELISA method.Results At 4 and 24 hours,brain water content in wild-type mice [(80.950 ± 0.184) ％,(82.178 ± 0.255)％ respectively] was higher than that in gene knock-out mice [(80.006 ± 0.199)％,(81.091 ± 0.295)％ respectively,P ＜ 0.01].Besides,brain water content in both wild-type and gene knock-out mice increased after injury (P ＜ 0.01).Plasma levels of ACTH and corticosterone were higher in geneknock-out sham mice than in wild-type sham mice [(120.214 ± 2.472) ng/L vs (91.767 ±7.395) ng/L,(27.814 ±0.888) μg/L vs (11.430 ±0.644) μg/L respectively,P ＜0.0l].At 4 and 24 hours,plasma levels of ACTH [(174.776-± 5.040) ng/L,(189.613 ± 4.802) ng/L respectively] in geneknock-out mice showed a higher increase than those in wild-type mice [(119.594 ± 6.945) ng/L,(124.93-± 11.001 7) ng/L respectively,P ＜ 0.05].Moreover,plasma levels of corticosterone [(40.138 ±-0.805) μg/L] at 4 hours and [(37.440-0.485)μg/L] at 24 hours in gene knock-out mice showed a same result as compared with that in wild-type mice [(19.702 ± 0.804) μg/L,(17.602 ± 0.743) μg/L respectively,P ＜ 0.05].Conclusions Knock-out of adenosine A2A receptor increases the release of ACTH and corticosterone in acute stage of moderate craniocerebral trauma and promotes pituitary-adrenal stress response.This may provide a novel explanation for the neuroprotective effect of A2A receptor deficiency.

Objective To obtain the expression pattern of imprint gene H19 in JEG-3 cell in order to explore the regulation mechanism of H19 on trophoblast cellular biological behavior .Methods After correct identification with sequencing for the recom-binant eukaryotic expression plasmid pRc/CMV which including the whole length of H19 cDNA ,the plasmid was transfected to the cell line JEG-3 .The expression of H19 mRNA was observed and the gene expression profile of three groups of JEG-3 cell were de-tected with Affymetri :U133 plus 2 .0 Array .Results After being transfected with target H 19 gene ,the expression of the mRNA level was significantly increased compared with control group .And the gene expression profile was changed significantly .19 genes were up-regulated ,77 genes were down-regulated .Expression levels of HES1 gene which being choosed as a different expression gene were detected by fluorescence quantitative PCR in severe preeclampsia placenta tissue and normal late pregnant placenta .The expression level of HES1 mRNA in severe preeclampsia placenta decreased significantly than normal late pregnant placenta tissues . Conclusion Many genes induced by H19 have been screened by high-throughput gene chip method .It provides the experimental ba-sis for advanced studying the regulation the cellular biological behavior with H 19 gene .