Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 M GTP, 1 M VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Halfmaximal stimulation of AC by VIP was observed at 26±10 nM (n=3) in OMCTi and at 19 nM (n=2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.     

Laser microdissection and pressure catapulting (LMPC) in paraffin sections mounted on glass slides. A methodological report

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.     

一种简便快捷的微小病变基因变异分析方法的建立

目的建立一种简便快捷的微小病变和微量细胞基因变异的检测分析方法。方法HE染色病理诊断后,在立体显微镜或生物显微镜下对病理诊断组织细胞进行显微解剖,直接回收组织细胞,进行DNA模板抽提,以目的基因为引物,进行PCR-SSCP分析。结果1例胆石症病例,显微解剖结合PCR-SSCP法成功获得了K-ras和P53基因产物,并且检测到在过度增生胆囊上皮细胞有K-ras基因变异。结论显微解剖结合PCR-SSCP法取材少、病变部位确定,省时、省力,是分子病理研究的可靠手段。     

Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-alpha, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real-time PCR on the original RNA samples as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation.     

两种TESA方法在腮腺炎致无精子症患者取精中的比较

目的探讨腮腺炎致无精症患者两种睾丸活检取精（TESA）即开放性睾丸活检和显微切割睾丸活检取精的优劣。方法每组24例患者,分别用开放性睾丸活检和显微切割睾丸活检取精,观察取精成功率、术后并发症发生率,术后并发症包括术后血肿的形成,术后3月睾丸萎缩、睾酮下降。结果开放性睾丸活检取精组,能找到活动的精子14例,取精成功率58．3％,术后并发症10例,约41．7％,分别为血肿2例、三月后睾丸萎缩3例、睾酮下降5例;显微切割睾丸活检取精组,能找到活动的精子18例,取精成功率75％,术后并发症5例,约20．8％,分别为血肿0例、三月后睾丸萎缩1例、睾酮下降4例。取精成功率和开放性睾丸活检取精组比较（P〈0．05）,术后并发症和开放性睾丸活检取精组此较（P〈0．01）。结论显微切割睾丸活检取精较开放手术睾丸活检取精成功率高、术后并发症少,值得推广。     

小型猪Barx1基因的克隆及其在牙胚中的定量表达

目的 观察小型猪牙齿发育相关基因Barx1片段在胚胎发育40 d(E40)不同牙胚中的定量表达,探讨Barx1基因空间表达量与牙齿形态的关系.方法 应用基因克隆技术克隆小型猪Barx1基因片段;应用激光捕获显微切割技术精确分离胚胎40 d小型猪第一乳切牙、乳尖牙、第三乳前磨牙、乳磨牙牙胚;提取微量RNA,以甘油醛-3-磷酸脱氢酶为对照,通过实时定量聚合酶链反应检测Barx1的相对表达量.结果 得到小型猪Barx1基因698个碱基的片段,Barx1在第一乳切牙、乳尖牙、第三乳前磨牙、乳磨牙中的相对表达量为0.000 249、0.000 715、0.026 096、0.1 12 656,在牙弓中由前向后呈递增趋势.结论 Barx1基因表达量与牙齿形态发育存在一定关系,推测小型猪Barx1基因可能与牙尖的分化或牙尖数目相关.     

弥漫性大B细胞淋巴瘤t(14;18)易位及bcl-2基因扩增的检测

目的探讨弥漫性大B细胞淋巴瘤（DLBCL）t（14;18）染色体异位及bcl-2基因扩增在其分类及临床分期、疗效评估中的作用。方法先对60例DLBCL的标本进行显微切割,获取相对比较纯的肿瘤组织,再使用细胞核芯片荧光原位杂交（FISH）对标本进行t（14;18）易位及bcl-2基因扩增检测,采用免疫组织化学SP法在组织微阵列上同步观测CD20、CD10、bcl-6、MUM1的表达,进行生发中心样（GCB）和非生发中心样（non-GCB）分类;通过病例分析得出治疗效果及临床分期的信息,并统计分析以上各因素之间的关系。结果在60例DLBCL中,10例bcl-2／IgH阳性,18例bcl-2基因扩增;GCB29例（48．3％）,non-GCB31例（51．7％）。经FISH检测t（14;18）阳性10例中,GCB8例,non-GCB2例,差异有统计学意义（P=0．031）。t（14;18）阳性及bcl-2基因扩增的病例bcl-2表达均增高。在36例正规CHOP治疗的病例中,bcl-2扩增13例,无扩增23例,bcl-2扩增的13例之治疗结果显效、部分有效、无效率分别为3例（23．1％）、4例（30．8％）和6例（46．2％）;临床分期情况为Ⅰ-Ⅱ期1例（7．7％）,Ⅲ-Ⅳ期12例（92．3％）,这两项指标与bcl-2不扩增的相比差异均有统计学意义（P=0．019、0．046）。结论t（14;18）易位及bcl-2基因扩增均是引起DLBCL之bcl-2蛋白表达的原因,bd-2阳性者与预后有关的原因难以确定,可能是由于引起其阳性表达的原因不同所致;bcl-2基因扩增与治疗效果较差及临床分期较晚有关;FISH检测t（14;18）染色体易位可用于DLBCL的分类。     

胃腺癌微环境caveolin-1的表达及其临床意义

背景：肿瘤的发生、发展是肿瘤微环境中肿瘤实质与间质成分共同作用的结果。目前仅少数研究探讨胃癌微环境小窝蛋白-1（caveolin-1）的表达情况、目的：探讨胃腺癌微环境caveolin-1的表达及其临床意义。方法：收集外科胃腺癌手术切除标本72例,32例正常胃窦黏膜组织作为对照,以免疫组化法检测实质和间质中的caveolin-1和原癌基因蛋白c-myc表达,分析胃癌实质caveolin-1表达与c-myc以及主要肿瘤临床病理特征的相关性。随机选取7例冰冻标本,激光捕获显微切割（LCM）技术获取纯净胃癌和癌旁组织实质和间质细胞,蛋白质印迹法检测caveolin-1和c-myc表达并行相关性分析。结果：常规标本中,胃癌实质和间质caveolin-1高表达率分别显著低于正常胃黏膜实质和间质（P〈0.05）：胃癌实质高表达c-myc,并与caveolin-1呈正相关（r=0.312,P〈0.05）胃癌实质caveolin-1高表达与肿瘤浸润深度、淋巴结转移、TNM分期呈负相关（P〈0.05）。LCM标本中,胃癌实质和间质caveolin-1表达分别显著低于相应癌旁组织实质和间质（P〈0.05）。caveolin-1与c-myc在胃癌实质中的表达呈正相关（r=0.752,P〈0.05）,在胃癌间质中的表达呈负相关（r=-0.975,P〈0.05）。结论：胃腺癌微环境低表达caveolin-1。胃癌实质caveolin-1高表达与肿瘤浸润、转移、临床进展呈负相关。c-myc可能通过不同机制调节胃癌实质和间质中的caveolin-1表达。胃癌间质caveolin-1低表达有望为胃癌预后判断提供依据。     

精浆AMH水平对非梗阻性无精症的临床预测价值

目的探讨抗缪勒管激素(AMH)水平对非梗阻性无精子症(NOA)的临床预测价值。方法釆用酶联免疫法和电化学发光免疫分析法检测NOA患者精浆AMH、血清促卵泡激素(FSH)以及睾酮(T)的浓度;B超检测其睾丸体积;通过睾丸显微取精术(M-TESE)检获精子;同时设置对照组,并进行统计学分析。结果 NOA组的AMH浓度分别低于OA组[(19.53±9.13)pmol/L,(52.34±15.13)pmol/L,P0.05]及NF组[(158.53±37.45)pmol/L,P0.05];NOA组的FSH浓度分别高于OA组[(18.36±8.95)U/L,(5.51±3.32)U/L,P0.05]及NF组[(6.12±3.02)U/L,P0.05];NOA患者的睾酮浓度[(15.32±5.43)nmol/L]分别与OA组[(15.63±6.23)nmol/L]、NF组[(15.81±5.73)nmol/L]比较无显著性差异(P0.05,P0.05);NOA组左右睾丸体积TV[(9.58±3.83)mL,(7.46±3.57)mL]分别与OA组[(16.97±2.56)mL,(15.32±3.63)mL]、NF组[(16.23±3.53)mL,(16.84±2.83)mL]存在显著性差异(P0.05,P0.05)。NOA组有10人检获精子,OA组19人检获精子(P0.05)。Logistic回归分析检验发现AMH预测NOA患者睾丸内是否存在精子的拟合优度最佳(Wald χ~2=26.198,P0.01);ROC曲线图显示精浆AMH的AUC值最大;精浆AMH的阳性似然比为7.36[81.82%/(1-88.89%)],阴性似然比为0.20[(1-81.82%)/88.89%]。结论精浆AMH浓度对于预测NOA患者M-TESE成功与否具有重要的意义。     

上皮性卵巢肿瘤中抑癌基因P53的表达及第8外显子突变的检测

目的：探索抑癌基因Ｐ５３基因的表达水平与卵巢肿瘤的临床和生物不行为之间是否存在一定的关系。方法：应用免疫组化ＳＰ法检测Ｐ５３在７１例乳巢上皮性肿瘤中的表达情况,并应用显微切割获得ＤＮＡ和聚全酶链式反应（ＰＣＲ）－单链构象多态性分析（ＳＳＣＰ）检测Ｐ５３第８外显子（ｅｘｏｎ８）突变的情况。结果：卵巢癌中Ｐ５３蛋白阳性率为５１．６％,正常及交界性肿瘤均为阴性,差异有极显著性意义（Ｐ〈０．００１）。Ｐ５