Objective:To analyze the clinical and genetic characteristics of late-onset Charcot–Marie–Tooth(CMT)type 2A.Methods:A pedigree survey combined with clinical and genetic testing was used for integrated analyses.Resul...Objective:To analyze the clinical and genetic characteristics of late-onset Charcot–Marie–Tooth(CMT)type 2A.Methods:A pedigree survey combined with clinical and genetic testing was used for integrated analyses.Results:The proband showed muscle atrophy in both hips and thighs,obviously in the posterior sides of both legs.The serum creatine kinase(CK)value was 272U/L.EMG:the median nerve conduction velocity was normal and the nerves of both lower extremities showed neurogenic damage(mainly axonal).Genetic testing revealed a heterozygous variant in exon 9 of the MFN2 gene NM 014874:c.839G>A(p.R280H).The Father of the proband had similar symptoms of peripheral neuromuscular and the same heterozygous mutation,but the mother did not show similar clinical symptoms and the genetic mutation.Conclusion:Pedigree investigation combined with clinical and genetic analysis is a kind of reliable method for late-onset CMT2A diagnosing and helping to identify different neuromuscular diseases.展开更多
Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the leading genetic cause of autosomal dominant familial Parkinson’s disease. We previously reported that two mutations within the ROC domain, n...Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the leading genetic cause of autosomal dominant familial Parkinson’s disease. We previously reported that two mutations within the ROC domain, namely R1441C and A1442P, exhibit increased protein degradation leading to lowered steady state LRRK2 protein levels in HEK293 cells. More recently, the common WD40 domain LRRK2 haplotype, Met2397, which is a risk factor for Crohn’s disease, has been shown to lower steady state protein levels in HEK293 cells. In view of recent evidence implicating LRRK2 and inflamemation in PD, we investigated the effects of Met2397 on LRRK2 expression, and compared them to the Thr2397 variant and other LRRK2 mutants. In this study, we transfected HEK293 cells with plasmid constructs encoding the different LRRK2 variants, and analyzed the resulting protein levels by Western blot and flow cytometry. Here we found that both the Met2397 and Thr2397 haplotypes yield similar levels of LRRK2 protein expression and do not appear to impact cell viability in HEK293 cells, compared to other LRRK mutants. Thus, we have concluded that the Met2397 haplotype is unlikely to play a role in LRRK2 mediated or idiopathic PD.展开更多
目的:构建敲除TGF-βRⅡ的靶向CLDN18.2的CAR T细胞,并探索其在胃食管交界癌中的治疗潜力;方法:通过CRISPR/Cas9敲除T细胞中的TGF-βRⅡ,并使用慢病毒感染法构建CLDN18.2 CAR T细胞;流式细胞术检测CAR T细胞的阳性率、食管腺癌细胞系中C...目的:构建敲除TGF-βRⅡ的靶向CLDN18.2的CAR T细胞,并探索其在胃食管交界癌中的治疗潜力;方法:通过CRISPR/Cas9敲除T细胞中的TGF-βRⅡ,并使用慢病毒感染法构建CLDN18.2 CAR T细胞;流式细胞术检测CAR T细胞的阳性率、食管腺癌细胞系中CLDN18.2的表达情况、CAR T细胞中Smad2/3的磷酸化水平以及PD-1的表达水平;免疫荧光法检测CAR T细胞中TGF-βRⅡ的表达;LDH释放实验检测对靶细胞的细胞毒性;裸鼠移植瘤模型检测肿瘤抑制能力;ELISA实验检测肿瘤组织中细胞因子的释放水平;结果:成功构建敲除TGF-βRⅡ的CLDN18.2 CAR T细胞(18.2BBZ/TGFBRKO)以及二代CLDN18.2 CAR T细胞(CLDN18.2);18.2BBZ及18.2BBZ/TGFBRKO在体外对CLDN18.2+的肿瘤细胞具有剂量依赖型细胞毒性;TGF-β1不能使18.2BBZ/TGFBRKO的Smad2/3发生磷酸化,在TGF-β1存在时,18.2BBZ/TGFBRKO与肿瘤细胞共孵育后PD-1的表达水平显著低于18.2BBZ(P<0.001);18.2BBZ/TGFBRKO较18.2BBZ对裸鼠移植瘤的抑制能力更强(P<0.0001),并且在肿瘤组织中保持更强的细胞因子分泌水平(P<0.01);结论:通过CRISPR/Cas9敲除CLDN18.2 CAR T细胞中的TGF-βRⅡ可以有效降低T细胞耗竭,并在小鼠移植瘤模型中产生更强的抑制肿瘤活性,并增加促炎性细胞因子的分泌。展开更多
proach change of CGRP with time in periodontal ligaments of rats in magnetic field, maxillae of 28 Wistar rats of 6 7 weeks were placed in magnetic fields of permanent magnets Nd 2 Fe 4 B. Changes of CGRP w...proach change of CGRP with time in periodontal ligaments of rats in magnetic field, maxillae of 28 Wistar rats of 6 7 weeks were placed in magnetic fields of permanent magnets Nd 2 Fe 4 B. Changes of CGRP with t ime in maxillary periodontal ligaments of the rats were studied by immunohistochemistry. The result showed that: magnetic field had dynamic effect and some promotional effect on recove?E F F E C T O F M A G N E T I C F I E L D O N C A L C I T O N I N G E N E- R E L A T E D P E P T I D E I N P E R I O D O N T A L L I G A M E N T S O F R A T S X. Chang , K. Qin and Y. L. Lu Orthodontic Department of Stomatology College , China Medical University , Shenyang 110001 , China Manuscript received 24 June 1999 In order to approach change of C G R P with tim e in periodontal liga m ents of rats in m agneticfield , m axillae of 28 Wistar rats of 6 7 weeks w ere placed in m agnetic fields of per m anentm agnets Nd 2 Fe4 B. Changes of C G R P with tim e in m axillary periodontal liga ments of therats were stu died by i m m unohistoche mistry . The result sho wed that : m agnetic field had dy na m ic effect an d so m e pro motional effect on recovering and reconstruction of the periodontaltissues in it . This provided a better theoretical evidence for clinical use of per m anent m agnets .展开更多
文摘目的利用CRISPR/Cas9系统构建稳定敲除4.1R基因的RAW264.7巨噬细胞株,为研究4.1R在巨噬细胞中的功能奠定基础。方法根据CRISPR/Cas9靶向原理设计并合成3个特异性识别4.1R基因的向导RNA(sgRNA),构建sgRNAlenti CRISPRv2重组质粒并转入293T细胞中制备sgRNA-Cas9慢病毒,慢病毒侵染RAW264.7细胞,嘌呤霉素筛选出阳性细胞并稀释至单克隆,Western blotting印记检测单克隆细胞中蛋白4.1R的表达,测序确认单克隆细胞中突变位点。结果 Western blotting印迹检测结果表明筛选出的1株单克隆细胞中蛋白4.1R的表达完全缺失;测序结果表明该细胞株中4.1R基因发生了19bp的缺失突变;并且4.1R基因敲除后,RAW264.7细胞的增殖能力显著增加。结论本研究利用CRISPR/Cas9系统成功的干扰了巨噬细胞系RAW264.7细胞中4.1R的表达,为研究4.1R在巨噬细胞中的功能提供了有效工具。
基金Fundamental research general project of local undergraduate universities(part)in Yunnan Province(No.2018FH001-082)Key project of Applied Basic Research Plan of Yunnan Province(No.2018FA010)+2 种基金National Natural Science Foundation of China(No.31571304)Yunnan Training Programme for the advanced Talents of Health and Medical Technology(No.D-2018013)the Scientific and technological talents and platform plan,high-level scientific and technological talents and innovative team selection project-Young and middle-aged academic and technical leaders reserve talent project(No.202005AC160011)。
文摘Objective:To analyze the clinical and genetic characteristics of late-onset Charcot–Marie–Tooth(CMT)type 2A.Methods:A pedigree survey combined with clinical and genetic testing was used for integrated analyses.Results:The proband showed muscle atrophy in both hips and thighs,obviously in the posterior sides of both legs.The serum creatine kinase(CK)value was 272U/L.EMG:the median nerve conduction velocity was normal and the nerves of both lower extremities showed neurogenic damage(mainly axonal).Genetic testing revealed a heterozygous variant in exon 9 of the MFN2 gene NM 014874:c.839G>A(p.R280H).The Father of the proband had similar symptoms of peripheral neuromuscular and the same heterozygous mutation,but the mother did not show similar clinical symptoms and the genetic mutation.Conclusion:Pedigree investigation combined with clinical and genetic analysis is a kind of reliable method for late-onset CMT2A diagnosing and helping to identify different neuromuscular diseases.
文摘Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the leading genetic cause of autosomal dominant familial Parkinson’s disease. We previously reported that two mutations within the ROC domain, namely R1441C and A1442P, exhibit increased protein degradation leading to lowered steady state LRRK2 protein levels in HEK293 cells. More recently, the common WD40 domain LRRK2 haplotype, Met2397, which is a risk factor for Crohn’s disease, has been shown to lower steady state protein levels in HEK293 cells. In view of recent evidence implicating LRRK2 and inflamemation in PD, we investigated the effects of Met2397 on LRRK2 expression, and compared them to the Thr2397 variant and other LRRK2 mutants. In this study, we transfected HEK293 cells with plasmid constructs encoding the different LRRK2 variants, and analyzed the resulting protein levels by Western blot and flow cytometry. Here we found that both the Met2397 and Thr2397 haplotypes yield similar levels of LRRK2 protein expression and do not appear to impact cell viability in HEK293 cells, compared to other LRRK mutants. Thus, we have concluded that the Met2397 haplotype is unlikely to play a role in LRRK2 mediated or idiopathic PD.
文摘目的:构建敲除TGF-βRⅡ的靶向CLDN18.2的CAR T细胞,并探索其在胃食管交界癌中的治疗潜力;方法:通过CRISPR/Cas9敲除T细胞中的TGF-βRⅡ,并使用慢病毒感染法构建CLDN18.2 CAR T细胞;流式细胞术检测CAR T细胞的阳性率、食管腺癌细胞系中CLDN18.2的表达情况、CAR T细胞中Smad2/3的磷酸化水平以及PD-1的表达水平;免疫荧光法检测CAR T细胞中TGF-βRⅡ的表达;LDH释放实验检测对靶细胞的细胞毒性;裸鼠移植瘤模型检测肿瘤抑制能力;ELISA实验检测肿瘤组织中细胞因子的释放水平;结果:成功构建敲除TGF-βRⅡ的CLDN18.2 CAR T细胞(18.2BBZ/TGFBRKO)以及二代CLDN18.2 CAR T细胞(CLDN18.2);18.2BBZ及18.2BBZ/TGFBRKO在体外对CLDN18.2+的肿瘤细胞具有剂量依赖型细胞毒性;TGF-β1不能使18.2BBZ/TGFBRKO的Smad2/3发生磷酸化,在TGF-β1存在时,18.2BBZ/TGFBRKO与肿瘤细胞共孵育后PD-1的表达水平显著低于18.2BBZ(P<0.001);18.2BBZ/TGFBRKO较18.2BBZ对裸鼠移植瘤的抑制能力更强(P<0.0001),并且在肿瘤组织中保持更强的细胞因子分泌水平(P<0.01);结论:通过CRISPR/Cas9敲除CLDN18.2 CAR T细胞中的TGF-βRⅡ可以有效降低T细胞耗竭,并在小鼠移植瘤模型中产生更强的抑制肿瘤活性,并增加促炎性细胞因子的分泌。
文摘proach change of CGRP with time in periodontal ligaments of rats in magnetic field, maxillae of 28 Wistar rats of 6 7 weeks were placed in magnetic fields of permanent magnets Nd 2 Fe 4 B. Changes of CGRP with t ime in maxillary periodontal ligaments of the rats were studied by immunohistochemistry. The result showed that: magnetic field had dynamic effect and some promotional effect on recove?E F F E C T O F M A G N E T I C F I E L D O N C A L C I T O N I N G E N E- R E L A T E D P E P T I D E I N P E R I O D O N T A L L I G A M E N T S O F R A T S X. Chang , K. Qin and Y. L. Lu Orthodontic Department of Stomatology College , China Medical University , Shenyang 110001 , China Manuscript received 24 June 1999 In order to approach change of C G R P with tim e in periodontal liga m ents of rats in m agneticfield , m axillae of 28 Wistar rats of 6 7 weeks w ere placed in m agnetic fields of per m anentm agnets Nd 2 Fe4 B. Changes of C G R P with tim e in m axillary periodontal liga ments of therats were stu died by i m m unohistoche mistry . The result sho wed that : m agnetic field had dy na m ic effect an d so m e pro motional effect on recovering and reconstruction of the periodontaltissues in it . This provided a better theoretical evidence for clinical use of per m anent m agnets .
