Objective To construct the eukaryotic expression plasmid of recombinant human hypoxia-inducible factor-1?(HIF-1?) and get ready for the coloning and expressing of HIF-1? gene. Methods The total RNA was isolated from blood cells,and cDNA library was constructed by reverse transcription PCR method.PIREGFP containing EGFP fragment and cDNA were used as templet,the three designed primers (EGFP-linker,HIF-1? upstream and HIF-? downstream)were put into,after amplification the target gene fragment was inserted into the shuttle vector T,and transfected with E.coli DH5?,the bacterial colonies containing recombinant plasmids were identified by LB/KANA-agar plate,and the recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.The correct sequences were cloned into the pVAX1 vector.Results The amplified fragments were about 870,1 199,672 bp by PCR,they were inserted into the shuttle vector T and sequenced. The result of sequencing was identical with that provided by GenBank.The final plasmid about 1 800 bp was obtained by the identification of enzyme digestion and it had same molecular size as expected.Conclusion The eukaryotic expression plasmid pVAX1-EGFP-linker-HIF-1? of recombinant human HIF-1? is successfully constructed.

Objective To observe the effect of heat stress-induced burst out of reactive oxygen on neuronal apoptosis and investigate pathogenesis of brain damage caused by severe heat stroke.Methods Neurons heat stress model is set up.Control group were incubated at 37 ℃,5%CO2 ,While heat stress group of cells were incubated at 43 ℃for 2 h,then all the cells were further incubated at 37 ℃for different time as indicated.The amounts of ROS were assayed by DCFH staining at 0 h,0.5 h,1 h,2 h after heat stress.Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining and expression of caspase-3 were determined by westen blotat 0 h、3 h、6 h、12 h after heat stress.In addition,MnTMPyP is the specificscavengers of ROS,which effect on apoptosis is also studied at 12 h after heat stress.Results Compared with control group,amounts of ROS was significant increased at 0 h after heat stress,the burst out of it was at 2 h after heat stress (P<0.05 ).Apoptosis was induced at 3h after heat stress ,it was significant increased at 12 h after heat stress (43.2%,P<0.05 ).The expression of caspase-3 was also significant increased at 12 h after heat stress (P<0.05 ),and its trend was consistent with apoptosis rate trend.In addition,the scavengers MnTMPyP significantly decreased the apoptosis (47.42% to 18.45%, P<0.05 )and expression of caspase-3 at 12 h after heat stress.Conclusions An upstream signaling molecules,ROS could mediate heat stress-induced neuronal apoptosis,but its intermediate mechanism needs for further studies.

Traumatic pseudoaneurysms (TPA) were surgically produced in the right common carotid artery of 24 rabbits. Three to four weeks later the survived rabbits 16 were randomly divided into 3 groups: (1)control group; (2)treatment group with TPAs embolized with microcoils(MC); (3)treatment group with occlusion of the by parent artery with MC. Three months after embolizaton, the TPAs were examined by digital subtraction angiography (DSA). The results showed that the TPAs embolized with MC were proved to be completely occluded, and the parent artery remained patent. Histopathological examination showed that TPAs were replaced by a mass of scar tissues. In the rabbits with occlusion of the parent artery embolization with MC, the TPA also disappeared. However, all the untreated rabbits died of rupture of TPAs. Statistic analysis showed that both methods of treatment were effective in the treatment of TPA.

Objective:To construct and evaluate a novel tissue-engineered bone composed of murine stromal cell-derived factor 1(mSDF-1), simvastatin (SIM) and collagen scaffold (Bio-Oss?), serving as a cell-homing approach for bone formation .Methods: In the study , 32 ICR mice were randomly divided into 4 groups,each group including 8 mice.The drug-loaded collagen scaffolds were implanted subcutaneously onto the cranium of each mouse according to the groups: ( 1 ) 1 ∶50 ( volume ratio ) dimethyl sulfoxide ( DMSO ) /phosphate-buffered saline ( PBS ) solution +collagen scaffold ( blank control group ); ( 2 ) 10 -3 mol/L SIM solution +collagen scaffold ( SIM group ); ( 3 ) 200 mg/L mSDF-1solution +collagen scaffold (mSDF-1 group); and (4) 10 -3mol/L SIM +200 mg/L mSDF-1 solution +collagen scaffold ( SIM +mSDF-1 group) .One week after implantation , the mice were trea-ted by injecting the same drug solution mentioned above around the scaffold once a day for two days .The specimens were harvested 6 weeks after implantation and the bone formation was evaluated by soft X-ray analysis , HE staining and immunohistochemical staining .Angiogenesis of each group was checked by calculation of vessels in each tissue section .Results:Six weeks after implantation , the collagen scaffolds were retrieved.The value of gray scale for the SIM +mSDF-1 group[(421 836.5 ±65 425.7) pixels] was significantly higher than that of the blank control group [(153 345.6 ±45 222.2)pixels, P<0.01], the SIM group [(158 119.2 ±100 284.2) pixels, P<0.01], and the mSDF-1 group[(255 529.5 ± 152 142.4) pixels, P <0.05 ]; HE staining analysis revealed that significant bone formation was achieved in the SIM +mSDF-1 group; The immunohistochemical staining showed the existence of os-teopontin and osteocalcin in the SIM +mSDF-1 group; There were more vessels in the SIM +mSDF-1 group[(46 ±8)vessels/mm2] than in the blank control group [(23 ±7) vessels/mm2, P<0.01], and the SIM group[(24 ±6) vessels/mm2 , P<0.01].Conclusion:The novel tissue-engineered bone com-posed of mSDF-1, SIM and collagen scaffolds has the potential to form bone subcutaneously in vivo.It re-presents a novel method of in vivo bone re-generation without seed cell delivery .

Early detection and treatment of high-risk adenomas and colorectal cancer (CRC) can reduce mortality of this disease.CRC screening is aimed at minimizing its harm and colonoscopy is presently the gold standard for it.However,colonoscopy needs bowel preparation and is invasive with high risk of intestinal perforation,causing a bad compliance,which is unfavorable to its popularization and application.Recently,non-invasive detection methods for CRC have gone through a rapid development.Tests based on CRC-related biomarkers in fecal and blood samples provide new options for non-invasive CRC screening.However,detection methods for these biomarkers still need further research and improvement because of the complex composition of feces and blood.In the two aspects of fecal tests and blood tests,the progress of recent studies on non-invasive screening methods for CRC was reviewed in this article.

Objective To compare anesthetic effect and safety of general anesthesia and combined spinal and epidural anesthesia used in autonomic nervous hyperresponsive surgery for patients with paraplegia.Methods 26 paraplegic patients were randomly divided into two groups-control group and treatment group from February 2011 to November,2015,each with 13 cases.The control group used general anesthesia,while the treatment group used combined spinal and epidural anesthesia,to observe onset time,duration,intraoperative hemodynamic changes and complications,Complications,length of stay and cost,Days and costs of hospitalization,satisfaction of patients and their families,of anesthesia in two groups.Results The dosage of narcotics and the onset time of the treatment group were better than that of the control group.The difference between the two groups was significant,and had statistical significance (P＜0.05) Two groups of patients after surgery,diastolic blood pressure,systolic blood pressure and heart rate were lower in the treatment group than in the control group,and had statistically significant difference (P＜0.05);The postoperative complications of the treatment group were significantly better than those of the control group,and had statistically significant difference (P＜0.05);There were statistically significant differences in postoperative pain degree between the two groups (P＜0.05);Two groups of patients in hospital days,hospital costs,satisfaction rate had statistically significant difference,Have statistical significance (P＜0.05).Conclusion In autonomic nervous hyperresponsive surgery for patients with paraplegia,anesthetic effect and safety of combined spinal and epidural anesthesia is significantly better than that of general anesthesia,featured by the rapid onset of action,long duration,fewer complications,strong safety and patients' great satisfaction.It is worth generalizing and applying clinically.

SUMMARY In this article , different methods to deal with teeth fractures were discussed by presenting a case of traumatic crown-root fracture in the anterior esthetic zone .The traumatic crown-root fracture is a common problem in clinic .When a fracture line locates in close proximity to or below the alveolar bone crest , the fracture most likely involve the junctional epithelium and the connective tissue attachment .This type of fracture becomes a challenge for restorative dentists because it involves biologic , functional , and es-thetic considerations , especially when the fracture occurs in an esthetic area .In this case , a young patient presented with two fractured upper anterior teeth to the Department of Periodontics , Peking University School and Hospital of Stomatology .After the comprehensive clinical evaluation , the right central incisor was decided to extract for implant therapy and the right lateral incisor was decided to retain by one modified crown lengthening surgery .The most common technique applied to save a retained root is a clinical crown lengthening procedure .However , the aggressive alveolar bone resection of both target and adjacent teeth to reestablish the bone width and periodontal health may compromise functional and esthetic outcomes .To re-duce loss of excessive osseous tissue during osteotomy procedure , the modified crown lengthening of the right lateral incisor was performed , including minor bone resection and root reshaping .Regarding the right central incisor , the retained root was all located below the alveolar bone crest .The extraction and implant procedure , combined with guided bone graft were performed to avoid the damage to neighbor teeth during traditional restorative therapy and to reshape a preferable buccal contour .At the last visit , the patient was recalled with healthy periodontium , normal tooth function and favorable esthetic results .

Objective:Human adipose-derived stem cells (hASCs)are a highly attractive source in bone tissue engineering.To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs ) in vitro,and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vi-vo.Methods:The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OCpro-Luc-Puro construct.Then,the OCpro-Luc-Puro construct together with three assistant vectors:pM-DLg/pRRE,pRSV-REV,and pVSVG,were transiently transfected into HEK293T cells followed by viral supernatants collection,filtration and concentration.Next,the hASCs stably expressing luciferase repor-ter gene driven by osteocalcin promotor were created with the lentivirus carrying OCpro-Luc-Puro cassette under puromycin selection.The OCpro-Luc-hASCs were then cultured in the absence or presence of osteo-genic differentiation medium.On the 7th and 1 4th days,after osteogenic induction,cellular extracts were collected and analyzed by luciferase reporter assay.Meanwhile,alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR)analysis of osteogenic associated genes osteo-calcin (OC),runt-related transcription factor 2 (Runx2)and alkaline phosphatase (ALP)were used to assess the osteogenic differentiation ability of OCpro-Luc-hASCs.Results:OCpro-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated.On the 7th and 1 4th days after osteogenic induction,the luciferase activity of the cellular extracts from OCpro-Luc-hASCs was dramatically increased.Consistently, the extracellular matrix mineralization,as shown by Alizarin red S (ARS)staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC,Runx2 and ALP, although to variable extent,was observed upon osteogenic differentiation.These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OCpro-Luc-hASCs.Conclusion:We established a luciferase reporter system that could be used to rapidly,quantitatively and specifically determine osteo-genic differentiation ability of hASCs.Comparing with the traditional time-consuming methods,the system we generated here was highly effective.This system not only can be used to examine ostogenic differentia-tion of hASCs in a high throughput manner,but also provides a way to monitor ostogenic differentiation of cells in vivo.

Objective To observe the effects of Qutantongluo decoction on the expression of TGF-β1 in renal tissue of diabetic nephropathy(DN) rats,which can help to understand the mechanism of action.Methods The rats models of diabetic nephropathy were established by injecting with streptozotocin intraperi-toneally.All Wistar rats were randomly divided into 6 groups:the normal control group,the model control group,Benazepril Hydrochloridec group ( 1 mg/kg ·d-1 ),Qutantongluo decoction low dose group (3 g/kg ·d-1 ),Qutantongluo decoction median dose group (4.5 g/kg · d-1 ),and Qutantongluo decoction high dose group (6 g/kg · d-1 ).Rats in the normal control group and the model control group were given celiac perfusion of distilled water once at the same time and dose.All the rats were gavaged once daily for 8 weeks.The expression of TGF-β1 at the level of gene and protein in renal tissue were tested by immunohistochemistry and RT-PCR methods.Results Compared with the model control group(2.79±0.22),the expression of TGF-β1 at the level of protein in renal tissue of Benazepril Hydrochloridec group (1.55 ±0.12)and the Qutantongluo decoction low dose group ( 1.54± 0.16),Qutantongluo decoction median dose group (1.49 ± 0.17),Qutantongluo decoction high dose group(l.39±0.25) decreased significantly (P＜0.05).Compared with the model control group (0.35±0.07),the expression of TGF-β1 mRNA in renal tissue of Benazepril Hydrochloridec group(0.35± 0.07)and the Qutantongluo decoction low dose group(0.39±0.03),Qutantongluo decoction median dose group (0.35± 0.06),Qutantongluo decoction high dose group (0.32± 0.07) decreased significantly (P＜0.05).Conclusion Qutantongluo decoction can down regulate the expression of TGF-β1 in kidney,which may be one of the mechanisms of Qutantongluo decoction effectiveness in clinical treatment of diabetic nephropathies.

BACKGROUND:Hyperpyrexia can induce a wide range of cel apoptosis in organisms, but no study has introduced the mechanism of heat stress-induced neuronal apoptosis.
OBJECTIVE:To observe the effect of nuclear factor kappa B (NF-κB) signal pathway on heat stress-induced neuronal apoptosis through reactive oxygen species.
METHODS:Heat stress model was established in the cel incubator. Heat stress group of cel s were incubated at 39,41,43℃for2hours,whilecontrolgroupofcelswereincubatedat37 ℃in5% CO2 for 2 hours. Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. The expression levels of caspase-3 and p-NF-κB65 were determined by western blot analysis. The amounts of intracel ular reactive oxygen species were assayed by DCFH staining. In addition, the effect of MnTMPyP and PTDC on heat stress-induced apoptosis was also studied.
RESULTS AND CONCLUSION:39 ℃ heat stress had no impact on the apoptosis, 41 ℃ heat stress induced a smal amount of apoptosis (10.19%), and 43 ℃ heat stress triggered a large amount of apoptosis (43.02%). The expression of caspase-3 and p-NF-κB65 was increased, in a temperature-dependent manner. In addition, both MnTMPyP and PTDC significantly decreased the heat stress-induced apoptosis and expression of caspase-3 and p-NF-κB65. Experimental findings indicate that, the increase of intracel ular reactive oxygen species may induce neuronal apoptosis, and NF-κB participates in the heat stress-induced neuronal apoptosis as the intermedial signal pathway.