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 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 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 assess the clinical effect and safety of human blood albumin in treatment of acute cerebral hemorrhage.Methods 88 patients with acute cerebral hemorrhage were randomly divided into 2 groups. Both group were given routine therapy and 45 cases in the treating group were given additional 10% human blood albumin 100 ml ivgtt once a day for 7 days.14 days served as a treatment course.Neurofunction was compared before and after treatment.Results Both the effective rate (P<0.05) and the neurofunction in the treating group were better than that of the control group (P<0.05).No advert effect happened.Conclusion Human blood albumin is an efiective and safe medicine for treatment of acute cerebral infarction.

[Objective] To explore how to deal with the paternity test of complex adoption cases. [Method] Samples from 13 families, in which adoptive parents were suspected related to biological parents, were genotyped using "Identifder + Sinofder + Powerplex 16" combined system (D8S1179, D21S11, D7S820, CSFIPO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818, FGA, D6S1043, D12S391, PentaD, PentaE) followed by further statistical analysis. [Result] Among all 13 cases, 2 were completely accordance with the Mendel law, PI > 10 000. There found more than 3 inconsistent loci in 8 cases. And found 1～2 inconsistent loci in 3 cases, needed to test more STR loci until PI≥10 000. The half sibling index (HSI) was also calculated with ITO method. The adoptive parents of 2 cases were not excluded from a full sibling with biological parents. In addition, Y-STR loci were tested for 4 cases (father/son). Two adoptive fathers of them were not excluded from the paternal relationship with biological fathers. [Conclusion] The most (76.9%) of all (13) complex adoptive cases of paternity test could be drawn a definite conclusion with combined system of "Identifder + Sinefiler + Powerplexl6". Minority (23.1%) of them was not definite yet and needed testing more STIR loci. Meanwhile, we suggested adding Y-STR tests and providing HSI for reference.

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 feasibility of green fluorescent protein (GFP) as a marker to trace the transplanted um-bilical cord mesenchymal stem cells (ucMSCs) in rats with cerebral ischemia-reperfusion .Methods ucMSCs were transfected by GFP-adenovirus .The rats were subjected to left middle cerebral artery occlusion using suture method .1 × 106 GFP-ucMSCs were transplanted with cerebral stereotaxic technique .Frozen sections of brain tissue were made at 7 d after cerebral ischemia .The ex-pression of GFP was observed by fluorescence microscope .Results In vitro ,the morphology of GFP in ucMSCs was fibrous ,and when fused ,the clusters were arranged in a radial or whirlpool shape ,The morphological feature of transfected ucMSCs was similar to that un-transfected ucMSCs .Under the fluorescence microscope ,the positive rate of GFP was more than 80% .In addition ,GFP could spread to the whole cells and show the completed form .No rejection was observed in the rats after transplantation ,and the GFP was found near the site of transplantation after 7 d ,and the fluorescence was not attenuated .Conclusion GFP is an effective tracer maker for ucMSCs transplantation in the treatment of ischemia-reperfusion ,which could provide experimental method for fur-ther study .

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.