油酸对人正常和瘢痕成纤维细胞增殖和分泌炎症介质的影响

目的 探讨油酸对人正常Fb和瘢痕Fb增殖和分泌炎症介质的影响. 方法 体外培养人正常Fb和瘢痕Fb,分别按照随机数字表法分为7组(各组样本数为8):空白对照组,除常规成分外培养液中不再添加其他物质;乙醇对照组,培养液中加入终浓度为体积分数2％无水乙醇;不同浓度油酸组,即0.25、0.50、1.00、2.00以及4.00 mmol/L油酸组,培养液中分别加入相应终浓度的油酸(以含体积分数2％无水乙醇培养液配制).采用锥虫蓝染色法观察各组细胞培养1～5d的生长情况.于倒置相差显微镜和透射电镜下观察2种细胞2个对照组、1.00 mmol/L油酸组细胞培养2d的细胞结构,采用流式细胞仪检测2种细胞2个对照组及1.00 mmol/L油酸组细胞培养2d的细胞周期.噻唑蓝法检测各组细胞培养2d的增殖情况.取各组细胞培养2d时的培养上清液,采用改良Griess法测定NO含量,ELISA法检测TNF-α、IL-6、IL-1β、IL-8含量.对数据进行多因素方差分析及重复测量设计的方差分析. 结果 (1)正常Fb和瘢痕Fb的空白对照组与乙醇对照组比较,各指标均无明显差别.(2)培养2～5d,正常Fb和瘢痕Fb 2.00、4.00 mmol/L油酸组细胞数量均低于对应的2个对照组(F值分别为13.773、11.344,P值均小于0.01).(3)培养2d,正常Fb和瘢痕Fb1.00 mmol/L油酸组细胞数量明显减少,部分细胞开始堆积、变圆易脱落;细胞膜不完整,线粒体空泡变性,核固缩,胞内可见脂滴.(4)正常Fb 1.00 mmol/L油酸组的G0/G1期和G2/M期细胞百分比[(93.56±9.98)％、(2.01士0.75)％]显著高于空白对照组[(84.23±10.96)％、(0.37±0.16)％],F值分别为3.026、34.751,P＜0.05或P＜0.01;S期细胞百分比为(4.42 ±0.87)％,明显低于空白对照组的(16.06±1.74)％,F=136.120,P＜0.01.瘢痕Fb 1.00 mmol/L油酸组G0/G1期和G2/M期细胞百分比分别为(93.86±13.90)％、(1.89±0.66)％,显著高于空白对照组[(83.88±10.42)％、(0.41±0.17)％],F值分别为3.529、32.710,P＜0.05或P＜0.01;S期细胞百分比为(3.87±0.63)％,明显低于空白对照组的(15.89±2.02)％,F=116.508,P＜0.01.(5)正常Fb和瘢痕Fb 0.50 ～4.00 mmol/L油酸组细胞增殖率显著低于对应的2个对照组(F值分别为215.945、194.555,P＜0.05或P＜0.01).(6)正常Fb各浓度油酸组细胞分泌NO水平明显高于2个对照组(F=30.240,P＜0.05或P＜0.01);瘢痕Fb 1.00～ 4.00 mmol/L油酸组细胞分泌NO水平明显高于2个对照组(F=12.495,P＜0.01).正常Fb和瘢痕Fb 2.00、4.00 mmol/L油酸组细胞分泌TNF-α、IL-6水平明显高于对应的2个对照组(F TNF-α值分别为6.911、3.818,FIL-6值分别为16.939、11.600,P ＜0.05或P＜0.01).正常Fb和瘢痕Fb各浓度油酸组细胞分泌IL-1β水平显著高于对应的2个对照组(F值分别为25.117、9.137,P值均小于0.01).正常Fb 1.00～4.00 mmol/L油酸组细胞分泌IL-8水平明显高于2个对照组(F=2.717,P＜0.05或P＜0.01),瘢痕Fb 2.00、4.00 mmol/L油酸组细胞分泌的IL-8水平明显高于2个对照组(F=3.338,P＜0.05).正常Fb和瘢痕Fb相同浓度油酸组各炎症因子水平比较无明显差异(F值为0.120 ～3.766,P值均大于0.05). 结论 高浓度油酸虽能抑制瘢痕Fb增殖,但同时也抑制正常Fb增殖,且高浓度油酸能同时促进正常Fb和瘢痕Fb分泌炎症介质,从而导致过度、持续的炎症反应,不利于创面愈合.     

没药提取物对人成纤维细胞增殖与胶原mRNA表达影响的实验研究

目的 观察没药提取物对人皮肤Fb生物学特性的影响,探讨其促进创面愈合的可能机制. 方法 从人正常包皮组织中分离并培养Fb,取第3～5代细胞用于实验.(1)将Fb接种至96孔板,按随机数字表法分为对照组,1×10-4、1 ×10-3、1×10-2、1 ×10-1、1、10、1×102 g/L没药水提取物组以及上述7种浓度的没药醇提取物组.对照组用含体积分数0.25％小牛血清的DMEM培养液(简称低浓度血清培养液)培养,各浓度没药水及没药醇提取物组分别用含相应终浓度2种没药提取物的低浓度血清培养液培养.培养48 h,用倒置相差显微镜观察细胞形态,噻唑蓝法测定各组Fb增殖活性(以吸光度值表示).(2)将Fb分别接种于培养瓶和培养皿中,均按随机数字表法分为2组:1 g/L没药水提取物组,用含终浓度1 g/L没药水提取物的低浓度血清培养液培养;对照组,采用低浓度血清培养液培养.培养72 h,分别采用流式细胞仪、实时荧光定量PCR法检测Fb周期与Ⅰ、Ⅲ型胶原mRNA的表达.对数据进行LSD-t检验. 结果 (1)各组细胞均呈长梭形生长,1 g/L没药水提取物组细胞融合较对照组更显著.1×10-3、1×10-2、1×10-1、1、10 g/L没药水提取物组Fb吸光度值分别为0.378±0.032、0.402±0.007、0.390±0.038、0.453±0.036、0.390±0.037,均高于对照组的0.332±0.044,t值分别为2.24、2.93、2.69、5.73、2.71,P值均小于0.05.1×10-4、1×102 g/L没药水提取物组吸光度值分别为0.312±0.048、0.154±0.009,前者与对照组比较,差异无统计学意义(t=2.84,P＞0.05);后者显著低于对照组(t =7.17,P＜0.05).1×10-3、1 ×10-1、1、10、1×102 g/L没药醇提取物组Fb吸光度值显著低于对照组(t值为2.30～24.79,P值均小于0.05).(2)1 g/L没药水提取物组G0/G1期细胞百分比为(74.3±6.3)％,明显少于对照组的(82.2±7.9)％,t=6.77,P＜0.05;S期及G2/M期细胞百分比分别为(16.6±3.4)％、(9.1±1.6)％,明显多于对照组的(13.3±2.3)％、(4.5±0.8)％,t值分别为7.53、6.34,P值均小于0.051 g/L没药水提取物组Fb中Ⅰ型胶原mRNA相对表达量(0.89±0.08)与对照组(1.00±0.06)比较,差异无统计学意义(t =1.17,P＞0.05);Ⅲ型胶原mRNA相对表达量(1.38±0.12)显著高于对照组(1.00±0.05,t=3.81,P＜0.01). 结论 没药水提取物能显著促进Fb增殖,加快Fb细胞周期进程,上调Fb中Ⅲ型胶原mRNA表达,可能与其促进创面愈合的机制相关.     

瘢痕疙瘩microRNA表达谱的筛选及miR-199a-5p生物功能的初步研究

目的 探讨并筛选出瘢痕疙瘩相关微小RNAs(miRNAs),检测其对瘢痕疙瘩成纤维细胞(keloid fibroblasts,KF)增殖的影响.方法 收集手术切除瘢痕疙瘩及正常皮肤组织标本各8例,采用基因芯片检测瘢痕疙瘩与正常皮肤组织差异表达的miRNA,并采用qRT-PCR验证,在瘢痕疙瘩成纤维细胞株中转染miRNA模拟物,模拟细胞中成熟miRNA的高表达,用EdU检测方法检测其对瘢痕疙瘩成纤维细胞增殖的影响.结果 ①通过芯片检测发现包括miR-199a-5p在内的17个差异表达的miRNAs.②qRT-PCR验证结果显示miR-199a-5p表达下调,与芯片检测结果相一致.③miR-199a-5p模拟物转染组与阴性对照组EdU的阳性率分别为(20.72±2.50)％和(27.68±4.92)％,miR-199a-5p模拟物转染组瘢痕疙瘩成纤维细胞的增殖率下降(t=2.183,P=0.047).同时,细胞的生长周期分布也发生改变,MiR-199a-5p模拟物转染组S期与G2/M期细胞百分比分别为(33.93±1.30)％和(10.87±0.80)％,阴性对照组分别为(31.39±0.79)％和(9.27±0.46)％,差异有统计学意义(P＜0.05).结论 ①瘢痕疙瘩中miRNA的差异表达有组织特异性;②miR-199a-5p在瘢痕疙瘩中的显著低表达,可影响瘢痕疙瘩成纤维细胞周期分布,抑制瘢痕疙瘩成纤维细胞增殖,提示miR-199a-5p可能参与了瘢痕疙瘩成纤维细胞增殖的调控.     

褪黑激素对人增生性瘢痕成纤维细胞增殖和凋亡的影响

目的 探讨褪黑激素对人增生性瘢痕Fb增殖和凋亡的影响与机制. 方法 采用组织块法分离培养人增生性瘢痕Fb.按随机数字表法将细胞分为低、中、高浓度组和对照组.前3组细胞分别采用含1 × 10-5、1×10-3、1 mmol/L褪黑激素的培养液培养,对照组不加褪黑激素常规培养.处理后24 h进行如下检测:对各组细胞进行形态学观察;用四氮唑复合物( XTT)-硫酸酚嗪甲酯(PMS)比色法检测细胞增殖活性;对细胞行膜联蛋白V-异硫氰酸荧光素(FITC)和碘化丙啶(PI)双染色后,用流式细胞仪分析细胞周期和凋亡情况;荧光定量RT-PCR法检测细胞周期蛋白E(cyclin E)、p53和Fas mRNA表达量.对数据行方差分析和LSD检验. 结果 形态学观察显示,对照组Fb为长梭形,呈集落分布;3个浓度组Fb随着褪黑激素浓度升高,细胞逐渐分散,胞体变形缩小,胞膜皱缩,核质比例减小.对照组、低浓度组、中浓度组、高浓度组Fb增殖活性(吸光度值)依次下降,分别为1.79±0.10、1.49±0.15、1.24±0.20、0.92±0.09(F=67.61,P ＜0.05);S期细胞百分比依次下降,分别为(16.9±1.3)％、(10.6±1.1)％、(6.1±1.2)％、(3.2±0.8)％(F=286.10,P ＜0.05);G2/M期细胞百分比依次下降,分别为(16.7±1.6)％、(13.5±1.1)％、(9.8±1.0)％(6.0±0.7)％(F=162.69,P＜0.05);早、晚期凋亡细胞百分比依次升高(F值分别为424.05、236.44,P值均小于0.05).对照组、低浓度组、中浓度组、高浓度组细胞cyclin E的mRNA表达量依次下降,分别为2.90±0.30、1.58±0.21、0.90±0.20、0.24±0.12(F=266.79,P＜0.05);p53和Fas mRNA表达量依次升高(F值分别为10.11、12.03,P值均小于0.05). 结论 褪黑激素可通过影响细胞cyclin E、p53和Fas基因的表达,抑制增生性瘢痕Fb增殖并诱导该细胞凋亡.     

Wnt/β连环蛋白信号在人真皮成纤维细胞表型转化中的作用及机制

目的 了解Wnt/β连环蛋白信号在人正常真皮Fb向肌成纤维细胞(MFb)表型转化中的作用及机制.方法 采用酶消化法分离培养人正常皮肤真皮Fb.(1)实验1.按随机数字表法将细胞分为4组:对照组,采用无血清DMEM培养液(以下简称培养液)培养;TGF-β1组,用含10 ng/mL重组人TGF-β1(浓度下同)的培养液培养;Wnt3a组,用含150 ng/mL重组人Wnt3a(浓度下同)的培养液培养;TGF-β1+Wnt3a组,用含重组人TGF-β1和重组人Wnt3a的培养液培养.48 h后,分别采用实时荧光定量PCR法和蛋白质印迹法,检测Fb β连环蛋白和α平滑肌肌动蛋白(α-SMA)的mRNA及蛋白表达水平.(2)实验2.按照随机数字表法将细胞分为4组:对照组、TGF-β1组,处理方法均同实验1;SB415286(糖原合酶激酶3β阻断剂)组,用含10 μmol/L SB415286(浓度下同)的培养液培养;TGF-β1+ SB415286组,用含重组人TGF-β1和SB415286的培养液培养.48 h后,分别采用实时荧光定量PCR法和蛋白质印迹法检测Fb α-SMA的mRNA和蛋白表达水平;用免疫荧光细胞化学法检测α-SMA阳性表达情况.各项检测重复3次,对数据进行方差分析及LSD-t检验.结果 (1)实验1.①β连环蛋白的mRNA表达水平:对照组、TGF-β1组、Wnt3a组和TGF-β1+Wnt3a组Fb组间比较,差异无统计学意义(F=0.302,P=0.823).②β连环蛋白的蛋白表达水平:4组比较,差异有统计学意义(F=16.713,P=0.001).与对照组表达水平(0.34±0.11)相比,TGF-β1组、Wnt3a组均显著上调(0.73±0.12、0.82±0.17,t值分别为3.028、3.727,P＜0.05或P＜0.01).TGF-β1+Wnt3a组(1.23±0.21)显著高于其余3组(t值分别为6.911、3.883、3.184,P值均小于0.01).③α-SMA mRNA表达水平:4组比较,差异有统计学意义(F=31.830,P=0.001);与对照组相比,TGF-β1组显著上调,Wnt3a组下调(t值分别为6.759、2.535,P＜0.05或P＜0.01);TGF-β1+Wnt3a组低于TGF-β1组(t=4.532,P＜0.01).④α-SMA蛋白表达水平:对照组、TGF-β1组、Wnt3a组、TGF-β1+ Wnt3a组分别为0.83±0.17、1.43±0.20、0.53±0.12、0.89±0.14(F=16.597,P=0.001).与对照组相比,TGF-β1组显著上调,Wnt3a组下调(t值分别为4.582、2.291,P＜0.05或P ＜0.01);TGF-β1 +Wnt3a组低于TGF-β1组(t =4.123,P＜0.01).(2)实验2.①α-SMA mRNA表达水平:4组比较,差异有统计学意义(F=34.101,P=0.001).SB415286组明显低于对照组(t =2.511,P＜0.05),TGF-β1+SB415286组明显低于TGF-β1组(t=3.587,P＜0.01).②α-SMA蛋白表达水平:4组细胞比较,差异有统计学意义(F =11.381,P=0.003).SB415286组显著低于对照组(t=2.364,P＜0.05),TGF-β1+ SB415286组低于TGF-β1组(t=2.556,P＜0.05).③免疫荧光细胞化学法检测显示,对照组Fb α-SMA表达微弱;TGF-β1组α-SMA表达较对照组显著增强(t =11.198,P＜0.01);SB415286组表达较对照组减少,TGF-β1+ SB415286组表达较TGF-β1组显著减少(t=5.902,P＜0.01).结论 Wnt/β连环蛋白信号可能参与Fb细胞表型转化,并且对TGF-β1所介导的促转化效应起负性调控作用.     

热损伤角质形成细胞培养上清对真皮成纤维细胞增殖与胶原mRNA表达的影响

目的 观察热损(创)伤后角质形成细胞(KC)培养上清对真皮成纤维细胞(Fb)增殖与胶原mRNA表达的影响.方法 建立KC热损伤模型,收集正常及热损伤12 h后培养上清配制不同浓度的细胞条件培养液;分离培养人正常Fb,实验组分别加入含不同浓度的细胞条件培养液,以单纯DMEM组为对照,采用噻唑蓝(MTT)比色法测定24 h后真皮成纤维细胞增殖;分别以流式细胞仪、实时荧光定量聚合酶链反应(Real-time PCR)测定DMEM组、50%浓度条件培养液组相同时间Fb生长周期及Ⅰ型胶原mRNA表达.结果 细胞增殖测定:热损伤上清(10%、30%、50%、70%)条件培养液组A值(0.151±0.004、0.165±0.009、0.195±0.006、0.202±0.008)均高于正常上清(10%、30%、50%、70%)条件培养液组(0.144±0.004,0.159±0.002,0.180±0.005,0.181±0.006)及对照组(0.144±0.007),除10%浓度组外各实验组A值与对照组比较差异均有统计学意义(t值分别为:4.01、11.42、11.41;P＜0.05),热损伤上清与正常上清条件培养液组比较:50%、70%浓度组间差异有统计学意义(t值分别为:2.03、1.94;P＜0.05);热损伤上清条件培养液50%与70%浓度组间差异无统计学意义(t值为1.34;P＞0.05).细胞周期测定见50%浓度热损伤上清组可明显促进Fb通过G1/S及S/G2限制点,S期及G2/M期细胞与对照组比较增多,G0/G1期细胞与对照组比较明显减少(t值分别为:5.87、11.3、4.86;P＜0.05).Ⅰ型胶原mRNA表达测定中,50%浓度热损伤上清组与对照组比较表达明显上调,差异亦有统计学意义(t=1.72;P＜0.05).结论 热损(创)伤后KC上清液可促进Fb的增殖,同时可上调Ⅰ型胶原mRNA的表达.

Abstract：

Objective To observe the effects of heat injured keratinocyte (KC) supematant on proliferation activity and collagen mRNA expression of the normal fibroblast (Fb).Methods A model of heat injured KC in vitro was reproduced,the supernatants of heat injured KC were collected and used as conditioned medium,and DMEM was used as control medium.Normal Fb were treated with different concentrations of conditioned medium.After treatment for 24 h,the proliferation activity,cell cycle and collagen Ⅰ mRNA levels in treated Fb were measured by using methylthiazol tetrazolium ( MTT),flow cytometry (FCM) and real-time polymerase chain reaction (PCR) techniques.Results As compared with the control group,the absorbance (A) values of MTT in treated groups were significantly increased (t = 4.01,11.42,11.41 ;P ＜0.05),except in 10% group (t = 1.34,P ＞0.05).As compared with normal supernatant treated group (0.159 ± 0.002,0.180 ± 0.005,0.181 ± 0.006),the A values in 50% and 70% conditioned medium group (0.195 ± 0.006,0.202 ± 0.008 ) were significantly increased ( t = 2.03,1.94;P ＜0.05).The 50% conditioned medium increased the percentage of G1 to S and S to G2 phase transit.As compared with control group,the number of cells in S and G2/M phase was increased significantly (t =5.87,11.3;P＜0.05 ) and that in G0/G1 phase decreased significantly ( t = 4.86;P＜0.05 ).Real-time PCR revealed that the collagen Ⅰ mRNA level in 50% conditioned medium group was significantly up-regulated (t= 1.72;P＜0.05 ) as compared with control group.Conclusion Heat injured KC supernatant may promote the proliferation of dermal Fb,and up-regulate the mRNA expression of collagen.     

血竭素高氯酸盐对成纤维细胞增殖与成纤维细胞生长因子mRNA表达的影响

目的 观察血竭素高氯酸盐(Dp)对人皮肤成纤维细胞(Fb)生物学特性的影响,探讨其促进创而愈合的机制.方法 分离培养人正常Fb,将Dp以不同浓度(0.063、0.125、0.250、0.625、1.250、2.500 mg/L)分别加入培养液,采用噻唑蓝(MTT)比色法检测Dp在不同浓度以及不同时间点对体外培养的Fb增殖作用的影响.通过流式细胞仪,实时荧光定量聚合酶链式反应( real-time PCR)分别检测最适浓度培养条件下Fb的细胞周期变化以及成纤维细胞生长因子(FGF) mRNA的合成表达.结果 Dp在0.625～1.250 mg/L浓度范围内,其吸光度值(0.237±0.012、0.243±0.017)均高于对照组(0.208±0.011)差异有统计学意义(t值分别为2.11、2.23,P＜0.05),此浓度范围可促进Fb增殖,且呈剂量依赖性,在浓度为1.250 mg/L时(A值0.243±0.017),促增殖作用最为显著(t =2.23,P＜0.01),流式细胞仪结果显示,在浓度为1.250 mg/L时,Dp可明显促进Fb通过G1/S及S/G2期限制点,S期及G2/M期细胞与对照组比较明显增多,G0/G1期细胞与对照组比较明显减少(t值分别为4.32、7.53、3.27,P＜0.05).细胞因子mRNA表达测定中,1.250 mg/L Dp组与对照组比较表达明显上调,差异亦有统计学意义(=1.48,p＜0.05).结论 Dp能显著促进Fb增殖,加快Fb周期进程,同时可促进FGF的mRNA表达,可能与血竭促进创面愈合的机制有关.     

La核糖核蛋白6对人血管内皮细胞增殖与凋亡的作用

目的 研究La核糖核蛋白6(Achn)在人血管内皮细胞增殖和凋亡中的调节作用.方法 (1)DMEM无血清培养基培养人血管内皮细胞株Eahy926细胞,按随机数字表法(下同)分为Achn抑制组(转染Achn抑制表达载体psi-Achn)、psi4.1空载体组(转染psi4.1)、Achn诱导组(转染Achn诱导表达载体pcDNA-Achn)、pcDNA3.1空载体组(转染pcDNA3.1)、Achn与钙/钙调蛋白依赖性丝氨酸蛋白激酶(CASK)共转染组(转染pcDNA-Achn与CASK抑制表达载体psi-CASK)、空白对照组(PBS处理),分别于转染后1、24、48、72 h用噻唑蓝法测定各组细胞570 nm波长下的吸光度值.(2)取Eahy926细胞,裂解细胞总蛋白,二辛丁酸法定量后分为蛋白质印迹组(总蛋白量为20μg)、Achn蛋白沉淀组、CASK蛋白沉淀组、IgG对照组,后3组细胞蛋白总量各为100μg,免疫共沉淀法检测各组Achn、CASK蛋白水平.(3)取Eahy926细胞分为LPS组(5 mol/L LPS处理)、氯化钾组(5 mol/L氯化钾处理)、空白对照组(5 mol/L PBS处理)、Achn诱导转染组(转染pcDNA-Achn)、Achn与CASK共转染组(转染pcDNA-Achn与psi-CASK),转染组转染24 h后加入LPS刺激12 h,免疫组织化学法检测各组半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达.(4)取Eahy926细胞分为Achn诱导组(转染pcDNA-Achn)、Achn抑制组(转染psi-Achn)、对照组(PBS处理),24 h后加入烧伤患者血清处理12 h,流式细胞仪检测各组细胞凋亡率.对实验数据行t检验和单因素方差分析.结果 (1)Achn抑制组细胞增殖水平从24 h开始低于psi4.1空载体组,48、72 h时差异均有统计学意义(t值分别为10.777、6.112,P值均小于0.05);转染后24、48、72 h Achn诱导组细胞增殖水平均显著高于pcDNA3.1空载体组(t值分别为5.367、6.053、9.831,P值均小于0.05);Achn与CASK共转染组细胞增殖水平48、72 h均显著低于Achn诱导组(t值分别为5.481、9.517,P值均小于0.05).(2)CASK蛋白沉淀组CASK抗体沉淀物中可同时检测到CASK蛋白和Achn蛋白,而Achn蛋白沉淀组Achn抗体沉淀物中可同时检测到Achn蛋白和CASK蛋白.(3)Achn诱导转染组血管内皮细胞caspase-3阳性表达率为(15.6±0.5)%,低于LPS组[(32.8±2.6)%,t=10.083,P＜0.05];Achn与CASK共转染组caspase-3阳性细胞表达率[(7.0±2.0)%]进一步降低,显著低于LPS组(t=9.827,P＜0.01).(4)Achn抑制组细胞凋亡率为(45.6±10.9)%,显著高于对照组的(13.2±4.3)%,t=7.043,P＜0.05;Achn诱导组的细胞凋亡率为(5.3±2.9)%,显著低于对照组(t=6.499,P＜0.05).结论Achn能促进入血管内皮细胞增殖,抑制LPS或烧伤血清诱导细胞凋亡并与CASK的作用相关联.

Abstract：

Objective To investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell. Methods ( 1 ) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4. 1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)] , blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 μg protein) , CASK antibody precipitation group ( 100 μg protein), IgG antibody group ( 100 μg protein), Western blot group (20 μg protein).Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn) , KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/LPBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group ( transfected with pcDNA-Achn vector), and blank control group ( treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test. Results ( 1 ) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10. 777, 6.112, P values all below 0. 05 ).The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3. 1 group (with t value respectively 5. 367, 6. 053, 9. 831, P values all below 0.05 ). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group ( with t value respectively 5.481, 9. 517, P values all below 0. 05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [( 15.6 ± 0. 5 ) %] as compared with that in LPS induction group [(32. 8 ±2.6)%, t = 10. 083, P ＜ 0. 05], and that in cotransfection group showed further inhibition [(7.0 ±2.0)%,t =9.827, P ＜0.01]. (4) Apoptosis rate in Achn inhibition group[(45.6 ± 10.9)%] was higher than that in blank control group [(13.2±4.3) %, t =7.043, P ＜0.05]; while that in Achn inductiongroup [(5.3 ±2.9)%] was lower than that in blank control group ( t =6.499, P ＜0.05).Conclusions Achn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.     

消瘢醑对瘢痕疙瘩成纤维细胞I、III型胶原蛋白的影响

目的研究复方中药制剂消瘢醑对体外培养的瘢痕疙瘩成纤维细胞(Keloid fibroblast,KFB)I、III型胶原蛋白的影响. 方法 6例瘢痕疙瘩成纤维细胞为实验组,6例正常皮肤成纤维细胞(Normal fibroblast,NFB)为对照组,采用成纤维细胞体外培养、ABC免疫细胞化学染色技术及积分光密度(IOD)分析,观察在10μg/ml消瘢醑浓度作用下,瘢痕疙瘩和正常皮肤成纤维细胞I、III型胶原蛋白的表达. 结果瘢痕疙瘩成纤维细胞I、III型胶原阳性染色表达强度均显著高于正常皮肤成纤维细胞(11113.1±1304.9 vs 3519.6±236.0与11157.7±1300.3 vs 2626.5±426.3,t值分别为14.42和13.47,P值分别为0.00003和0.00004).10μg/ml浓度的消瘢醑作用48小时后:1.瘢痕疙瘩成纤维细胞I、III型胶原阳性染色表达强度均显著高于正常皮肤成纤维细胞(7675.4±825.5 vs 2305.2±320.4与10595.2±1311.5 vs 2434.8±356.9,t值分别为13.37和12.66,P值分别为0.00004和0.00005);2.瘢痕疙瘩和正常皮肤成纤维细胞呈阳性表达的I、III型胶原染色强度明显下降,与未经药物处理的相应空白对照相比具有显著性差异(7675.4±825.5 vs 11113.1±1304.9与10595.2±1311.5 vs 11157.7±1300.3,t值分别为10.31和4.68,P值分别为0.0001和0.0054).瘢痕疙瘩组I型胶原蛋白合成抑制率30.7%,III型胶原蛋白合成抑制率为5.1%. 结论在I、III型胶原蛋白表达方面,体外培养的瘢痕疙瘩成纤维细胞明显高于正常皮肤成纤维细胞;"消瘢醑"能显著抑制体外培养瘢痕疙瘩成纤维细胞和正常皮肤成纤维细胞I、III型胶原蛋白的表达,并以抑制I型胶原表达为主.     

过氧化物酶体增殖物激活受体γ在瘢痕疙瘩组织中的表达及意义

目的探讨过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPAR-γ)在瘢痕疙瘩组织中的表达及其意义。方法取23例患者自愿捐赠的瘢痕疙瘩标本进行观察,以同一患者手术取皮修剪后的剩余正常皮片作为正常对照。将标本行免疫组织化学染色,观察PPAR-γ蛋白的表达,参照Shimizu免疫组织化学评分标准进行评分,计算标本阳性率以及阳性细胞率。结果免疫组织化学染色示,PPAR-γ蛋白在瘢痕疙瘩和正常皮肤中均有表达。在瘢痕疙瘩中,其位于表皮的棘细胞层、颗粒层以及真皮血管,染色较浅;在正常皮肤中,位于表皮的基底层以及真皮血管壁、汗腺、皮脂腺,染色较深。瘢痕疙瘩组免疫组织化学染色评分为(2.65±0.78)分,低于正常对照组的(3.65±1.19)分;标本阳性率为52.17%(12/23),显著低于正常对照组的82.61%(19/23);阳性细胞率为46.04%±8.61%,显著低于正常对照组的59.39%±11.26%。以上指标组间比较,差异均有统计学意义(t=5.030,P=0.000;χ~2=4.847,P=0.028;t=5.974,P=0.000)。结论与正常皮肤相比,PPAR-γ在瘢痕疙瘩中呈下调表达状态,提示其可能与瘢痕疙瘩的形成有关。     

胸腰椎骨折椎弓根内固定断裂与植骨融合方式的相关性分析

[目的]探讨胸腰椎骨折椎弓根螺钉内固定系统内固定术后,椎弓根螺钉断裂与植骨融合方式之间的关系,以探讨胸腰椎骨折植骨融合的最佳方式。[方法]回顾性研究1995年5月~2005年12月本院脊柱外科收治的胸腰椎骨折病人197例,其中A组单纯内固定(不植骨)患者14例,B组“H”形椎板植骨21例,C组横突间植骨67例,D组椎间、椎内联合横突间植骨95例。[结果]术后随访6~32个月,内固定断裂12例,其中A组4例,B组3例,C组5例,D组0例,4组中D组内固定断裂率显著低于其他3组(P<0.05)。[结论]椎间、椎体内联合横突间植骨重建脊柱三柱的稳定性,符合人体生物力学原理,能有效降低内固定断裂的发生。     

On the measurement of the functional properties of the CFTR

A number of methods are currently employed to assess the functional properties of CFTR channels and their response to pharmacological potentiators, correction of the defective CFTR trafficking, and vectorial introduction of new proteins. Here we review the most common methods used to assess CFTR channel function. The suitability of each technique to various experimental conditions is discussed.     

The Measurement of the Inclination Angle of the Hamate and Analysis of the Inclination Angle for the Rotation Deformity of the Little Finger in the Fixation of the Carpometacarpal Joint

ObjectiveComplex base fractures of the fifth metacarpal bone and dislocation of the fifth carpometacarpal joint are more prone to internal rotation deformity of the little finger sequence after fixation with a transarticular plate. In the past, we have neglected that there is actually a certain angle of external rotation in the hamate surface of transarticular fixation. This study measured the inclination angle of the hamate surface relative to the fifth metacarpal surface for clinical reference.MethodsIn a prospective single‐center study, we investigated the tilt angle of 60 normal hamates. The study included thin‐layer computed tomography (CT) data from 60 patients from the orthopaedic clinic and inpatient unit from January 2017 to March 2020, including 34 men and 26 women who were 15~59 years old, average 35 years old. The CT data of 60 cases in Dicom format of the hand was input into Mimics and 3‐Matics software for three‐dimensional (3D) reconstruction and measuring the angle α between hamate surface and the fifth metacarpal surface. According to the possible placement of the transarticular plate on the fifth metacarpal surface, we measured the angle β between the hamate surface 1 and the fifth metacarpal surface and the angle γ between the hamate surface 2 and the fifth metacarpal surface.ResultsThe average angle between the hamate surface and the fifth metacarpal surface was 11.66°. The hamate surfaces 1 and 2 have an external rotation angle of 7.30° and 7.51° on average with respect to the fifth metacarpal surface, respectively. There is no statistically significant difference in the angles between the two groups (P > 0.05).ConclusionsThe horizontal angle of the dorsal side of the hamate is different from the back of the fifth metacarpal surface, and the hamate has a certain external rotation angle with respect to the fifth metacarpal surface. No matter how the transarticular plate is placed, the plate always has a certain external rotation angle relative to the fifth metacarpal surface. When the fixation is across the fifth carpometacarpal joint, if the plate does not twist and shape, it will inevitably cause internal rotation of the fifth metacarpal, resulting in internal rotation deformity of the little finger sequence.     

静脉输注甘露醇开放血脑屏障对抗生素透过血脑屏障的影响

目的　通过快速静脉输注甘露醇可逆性开放血脑屏障 (BBB) ,探知此方法能否增加抗生素透过BBB的量 ,在何时达到最高峰 ,其通透量增加后临床上有无不良反应。方法　采用自身配伍设计 ,共 6个样本组。对照组仅使用抗生素 ;其余 5组分别在使用甘露醇前 60、3 0min ,同时使用甘露醇后 3 0、60min使用抗生素 ,各组皆取使用抗生素后 1h的脑脊液测其抗生素浓度。抗生素选用头孢三嗪。结果　测量值经过q检验 ,经 2 0 %甘露醇处理前后的CSF中的头孢三嗪浓度差异有非常显著性。全组患者经临床观察未出现神经系统的不良反应。结论　经静脉快速输注2 0 %甘露醇后可以使透过BBB的水溶性抗生素的量增加 ,两者使用的顺序是在抗生素使用 3 0min内即给予甘露醇快速滴注。该方法不会增加低神经毒性抗生素在中枢神经系统的不良反应。     

三角韧带损伤的手术治疗

[目的]探讨踝关节三角韧带损伤的手术治疗及效果。[方法]2002年4月-2005年4月治疗伴有三角韧带损伤的踝关节骨折40例，均采用切开复位和坚强内固定，并修复重建三角韧带，恢复踝关节内外侧结构的稳定性。下胫腓联合分离仍不稳定者，给予皮质骨螺钉横向内固定。[结果]全部病例得到16个月-3a随访，平均1．5a。按齐氏疗效评定标准：优良30例，可8例，差2例，优良率75％。[结论]强调踝关节骨折切开解剖复位，坚强内固定的同时，应充分重视修复重建三角韧带。     

Preservation of the pylorus and resection of the head of the pancreas

The historical evolution of the pylorus-preservation resection of the head of the pancreas is traced from the first resections early in this century to relative standardization of the operation, to a lowering of the operative mortality, and to an interest in improving nutritional status after resection. There are many theoretical advantages for the function of the upper gastrointestinal tract after pylorus and gastric preservation, such as maintenance of gastric capacitance and equilibration of osmotic pressure in gastric digestants, foodstuff digestion and absorption, and bowel motility. After the pylorus-preserving resection, gastric emptying is normal, pyloric function to prevent duodenal reflux is often normal, and gastric acids and serum levels of duodenal hormones are at normal levels, whereas after standard pancreatoduodenectomy, all of these are often abnormal. No prospective blinded studies have been published comparing nutritional values after the two operative procedures, but evidence is presented of a satisfactory result with regard to gastric capacitance, body weight gain, and lack of postgastrectomy symptoms. An undoubted advantage of the pylorus-preserving feature is a simplification of the operation. These gains are achieved without increase in operative mortality, without increase in the incidence of jejunal ulcer, and without theoretical or actual decrease in value of the procedure as a cancer operation, except in patients with duodenal carcinoma proximal to the ampulla of Vater.     

下颌全牙弓后移量及最后界的测量

目的：研究下颌牙弓的有效后移量及找寻下颌牙弓移动的后界。方法：选取涉及拔除下颌第三磨牙或下颌第三磨牙缺失的病例18例（男6例,女12例）。采用种植支抗牵引下牙弓向远中,治疗完成时所有病例均明确到达下颌牙弓后界,即下颌第二磨牙远中到达下颌升支前缘软组织交界处。应用治疗前后的曲断片测量下颌第二磨牙远中到升支前缘的距离。结果：下颌第二磨牙后移量为（3.49±1.21）mm;治疗后磨牙后间隙的长度为（4.43±0.97）mm。结论：下颌牙弓可确定性地实现整体后移;最大后移量由磨牙后间隙的长度决定;其最后界止于下颌第二磨牙远中与下颌升支前缘软组织交界处。