Objective To investigate the feasibility of using clustered regularly interspaced short palindromic repeats ( CRISPR)-mediated genome editing to downregulate the expression of programmed cell death protein 1 (PD-1) on primary T cells by using a lentivirus delivery system. Methods Lentivirus vec-tors pLentiCRISPR A1-A6 containing different PD-1 genomic DNA sequences as single guide RNA ( sgRNA) for Cas9 targeting were constructed individually. The lentivirus vectors were tranduced into primary CD4 T cells. Flow cytometry analysis was performed to detect the expression of PD-1 for evaluating the knockout ef-ficiency. Results The lentivirus vectors pLentiCRISPR A1-A6 carrying six different target sites were con-structed and respectively tranduced into primary CD4 T cells. The expression of PD-1 accompanied with the activation of T cells. Co-expression of CD25 and PD-1 was observed on activated T cells. All of the six sites could be targeted by Cas9, of which A2 and A6 sites were more efficient in knocking out the gene encoding PD-1 with a rate of 19% and 29%, respectively. Conclusion This study suggests that it is feasible to knock out the expression of PD-1 on primary T cells by using CRISPR.

Objective To study the optimal enhancement phase and exact triggering scan time on spiral CT angiography of vertebral and carotid artery,and to improve the rate of 3D-CT display in vertebral and carotid artery.Methods One hundred and thirty-three cases of CT angiography of vertebral and carotid artery(87 vertebral and 46 carotid artery)were examined with SmartPrep software,which controled the scan at real time after 100 ml contrast medium(350 mg I/ml Omnipaque)were quickly injected through foot back vein.One hundred and thirteen cases of control group were examined by triggeing off the scanning with a delay time of 18—22 s after blous injection through foot back vein.The vertebral and carotid arteries were demonstrated with 3 dimensional reconstruction.Results The successful rate in SmartPrep group was 100% with the enhancement situated in best(n=130)or good(n=3)scanning phase.The threshold concentration values of target vessel were kept on 100 HU in the whole scan period[(18?4)s].However, the triggering time of enhancement scanning was not equal(15—43s)and the deviation was up to 28 s.The average concentration of target vessel was obviously higher in SmartPrep group[(161.4?2.0)HU]than in the control group[(133.3?2.2)HU](t=-9.456,P

Objective To evaluate MRI in the diagnosis of the bone contusion of the knee joint and its clinical significance.Methods Using special coil for knee joint,coronal,sagittal,axial and oblique sagittal plane scanning with fast spin-echo sequence(T_1WI,T_2WI,PDWI+FS)was performed on knee joint in 205 patients in three days after injury.According the distributing bone marrow edema and injury mechanism,bone contusion were classified five types as pivot shift injury,clip injury,dashboard injury, hyperextension injury and lateral patellar dislocation.Results One hundred and forty-five cases of the 205 patients were found bone marrow edema without fracture on X-ray films.Among them,pivot shift injury was found in 43 cases accompanied with anterior cruciate ligament rupture in 30 cases,tear of the posterior horn of the lateral or medial meniscus in 12 and tears of the medial collateral ligament in 8 cases;clip injury in 53 cases accompanied with anterior cruciate ligament rupture in 10 cases,tear of the posterior horn of the lateral or medial meniscus in 15 and tears of the medial collateral ligament in 38 cases;dashboard injury 40 cases accompanied with posterior cruciate ligament rupture in 16 cases,hyperextension injury 9 cases accompanied with anterior cruciate ligament rupture in 2 cases,posterior cruciate ligament rupture in 5 cases.No lateral patellar dislocation was found.Forty-eight of 145 patients had undergone arthroscopy, 43 cases(89.6%)of them were in accordance with MRI diagnosis.Bone contusion were defined as geographic regions of abnormal signal intensity,that is,low signal intensity in T_1-weighted images and high signal intensity in PD-weighted or T_2-weigeted images with fat saturation.Conclusion MRI can accurately display the location and area of bone contusion of the knee joint as well as its adjunctive structure injury and deduce their injury mechanism.MRI should be used routinely for knee trauma.

To investigate the metabolic rate and metabolites of 9-dehydro-17-dehydro-andrographolide, which is the main active ingredient in Xiyanping injection, by using the in vitro rat liver microsome incubation system. 9-dehydro-17-dehydro-andrographolide was incubated together with liver microsome mixed with NADPH. Its metabolic rate was studied by determining its residual concentrations with the UHPLC-MS/MS method; Its metabolites were identified by the UPLC-TOF-MS(E) method. The results showed that 9-dehydro-17-dehydro-andrographolide was metabolized faster than rat liver microsomes mixed with coenzymes, with t½ and CL of (19.7 ± 0.5) min and (35.1 ± 0.8) mL x min(-1) x g(-1) (protein), respectively. Based on the high resolution mass spectrum data and information from literatures, altogether nine metabolites of 9-dehydro-17-dehydro-andrographolide were identified in the incubation system, particularly hydroxylated and dehydrogenized products. The results of identification would provide a basis for screening out more active andrographolide derivatives.
Animals ; Chromatography, High Pressure Liquid ; Diterpenes ; chemistry ; metabolism ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Microsomes, Liver ; chemistry ; metabolism ; Molecular Structure ; Rats ; Tandem Mass Spectrometry

OBJECTIVETo explore the reasons of secondary fracture after percutaneous vertebroplasty (PVP) for osteoporotic vertebral compression fractures (OVCFs) and discuss the measure of prevention and cure.

METHODSFrom January 2011 to January 2013, the clinical data of 180 patients with primary OVCFs treated by PVP were retrospectively analyzed. There were 75 males and 105 females, aged from 68 to 95 years old with an average of (79.50 ± 5.45) years. The involved vertebrae were identified according to the clinical symptoms and imaging data. PVP were performed in 362 vertebrae and the patients were followed up with an average of 12 months. Subsequent vertebral fractures were found through the pain's reappearance and MRI or bone scan. The patients were divided into secondary fracture group and no-secondary fracture group according to the subsequent fractures or no. Secondary fracture group was divided into two groups according to gender, and the patients with secondary fracture were also categorized into the original surgical vertebral fractures, adjacent vertebral fracture and remote vertebral fractures. The age, gender, the cement volume, the cement leakage, secondary fracture site, the incidence and type of secondary fracture were observed and compared among different groups.

RESULTSAmong the 362 vertebrae of PVP, there were 109 vertebrae in male and 253 vertebrae in female. And 27 vertebrae (10 in male and 17 in female) of 22 cases (9 males and 13 females) occurred secondary fracture. The second PVP were performed in 13 cases (16 vertebrae) and the third PVP in 2 cases (4 vertebrae); 7 cases (7 vertebrae) were treated with conservative therapy. There was no statistically significant difference on age, gender, cement volume and leakage between secondary fracture group and no-secondary fracture group (P > 0.05). There was no statistically significant difference on the incidence and type of secondary fracture between male and female (P > 0.05). No significant difference was found on the adjacent and remote vertebral fractures (P > 0.05). Most of secondary fracture occurred in 6 months, and whether the single and double side injection, cement leakage had no obvious relation.

CONCLUSIONThere is no significant difference in the subsequent fracture after PVP for the OVCFs different gender and fractured site, and also no significant difference in the adjacent and remote vertebral fractures. The report didn't support the biomechanical viewpoint that vertebral body stiffness increasing after PVP would lead to adjacent vertebral stress increasing and result easily in adjacent vertebral fracture. Secondary fracture occurs always in 6 months after operation, which is the natural course of osteoporosis.

Aged ; Aged, 80 and over ; Female ; Fractures, Compression ; surgery ; Humans ; Male ; Osteoporotic Fractures ; surgery ; Postoperative Complications ; etiology ; Recurrence ; Retrospective Studies ; Spinal Fractures ; surgery ; Vertebroplasty

BACKGROUND: It has been reported that in China, human bone marrow mesenchymal stem ceils are mostly harvested from adults. Studies on bone marrow mesenchymal stem cells in children are few.OBJECTIVE: To isolate and expand bone marrow mesenchymal stem cells from children, and to analyze the biological characteristics of bone marrow mesenchymal stem cells and their potential of differentiating into osteoblasts, adipocytes and neural like cells.DESIGN: Observational comparative study.SETTING: Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Experiments were performed at the Laboratory of Department of Orthopaedics of Wuhan Tongji Hospital from March to September 2006. Bone marrow mesenchymal stem cells were collected from one boy patient and two girl patients aged 5-8 years, who received pelvis osteotomy for dysplasia of the hip joint. The experimental procedures were approved by the Hospital Ethics Committee and family members of all children patients singed the informed consent.Dexamethasone, vitamin C, β-sodium glycerophosphate, 3-1sobutyl-1-methylxanthine, insulin, indometacin and butylated hydroxyanisole were bought from Sigma Company. Dimethyl sulphoxide was purchased from Amersco Company.METHODS: Bone marrow mesenchymal stem cells were cultured from mononuclear cells isolated over a Percoll gradient.Bone marrow mesenchymal stem cells were observed under an inverted phase contrast microscope. Bone marrow mesenchymal stem cells could differentiate into osteoblasts, adipocytes and neural like cells with osteoblast inductor (β-sodium glycerophosphate, dexamethasone, vitamin C), lipoblast inductor (dexamethasone, 3-isobutyl-1-methylxanthine,bovine insulin, indometacin) and serum-free medium inductor (dimethyl sulphoxide, butylated hydroxyanisole) respectively.Osteoblast marker (alkaline phosphatase, osteocalcin mRNA, calcium node), adipocyte marker (lipid droplet, PPAR γ-2mRNA) and neural ceil-like marker (nissl body, neuron specific enolase, neurofilament protein) were respectively determined by the immunohistochemical method, polymerase chain reaction and immunocytochemical method.MAIN OUTCOME MEASURES: ①Appearance and proliferation of bone marrow mesenchymal stem ceils from children,and ②determination results of osteoblast, adipocyte and neural cell markers.RESULTS: ①Children bone marrow mesenchymal stem cells could easily adhere to the wall, appeared fusiform, had high reproductive activity and arranged vortically after fusing. ②Appearance of bone marrow mesenchymal stem cells changed after receiving inductor. Osteoblast marker, adipocyte marker and neural cell-like marker were positive after chemical staining, polumerase chain reaction and immunocyte staining.CONCLUSION: Children bone marrow mesenchymai stem cells show stable proliferation, passage and multi-direction differentiation towards osteoblasts, adipocytes and neural like cells.

Objective To study the changes of plasma normetanephrine(NMN) and metanephrine(MN) during the resection of adrenal pheochromocytoma. Methods Fourteen patients with adrenal pheocromocytoma and 9 patients with adrenal cortex tumor were recruited in our study. Blood samples were obtained at these time points: after anesthesia induction,the beginning of incision of skin, when exploring the tumor,resection of the tumor, and the end of anesthesia. The NMN and MN were determined by high performance liquid chromatogram (HPLC). Results NMN were obviously different among 5 time points in the patients with adrenal pheocromocytoma (P0.05). No significant difference was found between NMN and MN in the patients with adrenal cortex tumor. Conclusion NMN has markedly changed during the resection of adrenal pheochromocytoma, while MN has been relatively stable. The anesthesia induction and exploring of the tumor are the key of a successful operation. MN is the stable index in the diagnosis of adrenal pheochromocytoma.

AIMTo evaluate the Rap1A mRNA expression and its significance in the testes of normal and azoospermic subjects.

METHODSA cDNA microarray that contained Rap1A and some other genes such as RBM, EIF1AY was used to identify the differential gene expression profiles between the normal and azoospermic testes. cDNA probes were prepared by labeling mRNA from azoospermic and normal testicular tissues through reverse transcription with Cy5-dUTP and Cy3-dUTP, respectively. The mixed cDNA probes were then hybridized with cDNA microarray (each containing 4096 unique human cDNA sequences). The fluorescent signals were scanned and the values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated. In situ hybridization was employed to detect the expression of Rap1A in the testes of 10 fertile and 39 azoospermic subjects.

RESULTSOne hundred and twenty-eight differentially expressed genes were found to be possibly related to azoospermia, of which 56 were up-regulated and 72, down-regulated genes. The mRNA expression of Rap1A in the spermatogenic cells of azoospermic was stronger than that in those of the fertile testes.

CONCLUSIONRap1A may play certain roles in the development of azoospermia.

Adult ; Gene Expression ; Humans ; In Situ Hybridization ; Male ; Oligospermia ; metabolism ; RNA, Messenger ; analysis ; Spermatozoa ; chemistry ; Testis ; chemistry ; rap1 GTP-Binding Proteins ; genetics ; physiology

Objective To study the genotypes of Mycobacterium tuberculosis(M.tuberculosis)strains isolated from Jiangsu province and to explore the relationship between the 'Beijing family' and the drug resistance of M.tuberculosis.Methods Two hundred and sixty M.tuberculosis strains were isolated from 30 drug surveillance sites in Jiangsu province.Susceptibility of the isolates to the first-line antituberculosis drugs(isoniazid,streptomycin,rifampicin and ethambutol)was tested by using the proportion method.Molecular typing of M.tuberculosis strains was determined by Spoligotyping and analyzed with BioNumerics software.Results Based on Spoligotyping fingerprint,260 strains showed 34 different genotypes,including 27 exclusive genotypes and 7 shared genotypes.These strains could be clustered into two groups:the Beijing family(80.4％,209/260)and the Non-Beijing family(19.6％,51/260).Data from logistic regression analysis revealed that infection by the Beijing family was related to an increased risk of multi-drug resistant M.tuberculosis,with the OR(95％ CI)of 11.07(1.45-84.50).Non-Beijing families including T1,T2,H3,H4,CAS,LAM,U and MANU2 families were also found.Among them,the CAS,LAM and MANU2 families were first reported in Jiangsu province.Conclusion It was revealed that the marked gene polymorphisms did exist in M.tuberculosis strains.The Beijing family had been the predominant strain circulating in Jiangsu province,which might be related to multi-drug resistant M.tuberculosis strains.

Objective To observe the brain activation of interictal epiletiform discharges(IEDs) and to localize the epileptogenic foci of epilepsy.Methods The electroencephalogram(EEG)and functional MRI data of 12 focal epileptic patients were acquired using a combination of EEG and functional MRI simultaneously.The IEDs onset time detected with EEG were set as the time parameters in an event- related paradigm of functional MRI analysis.The spatial and temporal characters of IEDs activation were analyzed in detail.In order to confirm the consistency of this method,all patients were scanned repeatedly and the results were correlated with clinical evaluation.Results Of the 12 patients,valid data from EEG- fMRI were obtained from 10 patients in a total of 18 sessions..Compared with the structural foci,the epileptic foci localization results of eleven sessions were good,five sessions were fairly good,and two sessions were poor.The results obtained from six patients in two separate sessions were concordant.respectively.Moreover,thalamic activation was detected in ten sessions,cerebellar activation was detected in all sessions,and the deactivation was found in the default mode loci in nine sessions. Conclusion The method of performing EEG and fMRI simultaneously can potentially be a useful tool in epilepsy research.