Aim To investigate the effect of bufalin on proliferation and expression of WT1 in K562 cells. Methods The colony number of K562 cell was detec-ted with semi-solid culture assay. The cell cycle was measured by flowcytometry, and the expression of WT1 was observed with immunocytochemistry. Subcutaneous tumor models established by K562 cells in BALB/C nu/nu mice were divided into three groups, including model group, bufalin group and positive control group. After 21 days, the subcutaneous tumors were removed for calculating the inhibitory rate of tumor growth. HE staining and immunohistochemistry were used to ob-serve the morphological changes and the expression of WT1 . Results ① Bufalin could significantly decrease the colony number of K562 cell, arrest it at G0/G1 phase and down-regulate its expression of WT1 in a dose-dependent manner. ② Compared with the model group, the tumor inhibitory rate was much higher, while the volume and the weight were obviously lower in the other two groups. ③Bufalin could induce apop-tosis, necrosis, hemorrhage and fibrosis with HE stai-ning, and down-regulate the expression of WT1. Con-clusion Bufalin could inhibit the proliferation, arrest the cell cycle at G0/G1 phase and down-regulate the expression of WT1 in vitro. Bufalin could inhibit the tumor inhibitory rate, the volume and the weight of the subcutaneous tumors, induce apoptosis, necrosis, hemorrhage and fibrosis with HE staining and down-regulate the expression of WT1 .

Objective To investigate the dynamic expression from gene and protein levels of hypoxia inducible factor-1α(HIF-1α) during the development of hypoxic pulmonary hypertension (HPH) in neonatal rats.Methods A neonatal rat model of HPH was established,normal neonatal rats were enrolled as the control group.At the 3th 、7th、10th and 14th days,we measured the mRVSP through catheterization of right ventricule,collected hearts to figure out the right ventricular hypertrophy index(RVHI),evaluated vascular remodeling by the percentage of medial thickness to outer diameter of the small pulmonary arteries (MT％) and the percentage of medial cross section on area to the total cross section area of the pulmonary small arteries (MA％),observed the expression of HIF-1α by immunochemistry,and measured the expression of HIF-1α in mRNA and protein by RT-PCR and Western blot respectively.Results The mRVSP increased at the 3th day,and sustained until the 14th day (P ＜ 0.01).RVHI MT％ and MA％ increased at the 7th day,and sustained until the 14th day (P ＜0.01).HIF-1α mainly expressed in endothelium and smooth muscle cells in the CHPH group and the HIF-1αmRNA increased significantly on the 3th and 7th days (P ＜ 0.05),and the HIF-1α protein increased significantly on the 7th、10th and 14th days in the CHPH group compared with the control group(P ＜ 0.01).Conclusion The mRVSP increased at the early stage after hypoxic exposure in neonatal rats,followed by vascular remodeling and right ventricular hypertrophy.Both mRNA and protein levels of HIF-1α sustained higher than control group during the vascular remodeling stage,indicating that HIF-1α might be a important factor contributing to the vascular remodeling.

Aim To observe mesenchymal stem cell differentiation into osteoblast induced by TSPG, and explore how TSPG enhances the promotion of hemato-poiesis of osteoblast differentiation from mesenchymal stem cell. Methods MSCs were cultured by TSPG combined with osteoinductive medium. The cellular vi-ability of proliferation was detected with MTT assay. The content of alkaline phosphatase in the cultural su-pernatant was tested with pNPP assay. The ability of MSCs to form calcium nodes was also observed after a-lizarin red stain. The protein expression of RUNX2 was analyzed with Western blot. The content of cytokines associated with hematopoiesis was tested with Elisa as-say. The ability of promoting hematopoiesis was detec-ted with hematopoietic colony forming assay. Results Both MTT and pNPP assay showed that optical den-sity ( OD) values were increased in response to TSPG treatment in a dose-dependent manner. The mineraliza-tion formation ability was enhanced with TSPG-treat-ment. Meanwhile, the expression of RUNX2 protein was up-regulated in TSPG-treated cell. Moreover, the content of cytokines associated with hematopoiesis and the number of hematopoietic progenitor colony were in-creased by TSPG-treatment compared with the control group. Conclusion TSPG could induce MSCs differ-entiation in to osteoblast and enhance the effect of oste-oblast differentiation from MSCs on promoting hemato-poiesis.

Objective To evaluate the changes in the expression of Toll-like receptor 4 (TLR4) mRNA and its down-stream cytokines IL-1β and TNF-α mRNA in spinal cord in a rat model of incisional pain.Methods Fifty-eight male Sprague-Dawley rats,weighing 180-220 g,were used in this study.A 1-cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of the hindpaw in isoflurane-anesthetized rats.Mechanical paw withdrawal threshold to yon Frey filament stimulation (MWT) on the operated and non-operated sides was measured before operation and at 0.5,1,2,6 and 12 h and 1,2,3,5 and 7 days after operation.Six rats were chosen and sacrificed before operation and at 2 and 8 h and 1,2,3,5 and 7 days after operation.Their lumbar segments (L4-6) of the spinal cord were removed for determination of the expression of TLR4,IL-1β and TNF-α mRNA by real-time PCR.Results Compared with the baseline value before operation,MWT on the operated side was significantly decreased at 0.5 h-5 days after operation,and the expression of TLR4,IL-1β and TNFα mRNA was up-regulated at 2 and 8 h and 1,2 and 3 days after operation (P ＜ 0.05),and no significant change was found in MWT on the non-operated side (P ＞ 0.05).MWT on the operated side was lowest at 2 h after operation and then gradually increased,the expression of TLR4 mRNA peaked on 1 day after operation,and the expression of IL-1 and TNF-α mRNA peaked at 8 h after operation (P ＜ 0.05).The TLR4 mRNA expression was negatively correlated with MWT on the operative side (r =-0.484,P ＜ 0.05),and IL-1 mRNA and TNF-α mRNA expression was positively correlated with TLR4 mRNA (r =0.294 and 0.540,respectively,P ＜ 0.05).Conclusion The expression of TLR4 mRNA and its down-stream cytokines IL-1β and TNF-α(mRNA in spinal cord is up-regulated,this change is involved in the maintenace of incisional pain,but it does not play an important role.

Objective To study the influencing factors of clinical outcome in elderly acute ischemic stroke (AIS) patients after intravenous thrombolysis.Methods One hundred and fifty-one AIS patients admitted to our hospital for intravenous thrombolysis were divided into good outcome group (n=77) and poor outcome group (n=74) according to their modified Rankin scale score 3 months after the onset of AIS.The baseline data,thrombolysis time window,NIHSS score and ischemic stroke typing before thrombolysis,early symptom improvement,cerebral hemorrhage after thrombolysis were compared between the two groups.Results The serum levels of blood glucose and CRP,NIHSS score≥9 before thrombolysis,incidence of AF and cerebral hemorrhage were significantly higher in poor outcome group than in good outcome group (P＜ 0.05,P＜0.01).Multivariate logistic regression analysis showed that NIHSS score,OCSP typing,blood glucose before thrombolysis,24 h symptom improvement were the independent influencing factors of clinical outcome in elderly AIS patients (OR =1.262,95 ％ CI:1.075-1.482,P =0.005;OR =0.203,95％CI:0.066-0.628,P=0.006;OR=1.264,95％CI:1.042-1.532,P=0.017;OR=25.764,95％CI:5.131-129.361,P=0.000).Conclusion NIHSS score,OCSP typing,blood glucose before thrombolysis and 24 h symptom improvement are the independent influencing factors of clinical outcome in elderly AIS patients after intravenous thrombolysis.

Objective:To evaluate the relationship between milk fat globular epidermal growth factor Ⅷ (MFG-E8)-mediated efferocytosis and sepsis-associated encephalopathy (SAE) in mice.Methods:Forty clean-grade healthy male C57BL/6 mice, aged 2-3 months, weighing 18-22 g, were divided into 4 groups ( n=10 each) using a random number table method: sham operation group (group Sham), cecal ligation and perforation (CLP) group (group CLP), sham operation+ phosphate buffer solution (PBS) group (group Sham+ PBS) and CLP+ recombinant mouse MFG-E8 group (group CLP+ rmMFG-E8). SAE was induced by CLP in anesthetized mice.PBS 1 μl and rmMFG-E8 1 μg were injected into the lateral cerebral ventricle in Sham+ PBS and CLP+ rmMFG-E8 groups after operation for 5 consecutive days.Novel object recognition test was performed at 6 days after operation, and the contextual fear conditioning test was performed at 7 and 8 days after operation.The percentage of time spent exploring a novel object, discrimination index and percentage of freezing time induced by condition were calculated.The animals were sacrificed after the end of behavioral tests, and the hippocampus was extracted for determination of the expression of MFG-E8 and GTP-Rac1 (by Western blot), the mRNA expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1β (using real-time reverse transcription-polymerase chain reaction) and the apoptosis rate in hippocampus (by TUNEL). Results:Compared with group Sham, the percentage of time spent exploring a novel object, discrimination index and percentage of freezing time were significantly decreased, hippocamcal MFG-E8 expression was down-regulated, GTP-Rac1 expression was up-regulated, mRNA expression of IL-6, TNF-α and IL-1β was up-regulated, and apoptosis rate was increased in group CLP ( P<0.05). Compared with group CLP, the percentage of time spent exploring a novel object and discrimination index were significantly increased, freezing time was prolonged, hippocamcal MFG-E8 and GTP-Rac1 expression was up-regulated, mRNA expression of IL-6, TNF-α and IL-1β was down-regulated, and apoptosis rate was decreased in group CLP+ rmMFG-E8 ( P<0.05). Conclusion:The reduction of hippocampal MFG-E8-mediated efferocytosis may be involved in the pathophysiological mechanism of SAE in mice.