Objective To study the feasibility of stem cell transplantation under mild hypothermia so as to provide a prerequisite for stem cell transplantation in patients with traumatic brain injuries (TBI) during mild hypothermia treatment. Methods After transfecting plasmid containing temperature-sensitive simian virus 40 large T-antigen (tsSV40LT) into temperature-sensitive umbilical cord mes-enchymal stem cells (tsUCMSCs) , the changes of cell morphology, nuclear proliferation index (PIx) and telomerase activity were detested when the tsUCMSCs were cultured at 33℃ and 37℃. After the mouse model with tTBI treated with mild hypothermia was established, tsUCMSCs were transplanted into semi-injury area to detect survival rate, proliferation and apoptosis indices and perform neurological deficit scoring. Results When cultured at 33℃, the tsUCMSCs displayed long spindle-shaped and highly refractive, with higher proliferation index and telomerase activity than those cultured at 37℃. Compared with control group (non-temperature-sensitive UCMSCs transplantation), tsUCMSCs in semi-injury area showed much higher cell survival and proliferating cell nuclear antigen expression ( P < 0.05 ) , with fewer apoptotic cells and better neurological function (P < 0.05). Conclusion The establishment of temperature-sensitive stem cell line enables stem cell transplantation during treatment of TBI with mild hypothermia, as provides us a new direction for treatment of TBI.

Objective To investigate the effects of HO/CO pathway on inflammation cytokines in a rat model of incisional pain. Methods Thirty-six rats were executed to collect ipsilateral spinal cord tissues for HO-1 detection by Western blot assay, and cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6 and high mobility group box (HMGB)1 were detected by ELISA before and at 1, 4, 8, 12 and 24 h after establishing incisional pain model. Additionally, 36 rats without establishment of incisional pain model were used as control group. A total of 144 model rats of incisional pain were divided into incisional pain (IP) group, IP+hemin group (100 mg/kg hemin was injected by i.p. before operation), IP+Znpp-IX group (45μmoL/kg Znpp-IX was injected by i.p. before operation) and IP+CORM-2 group (10 mg/kg CORM-2 was injected by i.p. before operation). Values of paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were detected, and expressions of TNF-a, IL-1 b, IL-6 and HMGB1 were measured by ELISA before and at 1, 4, 8, 12 and 24 h after operation. Results Compared with pre-operation of incisional pain in rats, expression levels of HO-1 protein and cytokines TNF-a, IL-1 b, IL-6 and HMGB1 were increased at 1, 4, 8, 12 and 24 h after operation (P<0.05). Compared with control group, values of PWMT and PWTL were obviously decreased, and expression levels of IL-1β, TNF-α, IL-6 and HMGB1 were increased at 1, 4, 8, 12 and 24 h after operation in IP groups (P<0.05). Compared with IP groups, values of PWMT and PWTL were significantly increased and cytokines TNF-a, IL-1 b, IL-6 and HMGB1 were decreased at 1, 4, 8, 12 and 24 h after operation in IP+hemin group and IP+CORM-2 group (P<0.05). Values of PWMT and
PWTL were decreased and cytokines TNF-α, IL-1β, IL-6 and HMGB1 were increased in IP+Znpp-IX group (P<0.05). Conclusion Incisional pain can increase the expression of HO-1, and HO-1/CO pathway exists the regulatory effect on inflammatory cytokines in the rat model of incisional pain.

Objective To evaluate the influence of autophagy on pain behavioristics and astrocytic activation in rats with inflammatory pain.Methods Seventy-eight clean male Sprague-Dawley (SD) adult rats were randomly divided into four groups: control group (n = 12), model group (n = 42), autophagy inducer rapamycin (Rap) pretreatment group (n = 12) and autophagy inhibitor 3 methyladenine (3-MA) pretreatment group (n = 12). The inflammatory pain rat model was reproduced by subcutaneous injection of freund's complete adjuvant (CFA) 100μL at foot sole, whilein control group, the same volume 0.9% normal saline 100μL was injected at the same site. One hour before modeling, Rap 10 mg/kg and 3-MA 15 mg/kg were intraperitoneally injected in rats in Rap and 3-MA pretreatment groups respectively, and the same volume of 0.9% normal saline was injected intraperitoneally in rats of control and model groups. Before modeling and 6, 12, 24 hours and 3 days after modeling, the L4-L6 spinal cord tissue was harvested from 6 rats in model group, and autophagy protein membrane microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and autophagy related gene Beclin-1 expressions were detected by Western Blot in the tissue; the changes of pain behavioral indexes mechanical withdraw threshold (MWT) and thermal withdrawal latency (TWL,n = 6), were observed at6, 12, 24 hours and 3 days, 7 days after modeling in the 6 rats taken from each group; in another 6 rats in each group, 24 hours after modeling, L4-L6 spinal cord tissue was collected, immunofluorescence staining was used to observe the changes of astrocytes and the positive expression of glial fibrillary acidic protein (GFAP) under a confocal microscopy, and the protein expression quantity of GFAP was detected by Western Blot in the tissue.Results ① The inflammatory pain could induce the increase of rat autophagy protein LC3Ⅱ and Beclin-1 expressions in spinal cord tissue, reaching their peaks at 24 hours (A value: 0.59±0.07, 0.51±0.06, respectively), and then they were gradually decreased. ② With the prolongation of time, in the model group MWT was gradually decreased, TWL was gradually shortened, they reached their valley values at 24 hours after modeling [MWT (g): 17.8±1.9, TWL (s): 6.8±0.4], and from 12 hours they were significantly decreased compared with those in control group [12hours MWT (g): 21.5±2.4 vs. 43.4±5.1, TWL (s): 12.0±1.1 vs. 17.6±1.2, bothP < 0.05], after modeling for 3 days they were increased; Compared with model group, 12 hours after autophagy inducer Rap was given, MWT was significantly increased (g: 36.8±4.9 vs. 21.5±2.4,P < 0.05), TWL was significantly prolonged (s: 14.3±1.1 vs. 12.0±1.1,P < 0.05); from 12 hours after autophagy inhibitor 3-MA was given, MWT was further reduced (g: 18.6±1.9 vs. 21.5±2.4, P<0.05), TWL was further shortened (s: 8.4±0.6 vs. 12.0±1.1,P < 0.05). ③ Confocal microscopic findings showed, there was no significant acstrocytic change, and only litter GFAP expression was seen in control group. In model group, the inflammatory pain induced astrocyte activation, manifesting glial cell hypertrophy, hyperplasia, gelatinousnetwork deformation, and GFAP expression was obviously increased compared with that in the control group (A value: 0.54±0.09 vs. 0.16±0.02,P < 0.05). Since autophagy inducer Rap can decrease astrocyte activation and inhibit GFAP expression, there was statistical significant difference between Rap pretreatment and model groups (A value: 0.33±0.06 vs.0.54±0.09,P < 0.05); autophagy inhibitor 3-MA can further aggravate astrocytes activation and up-regulate GFAP expression in 3-MA pretreatment group (A value: 0.73±0.08 vs. 0.54±0.09,P < 0.05).Conclusion Autophagy participates in the process of astrocytic activation and pain behavioristics in rats with inflammatory pain.

Background Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to have the ability of migrating after transplantation into rat model of traumatic brain injury. In this study, MSCs'characteristic of tropism for intracranial glioma was explored following being labeled and transplanted into brain and blood of gliomabearing rats.Methods Cellulae medullares were cultured to get pure MSCs. The cell surface antigen and cell cycle of MSCs were detected to confirm their identity by flow cytometry. MSCs were marked with BrdU and then were injected into contralateral brain or collateral internal carotid artery of rats bearing glioma. 2 weeks later, the brains were resected and pathological sections were made. Immunohistochemistry and immunofluorescence technology were performed to detect MSCs.Results MSCs displayed extensive tropism for intracranial glioma. MSCs which were injected in the contralateral brain of the glioma scattered densely in the juncture between brain tissue and tumor and there were a few MSCs in the glioma; MSCs which were injected into the collateral internal carotid artery scattered widespread in the glioma and there were a few MSCs in the juncture area.Conclusions MSCs have the ability to migrating toward glioma and penetrating blood brain barrier. MSCs may be ideal gene therapy vehicles against glioma.

ObjectiveTo investigate the roles of 11 β-hydroxysteroid dehydrogenase type 1 ( 11 β-HSD1 )on hippocampus of rat with chronic unpredictable mild stress (CUMS).MethodsTwenty-four male SpragueDawley rats were randomly divided into control group and depressive model group. Chronic unpredictable mild stress (CUMS) was used to make up depressive animal model.Behavioral changes were recorded by body weight measuring,sucrose consumption test (SCT) and open field test (OFT),respectively.The mRNA transcription of 11β-HSD1 in hippocampus tissues of the rats were detected by real-time RT-PCR,and the protein expression of 11β-HSD1 were detected by western blot and immunofluorescence.ResultsBcforc starting CUMS protocol,the rats exhibited equivalent weight and sucrose consumption.Twenty-eight days after CUMS protocol,behavior parameters such as body weight,sucrose consumption,nunber of crossing,and number of rearing were significantly decreased in rats exposed to CUMS group compared with control group (P ＜ 0.05,P ＜ 0.01 ).Correspondingly,realtime RT-PCR assays showed the mRNA expression of 11 β-HSD1 in the hippocampus of CUMS group,which was (31 ±9) ％ lower than that of control group.Meanwhile,the protein expression of it in CUMS group was lower than that of control group (P ＜ 0.05 ).Inmunofluorescence revealed that the number of positive 11 3-HSD1 cells was high (223 ± 13) in the control group,while the number was decreased prominently (92 ± 11 ) in the CUMS group (P ＜ 0.01 ).ConclusionDepressive behavior of rats is induced and the expression of 11 β-HSD1 in the hippocampus is decreased prominently by CUMS,the mechanism of which is at least related to the low expression of 11β-HSD1 and disturbance of glucocorticoid metabolism caused by CUMS.

Objective To study the effect of brain-derived neurotrophic factor (BDNF) on the environmental nutrition and neural differentiation of the transplanted stem cells under hypothermia.Methods The BDNF gene mediated by liposome was transfected into 293T cell line, and ELISA assay was applied to find the peak time of BDNF expression. When BDNF was highly expressed, the supernatant was collected for establishment of SD rat models of brain injury. The rats were divided into Group A (stem cell transplantation group) and Group B (stem cell transplantation and BDNF group). Rats in both groups were under hypothermia treatment for five days. Four and eight days later ( three days from rewarming), rat brain tissues were obtained to detect the expressions of proliferating cell nuclear antigen (PCNA), nestin, neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) by immunohistochemical method and to detect the apoptosis by in situ hybridization. Finally, the nerve function scores were obtained for evaluation of the nerve function. Results The ELISA showed that the high level of BDNF expression was at 48 to 60 hours after gene transfection. PCNA and nestin were highly expressed, while NES and GFAP showed nil or low level of expression in both groups at the fourth day after hypothermia, with little apoptotic cells especially in the Group B (P ＜0.05). The expressions of PCNA and nestin were decreased, but the expressions of NSE and GFAP were increased at the third day after rewarming. The positive rate of NSE expression in the Group B was much higher and the apoptotic cells were much less compared with the Group A ( P ＜ 0. 05 ). A better nerve score was obtained in the Group B. Conclusion BDNF can enhance the survival rate of the transplanted stem cells and induce their differentiation into neurons under hypothermia.

Objective To investigate the signal pathway of platelet activation in acute respiratory distress syndrome (ARDS). Methods Thirty healthy Sprague-Dawley (SD) rats were randomly divided into control group (n = 6) and model group (n = 24). The model of ARDS was reproduced by intravenous injection of oleic acid (0.25 mL/kg), and the rats in control group were injected with the same amount of normal saline. The blood of abdominal aorta was collected at 2, 6, 24, and 72 hours after model reproduction, the platelets were separated, and c-Jun N-terminal kinase phosphorylation (pJNK) levels which was one of major protein kinases in the mitogen-activated protein kinases (MAPKs) signal pathway was determined by Western Blot. The rats were sacrificed, the lung tissues were harvested, and lung coefficient (lung weight/body weight ×100%) and lung wet/dry (W/D) ratio were calculated. Pathological changes in the lung tissue were observed with hematoxylin-eosin (HE) staining in light microscope. Results Comp ared with the control group, platelet pJNK level in ARDS model group was significantly increased at 2 hours after model reproduction (gray value: 0.72±0.09 vs. 0.22±0.01), and peaked at 6 hours (gray value: 0.91±0.03 vs. 0.22±0.01), then it was decreased gradually. It was also significantly higher than that of control group till 72 hours after model reproduction (gray value: 0.39±0.06 vs. 0.22±0.01, all P < 0.05). Lung coefficient and lung W/D ratio in ARDS model group were significantly increased at 2 hours after model reproduction as compared with those of control group [(1.30±0.20)% vs. (0.60±0.10)%, 6.00±0.60 vs. 3.30±0.30], then they were decreased gradually. They were also significantly higher than those of control group till 72 hours after model reproduction [(0.90±0.10)% vs. (0.60±0.10)%, 4.80±0.70 vs. 3.30±0.30, all P < 0.05]. It was showed by light microscopy that lung tissue of rats in the control group had no significant pathological changes. At 2 hours after model reproduction in model group, clearly visible alveolar edema and interstitial edema, interstitial lung infiltration of inflammatory cells, small blood vessels dilation and congestion were found, and the re were a lot of protein exudates. The lesions of lung peaked at 24 hours. At 72 hours, absorption of most of fluid leaking in alveolar, alveolar space narrow, alveolar septum thickening, the reduction of inflammatory cells infiltration, fibrous tissue proliferation, and micro thrombosis formation were found. Conclusion In ARDS, in addition to pathological changes in the lung tissue, platelet activation occurs, and its activation process is related to the priming of JNK signal transduction pathways.

Objective To study the function of the nano-molecular polyamide-amine (PAMAM) as microRNA(miR) carrier targeting gastric adenocarcinoma, and the foundation of developing an efficient delivery of small molecule drugs tar-geting gastric cancer thereof. Methods The folic acid (FA)/PAMAM comoles compound was prepared by dialysis method. After transfection of miR-7 or liposomes into SGC-7901 cell line, fluorescence microscope was used to detect the gene trans-fect efficiency. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the miR-7 level. The immu-nocytochemistry assay was used to test the protein expressions of epidermal growth factor receptor (EGFR), protein kinase B (PKB) and proliferating cell nuclearantigen (PCNA). The transwell system was utilized to explore the migration ability of tu-mor cells. Results Compared with liposme, FA/PAMAN complex compound can significantly improve the level of miR-7 in SGC-7901 cells,reduce the protein levels of EGFR, PKB and PCNA in SGC-7901 cells, and also reduce the percentage of cancer cell migration (P<0.05). Conclusion PAMAM can effectively transfect miR into gastric cancer cells, which is expected to become an efficient delivery of small molecule drugs.

Objective To judge injury severity of severe traumatic brain injury (sTBI) by using surface enhanced laser desorption-ionization (SELDI) protein chip technique. Methods Serum sam-ples from sTBI patients were used to detect expression of differential proteins by protein chip CM10 and SELDI to analyze the correlation between expression peak intensity and GCS. Results We obtained 101 protein peaks, with statistical difference upon expression of 27 protein peaks, when negative correla-tion was found between two peaks ( m/z 4 972 and m/z 5 322 ) and GCS score and positive correlation be-tween six peaks (m/z 3 941, m/z 4 295, m/z 8 714, m/z 8 792, m/z 14 020 and m/z 28 148) and GCS score. Conclusion SELDI protein chip technique may become a new and objective detection method in judging injury severity of sTBI.

Objective To study the changes of proteome expression in brain tissues from rats with severe traumatic brain injury(sTBI). Methods Total protein of brain tissues were obtained at days 3,7 and 14 for two-dimensional gel electrophoresis to screen and identify differential protein spots.Results We screened 17 differential protein spots that were involved in cellular metabolism,stress and inflammatory reaction. Conclusion Some differential proteins involved in sTBI can be found by twodimensional gel electrophoresis.