两种不同方法培养大鼠肾上腺细胞的比较

目的 观察胶原凝胶三维培养对肾上腺细胞形态和功能的影响.方法 分离SD大鼠乳鼠肾上腺细胞,于自制鼠尾型Ⅰ胶原凝胶中培养14 d,与传统平面培养的肾上腺细胞相对比,比较两者在细胞形态、细胞增殖率、蛋白表达和分泌功能方面的差异.结果 两种培养方式下,肾上腺细胞均能表达其特异性细胞色素氧化酶CYP11B1和CYP11B2.原代培养8 d后,三维胶原凝胶培养大鼠肾上腺细胞的增殖率显著高于平面培养,至第14天,胶原凝胶中培养的肾上腺细胞增殖率为16.6%,而传统平面培养的仅为2.9%.胶原凝胶中培养的肾上腺细胞分泌功能维持时间更长,第14天其培养液中皮质酮和醛固酮浓度分别为(278.33±38.20)μg/L和(133.85±38.20)ng/L,而相应的平面培养肾上腺细胞分泌皮质酮和醛固酮浓度分别为(160.35 ±28.12)μ/L和(74.03±12.12)ng/L,差异有统计学意义(P＜0.05).结论 胶原凝胶中三维培养肾上腺细胞更有利于细胞功能的维持.     

血小板源性生长因子-BB影响下大鼠血管平滑肌细胞增殖及其ras蛋白的表达

目的 观察血小板源性生长因子-BB(PDGF-BB)对大鼠血管平滑肌细胞(VSMC)的增殖及其ras蛋白表达的影响.方法 体外培养大鼠VSMC并传代,将第4代VSMC分4组,设空白对照组,另设3组分别加入1、2、4 μg/L PDGF-BB进行干预,采用细胞计数、免疫组织化学及免疫荧光等方法,观察干预后1、3、7 d 3个时间点的VSMC计数、增殖细胞核抗原(PCNA)表达及相应时段ras蛋白的表达,并分析两者之间的相关性.结果 (1)在7 d内各组VSMC计数均逐渐增加,其中2 μg/L和4 μg/L PDGF-BB组的计数增长较对照组快(P＜0.05),4 μg/L PDGF-BB组较1 μg/LPDGF-BB组快(P＜0.05);(2)免疫组织化学和免疫荧光检测显示,干预3 d后各组VSMC增殖细胞百分比均表现为高PDGF-BB浓度组高于低浓度组(P＜0.05),ras蛋白荧光表达强度亦呈现相同趋势(P＜0.01).PDGF-BB干预后的VSMC的PCNA表达和ras蛋白表达呈显著正相关(r=0.735,P＜0.05).结论 PDGF-BB可以刺激体外培养大鼠VSMC增殖,并可促进其ras蛋白的表达.     

己酮可可碱对肾小管上皮细胞转分化抑制作用的实验研究

目的:探讨己酮可可碱(PTX)对单侧输尿管梗阻(UUO)模型大鼠肾间质平滑肌肌动蛋白(α-SMA)表达的影响,并在体外研究其对肾小管上皮细胞转分化及其对Smad 7表达的作用.方法:将实验大鼠分为假手术组、UUO对照组和UUO PTX治疗组.用免疫组化法测定各组动物肾组织α-SMA表达.体外培养的人肾小管上皮细胞(HK-2),经TGF-β1及PTX干预后,用蛋白印迹检测α-SMA和Smad 7表达,用ELISA检测培养上清中Ⅰ型胶原含量.结果:假手术组大鼠肾组织仅血管壁α-SMA免疫染色阳性.UUO对照组第3 d、第7 d、第14 d,α-SMA表达程度分别为(2.90±0.71)%、(8.50±0.97)%、(17.9±2.70)%,PTX治疗组则分别为(2.70±0.62)%、(6.90±0.83)%、(13.8±1.9)%,术后第7 d(P<0.05)、第14 d(P<0.01)两组有统计学差异.TGF-β1(8 ng/ml)刺激HK-2细胞72 h后,α-SMA表达强度为(87.7±7.0)%,培养上清中Ⅰ型胶原浓度为(2 560.49±95.03) ng/ml;而加入PTX 10、50、100、300、500 μg/ml干预72 h后,α-SMA表达强度(F＝174.998,P<0.001)和培养上清中的Ⅰ型胶原(F＝460.883,P<0.001)呈浓度依赖性减少.在TGF-β1刺激前24 h、TGF-β1刺激后0 h、12 h、24 h、36 h或48 h后分别加入PTX(500 μg/ml),α-SMA表达强度(F＝144.131,P<0.001)和培养上清中的Ⅰ型胶原(F＝444.557,P<0.001)则呈时间依赖性减少.TGF-β1刺激的同时,分别加入PTX 10、50、100、300、500 μg/ml干预72 h后,Smad 7表达强度分别为(50.6±3.1)%、(49.4±2.9)%、(48.9±2.5)%、(51.4±1.8)%、(49.5±3.0)%,与阳性对照组(46.9±3.5)%比较无统计学差异(P＞0.05).结论:(1)PTX可减少UUO大鼠肾间质α-SMA表达;(2)在体外,PTX可抑制TGF-β1诱导的肾小管上皮细胞转分化,该作用与Smad 7表达无关,其机制有待于进一步研究.     

Foxo3a和p27kip1在大鼠坐骨神经损伤后背根神经节中的表达

目的 探讨坐骨神经夹伤后Foxo3a和p27kip1在腰段背根神经节(DRG)中表达变化及其意义.方法 将成年SD大鼠随机分为正常对照组和实验组.对实验组实行坐骨神经夹伤术.运用蛋白质印迹、免疫荧光双标,研究大鼠坐骨神经夹伤后,腰段DRG中Foxo3a和p27kip1的表达、分布以及细胞增殖和轴突再生情况.结果 Foxo3a在坐骨神经夹伤后1 d表达值(7.0±3.5)开始明显下降,2 d表达值(6.0±3.8)达到最低值,随之逐渐升高,p27kip1在坐骨神经夹伤后2 d表达值(29.0±3.5)表达开始明显下降,7 d表达值(21.0±3.0)达到最低值,随之逐渐升高;Foxo3a和p27kip1分布于神经元和神经胶质细胞内且在坐骨神经夹伤后2d DRG中,Foxo3a和p27kip1在神经元细胞[(37.8±5.7)%、(43.3±4.3)%]和神经胶质细胞[(22.4±3.9)%、(13.8±3.2)%]中表达较在正常组神经元细胞[(73.6±2.5)%、(84.1±3.7)%]和神经胶质细胞[(61.3±4.4)%、(68.7±5.6)%]减少;细胞核增殖抗原(proliferating cell nuclear antigen,PCNA)和神经元生长相关蛋白(GAP-43)在坐骨神经夹伤后2 d DRG中表达值[12±2.6,15±1.9]均开始上调,PCNA在7 d表达值(25.0±3.2)达到最高点,GAP-43则保持较高水平;此外,PCNA与神经胶质细胞共定位明显,与神经元细胞几乎无共定位.结论 大鼠坐骨神经损伤后,Foxo3a和p27kip1在腰段DRG中的表达减少与神经胶质细胞增殖和轴突再生密切相关.     

人胚胎神经干细胞延迟移植对脊髓损伤大鼠运动功能的影响及其机制

目的:初步探讨人胚胎神经干细胞(human fetal neural stem cells,hfNSCs)延迟移植对脊髓损伤大鼠运动功能的影响及其机制。方法:体外分离、培养和鉴定hfNSCs,选取200~250g的Wistar大鼠28只建立T9~T11节段脊髓挫伤模型,损伤后第9天取20只BBB评分为4~5分的大鼠随机分为对照组及移植组(每组10只),同时在T10水平脊髓中线两侧1.5mm处分别给予2μl不含hfNSCs的杜氏磷酸缓冲液(DPBS)及2μl含有105个体外培养第2代hfNSCs的DPBS,细胞移植后第7、14、28、42、56天用BBB评分评价两组后肢运动功能,移植后第28天及第56天用免疫组织化学法检测移植组大鼠损伤脊髓局部神经胶质纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP2)和2′,3′-环腺苷酸-3′-磷酸二酯酶(CNPase)的表达情况,以评价大鼠损伤脊髓局部hfNSCs的存活及分化情况。移植后第56天髓磷脂碱性蛋白(MBP)染色及透射电子显微镜扫描观察移植组与对照组损伤局部髓鞘形成情况。结果:体外培养的hfNSCs表达Nestin,并可分化为星形胶质细胞[(61.2±1.6)%]、神经元[(27.6±3.4)%]及少突胶质细胞[(6.0±5.5)%]。移植前及移植后第7、14天两组BBB评分比较无统计学差异(P>0.05),移植组在移植后第28、42、56天评分(分别为8.30±1.07分,11.30±1.05分,15.10±1.12分)较对照组(分别为7.00±0.98分,8.30±1.02分,9.10±0.96分)明显提高(P<0.05)。在移植后第28天hfNSCs可分化为GFAP阳性的星形胶质细胞、MAP2阳性的神经元和CNPase阳性的少突胶质细胞,在移植后第56天hfNSCs仍在损伤局部存活并可分化为星形胶质细胞和少突胶质细胞,但未见神经元分化;移植后第56天星形胶质细胞的积分光密度(IOD)值(1502.31±131.92)大于移植后第28天(628.98±119.31)(P<0.05),而少突胶质细胞在移植后第28天及第56天的变化无统计学差异。移植后第56天MBP染色证实人源性少突胶质细胞参与损伤区髓鞘的形成,透射电子显微镜扫描结果显示移植组有完整和连续的髓鞘形成,对照组髓鞘呈断裂及畸形表现。结论:hfNSCs延迟移植可促进脊髓损伤大鼠运动功能的恢复,其机制可能与促进损伤局部髓鞘的形成有关。     

急性胰腺炎大鼠肾上腺损伤中bax与bcl-2表达的研究

目的 观察急性胰腺炎(AP)大鼠肾上腺损伤时细胞凋亡及调控基因bcl-2和bax蛋白的表达及作用.方法 采用牛磺胆酸钠逆行胰胆管注射法建立AP模型.术后3、6、12 h检测血皮质酮水平,观察肾上腺病理,TUNEL检测细胞凋亡,免疫组织化学检测bcl-2和bax蛋白表达.结果 与SO组比较,AP3 h组血清皮质酮显著增高,随后逐渐降低(P＜0.05).随病程延长,肾上腺细胞凋亡指数增高,bax蛋白表达增强,bax/bcl-2比值亦逐渐升高(P值均＜0.05),并与凋亡指数(AI)呈正相关(r =0.759,P＜0.05).结论 bax和bcl-2可能参与了AP肾上腺损伤的发病过程.通过激活bcl-2和抑制bax的蛋白表达,减少细胞凋亡发生从而保护肾上腺结构和功能,可能为今后AP及相关肾上腺损伤的药物及基因治疗提供依据和新思路.     

不同浓度γ干扰素诱导大鼠脾脏来源树突状细胞表达吲哚胺2,3-双加氧酶的变化及对T淋巴细胞增殖的影响

目的 观察不同浓度γ干扰素(IFN-γ)作用下大鼠脾脏来源的树突状细胞(DC)中吲哚胺2,3-双加氧酶(IDO) mRNA和蛋白表达的变化及IDO对同种异体T淋巴细胞增殖的影响.方法 应用细胞因子体外诱导培养大鼠脾脏来源DC,应用流式细胞仪测定大鼠DC特异性分子OX62 和表面分子CD80、CD86的表达.分别用不同浓度(0、100、300、500 U/ml)的IFN-γ诱导作用DC后,实时聚合酶链反应( Real-time PCR)测定DC中IDO mRNA的相对表达水平,Westemblot检测DC中IDO蛋白在DC中的表达水平.同种异体混合淋巴细胞反应(MLR)检测不同浓度IFN-γ诱导后的DC对同种异体T淋巴细胞增殖的影响.结果 体外诱导培养的DC光学显微镜下观察,具有典型的树枝状突起.0X62表达率达到80％以上,CD80、CD86的阳性表达也在80％左右;DC的IDOmRNA(2 -△△Ct值分别为:1.010 ±0.094、1.340±0.114、1.700±0.087、2.080±0.150)和蛋白的相对表达量(分别为:0.861 ±0.612、1.155±0.059、1.308±0.068、1.755±0.118)随着IFN-γ作用浓度的增大逐渐增大,分别为:不同浓度组间差异有统计学意义(P＜0.05);各组IFN-γ作用后DC与对照组比较,T淋巴细胞的增殖率显著降低(P＜0.05);且随着IFN-γ作用浓度的增大T淋巴细胞的增殖率逐渐降低,A值分别为:0.458±0.041、0.423±0.030、0.349±0.019、0.312±0.036,各组间差异有统计学意义(P＜0.05).结论 采用改良的培养黏附法体外获得纯度较高的大鼠脾脏来源的DC;IFN-γ可以诱导大鼠脾脏来源DC表面活性IDO的表达增加,减弱脾脏DC对同种T细胞增殖的刺激能力.     

肝细胞生长因子在单侧输尿管梗阻大鼠肾脏中的表达及其意义

目的　检测肝细胞生长因子 (HGF)在单侧输尿管梗阻 (UUO)大鼠肾脏中的表达并探讨其与小管细胞增殖与凋亡的关系。方法　免疫组织化学和脱氧核苷酸末端转移酶介导的核苷酸缺口末端标记 (TUNEL)法分别检测了第 0、3、6、9、1 2天UUO大鼠肾脏小管间质α 平滑肌肌动蛋白 (α SMA)、转化生长因子 β1 (TGF β1 )、HGF的表达以及小管细胞增殖性细胞核抗原 (PCNA)的表达和细胞凋亡的情况。噻唑蓝 (MTT)法检测重组HGF(rHGF)对培养的小管上皮细胞促增殖作用。结果　随着梗阻时间的延长 ,UUO大鼠肾脏α SMA、TGF β1蛋白的表达逐渐增多 ,HGF与PCNA蛋白的表达在第 6天达到高峰 ,随后逐渐下降 ,且它们之间呈正相关 (r =0 .870 5 ,P <0 .0 1 ) ,而细胞凋亡数随着时间的延长也逐渐增多。rHGF可促进体外培养的小管上皮细胞增殖。结论　HGF具有促进小管上皮细胞增殖的作用 ,同时它可能具有抗凋亡的作用     

染料木黄酮对去势抵抗性前列腺癌VCaP细胞增殖的影响

目的:研究染料木黄酮(GDN)对去势抵抗性前列腺癌细胞VCa P增殖的抑制作用及可能机制。方法:以不同浓度(0、12.5、25、50、100、200μmol/L)的GEN处理去势抵抗性前列腺癌细胞VCaP,分别在24、48、72 h以CCK-8法测定细胞增殖;以流式细胞术检测细胞周期;以免疫荧光法检测Ki-67表达水平;以Western印迹法检测Cyclin D1、PCNA、P53及PSA表达水平。结果:12.5、25、50、100、200μmol/L GEN作用于VCaP细胞72 h后,对VCaP细胞的抑制率分别为(25.38±0.02)%、(31.14±0.29)%、(45.27±0.03)%、(52.19±0.05)%与(68.21±0.19)%,与对照组[(10.08±0.02)%]相比差异有统计学意义(P0.05)。流式细胞术显示GEN可导致VCa P细胞G2/M期阻滞(P0.05)。免疫细胞化学法检测显示GEN能够抑制细胞中Ki-67的表达。Western印迹显示去势抵抗性前列腺癌细胞内PSA、Cyclin D1、PCNA随着GEN浓度的增加逐渐下调,P53随着GEN浓度的增加逐渐上调(P0.05)。结论:GEN能够抑制体外培养的去势抵抗性前列腺癌细胞VCaP增殖,其作用机制可能与阻滞细胞周期并伴随相关周期蛋白表达的改变有关。     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

目的 观察胰腺癌微环境对树突状细胞(DC)成熟的影响及功能变化并探讨胰腺肿瘤细胞免疫逃逸的机制.方法 培养树突状细胞,加入粒细胞巨噬细胞集落刺激因子(rmGM-CSF)40μg/L、白细胞介素(IL)-4 40μg/L,培养到第6天时得到大量的未成熟树突状细胞(imDC),加入大鼠胰腺癌细胞(AR42J cell)培养上清液诱导,流式细胞术检测DC的表面分子CD86、CD80的表达(n=6),观察能否延缓或阻断imDC的成熟及其在脂多糖(LPS)刺激后这种作用能否被逆转.并观察AR42J细胞培养上清诱导的Dc对同种异体混合淋巴细胞增殖.结果 加入胰腺癌癌细胞上清液培养的DC,与正常成熟的DC比较,CD80+CD86+阳性率由(70.88±3.60)%降至(7.15±0.71)%,LPS刺激后DC细胞的表面分子CD80+CD86+表达仍然较低(7.43±1.05)%,表明胰腺癌细胞培养上清液对DC的成熟有阻断作用.AR42J细胞上清诱导培养的imDC组刺激同种异体混合淋巴细胞增殖的强度显著低于正常培养的imDC组刺激同种异体混合淋巴细胞增殖的强度.结论 体外大鼠胰腺癌细胞培养上清液可以诱导DC处于不成熟状态,且这种不成熟状态不容易被逆转.     

2015年乳腺癌辅助化疗进展

【摘要】〓乳腺癌是危害我国女性健康的头号杀手,尽管近年来辅助化疗的研究进展突飞猛进,但临床中仍有不少问题未能明确,如辅助化疗的合适人群、化疗的开始时间、蒽环及紫杉类的地位和用法、强化维持治疗的作用、疗效及预后的生物标志物等。本文结合乳腺癌辅助化疗在临床上的常见问题和2015年各大乳腺癌会议阐述乳腺癌辅助化疗的最新进展。     

Alterations in T-Cell Subset Frequency in Peripheral Blood in Obesity

Background: Obesity affects the regulation of immune and inflammatory responses. This study characterizes differences in peripheral blood lymphocyte phenotype in obese humans. Methods: Frequencies of lymphocyte subsets among peripheral blood mononuclear cells were compared between 10 obese (BMI ≥35) and 10 lean subjects, as determined by antibodies directed against cluster differentiation (CD) markers. Results: Obese patients demonstrated an increased frequency of CD3+CD4+ T-cells (mean difference 12%, P=0.004), a decreased frequency of CD3+CD8+ T-cells (mean difference 9.4%, P=0.016) and an increased frequency of CD3+CD8+CD95+ T-cells (mean difference 13.3%, P=0.032). No other differences among T-cell or monocyte subsets were noted. Conclusions: Obesity is associated with alterations in frequencies of peripheral CD4+ and CD8+ T-cells and aberrations in the expression of CD95 among CD8+ T-cells. These data suggest both CD4+ and CD8+ T-cell compartments, as well as the regulation of CD95 expression on CD8+ T-cells, as targets for further study into obesity's effects on the immune system.     

西宁地区烧伤病人动脉血气分析观察

对高海拔地区的27例烧伤病人动脉血气变化进行了分析和观察。结果证明:无论是存活病人还是死亡病人伤后均存在有低氧血症问题。并且在死亡病人和烧伤合并吸入性损伤病人其低氧血症的发生早于单纯烧伤病人。提示:吸入性损伤病人应立即行气管切开术以保障氧气供给,单纯烧伤病人可常规吸氧以维持正常血 PaO_2,ARDS 均发生在合并吸入性损伤的病人,高频喷射通气技术对纠正低氧血症有一定效果。     

Controversies in Fistula in Ano

Managing a complex fistula in ano can be a daunting task for most surgeons; largely due to the two major dreaded complications—recurrence & fecal incontinence. It is important to understand the anatomy of the anal sphincters & the aetiopathological process of the disease to provide better patient care. There are quite a few controversies associated with fistula in ano & its management, which compound the difficulty in treating fistula in ano. This article attempts to clear some of those major controversies.     

β-半乳糖苷酶在体内外成骨细胞中的表达

目的 研究β—半乳糖苷酶(β—gal)在成骨细胞中的表达状况，为阐明MorquioB综合征的发病机制提供依据。方法 裸鼠各器官和骨组织标本行X-gal染色检测。抽取羊和人骨髓行骨髓基质细胞(BMSCs)培养，分为4组：I：Adv-hBMP-2转染组；Ⅱ：Adv—β—gal转染组；Ⅲ：未转染组；Ⅳ：地塞米松诱导组。分别行X-gal染色和RT-PCR检测β—gal的表达。结果 裸鼠骺板两侧、骨膜内面及松质骨的成骨细胞和破骨细胞可见多量β—gal的表达。未转染BMSCs组有少量β—gal的表达，其他3组细胞的β—gal表达增高。结论成骨细胞和破骨细胞可表达多量β—gal，该两种细胞的β—gal缺乏可能是MorquioB综合征骨骼异常的直接原因。     

Fluid-phase transcytosis in the primate epididymis in vitro and in vivo

Ligated tubules from the corpus epididymidis of men and monkeys were incubated in medium containing horseradish peroxidase (HRP) as a marker for fluid-phase endocytosis. HRP was localized by light and electron microscopy after 0, 15, 30 and 60 min of incubation. Movement between the cells was prevented by tight junctions, but bypass of this barrier was apparently achieved by an intracellular vesicular mechanism leading to a time-dependent appearance of HRP in the lumen. Uptake of HRP into basal cells and capture by the lysosomal apparatus of principal cells were also observed. HRP-filled vesicles also appeared in the basal, mid and apical cytoplasm of epithelial cells in the caput 1 h after injection of the tracer into the epididymal circulation of the monkey, suggesting that this pathway also operates in vivo.     

Surgical management of ductal carcinoma in situ in Australia in 1995

Background: In the present paper we describe the presentation and management of ductal carcinoma in situ (DCIS) of the breast in women in Australia in 1995. This representative, national data set provides a historical comparator for studies examining DCIS management that follow. Methods: Surgeons identified by population‐based cancer registries as having treated a new diagnosis of DCIS between 1 April and 30 September 1995 completed a questionnaire on the presentation and management of each case. Results: Two hundred and five surgeons supplied treatment details on 418 DCIS tumours in 415 women . Half of all tumours were detected at BreastScreen clinics and a further 25% were detected at other mammography centres. Twenty‐six percent of tumours were palpable at presentation, 33% were multifocal and 55% were high grade (including comedocarcinoma). Breast conserving therapy (BCT) rather than mastectomy was utilized in 260 (62%) of cases. Tumours that were of low grade, small in size and not multifocal were more likely to be treated by BCT. Surgeons seeing six or more DCIS cases in the 6‐month period were more likely to utilize BCT. Of the conservatively treated cases, 22% were referred for a radiation oncology consultation. The most common reasons for treating DCIS with mastectomy were that the tumour was too extensive or multifocal (63%), it extended to margins of the specimen (42%), or patient concerns about recurrence (34%). Conclusions: In 1995 the majority of DCIS was treated with breast conserving surgery alone. Surgeons treating more DCIS cases were more likely to perform conservative surgery than surgeons treating only one DCIS case in the study period.     

Smoking-Attributable mortality in Spain in 2016

IntroductionSmoking-attributable mortality (SAM) is a valuable indicator that can be used to characterize the course and health burden of the smoking epidemic. The aim of this paper was to estimate SAM in Spain in 2016 in the population aged 35 and over, using the best available evidence.MethodsA smoking prevalence-dependent analysis based on the estimation of population-attributable fractions was performed. Smoking prevalence (never, former, and current smokers) was calculated from a combination of the Spanish Health Survey (2016) and the European Health Survey (2014); the relative risk of death among current and former smokers was taken from the follow-up of various cohorts; and mortality rates were obtained from National Center for Statistics data. SAM estimates are presented globally, and by sex, age groups, and major disease categories: cancer, cardiometabolic diseases and respiratory diseases.ResultsIn 2016, 56,124 deaths were attributed to tobacco consumption, 84% in men (47,000), and 50% in the population aged over 74 (27,795). Overall, 50% of SAM was due to cancer (28,281), 65% of which was lung cancer. One in 4 attributable deaths (13,849) occurred before the age of 65.ConclusionsOne in 7 deaths in Spain in 2016 were attributable to smoking. This estimation of SAM clearly highlights the great impact of smoking on mortality in Spain, mainly due to lung cancer and chronic obstructive pulmonary disease.