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1.
目的 基于重叠延伸PCR(SOE PCR)技术构建转录因子EB(TFEB)丝氨酸114位点突变体原核表达质粒,并进行体外诱导表达和纯化。 方法 根据SOE PCR技术原理设计突变引物,以pGEX-6p-1-TFEB质粒为模板,分别采用外侧引物F和R及突变引物Fn和Rn进行第1步PCR扩增,获得含突变位点的产物1和产物2。经第2步PCR退火延伸对产物1和产物2进行重叠拼接,以拼接后DNA片段为模板,采用外侧引物F和R进行第3步PCR扩增获得含突变位点的目的DNA;将其克隆至pGEX-6p-1载体,采用BamH Ⅰ和Sal Ⅰ进行酶切鉴定,DNA测序验证突变结果。于大肠杆菌(E.coli)中分别采用不同浓度异丙基-β-D-硫代半乳糖苷(IPTG)在不同条件下诱导TFEB及其突变体重组蛋白表达。Glutathione-Sepharose 4B琼脂糖凝珠分离纯化蛋白,SDS-PAGE凝胶电泳检测纯化产物蛋白浓度和相对分子质量。 结果 第1步PCR扩增获得均含有突变位点的2条DNA片段,经第2步PCR退火延伸和第3步PCR扩增后获得大量含有突变位点的完整DNA片段。双酶切鉴定,连接的DNA片段与目的片段大小一致。DNA测序显示TFEB的114位丝氨酸(TCT)成功突变为丙氨酸(GCT)。在0.5?mmol·L-1 IPTG、16?℃诱导过夜条件下获得TFEB及其突变体的可溶性蛋白表达。SDS-PAGE凝胶电泳,纯化后蛋白浓度较高,分子大小正确。 结论 利用SOE PCR技术成功实现了TFEB丝氨酸114位点的定点突变,并且TFEB及其突变体基因在E.coli中成功表达。  相似文献   
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【摘要】 目的:比较自体髂骨、同种异体骨和羟基磷灰石(hydroxyapatite,HA)人工骨在前路颈椎间盘切除融合术(anterior cervical discectomy and fusion,ACDF)应用的临床效果和影像学结果。方法:回顾性分析2015年1月~2019年12月于南方医科大学第三附属医院接受ACDF患者的临床资料。共纳入107例患者、164节段。根据使用的植骨材料不同分为自体髂骨组(37例、53个节段)、同种异体骨组(46例、70个节段)和HA人工骨组(24例、41个节段)。术前和末次随访时采用疼痛视觉模拟量表(visual analogue scale,VAS)评分、颈椎功能障碍指数(neck disability index,NDI)、日本骨科协会(Japanese Orthopedic Association,JOA)评分评估患者颈椎疼痛和功能情况。在颈椎矢状面X线片测量手术节段高度和矢状面参数[C2-C7矢状面轴向距离(SVA)和C2-C7 Cobb角],评估融合器下沉情况。根据Odom标准进行满意程度评估,采用Brantigan评分评估颈椎椎间融合状态,采用多因素Logistic回归分析植骨不融合的危险因素。结果:三组患者的性别比例、年龄、病程、体质量指数(BMI)、合并糖尿病和终板异常比例、单/非单节段比例、手术时间和出血量均无统计学差异(P>0.05)。三组临床随访时间有统计学差异(自体髂骨组41.6±18.4个月,同种异体骨组26.9±15.7个月,HA人工骨组 31.3±8.4个月,P<0.05)。三组患者末次随访时的颈痛VAS评分、NDI和JOA评分均较术前显著性改善(P<0.05),HA人工骨组末次随访时的VAS评分和NDI显著性高于自体髂骨组和同种异体骨组(P<0.05);三组患者VAS评分改善程度无显著性差异(P>0.05),同种异体骨组的NDI和JOA评分改善程度显著性优于自体髂骨组和HA人工骨组(P<0.05)。末次随访时自体髂骨组、同种异体骨组和HA人工骨组患者满意程度优良率分别为81.1%(30/37)、84.8%(39/46)和54.2%(13/24),HA人工骨组的满意率较低(P<0.05);融合率分别为94.3%(50/53)、81.4%(57/70)和31.7%(13/41),融合器下沉发生率分别为3.8%(3/53)、4.3%(3/70)和58.5%(24/41),HA人工骨组的融合率显著性较低(P<0.05),融合器下沉发生率显著性较高(P<0.05)。末次随访时,HA人工骨组的手术节段高度显著性较低(P<0.05),三组患者的SVA和C2-C7 Cobb角无显著性差异(P>0.05)。多因素Logistic回归分析显示同种异体骨和人工骨的使用为植骨不融合的危险因素。HA人工骨组有2例患者(8.3%)因假关节形成接受翻修手术治疗。结论:同种异体骨植骨的临床效果良好,患者满意率和植骨融合率较高,融合器下沉风险较低,可作为自体骨替代材料应用于ACDF;HA人工骨植骨的临床效果相对较差,患者满意率和植骨融合率较低,融合器下沉率较高,且有较高的翻修手术风险。  相似文献   
4.
目的探讨MRI融合图像技术对凶险性前置胎盘(PAS)的诊断价值。资料与方法回顾性分析2018年12月—2020年11月于南京医科大学康达学院第一附属医院经手术或病理证实的67例PAS患者的MRI图像,比较T2WI、扩散加权成像(DWI)及T2WI-DWI 3种成像技术对PAS的诊断差异及效能。结果67例PAS中,无植入14例,胎盘粘连26例,胎盘植入27例。T2WI诊断无植入28例,胎盘粘连14例,胎盘植入25例;DWI诊断无植入27例,胎盘粘连10例,胎盘植入30例;T2WIDWI诊断无植入15例,胎盘粘连23例,胎盘植入29例。3种成像技术诊断无植入和胎盘粘连差异有统计学意义(χ2=10.645、10.976,P均<0.05),诊断胎盘植入差异无统计学意义(χ2=1.139,P>0.05)。T2WI-DWI诊断无植入的准确度、敏感度、特异度分别为95.52%、92.86%、96.23%,诊断胎盘粘连的准确度、敏感度、特异度分别为83.58%、73.08%、90.24%,诊断胎盘植入的准确度、敏感度、特异度分别为88.06%、88.89%、87.50%。与T2WI和DWI比较,T2WI-DWI诊断无植入、胎盘粘连和胎盘植入的准确度和敏感度较高,诊断无植入和胎盘植入的特异度较高。结论MRI融合图像技术可以提高PAS的诊断准确度、敏感度、特异度,有助于为临床提供有效的手术方案。  相似文献   
5.
《Neuro-Chirurgie》2023,69(3):101428
BackgroundRodent models are commonly used experimentally to assess treatment effectiveness in spinal fusion. Certain factors are associated with better fusion rates. The objectives of the present study were to report the protocols most frequently used, to evaluate factors known to positively influence fusion rate, and to identify new factors.MethodA systematic literature search of PubMed and Web of Science found 139 experimental studies of posterolateral lumbar spinal fusion in rodent models. Data for level and location of fusion, animal strain, sex, weight and age, graft, decortication, fusion assessment and fusion and mortality rates were collected and analyzed.ResultsThe standard murine model for spinal fusion was male Sprague Dawley rats of 295 g weight and 13 weeks’ age, using decortication, with L4-L5 as fusion level. The last two criteria were associated with significantly better fusion rates. On manual palpation, the overall mean fusion rate in rats was 58% and the autograft mean fusion rate was 61%. Most studies evaluated fusion as a binary on manual palpation, and only a few used CT and histology. Average mortality was 3.03% in rats and 1.56% in mice.ConclusionsThese results suggest using a rat model, younger than 10 weeks and weighing more than 300 grams on the day of surgery, to optimize fusion rates, with decortication before grafting and fusing the L4-L5 level.  相似文献   
6.
《Vaccine》2022,40(6):934-944
Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.  相似文献   
7.
目的探究DRG付费对医生诊疗行为和医疗质量的影响。方法从11家实施DRG付费或模拟测算的三级综合医院中选取12名骨科医生进行半结构化访谈,采用Colaizzi 7步法分析。结果共识别重要陈述308条,形成治疗决策、环节把控、患者管理、医疗质量4项基本结构。DRG付费后,医生更关注住院费用,以检查和手术为代表的诊疗行为发生了变化;医生诊疗行为的改变可能引起医疗质量的变化。结论医生应主动控制成本,制定合理的诊疗方案;医院需强化临床指南的使用,加强对医疗质量的考核,以防范医生不规范诊疗行为的发生。  相似文献   
8.

BACKGROUND CONTEXT

Cervical laminectomy and fusion (CLF) is a common surgical option for multilevel cord compression. Postoperative C5 palsy occurrence after CLF has been a vexing problem for spine physicians. The posterior shift of the cord following laminectomy has been implicated as a major factor for postoperative C5 palsy, but attempts by spine surgeons to mitigate excessive shift while providing sufficient decompression have not been well reported.

PURPOSE

To compare the incidence of postoperative C5 palsy after performing selective blocking laminoplasty concurrently with CLF to those of conventional CLF.

STUDY DESIGN

A retrospective comparative study of prospectively collected data.

PATIENT SAMPLE

Of 116 cervical myelopathy patients with degenerative cervical myelopathy, ossification of the posterior longitudinal ligament, and multilevel disc herniation, 93 patients (69 in group A [CLF group] and 24 in group B [selective blocking laminoplasty with CLF, CLF-S group]) were included in the study.

OUTCOME MEASURES

The primary outcome measure was the occurrence of postoperative C5 palsy. Secondary end points included (1) clinical outcomes based on pain intensity, neck disability index (NDI), Japanese Orthopaedic Association (JOA) score, (2) radiologic outcomes including cervical alignment and fusion rate at 1 year and hardware complications, and (3) perioperative data (hospital stay, blood loss, and operative times).

METHODS

We compared the occurrence of postoperative C5 palsy, as well as clinical, radiologic, and surgical outcomes, between the two groups at 1-year follow-up.

RESULTS

The patients in both groups were statistically similar between the groups with respect to demographic characteristics such as age, sex, smoking status, body mass index, preoperative pathology, surgical segments, and the degree of the cervical lordosis. Postoperative C5 palsy developed in 9 of 61 patients (14%) in group A and in 0 of 24 patients (0%) in group B (CLF-S group) (p=.03). Postoperative neck pain, NDI, and JOA improvement were not significantly different between the two groups (p=.93, 0.90, and 0.79, respectively). Perioperative data did not differ significantly between the two groups.

CONCLUSIONS

This study showed that performing selective blocking laminoplasty might lead to reducing the incidence of postoperative C5 palsy in CLF surgery.  相似文献   
9.
Tumor cells/dendritic cells (DCs) fusion cells (tumor/DC) represent a promising immunotherapeutic strategy but are still under performed in clinical trials for cancer treatment. To further boost their anticancer efficacy, here we developed a novel design for fusing dendritic cells with MDA-MB-231 cells expressing the heterologous α-galactose (α-gal) epitope and assessed its anticancer activities both in vitro and in vivo. The high expression of α-gal in MDA-MB-231 (Gal+)/DC correlated with enhanced DC activation. When applied to T cells, MDA-MB-231 (Gal+)/DC significantly stimulated T-cell proliferation and activation, promoted productions of cytokines IL-2 and IFN-γ, and enhanced T-cell-mediated cytotoxicity against MDA-MB-231 cells. MDA-MB-231 (Gal+)/DC inhibited proliferation and promoted apoptosis of tumor cells in vivo, prolonged mouse survival, and significantly boosted anticancer immunity by increasing CD4+ and CD8+ T cells systemically and elevating serum levels of cytokines and IgG. These results suggested that fusing dendritic cells with tumor cells expressing the heterologous α-gal epitope provides a novel therapeutic strategy for cancer treatment.  相似文献   
10.
Breast cancer image fusion consists of registering and visualizing different sets of a patient synchronized torso and radiological images into a 3D model. Breast spatial interpretation and visualization by the treating physician can be augmented with a patient-specific digital breast model that integrates radiological images. But the absence of a ground truth for a good correlation between surface and radiological information has impaired the development of potential clinical applications.A new image acquisition protocol was designed to acquire breast Magnetic Resonance Imaging (MRI) and 3D surface scan data with surface markers on the patient’s breasts and torso. A patient-specific digital breast model integrating the real breast torso and the tumor location was created and validated with a MRI/3D surface scan fusion algorithm in 16 breast cancer patients.This protocol was used to quantify breast shape differences between different modalities, and to measure the target registration error of several variants of the MRI/3D scan fusion algorithm. The fusion of single breasts without the biomechanical model of pose transformation had acceptable registration errors and accurate tumor locations. The performance of the fusion algorithm was not affected by breast volume. Further research and virtual clinical interfaces could lead to fast integration of this fusion technology into clinical practice.  相似文献   
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