Objective To explore the transfection efficiency of primary lymphocytes from human peripheral blood by different methods to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using Ficoll-Hypaque.Cell viability was detected by Trypan blue staining.Suspending lymphocytes were sucked out and were incubated in 24-well plate after cultured in 6-well plate for 2 h.Activated lymphocytes were transfected by electroporation with plasmid(PEGFP-N1).Resting or activated lymphocytes were transfected by lentivirus vector(LVGFP) single infection or repeated infection,respectively.Green fluorescence protein (GFP) was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry at different time points after infection.At the same time,the effectiveness of lentivirus infection was compared under different conditions.Results Purity of mononuclear cells isolated by Ficoll-Hypaque was 95 ％ and its viability was over 95 ％.The percentage of lymphocytes obtained with a uniform shape was 90 ％-95 ％.Scattered fluorescence was observed by electroporation under the conditions of voltage 2 100 V,pulse width 10 ms,pulse number 1 for lymphocyte,while fluorescent became weaker over time and no green fluorescent was observed after transfection for 72 h.After resting lymphocytes were infected once for 48 h by lentivirus vector,green fluorescent was not found and positive cells were less than 1％.1％-5 ％ of activated lymphocytes could express GFP after single lentivirus infection and the expression levels were enhanced with concentration increasing,while 5 ％-10 ％ of activated lymphocytes showed strong green fluorescent by repeated lentivirus infection.In contrast with electroporation,the fluorescent with lentivirus infection was stronger over time.Conclusion Repeated lentivirus infection could efficiently transfect exogenous genes into activated lymphocytes for stable expression.

Objective To analyze the relationship between cervical lordosis and the development of dysphagia after anterior cervical spine surgery.Methods From June 2007 to May 2010,data of 172 successive patients who had undergone ACDF operation in our hospital were reviewed in this study.The presence and duration of postoperative dysphagia were recorded via face-to-face questioning or telephone interview at least one year after the procedure.Plain cervical radiographs before and after surgery were collected.The C2-7 angle was measured.The change of C2-7 angle was defined as dC2-7 angle=postoperative C2-7-preoperative C2-7 angle.The correlation between postoperative dysphagia and dC2-7 angle was studied.Results There were 22 patients in dysphagia group,including 17 males and 5 females.Their age ranged from 25 to 70 years old,and average was 47.7±5.4.The average of BMI was 25.0±2.9 kg/m2.150 patients were in non-dysphagia group,including 101 males and 49 females.Their age ranged from 18 to 72 years old,and average age was 49.2±4.8.The average of BMI was 24.4±3.4 kg/m2.There was no statistical difference in gender,age,and BMI between two groups.The dC2-7 angle of dysphagia group ranged from-1 °-20.5°,and average was 8.6°±4.0°.The dC2-7 angle of non-dysphagia group ranged from-13°-28.5°,and average was 5.0°±4.3°.There was significant difference in dC2 7 angle between dysphagia and non-dysphagia group.Spearman Analysis revealed that there was strong correlativity between dC2-7 angle and postoperative dysphagia.When dC2-7 angle was greater than 5°,the chance of developing postoperative dysphagia significantly increased (19.3％ [17/88] vs 6.0％ [5/84]).What's more,Spearman Analysis also revealed that there was no correlativity between dC2 7 angle and degree of operative dysphagia.There was no significant difference in gender,age,and BMI between dysphagia and non-dysphagia group.There was no statistical difference in operative time,blood loss revision surgery,revision surgery ratio,most cephalic operative level and number of operative levels between dysphagia with non-dysphagia group.Logistic regression model showed that an increased likelihood of postoperative dysphagia persists with increasing dC2-7 angle,but had no relationship with operative time,blood loss,revision surgery,most cephalic operative level and number of operative levels.Conclusion dC2-7 angle may play an important role in the development of postoperative dysphagia.We found no statistical difference in operative time,blood loss revision surgery,revision surgery ratio,most cephalic operative level and number of operative levels between dysphagia and non-dysphagia group.Intraoperative measurement of the dC2-7 angle is practical and essential for reducing the postoperative dysphagia.

Pro-inflammatory macrophages play key regulatory role in the occurrence and development of rheumatoid arthritis (RA). In this study, we constructed a celastrol (Cel)-loaded polyamide-amine dendrimer (PAMAM) drug delivery system, which could target folate receptor and mitochondria. It could target inflammatory macrophages and realize chemo-photothermal synergistic therapy. Using PAMAM as the nano-carrier, folate receptor-targeting group folic acid (FA) and mitochondria-targeting group IR808 (also known as the photothermal agent) were conjugated with PAMAM through amide reaction, and then complexed with anti-inflammatory drug Cel to prepare the FA-PAMAM-IR808/Cel nanocomplex. In vitro characterization results showed that the drug loading efficiency of the nanocomplex was 50.90%, particle size was between 130 and 160 nm, average potential was between 1.0 and 3.5 mV, the drug release showed pH sensitivity, temperature reached to 42.5 ℃ after near-infrared (NIR) light irradiation for 10 min. In vitro cellular uptake experiments showed that the nanocomplex had obvious folate receptor-targeting and mitochondria-targeting ability. Following irradiation with NIR light, the cytotoxicity and cellular apoptosis enhanced. The secretion of pro-inflammatory factors tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and nitric oxide (NO) decreased in a concentration-dependent manner. This study provided insights for the development of novel anti-RA nanomedicines.

Clean needle technique (CNT) examination is required for all applicants seeking National Certification Commission for Acupuncture and Oriental Medicines' (NCCAOM) certification. CNT examination is one of compulsory subjects of certification of professional competence to practice acupuncture safely and effectively for the sake of the health and safety of the public, which is developed by the Council of Colleges of Acupuncture and Oriental Medicine (CCAOM). The uniform standard of practice for acupuncture, the content of CNT course, application procedure and the requirements of CNT examination are introduced in this paper.
Acupuncture ; manpower ; organization & administration ; Acupuncture Therapy ; instrumentation ; methods ; standards ; Certification ; organization & administration ; standards ; Humans ; Licensure, Medical ; standards ; Professional Practice ; legislation & jurisprudence ; organization & administration ; standards ; United States

Objective To evaluate the mid-term tollow-up results of cervical artificial disc replacement (CADR) for cervical degenerative disc disease,and to explore whether it can reduce the occurrence of adjacent segment degeneration (ASD).Methods A prospective comparative study of 93 patients who underwent CADR or anterior cervical decompression and fusion (ACDF) for cervical degenerative disc disease were conducted.All patients were followed up for more than 6 years.The Japanese Orthopaedic Association (JOA) score,neck disability index (NDI),Odom's scale,X-rays and magnetic resonance imaging (MRI) were used to evaluate the clinical and radiologic results.Results Twenty eight patients who underwent CADR and 35 patients who underwent ACDF had complete follow-up data.At final follow-up,the JOA score and NDI improved significantly in both groups.Between the two groups,there was no significant difference in terms of JOA score,NDI and Odom's scale.The sagittal alignment was well maintained in both groups.The total cervical spine range of motion (ROM) had no significant change for the CADR group,whereas,it significantly decreased for the ACDF group.The ROM at the replacement level of CADR patients decreased from 9.5° ± 3.7° before operation to 7.0° ± 3.0° 3 months after operation,and it was maintained to 6.6° ± 4.1° at final follow-up without significant decrease.Lateral radiographs and T2-weighted MRI showed the incidence of ASD in CADR group was significantly lower than that in ACDF group.Conclusion The six-year follow-up results of CADR are basically satisfactory.Compared with ACDF,it could better preserve physiological motion and biomechanics of cervical spine,and reduce the incidence of ASD.

Adult ; Female ; Humans ; Laryngeal Diseases ; microbiology ; Male ; Middle Aged ; Mycosis Fungoides ; Retrospective Studies

Objective To explore the change of nerve growth factors(NGF) through the blood brain barrier(BBB) after distal intravenous injection of mannitol into the experimental rats and the effect of exogenous NGF on the expression of growth associated protein-43 in hypoxic-ischemic brain.Methods One hundred cases of 7 days rats were divided into 2 units.One unit was divided into 3 groups:treatment group,control group and sham operated group,20 rats in each group.The other unit was divided into 4 groups:mannitol and NGF treated group,NGF treated group,control group,and sham operated group,there were 10 rats in each group.The model rats with perinatal hypoxic-ischemic brain damage(HIBD) rats were prepared by ligation of left common carotid artery with a temporary systemic hypoxia(inhaling 80 mL/L O2 and 920 mL/L N2).The sections of brains were processed by immunochemistry with antibodies against GAP-43,and the study and memory ability of rats were tested by maze test.The effect of osmotic opening of BBB on the facilitation of NGF′s passage was tested by ELISA.Results The expression of GAP-43 increased after NGF treatment,and the differences were remarkable(P

Objective To investigate the effects of B lymphocyte-induced maturation protein-1 ( Blimp-1) on the number and function of splenic lymphocytes.Methods The mice with defective Blimp-1 in T cells were generated by cross-breeding B6.Blimp-1flox/flox mice with B6.Lck-Cre mice.The mononuclear lymphocytes isolated from spleen of T cell conditional Blimp-1 knockout (Blimp-1CKO) mice and wild type ( WT) C57/B6 mice were comparatively analyzed.Alterations of CD4+T and CD8+T cell subsets, the secre-tion of cytokines as well as the expression of C-C chemokine receptor type 7 ( CCR7 ) and Sphingosine-1-phosphate receptor 1 (S1P1) in mice from the two groups were analyzed by flow cytometry.The changes of CD19+B cell subsets were also detected.Results Compared with WT mice, the total numbers of mononu-clear cells, T and B lymphocytes were all significantly increased in Blimp-1CKO mice ( P<0.05) .The ab-solute numbers of CD4+T, CD8+T and CD19+CD5+CD1d+B cells in mice form Blimp-1CKO group were higher than those of the control group (P<0.05), however, no significant differences with the percentages of these cell populations were observed between two groups.Higher numbers and percentages of CD19+CD5+B cells were detected in mice from Blimp-1CKO group (P<0.01).The Blimp-1CKO mice showed increased secretion of IFN-γ, TNF-α, IL-17 and IL-2, but decreased expression of CCR7 on CD8+T cells as com-pared with WT mice (P<0.05).No significant differences with the changes of S1P1 were found between the two groups.Conclusion Blimp-1 played an important role in the maintenance of number, phenotype and function of T cells.Furthermore, not only T cells but also B cell subsets in mice were affected by the dele-tion of Blimp-1 in T cells.

ObjectiveTo Compare the clinical results between microscope and loupes which used in microsurgical discectomy.MethodsA prospective randomized controlled trial of 93 patients who had undergone microsurgical discectomy from January 2007 to December 2010 was performed.Clinical results were assessed by comparing the following parameters between patients who had undergone the surgery by microscope and loupes:length of stay,hospitalization cost,operative time,estimated blood loss,Japanese Orthopaedic Association (JOA) score and JOA recovery rate,Odom's standard.ResultsForty-nine patients underwent surgery by microscope,and forty-four patients underwent surgery by loupes.Eighty patients received outpatient or telephone follow-up.The follow-up period was 6.17 to 52.90 months with an average of (29.64±13.05) months,and the follow-up rate was 86.02％.According preoperative data,the two groups didn't differ with respect to age,gender,level of radiculopathy,or preoperative JOA score and JOA recovery rate.No statistically significant differences were identified in postoperative JOA score and JOA recovery rate,length of stay,hospitalization cost,length of follow-up,or relapse rate.Statistically significant differences were identified in operative time,estimated blood loss,and follow-up JOA score and JOA recovery rate.Conclusion Microscope can provide relatively more clear and comfortable vision for the surgery.It can short the operative time,decrease blood loss,reduce the potential risk of nerve injury,and retain more normal tissue,which can ensure better clinical results.

BACKGROUNDParkinson's disease (PD) is a common neurodegenerative disease, which occurs mainly in the elderly. Recent studies have demonstrated that apoptosis plays an important role in the occurrence and development of PD. Subthalamic nucleus deep brain stimulation (STN-DBS) has been recognized as an effective treatment for PD. Recent clinical observations have shown that STN-DBS was able to delay early PD progression, and experiments in animal models have also demonstrated a protective effect of STN-DBS on neurons. However, the correlation between the neuron-protective effect of STN-DBS and the progression of substantia nigra pars compacta (SNc) neuronal apoptosis is still unknown. The aim of this study was to investigate the protective effect and potential mechanism of STN-DBS on SNc neurons in PD rats.

METHODSAfter the establishment of a PD rat model by unilateral/2-point injection of 6-hydroxydopamine in the medial forebrain bundle of the brain, DBS by implanting electrodes in the STN was administered. Behavioral changes were observed, and morphological changes of SNc neurons were analyzed by Nissl staining and DNA in situ end-labeling. Through extracellular recording of single neuron discharges and microelectrophoresis, the causes of and changes in SNc excitability during STN-DBS were analyzed, and the protective effect and potential mechanism of action of STN-DBS on SNc neurons in PD rats was investigated.

RESULTSSNc neuron apoptosis was significantly decreased (P < 0.05) in the stimulation group, compared with the sham stimulation PD group. Spontaneous discharges of SNc neurons were observed in normal rats and PD model rats, and the mean frequency of spontaneous discharges of SNc neurons in normal rats ((40.65 ± 11.08) Hz) was higher than that of residual SNc neurons in PD rats ((36.71 ± 9.23) Hz). Electrical stimulation of the STN in rats was associated with elevated excitation in unilateral SNc neurons. However, administering the gamma-aminobutyric acid receptor blocker, bicuculline significantly reduced SNc neuron excitation, but the change in SNc neuron excitation was not present when MK801, a glutamate receptor blocker, was administered.

CONCLUSIONSHigh-frequency stimulation of the STN has a protective effect on SNc neurons in PD rats. The possible molecular mechanism may be related to changes in the distribution and metabolism of neurotransmitters in the SNc region.

Animals ; Deep Brain Stimulation ; methods ; Disease Models, Animal ; Male ; Neurons ; cytology ; metabolism ; Parkinson Disease ; pathology ; therapy ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; cytology