Objective:To introduce a programmed death-1 (PD-1) inhibitor nivolumab used as a new antitumor agent. Methods:According to the literatures, the action mechanism of nivolumab and the clinical trial results on the main indications approved or being investigated in phase III trials were reviewed and evaluated. Results:Nivolumab could restore the antitumor activity of T cells through binding with PD-1 and consequently blocking its interaction with the key ligands of PD-L1 and PD-L2. Several completed and ongoing clinical trials showed that nivolumab used alone or combined with chemotherapy or CTLA-4 inhibitor ipilimumab exhibited better effica-cy when compared with current clinical used chemotherapy drugs in the treatment of melanoma, non-small cell lung cancer and renal cell carcinoma. Nivolumab was well tolerated during the treatment with such main grade 3-4 adverse events as immune-mediated pneu-monia, abnormal liver functions and fatigue. Conclusion:Through its anti-tumor immune response, nivolumab can improve the clinical efficacy in the treatment of various tumors including melanoma, non-small cell lung cancer and renal cell carcinoma.

BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei appearances. ②The growth curve showed that the growing adaptive phase was the first day after AAV-293 cells were passaged, the actively growing phase was from the second day to the fifth day, and the growing platform phase was after the sixth day. ③ AAV-293 cells with green fluorescence observed by fluorescence inverted microscope, and cotransfection of the there AAV system plasmids was successful. AAV-293 cells gave out green fluorescence after infected with AAV supernatant, and AAV package succeeded.CONCLUSION: The culture method established by the authors in the experiment is simple and useful, and the cultured AAV-293 cells remain a good function of AAV package.

Objective To explore the effects of tumor necrosis factor-α induced protein 6 (TSG-6) on acute kidney injury (AKI) following paraquat poisoning in rats.Methods Twenty-four male Sprague-Dawley (SD) rats were randomly divided into sham group (n=8),model group (n=8) and TSG-6-treated group (n=8) using a randomized number table.Rats were given an injection of 50 mg/kg of paraquat intraperitoneally (total volume was equalled to sterile normal saline) in model and TSG-6-treated groups.Rats in sham group were given 2 mg/kg of sterile saline.Mter 1 hour of paraquat administration,rats were treated with 30 μg of recombinant human TSG-6 intraperitoneally in TSG-6-treated group.After 6 hours of paraquat administration,serum was collected to assess renal function,then rats were sacrificed and renal tissues were immediately harvested.AKI score was evaluated by renal histopathology and gene expression of pro-inflammatory cytokines including interleukins (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) in kidney was assayed with real-time reverse transcription-polymerase chain reaction (RT-PCR).Results Compared with sham group,blood urea nitrogen (BUN),creatinine (Cr) and AKI score were significandy increased in model group [BUN (mmoUL):22.64 ±2.36 vs.7.09 ±0.65,t=6.986,P=0.000; Cr (μmol/L):177.28 ± 18.67 vs.60.32 ± 3.11,t=7.134,P=0.000; AKI score:9.14 ± 0.28 vs.0.30 ± 0.23,t=9.013,P=0.000].Moreover,the mRNA expressions of IL-1β,IL-6 and TNF-α were significantly elevated in model group (IL-1β mRNA:3.23 ± 0.28 vs.1.00 ±0.07,t=5.874,P=0.000; IL-6 mRNA:4.16 ±0.37 vs.1.00 ±0.08,t=7.125,P=0.000; TNF-α mRNA:3.85 ±0.31 vs.1.00 ±0.10,t=6.342,P=0.000).However,serum BUN,Cr,AKI score and the mRNA expressions of IL-1β,IL-6 and TNF-α in TSG-6-treated group were significantly lower than those in model group [BUN (mmol/L):14.07 ± 5.23 vs.22.64 ± 2.36,t=2.533,P=0.026; Cr (μmol/L):112.76 ± 14.81 vs.177.28 ± 18.67,t=2.778,P=0.016; AKI score:5.35 ±0.19 vs.9.14 ±0.28,t=2.885,P=0.013; IL-1β mRNA:2.26 ± 0.19 vs.3.23 ±0.28,t=2.457,P=0.023; IL-6 mRNA:2.92 ±0.29 vs.4.16 ±0.37,t=2.975,P=0.011; TNF-α mRNA:2.58 ± 0.23 vs.3.85 ± 0.31,t=2.564,P=0.019].Conclusion TSG-6 attenuates AKI following paraquat poisoning by suppressing inflammatory response.

Objective:To explore the effects of necroptosis specific inhibitor-1 (Nec-1) on brain injury in rats after cardiac arrest and its mechanism.Methods:A total of 24 Sprague-Dawley (SD) rats were divided into Sham group, model group and Nec-1 group ( n = 8 per group) according to random number table method. In the Sham group, only general surgical procedures were underdone without inducing cardiac arrest. In the model group, the rats were subjected to asphyxial cardiac arrest followed by cardiopulmonary resuscitation (CPR) at 6 minutes after cardiac arrest. In the Nec-1 group, Nec-1 of 1 mg/kg was administered after cardiac arrest, and CPR was performed at 6 minutes after cardiac arrest. At 72 hours after CPR, neurological deficit scores (NDS) were assessed, serum S100B levels were measured by enzyme linked immunosorbent assay (ELISA), receptor-interacting protein 3 (RIP3) expression in cerebral cortex and hippocampus was observed under immunofluorescence and positive rate was calculated, and the levels of RIP3 protein expression in brain were analyzed by Western blotting. Results:At 72 hours after CPR, the rats in the model group showed obvious necroptosis and injury in brain. Compared with the Sham group, the NDS scores in the model group were significantly decreased [57.0 (52.7, 60.0) vs. 80.0 (80.0, 80.0), P < 0.05], the serum S100B was significantly increased (ng/L: 44.9±4.5 vs. 18.6±1.5, P < 0.05), the percentages of RIP3 positive cells in cerebral cortex and hippocampus were significantly elevated [cerebral cortex: (31.7±4.8)% vs. (11.6±3.2)%, hippocampus: (28.4±0.8)% vs. (10.9±0.6)%, both P < 0.05], and the levels of RIP3 protein expression in brain were significantly increased [RIP3 protein (RIP3/GAPDH): 0.708 (0.642, 0.722) vs. 0.408 (0.253, 0.504), P < 0.05]. After Nec-1 intervention, necroptosis and injury in brain were obviously improved. Compared with the model group, the NDS scores at 72 hours after CPR in the Nec-1 group were significantly increased [70.5 (68.5, 71.7) vs. 57.0 (52.7, 60.0), P < 0.05), the serum S100B was significantly decreased (ng/L: 31.9±2.7 vs. 44.9±4.5, P < 0.05), the percentages of RIP3 positive cells in cerebral cortex and hippocampus were significantly lowered [cerebral cortex: (23.7±4.1)% vs. (31.7±4.8)%, hippocampus: (20.4±0.4)% vs. (28.4±0.8)%, both P < 0.05], and the levels of RIP3 protein expression in brain were significantly declined [RIP3 protein (RIP3/GAPDH): 0.437 (0.379, 0.507) vs. 0.708 (0.642, 0.722), P < 0.05]. Conclusion:Nec-1 attenuated necroptosis of brain cells by inhibiting the expression of RIP3 protein, so as to reduce brain injury after cardiac arrest in rats.

Objective To investigate the mechanism that bFGF promotes the regeneration of injured optic nerve and induces dedifferentiation of glial cells in it. Methods Fifty-five adult male SD rats were randomly divided into 3 groups as normal control group, injury group and bFGF group. At day 7 post operation, optic nerves from injury group and bFGF group were detected by gene chip and real-time PCR. At day 7, 14 post operation, optic nerves were harvested and detected by HE staining and immunohistochemistry. Results Compared with the injury group, there were 645 genes expression up-regulated and 458 genes down-regulated including genes related neural stem cell or precursor cell neural development, proliferation, apoptosis, chromatin configuration, transcription regulation, signal transduction, neural growth and so on in the bFGF group. There were bigger nuclei, more cells, more immunoreactivity of nestin, extracellular signal-regulated kinase(Erk1/2), glial fibrillary acidic protein(GFAP), and myelin basic protein(MBP) in the distal optic nerves and more immunoreactivity of neurofilament(NF) in the proximal optic nerves in the bFGF group than that in the injury group.Conclusion bFGF could promote the proliferation of neuroglia cells, dedifferentiation of neural glias and improve the microenvironment to favour the regeneration of injured optic nerve.

Objective To investigate the changes of serum bilirubin level in elderly patients with acute cerebral infarction and its significance.Methods 164 hospitalized elderly patients,who suffered from acute cerebral infarction within 1 week after onset,were divided into 2 groups according to age:group A aged over 60 years(n=85) and group B aged 40-60 years(n=79),and 66 healthy subjects aged over 40 years were collected as controls(group C).Serum bilirubin levels in all subjects were determined.The ratio of pulse pressure over mean arterial pressure(PP/MAP) in group A and B was calculated.Nerve function scores in the three groups were detected before and after 2 weeks of treatment.Meanwhile,the data of risk factors including blood glucose,blood pressure,blood lipids,smoking and drinking in group A and B were collected.Results Compared with group C,serum total,direct,indirect bilirubin levels were increased in group A and B(both P＜0.01),and the change was smaller in group A than in group B(P＜0.05).The nerve function scores was lower in group A than in group B before and after treatment [(35.2±12.6) vs.(44.3±7.9),(40.7±9.1) vs.(51.3± 4.1),t=5.58,9.73,both P＜0.01],but PP/MAP and the numbers of risk factors were higher in group A than in group B [(0.46±0.06) vs.(0.38±0.06),93.01 vs.71.20,both P＜0.01].There were no significant correlations of serum total,direct and indirect bilirubin levels with nerve function scores in group A or B(all P＞0.05).Conclusions Serum bilirubin level is increased in patients with acute cerebral infarction,but the endogenous antioxidant capacity is decreased because of aging,multiple risk factors and more serious atherosclerosis in elderly patients,and the increment of bilirubin level is relatively smaller in acute cerebral ischemia,leading to the reduced protective effect against stress.Serum bilirubin level may influence the prognosis in patients with acute cerebral infarction.

Objective: To compare regeneration of motor fibers with that of sensory fibers by end to side neurorrhaphy and compare the effect of end to side neurorrhaphy with that of end to end neurorrhaphy. Methods: 20 SD rats were randomly divided into two groups: group A and group B. In group A, the right peroneal nerve was sectioned and the distal end was sutured laterally to the tibial nerve; in group B, the right peroneal nerve was sectioned and sutured with end to end neurorrhaphy. All left sides of two groups were used as control. Retrograde transportation of HRP was observed after 28 weeks. Results: The labelled neurons were also found in the anterior horn of the spinal cord and the spinal ganglia. Conclusion: The regenerative nerve fibers had motor fibers as well as sensory fibers by end to side neurorrhaphy. The effect of end to side neurorrhaphy is not as good as that of end to end neurorrhaphy. [

Through various common domestic and foreign electronic sphygmomanometers to test blood pressure, we find that when measuring high blood pressure or low blood pressure, there is a mismatch between the maximum inflation pressure and the blood pressure measurement, which often results in repeatedly inflating and deflating as well as the problem of high inflation pressure. In order to solve these problems and find a suitable maximum inflation pressure, two intelligent pneumatic solutions based on identifying of pulse wave are suggested and 700 groups of blood pressure experiments are done, then the two solutions are verified by experiments. The experiment proved that these solutions proposed have good stability and accuracy, they can solve the problems appeared in measuring blood pressure effectively, at the same time, the second solution that estimate the maximum inflation pressure during inflation is considered as the best one.
Blood Pressure ; Blood Pressure Determination ; instrumentation ; Heart Rate ; Humans ; Hypertension ; Sphygmomanometers

Objective To compare the myocardial protective effects of post-treatment with sevoflurane and isoflurane on myocardial ischemia-reperfusion injury(MIRI) in adult rats.Methods Twenty-four adult male SD rats were divided into four groups (n =6) by using the random number table,control group (C),isehemia-reperfusion group (R),sevoflurane post-treatment (S) and isoflurane post-treatment group(I).The Langendorff isolated heart perfusion model was established.The heart rate(HR),left ventricular end-diastolic pressure(LLVEDP),left ventricular developed pressure(LVDP),maximum rate of rise of left ventricular pressure(LV+-dp/dtmax),and maximum rate of decrease of left ventricular pressure(LV-dp/dtmax) were recorded at the end of equilibrium perfusion,and at 30,90 min of reperfusion,respectively.At the end of infusion,1 mm3.of apical myocardial tissue was removed for observing mitochondrial structure under electron microscopy and scoring.The myocardial infarct size(MIS) in the remaining heart tissue was measured by TTC staining.Results Compared with the R group,the S and I groups showed improved cardiac function indicators,decreased MIS,and reduced mitochondrial damage after reperfusion(P＜0.05).Compared with the S group,the I group showed worse heart function,increased MIS,and more severe mitochondrial damage after reperfusion(P＜0.05).Conclusion Post-treatment with sevoflurane and isoflurane has a protective effect on MIRI in adult rats.Post-treatment with sevoflurane has a better cardioprotective effect than that with isoflurane.

Objective To investigate the dedifferentiation of neuroglial cells and its induction after optic nerve injury. Methods Adult male SD rats were randomly divided into 4 groups the normal control group,the injury group,the transplantation group and the microcrush and transplantation group.Optic nerves were harvested at days 3,7,14 and 28 after the operation.HE staining was used to count the number of neuroglial cells.Immunohistochemistry,Western blotting and in situ hybridization histochemistry were employed together with computerized image analysis to evaluate the expressions of Nestin,GFAP,MBP,NF,BDNF,Nogo-A and Nogo-A mRNA.Immunofluorescence double staining was used to detect the co-expression of Nestin and GFAP or Nestin and MBP. Results The number of cells only increased at day 7 after the nerve injury, the expressions of Nestin,MBP,Nogo-A and Nogo-A mRNA were up-regulated,the expressions of GFAP,NF and BDNF were down-regulated,and some Nestin-GFAP positive cells and a few of Nestin-MBP positive cells were detected in the injury group.Compared with the injury group,the number of cells was increased sometime after the nerve injury;the expressions of Nestin,GFAP,BDNF and NF were up-regulated,the expressions of MBP,Nogo-A and Nogo-A mRNA were down-regulated,and the number of Nestin-GFAP positive cells increased in the transplantation group and the microcrush and transplantation group.Conclusion After optic nerve injury,some astrocytes undergo dedifferentiation while the macroglial cells display a gene expression pattern that is unfavorable for nerve regeneration.Pre-degenerated peripheral nerves could enhance the dedifferentiation of astrocytes and induce the gene expression pattern of macroglial cells that is favorable for nerve regeneration.