Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

BACKGROUND:The infection after spinal internal fixation was its serious complications. A number of studies have shown that erythrocyte sedimentation rate and C-reactive protein are of great importance in judging infections. OBJECTIVE:To analyze the trend of change of erythrocyte sedimentation rate and C-reactive protein for patients without infection after the cervical fixation. METHODS:Total y 56 patients, who underwent cervical fixation from October 2013 to July 2014, were retrospectively analyzed, and then divided into anterior cervical group (n=29) and posterior cervical group (n=27). Patients in the anterior cervical group underwent anterior cervical decompression bone graft internal fixation. Patients in the posterior cervical group underwent posterior cervical unilateral open door decompression internal fixation. The peripheral blood was col ected before fixation and at the early morning of the 1, 3, 6, 9 days after fixation. Erythrocyte sedimentation rate and C-reactive protein values were determined. The fol ow-up of patients was more than one year. Signs of infection did not appear. RESULTS AND CONCLUSION:(1) General rule:After the cervical fixation, the erythrocyte sedimentation rate was increased significantly and reached a peak on postoperative day 6. The peak level gradual y decreased but has not returned to normal at the 9 postoperative days. The C-reactive protein increased significantly on the first postoperative day and reached a peak on postoperative day 3. The peak level rapidly decreased but has not returned to normal at the 9 postoperative days. The level of erythrocyte sedimentation rate of patients in the posterior cervical group was significantly higher than that in the anterior cervical group at 3, 6 and 9 days after internal fixation (P<0.05). There was no significant difference in the C-reactive protein between these two groups (P>0.05). (2) These results demonstrate that C-reactive protein is an important indicator of monitoring the inflammatory response of patients after cervical internal fixation, which was conductive to the judgment of early infection after internal fixation. The abnormal inflammatory indices of erythrocyte sedimentation rate and C-reactive protein do not suggest a presence of blade infection after internal fixation. C-reactive protein can reach the peak at 3 days after fixation. It is recommended to check blood at 2 and 3 days. If there is no apparent rebound, then the possibility of infection is smal . It may indicate the presence of infection if the inflammatory indices increased again or decreased slowly after the decrease.

Objective To investigate the effects of Dangshen (DS) extract saponins alleviating apoptosis in ischemia-reperfusion injury (I/R) of renal grafts and the mechanism. Methods The I/R injury model in SD rats was established after kidney transplantation. The SD rats were randomly divided into the following 3 groups (n = 20): sham operation group, I/R model group, DS saponin intervention group. Blood urea nitrogen (Bun) and creatinine (Scr) levels were determined at the 24th h after operation; apoptosis index (AI) was detected by using TUNEL method; the expression of Bcl2 and bax mRNA was detected by RT-PCR. Results As compared with the sham operation group,the blood Bun and Scr levels were significantly increased in the I/R model and DS saponin intervention groups (P＜0. 05). As compared with the DS saponin intervention group, the blood Bun and Scr levels were significantly increased in the I/R group (P＜0.05). The AI was significantly increased in the I/R model group and DS saponin intervention group. After DS saponin intervention, the AI was decreased from 40. 28 % in the I/R model group to 28. 45 % in the DS saponin intervention group (P＜0. 05). The expression of Bcl-2 mRNA in DS saponin intervention group and I/R model group was significantly decreased as compared with the sham operation group (P＜0. 05), but that of Bax mRNA was significantly increased in the I/R model group and DS saponin intervention group as compared with the sham operation group (P＜0. 05). After DS saponin intervention, the expression of Bcl-2mRNA was increased from 0. 25 in the I/R model group to 0. 391 (P＜0. 05), and that of Bax mRNA was decreased from 0. 565 in the I/R model group to 0. 473 (P＜0. 05). Conclusion DS extract saponin could significantly alleviate apoptosis in the I/R injury of renal grafts possibly by up-regulating the expression of Bcl-2 mRNA and down-regulating the expression of Bax gene.

BACKGROUND:Pathological examination and MRI have been widely used in clinic, but their combination is rarely reported in discrimination of early spine infections.
OBJECTIVE:To determine the accuracy of pathology and MRI for discrimination between early pyogenic spondylitis and brucella spondylitis.
METHODS:Twenty-two patients with pyogenic spondylitis and 20 patients with brucella spondylitis who had CT-guided percutaneous biopsy and MRI of the spine were retrospectively reviewed. Pathological observations included structure and activity of bone lesions, tissue cells and their main components;MRI observations included signal and sign changes at lesion sites. Statistical analysis was performed with the chi-square test.
RESULTS AND CONCLUSION:The patients with pyogenic spondylitis had a significantly higher incidence of pathological and MRI findings as fol ows (P<0.05):neutrophil infiltration;intervertebral disc abnormal signal, location of vertebral body lesions anterior+posterior, obviously shape change in the vertebral body, paraspinal abnormal signal, presence of intraosseous or paraspinal abscess. Pathological and MRI examination was accurate for early differentiation of pyogenic spondylitis from brucella spondylitis.

BACKGROUND:The reasons for spinal imbalance include spinal deformity, spinal degenerative disease osteoporotic vertebral compression fractures. We believe that the power factor (back muscle) plays a key role in spinal sagittal imbalance. OBJECTIVE:To analyze the reasons for spinal sagittal imbalance by observing clinical manifestations and therapeutic outcomes in patients with osteoporotic vertebral compression fractures. METHODS:A total of 41 patients with osteoporotic compression fractures combined with spinal sagittal imbalance were retrospectively analyzed from January 2012 to May 2013. Al patients were subjected to percutaneous bal oon vertebroplasty under local anesthesia. Before treatment, they received bone density, standing ful-spine lateral X-ray, CT and MR imaging with injured vertebrae as the center. Using standing ful-spine radiographs, the height of anterior border of the injured vertebrae, Cobb angle of kyphosis and improved angle, wedging angle of the injured vertebrae and improved angle were measured. The patients underwent weight loading test and walking test. Preoperative and postoperative data were compared. RESULTS AND CONCLUSION:The patients affected spinal sagittal imbalance symptoms, so the walking distance was significantly shorter than that postoperatively (P<0.05). Moreover, the time of weight loading test was significantly shorter than that postoperatively (P<0.05). In standing ful-spine radiographs, the average difference of Cobb angle was (10.01±0.76)°. The mean difference of vertebral wedging improvement was (4.84±0.40)° (P<0.05). Al patients were fol owed up. Low back pain and sagittal imbalance symptoms were relieved. No severe complications appeared after percutaneous bal oon vertebroplasty. Results indicated that patients with osteoporosis compression fractures can affect the symptoms of spinal sagittal imbalance, which is not only induced by wedging of the injured vertebra. In addition, after percutaneous bal oon vertebroplasty, imbalance symptoms are apparently improved, suggesting that back pain after spinal fracture limits back muscle strength and is an important cause for spinal sagittal imbalance.

Objective To study the effect of Rho-kinase inhibitor on experimental allergic neuritis. Methods 54 female Lewis rats were divided into three groups; EAN group, EAN + Rho-kinase inhibitor group, and CFA group. The rats were sacrificed on the 9th day, 17th day, and 26th day after immunized. The changes of weight, EAN incidence, and mean day of onset, mean maximum clinical score, and histopa-thology were observed. Results The clinical course in EAN group reached peak on the 17th day. Compared with EAN group, the weights of Rho - kinase inhibitor group were increased, while EAN incidence, mean day of onset delay, and the clinical scores in Rho-kinase inhibitor group were significantly decreased, ( P < 0.01) , and the demyelization and inflammation cells infiltrating was ameliorated in spinal nerve. CFA group didnt show any clinical manifestation. Conclusions Rho - kinase inhibitor may ameliorate the development of EAN through inhabiting the Rho/ROK signal pathway.

Objective To study the expression of mRNA of OX40 and OX40L in the sciatic nerve,spleen,peripheral blood mononuclear cells and lymph nodes of EAN under the influence of Rho-kinase inhibitor.Methods All 54 female Lewis rats were divided into 3 groups:the EAN group,the EAN+ Rho-kinase inhibitor group and the complete Freund's adjuvant(CFA)group.The rats were sacrificed at 9,17 and 26 days after immunized.Ox40 and OX40L mRNA were detected by RT-PCR which came from spleens,sciatic nerves,peripheral blood mononuclear cells and lymphonodes.Results In EAN+ Rho-kinase inhibitor group,the mRNA expression of OX40 were 0.266±0.031,0.298±0.024 and 0.113±0.018 at 9.17 and 26 days in the sciatic nerve,the expression were 0.453±0.030,0.496±0.100 and 0.220±0.016 in the lymph nodes.The mRNA expression of OX40L were 0.247±0.018.0.298±0.026 and 0.165±0.013 in the sciatic nerve,the expression were 0.283±0.027,0.306±0.011 and 0.161±0.012 in the lymph nodes.The mRNA expression of OX40 and OX40L in EAN+Rho-kinase inhibitor group was lower than EAN group at the three time points(t=2.24-4.89,P＜0.05),and the demyelination and inflammation cells infiltrating were ameliorated in spinal nerve.CFA group didn't show any clinical manifestation.Conclusion Rho-kinase inhibitor may ameliorate tlIe development of EAN through inhabiting the OX40 and OX40L activation.

This study is to report the establishment of an UPLC-MS/MS method for the determination of plasma concentration of UA carried in self-microemulsifying drug delivery system (SMEDDS) and its pharmacokinetics in rats. It was used for determination and analysis when serum with internal standard was extracted from C18 solid-phase column. Acquity UPLC BEH C18 column (100 mm x 2.1 mm, 1.7 microm) was used for separation. The mobile phase was acetonitrile -0.1% ammonia with gradient elution at the flow rate of 0.2 mL x min(-1). The column temperature was 40 degrees C and the detection wave length was 210 nm. It was detected by negative ion using electrospray ionization source (ESI) and scanned by multiple reaction ion monitoring (MRM) mode. The liner relationship of UA was very good in the range of 1.19-3 815.00 ng x mL(-1) (r = 0.999 0). Recovery rate of different concentrations were 87.42%-89.95%. The precision of inter-day and intra-day were less than 11%. The method developed in our study was proved to be sensitive, rapid and simple. It is suitable for the pharmacokinetic study of UA-SMEDDS in rats.

Objective To evaluate the effect of hypoxemia factor on hippocampal long-term potentiation(LTP)in newborn rats undergoing propofol anesthesia. Methods Forty-two pathogen-free healthy Sprague-Dawley rats(21 males,21 females),aged 7 days,weighing 14-18 g,were divided into 3 groups(n=14 each)using a random number table:propofol plus air group(group PA),propofol plus pure oxygen group(group PO)and intralipid plus pure oxygen group(group IO).Propofol 50 mg/kg was injected intraperitoneally once a day for 7 consecutive days in PA and PO groups. Intralipid 5.0 ml/kg was injected intraperitoneally once a day for 7 consecutive days in IO group. The rats were exposed to air or pure oxygen for 6 h after the end of each injection. The arterial oxygen saturation and respiratory rate were determined after administration. The rats were returned to the cage after recovery of righting reflex. Six rats in each group were selected for preparation of hippocampal slices at 24 h after the last injection on 7th day,and the electric stimulation-induced field excitatory post synaptic potential(fEPSP)and success rate of LTP induction were recorded. Morris water maze test was performed in the other rats at 2 weeks after administration to assess the cognitive function. Results Compared with group IO,the respiratory rate,amplitude of fEPSP and success rate of LTP induction were significantly decreased,and the escape latency was prolonged in group PO(P<0.05).Compared with group PO,the arterial oxygen saturation,amplitude of fEPSP and success rate of LTP induction were significantly decreased,the escape latency was prolonged,and the number of crossing the original platform was decreased in group PA(P<0.05).Conclusion Hypoxemia factor increases propofol-induced neurotoxicity in the central nervous system of newborn rats.