Retrieval of ancient DNA (aDNA) sequences from organism remains provide direct view of their evolutionary history. However, researches on aDNA have suffered from lots of technical problems. Specifically, discredited sequences were generated from damaged aDNA templates, and expensive and time-consuming methods were employed. Here, a method which could recover the endogenous aDNA as well as to reduce the cost and research period is described. This is achieved by improving the ancient DNA extraction method of isopropanol precipitation, and reevaluating the method of PCR after N-glycosylase (UNG) treatment, which could remove the damaged DNA from the aDNA extract. The efficiency of these methods were tested by comparing with traditional methods using ancient specimens of pig teeth aged between 4 300 years before present (BP) and 3 900 BP. The results showed that: firstly, the extraction efficiency of the improved method of isopropanol precipitation and current method with silica-based spin column were all 60%. Furthermore, the research period at least could be reduced by half with the application of the improved methods and the cost to 1/10 of the current method. Secondly, sequences obtained through the method of PCR after UNG treatment were 100% authentic. In contrast, 66%～ 88% sequences were authentic based on the results obtained with the method of multiple PCRs without UNG treatment. And the research cost and period needed by the method with UNG treatment were only half of the later one. These results demonstrate that the improved extraction method of isopropanol precipitation combined with the method of PCR after UNG treatment could increase the success rate of authentic DNA amplified and at least reduce the research cost and period by half. Therefore, this method can be applied in the large-scale detection of ancient specimens.

Objective:To study the influence of mesenchymal stem cells(MSC) infected by AdIL-12 on the proliferation of C6 glioma cells.Methods: The MSCs were cultured from rat bone marrow and verified by immunohistochemistry and flow cytometry.Recombinant adenovirus vectors harboring IL-12(AdEasy-IL-12) were infected into MSCs to construct AdIL-12-MSC containing exogenous IL-12.RT-PCR and Western Blotting were used to detect IL-12 mRNA and protein expression in AdIL-12-MSC.MTT method was used to detect the effect of AdIL-12-MSC supernatant on the proliferation of C6 glioma cells.Results: Exogenous IL-12 gene was effectively transfected into MSCs by recombinant adenovirus vectors.IL-12 was expressed in MSCs at both mRNA and protein levels as detected by RT-PCR and Western Blotting.The supernatant of AdIL-12-MSC significantly inhibited the proliferation of C6 glioma cells compared with MSC supernatant(P

Objective High mobility group box-1 protein ( HMGB1) plays an essential role in regulating energy metabolism of tumor cells via affecting mitochondrial autophagy .The aim of this study was to explore the effect of HMGB 1 on mitochondrial biogene-sis and cell energy metabolism in anoxia environment . Methods HepG2 cells were divided into normoxia control group ( cells were cultured in a culture box containing 5%CO2) , hypoxia control group ( cells were cultured in a culture box containing 1%O2+5%CO2+94%N2 ) , hypoxia HMGB1 siRNA group ( cells were cultured in a culture box containing 1% O2+5% CO2+94% N2 after transfected with HMGB1 siRNA) and hypoxia NC siRNA group ( cells were cultured in a culture box containing 1%O2+5%CO2+94%N2 after trans-fected with negative control siRNA ) .MTS assay was carried out to measure cell proliferation rate .The alterations of mitochondrial bio-genesis associated proteins were detected by RT-PCR and western blot.Mitochondrial density and morphology were determined by transmission electron microscopy (TEM).The ATP content in whole cell extracts was determined with a colorimetric ATP detection kit . Results Compared with the hypoxia control and hypoxia NC siR-NA group, the proliferationof hypoxia HMGB1 siRNA group was significantly inhibited, especially in 48 h and 72 h(P<0.05).Com-pared with hypoxia control group and hypoxia NC siRNA group , the expression of PGC 1α, NRF1and TFAM in hypoxia HMGB1 siRNA group were decreased significantly ( P<0.05) .Western blot results showed that , compared with normoxia control group , the expressions of PGC1α(0.494±0.210 vs 0.090±0.020), NRF1(1.080±0.470 vs 0.581±0.190)and TFAM(1.585±0.340 vs 0.792±0.350) protein in hypoxia 24 h group were increased obviously ( P<0.05) .Compared with hypoxia control group and hypoxia NC siRNA group , the expres-sions of PGC 1α, NRF1 and TFAM protein in hypoxia HMGB1 siRNA group were significantly decreased (P<0.05).Compared with hypoxia control group , the content of ATP in the HMGB 1 siRNA hypoxia group was significantly decreased , and hypoxia 12 h and 24 h were the most obvious ( P<0.05) . Conclusion HMGB1 could maintain cell energy metabolism by regulating mitochondrial biogene-sis so that cells could continue to proliferate in adverse anoxia environment .

AIM:To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcino-ma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells.METHODS:The RNA from SMMC-7721 cells and HL-7702 cells was extracted.SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine.After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation.Flow cytometry was used to detect cell cycle and apoptosis.The changes of migration ability were investigated by Transwell invasion assay.RESULTS:The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells ( P<0.05 ) .Com-pared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells trans-fected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P<0.05).The percenta-ges of G1 phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migra-tion ability was inhibited (P<0.05).Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P<0.05).When miR-141 was down-regulated, the percentages of G1 phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P<0.05).CONCLUSION:miR-141 is down-regulated in human hepatocarcinoma cell line.Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells.miR-141 may function as a tumor suppressor gene during HCC development.

Objective To investigate the effect of ligustrazine on the intracellular translocation of Smad protein in HSC-T6 cell line.Methods HSC-T6 cell was cultured with ligustrazine at the dose of 10-5 mol/L in the culturing dish for 2 hours.After the culturing,the translocation of Smad-2 and Smad-4 proteins was detected by immunofluorescence assay.Results There was no evidence of the translocation of Smad-2 and Smad-4 proteins in HSC-T6 cell after the culturing.Conclusion Ligustrazine can block the signal pathway of TGF-?/Smad,which may be one of its important mechanisms of inhibiting the proliferation of HSC-TS cell.

Objective: To investigate a new method for improving the therapeutic effect on malignant glioma. Methods: A new type of killer cells, named GS-LAK, was induced by means of costimulating the peripheral ginsenoside(GS) and interleukin-2 (IL-2). Comparing with control group-LAK cells, cytotoxicity of GS-LAK cells against malignant glioma cells(BT325) was examined with MTI method. Results: It showed that GS-LAK cells exhibited some advantages over LAKcells in proliferation, cytotoxicity, as well as the utilizing of IL-2. Conclusion: The application of GS-LAK cells mightopen a new prospect to clinical therapeutic approach to malignant glioma.

As China has not yet established a sound regional trauma treatment system and standardized trauma centers at all levels, the trauma treatment capability in China is poorer than that in the developed countries. At present, Shaanxi Province has not established a regional trauma treatment system and standardized trauma centers at all levels. Based on the analysis of the characteristics of geography, population and social environment in Shaanxi Province, the authors explore the concept of the trauma treatment system and the construction of trauma centers at all levels in Shaanxi Province on the platform of the trauma center of Shaanxi People's Hospital ( Grade I trauma center) . The authors clarify the respective hardware facilities, team structure, treatment process and quality control goals, training and management system of professional trauma teams in trauma centers at all levels, so as to provide reference for improving the overall level of trauma treatment in Shaanxi Province.

BACKGROUND: Bone defect difficult to manage clinically and it is a big challenge to repair it. Secondary metabolites source from herb has shown potential for the treatment of bone defect. METHODS: Mesenchymal stem cells (MSCs) were isolated from mice and incubated with urolithin A (UA) (10, 25, and 50 lg/mL). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to estimate apoptosis and mineralisation was evaluated by alkaline phosphatase assay and alizarin red S staining. A middle femoral defect was induced in mice and bone tissue was prepared for endochondral ossification by treating with UA. The effect of UA was estimated by determining markers of osteoblast proliferation in serum and micro-computed tomography to analyse bone defects. RESULTS: UA enhanced mineralisation of MSCs and osteogenic gene markers in MSCs in vitro. Also, the bone defect score and bone mineral density were improved by UA. Moreover, UA ameliorated the altered Wnt3a protein and histopathological changes in bone defect mice. CONCLUSION Presented report conclude that UA enhances osteoblast proliferation in bone-defect mice by activating the Wnt pathway.

Objective To study the role of high-mobility group protein 1 (HMGB1) in the promotion of diethylnitrosamine-induced liver cancer formation in C57BL/6 mice and its mechanism.Methods HMGB1loxp/loxp/Alb-Cre+/-were used as a liver-specific knockout (KO) of HMGB1 gene in mice.HMGB1loxp/loxp/Alb-Cre-/-,HMGB1loxp/WT/Alb-Cre+/-and HMGB1loxp/WT/Alb-Cre-/-born in the same litter were wild-type mice.Six 12-day-old male WT and KO mice were separated and given a single intraperitoneal injection of diethylnitrosamine (25 mg/kg).Six months later,HE staining was used to evaluate the histopathological changes and then the incidence of liver cancer in each mice group was calculated.Serum samples were taken from each mice group to determine alanine aminotransferase levels.Immunohistochemical staining was used to detect the expression and intracellular localizations of HMGB1 protein status in tumor tissue of the two groups of mice.Western blot was used to detect the expressional condition of mitochondrial biogenesis in tumor tissue of the two groups of mice.RT-PCR was used to detect mitochondrial DNA copy number of tumor tissue and normal liver tissue in the two groups of mice.Intra and inter group data comparison was compared using t-tests and one one-way analysis of variance.Results Compared with WT mice,the liver/body weight ratio of KO mice was decreased significantly (t =2.634,P =0.0225).Serum alanine aminotransferase levels in both groups of mice were increased,and the difference was not statistically significant (t =0.4062,P =0.6932).There were many visible gray-white nodules of different sizes on the liver surface of WT mice,and the histological type was hepatocellular carcinoma.There was no statistically significant difference in the incidence of liver cancer among different genotypes of WT mice (P ＞ 0.05).The incidence rate of liver cancer in KO mice was significantly reduced (t =8.521,P ＜ 0.001).Compared with WT mice,the expression levels of HMGB1 and mitochondrial biogenesis (PGC-1α and NRF1) was significantly reduced (t =6.238,4.852,P =0.0335,0.041) in tumor tissue of KO mice.Mitochondrial DNA copy number was decreased significantly (t =9.211,P ＜ 0.01).Mitochondrial DNA copy number in tumor tissue of WT mice was significantly higher than that in normal liver tissue (t =8.305,P =0.0142).Conclusion HMGB1 promotes the formation of diethylnitrosamine-induced liver cancer by inducing mitochondrial biogenesis.

OBJECTIVETo provide practical method for microscopic authentication of traditional Chinese medicine Gusuibu and its adulterants.

METHODBy means of light microscope, scanning electron microscopy and tissue section techniques, the morphology, the size of the rhizome scales and their bearing position in the original plants of Gusuibu and its adulterants, i. e. Drynaria roosii, D. delavayi, D. quercifolia and Pseudodrynaria coronans were analyzed.

RESULTThere were significant differences between scales length of D. roosii, D. delavayi and P. coronans, while there was no significant difference between that of D. roosii and D. quercifolia. The scale teeth of D. delavayi were usually curved, bifid and uneven distributed at the scale fringe, which was different from that of the other three species. The base of the scales sinks in epidermis in D. roosii, D. quercifolia, and P. coronans, while it bore at the raised part of epidermis in D. delavayi.

CONCLUSION[corrected] Morphology, size and bearing position of the rhizome scales have significant differences in the several species. Therefore, these characteristics can be applied to the identification of Gusuibu and its adulterants.

Medicine, Chinese Traditional ; methods ; Microscopy ; Microscopy, Electron, Scanning ; Polypodiaceae ; anatomy & histology ; classification ; Rhizome ; anatomy & histology ; classification