Objective To analyze whether tetramethylpyrazine could protect PC12 cells from injuries induced by caffeine,and to explore the mechanism of tetramethyipyrazine in the treatment of cerebral ischemia-reperfusion injury. Methods Caffeine was added to induce apoptosis of PC12 cells. Cytotoxicity was detected by CCK-8 assay. The electric potential of mitochondrial membrane was determined by flow cytometry. HMGB1 was detected by Western blotting. Oxidative stress was detected by ELISA. We observed toxicity of caffeine and the protective effects of tetramethylpyrazine. Results After the pre-treatment,tetramethylpyrazine significantly improved PC12 cell survival. Mitochondrial membrane potential was increased,the expression of HMGB1 decreased,SOD increased,LDH and MDA decreased,and GSH elevated. Conclusion Tetramethylpyrazine exerts a significant protective effect on PC12 cell injury caused by caffeine. The protective effect may be related to inhibition of apoptosis and regulation of the expression level of mediators involved in inflammation and oxidative stress.

Objective To observe the efficiency and toxicity of Vinorelbine(NVB) combined with Cisplatin (DDP) in the treatment of advanced breast cancer patients resistent to Adriamycin (ADM) or Taxol treatment.Methods 34 patients with 8 cases simplex carcinoma,20 cases infiltrating duct carcinomas,3 cases medullary carcinoma,1 case large sweat gland-like carcinoma and 2 cases scirrhous carcinoma,who were relaped and refractory after ADM or Taxol treatment,were treated with NVB 25mg/m2,DDP 25mg/m2,Ⅳ.Both drugs were given in a 21-days cycle.The efficacy was evaluated every 2 or 3 cycles by using response evaluation criteria in solid tumor.Results 2 cases of 34 assessable patients achieved complete response (CR),18 patients had partial response (PR),8 cases had stable disease(S D),6 cases had progressive disease(PD).The total effective rate was 58.8％ (CR + PR).The medium duration of response and medium survival time were 8.5 months and 18.3 months,respectively.The predominant toxicity was hematological,with grade Ⅲ～ Ⅳ leukopenia occurring in 50.0％ (17/34) patients.Other toxicities were nausea,vomiting,anemia,phlebitis and so on.Conclusion The regimen of NVB combined with DDP is active and well tolerated,with an acceptable price,in treatment of advanced breast patients with refractory and relapsed after ADM or Taxol treatment.

Objective To evaluate the normalized correction effect of the ROX reference fluorescence on the real-time quantitative RT-PCR assessment for the Nrf2 mRNA.Methods The Taq-man probe was utilized in the real-time quantitative RT-PCR method to assess the Nrf2 mRNA; The reaction specificity of the Nrf2 PCR products obtained from the rat cDNA was assessed using gel electrophoresis and sequencing identification.The PCR calibrators with different concentrations varied at 10 gradients(from 2.0 × 109 to 2.0 × 100) were prepared for real-time quantitative RT-PCR assessment,and then the influence of the ROX reference fluorescence correction on the linear range of the PCR standard curve was investigated experimentally.The recombinant plasmids with different concentration levels(e.g,high,medium or low concentration) were assessed by the real-time RT-PCR and 20 replicate measurements were performed for all the samples in both intra- and inter-assays.The repeatability differences of both the inter- and intra-assays were analyzed systemically with/without the ROX reference fluorescence correction.Results The primers and probes required for Nrf2 mRNA real-time quantitative RT-PCR assessments were constructed,and the reaction system and conditions were also established.The length of the amplified fragment agreed well with the expected length(122 bp),and the sequencing analysis showed the amplified fragment was verified by the sequencing identification.After applying the ROX reference fluorescence correction,the linear range ofthe PCR standard curve was measured as 2.0 × 109 -2.0 × 100 copies/μl(R2 ＞ 0.99),which was 100times wider than that measured without ROX reference fluorescence correction(2.0 × 109 - 2.0 × 102copies/μl,R2 ＞ 0.99).Without the ROX reference fluorescence correction,the intra-assay CVs of the Ct value were measured as 4.1%,2.7% and 2.1% for the recombinant plasmids with high-,medium-,and low-level concentration,respectively.The inter-assay CVs of the Ct value were 4.3% 、3.0% 、2.4%.By contrast,with the ROX reference fluorescence correction,the intra-assay CVs of the Ct value became 0.7%,0.5%,0.4% and the inter-assay CVs of the Ct value turned to 1.0%,0.8% and 0.7%.The discrete degree of the Ct value with the correction was lower than that without the correction.The intra- and interassay Ct values of the high- and medium-level samples exhibited statistic significance for the measurements with/without the correction.In the high-level group,the t values of the intra- and inter-assays were 4.843 and 2.566,with P＜0.05.In the medium-level group,the t values of the intra- and inter-assays were 4.293 and 4.423,with P＜0.05.However,no statistic significance was observed the low-level group(the t values of the intra- and inter-assays were 0.753 and 1.279,with P ＞.0.05).Conclusions The utilization of the ROX reference fluorescence in the real-time quantitative PCR assessment for Nrf2 mRNA could widen the linear range of the standard PCR calibration curve and improve the repeatability of the assessment,which should be quite helpful for accurately assessing the expression level of the Nrf2 mRNA.

Objective To compare the transfection efficiency of cationic polyethylenimine(PEI)with Lipofectamine 2000TM by using the plasmid DNA encoding vascular endothelial cell growth factor (VEGF165 )gene in human embryonic kidney cell line 293T.Methods PEI of different N/P ratio and Lipofectamine 2000TM were used to deliver the vector containing VEGF165 to 293T cells,respectively.Green fluorescent protein(GFP)gene was inserted into the vector as a report gene.Evaluation of cytoactive was performed by CCK-8 assay 24 h after transfection.The cells were observed by fluorescent microscope and the presence of VEGF165 in cell supernatant was detected by ELISA 48 h after transfection.The transfection efficiency was calculated and com-pared.Results Similar cytoactive and best transfection efficiency could be obtained when N/P ratio was 9,the transfection efficien-cy was around 70%.Furthermore,the presence of VEGF165 increased significantly after transfection(P <0.05),but there was no significant difference between the two groups in which different transfection methods were adopted.Conclusion PEI as a novel oli-gofectamine reagent could mediate more efficient transfection compared with lipofectamine.It also has low cell-toxicity and low price and could be an ideal vector in gene delivery technology.

Objective:To investigate the effect of Tongrnai Yangxin Pill on the serum hepcidin level of patients with Coronary Atherosclerotic Heart Disease and Anemia of Chronic Disease.Methods:Seventy patients with Coronary Atherosclerotic Heart Disease and Anemia of Chronic Disease whose age were above 40 years old were enrolled as the research group and divided into the medication group (group Ⅱ) (n=35) and the nonmedication group (group Ⅲ)(n=35),while 40 CAD patients were enrolled as the normal control group (group Ⅰ).Before and after medication,the Hepc,Hb,etc levels were compared between two groups.Results:Before medication,the levels of Hepc in the two research subgroup were significantly higher than that of the control group (P＜0.05).At 8 weeks after treatment,the Hepc level of group Ⅱ was significantly declined,and the level of Hb was increased than those before treatment (P ＜0.05);the Hepc levels of group Ⅰ and group Ⅲ showed no significant difference (P＞0.05),the Hepc levels of group Ⅱ and group Ⅲ were obviously higher than that of the control group (P ＜0.05),the Hepc levels of group Ⅲ was obviously higher than that of the group Ⅱ(P＜0.05),the Hb level of group Ⅲ was obviously lower than those of group Ⅰ and group Ⅱ (P＜0.05).Conclusion:Tongmai Yangxin Pill could reduce the level of Hepc and enhance the Hb levels of patients with Coronary Atherosclerotic Heart Disease and Anemia of Chronic Disease.It was useful to the patients with Coronary Atherosclerotic Heart Disease and Anemia of Chronic Disease,especially patients with mild anemia.

Objective To investigate the effect of hyperbaric-oxygen therapy on osteoprotegerin (OPG) and integrin β1 (Itgβ1 ) expression during mandibular distraction osteogenesis .Methods Forty New-Zealand rabbits were used .The animals were randomly divided into two groups (experimental group and control group) with 20 animals each ;after accomplished osteotomy and implant distraction devices on mandible bilaterally for 3 days of latency period ,the device was activated at the rate of 0 .8 mm per day for 10 days .All animals in the experimental group were subjected to hyperbaric oxy -gen for 90 minutes once a day since the beginning of distraction ,and lasted for four weeks .In control group ,all the distractors were activated following the same distraction protocol as the experimental group ,but without hyperbaric oxygen therapy .Five animals of each group were sacrificed at 10th day after distraction ,7th ,14th and 28th day of consolidation , respectively .The lengthened mandibles were harvested and processed for immunohistochemical examinations to detect OPG and Itg β1 expres-sion in the distraction gap .Semi-quantitative analysis was carried out by image analysis software . Results OPG staining was mainly located in the membrane and cytoplasm of osteoprogenitor cells and osteoblasts in the distraction zones .The expression of OPG increased after distraction accomplished and reached to the peak at 7th day of consolidation ,and then decreased gradually .At every time point , the level of expression of OPG in the experimental group was remarkably higher than those in control group .There were significant differences between the experimental group and control group ( P <0 .01) .Itgβ1 mainly located in actively proliferating osteoblasts ,fibroblasts and mesenchymal cells . The expression of Itgβ1 decreased significantly after reached to the peak at the 10th day distraction .At 10th day distraction ,7th and 14th day of consolidation ,Itgβ1 expressed more strongly than that in the experimental group ,which was remarkably higher than those in control group .There were significant differences between experimental group and control group (P < 0 .05) .At 28th day of consolidation , Itgβ1 expressed weakly ; there was no significant difference between the two groups ( P > 0 .05 ) . Conclusions Hyperbaric oxygen therapy can up-regulate the expression of OPG and Itgβ1 in the dis-traction gap ,which may promote osteoblast differentiation ,proliferation ,enhance osteoblast func-tion ,and new bone formation in distraction gap .

Objective: To explore the role of β-catenin in distraction osteogenesis of new bone formation, the expression of β-catenin in the distraction gap callus was detected during rabbit mandibular distraction osteogenesis. Methods: 26 New-Zealand rabbits were randomly divided into 3 groups. Group A is a normal control group with 2 rabbits, group B is the mandibular defect control group, and group C is the distraction group. Group B and C with 12 rabbits, respectively. The two rabbits in group A without surgery, their mandibles are normal control. In group B, vertical osteotomy was performed between the first molar and the mental foramen on the mandibles bilaterally, followed by rigid internal fixation with titanium plates and screw with 5 mm gap immediately. In group C, after the same osteotomy was performed, the fragments of mandibles were reduced and fixed with mandibular distractors bilaterally. On the fourth day postoperatively, the distraction started at a rate of 0.8 mm/d and lasted for 7 days, followed by consolidation period. Two rabbits of group B and C were sacrificed at 6th, 10th, 17th, 24th, 31st, 38th day postoperatively, respectively. The newly formed callus in the distraction gap of mandibles was harvested for Western blotting and immunohistochemistry examination to detect the distribution and expression of β-catenin. The experimental data were analyzed by SPSS 22.0 statistical software using Spearman function. Results: The result of Western blotting showed that the expression of β-catenin gradually increased at distraction period(6-10 days after surgery) and reached the peak at the end of the distraction period(10th day postoperatively), it gradually decreased during the consolidation period. However, the expression of β-catenin in group C was higher than that of group B. Immunohistochemistry stain showed that the expression of β-catenin mainly located in inflammatory cells(eg. monocyte), fibroblast of the granulation tissue, the osteoblasts, osteocyte on the surface of new formed trabecular, and the connective tissues surrounding the new bone in the new formed callus. Cytoplasmic and nuclear staining were positive. In group C, the expression of β-catenin was strong (3.245 8±0.132 3) after distraction (6th day postoperatively), and reached a peak (4.602 8±0.021 9) on the 10th day postoperatively. With the disappearance of the distraction stress, the expression of β-catenin gradually decreased since17th day postoperatively(3.639 8±0.125 5), but the staining was still positive. In group B, the strong positive staining of β-catenin on the 6th day after surgery (2.734 0±0.134 7), the strongest staining on the 10th day after surgery (3.101 3±0.104 8), and the expression of β-catenin on the 17th day after surgery (2.542 8±0.211 1) was weaker than that on the 10th day after surgery. At each time point, the expression of β-catenin in group C was significantly higher than that in group B, and the difference was statistically significant (r=0.943, P=0.005 6). Conclusions During mandibular distraction osteogenesis, the distraction stress activates the Wnt signal pathway to enhance the expression of β-catenin, it suggests that β-catenin plays an important role in the transformation of the mechanical signal to chemical signals during the process of distraction osteogenesis, and participates in the regulation of new bone formation during distraction osteogenesis.