华南、华北人群HLA-DQB1等位基因多态性

目的从基因水平调查了中国华南、华北地区人群HLA-DQB1等位基因频率，并研究比较两地区人群HLA-DQB1多态性分布。方法采用深圳益生堂生物企业有限公司研制开发的“HLA-DQB1低分辨率分型基因芯片检测试剂盒”，应用聚合酶链反应．序列特异性引物＋序列特异性寡核苷酸探针芯片检测技术，对700名南方地区的中国人和320名北方地区的中国人进行基因分型。结果鉴定了10个HLA-DQB1等位基因，获得了一组准确、科学的统计数据。结论得到了中国华南、华北地区人群HLA-DQB1等位基因频率差异的数据，证明中国人群HLA-DQB1＊02，05，0601，0602，0603的分布南北差异有统计学意义（P〈0．05），为疾病相关性研究、人文科学研究提供了可靠的遗传学数据。     

Nitric Oxide Synthase Expression in Macrophages of Histoplasma capsulatum-Infected Mice Is Associated with Splenocyte Apoptosis and Unresponsiveness

Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of N^G-monomethyl-l-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice.

Nitric oxide (NO) has been reported to induce apoptosis in many cells including smooth muscle cells (20), oligodendrocytes (27), pancreatic β cells (11), melanoma cells (35), thymocytes (7), B lymphocytes (4), and macrophages (2). Fehsel et al. recently demonstrated apoptosis in freshly isolated thymocytes after exposure to NO (7). In the same report, they also showed apoptotic foci in close proximity to blood vessels after lipopolysaccharide treatment. Capillary endothelial and dendritic cells adjacent to apoptotic foci stained strongly for inducible nitric oxide synthase (iNOS), suggesting that NO may be the mediator for thymic apoptosis (7). Data from another laboratory also showed that cloned thymic stromal cell monolayers eliminate thymocytes in vitro through production of NO (26). Furthermore, apoptosis has been suggested as a mechanism by which the immune system replenishes itself and maintains homeostasis (30).The dimorphic fungus Histoplasma capsulatum is a facultative intracellular pathogen of the macrophage (32). Although it is not an obligate intracellular pathogen, the organism is found almost exclusively inside host cells during histoplasmosis (5). In our in vitro studies, H. capsulatum exhibits uninhibited growth in normal unstimulated murine macrophages (32). In activated macrophages, either peritoneal macrophages and cells from the Raw 264.7 line stimulated by gamma interferon (IFN-γ) or splenic macrophages stimulated by IFN-γ and lipopolysaccharide, growth of the fungus is inhibited (13, 18, 32). Furthermore, the anti-histoplasma activity of macrophages is dependent on the expression of iNOS and the production of NO (14, 18). However, the significance of NO production in immunoregulation of histoplasmosis is not clearly defined.In this study, we examined whether NO can act as a regulator of apoptosis in lymphoproliferative responses of splenocytes from H. capsulatum-infected mice. We showed that iNOS was induced in splenic macrophages during active infection and the expression of iNOS coincided with active infection. We also observed by in situ terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) of spleen sections that apoptosis occurred in immune cells in the spleens of infected mice but was minimal in control mice. The link between apoptosis and NO production was established by inclusion of N^G-monomethyl-l-arginine (NMMA) in the culture medium. Inhibition of NO production reduced the amount of apoptosis in splenocyte culture. Thereby, we also confirmed the findings of Zhou et al. (36) that production of NO by splenocytes of H. capsulatum-infected mice suppressed the splenic lymphocyte proliferative response. In addition, we showed that macrophages were mediators of splenocyte unresponsiveness through the NO that they produced and that NO production was associated with apoptotic changes in cultured splenocytes from infected mice.     

HLA-E的表达对RSA及PIH患者外周血γδT细胞细胞毒效应的影响

探索HLA-E分子的表达对习惯性流产(recurrent spontaneous abortion,RSA)及妊高症(pregnancy-induced hypertension,PIH)患者外周血γδT细胞细胞毒效应的影响。通过固相抗体法体外分离并扩增外周γδT血细胞作为效应细胞,以HLA-E转染的LcL721.221细胞(.221E)及滋养层细胞JAR作为靶细胞,采用4 h乳酸脱氢酶(LDH)释放试验观察NK细胞对靶细胞的细胞毒效应。结果显示,来自RSA、PIH及正常对照组的γδ细胞均不能有效杀伤HLA-E转染的.221E细胞及JAR细胞,但未经HLA-E转染的LcL721.221细胞则被溶解;抗HLA-E单抗3D12及抗CD94单抗HP-3B1的阻断可以分别部分恢复效应细胞对靶细胞LcL721.221E的杀伤,但对JAR细胞没有影响;与正常对照组相比,RSA及PIH患者外周血γδT细胞对.221、.221E及JAR细胞的细胞毒活性没有显著性差异(P>0.05)。这说明HLA-E分子的体外表达可以保护靶细胞防止γδT细胞的杀伤,该保护机制主要是通过γδT细胞受体CD94/NKG2对靶细胞表面HLA-E分子的识别来实现的;滋养层细胞对γδT细胞杀伤的抵抗可能存在MHC I类非依赖的机制。     

甲型副伤寒沙门氏菌体抗原抗血清的简易提纯

目的 单克隆抗体以其结合抗原的专一性用于血清学检验是一种必然趋势,而多克隆机体则以其简易的制备方式在目前仍得到广泛的应用,但多克隆抗体的提纯仍有许多繁琐之处,使其优势受到影响。本试验以甲型副伤寒沙门氏菌体抗原多克隆抗体的制备及提纯为例,提出可行的更简易的提纯方法。方法 利用产G蛋白链球菌,以一种较为简易的特异性结合和解离的方法来纯化甲型副伤寒沙门氏菌体抗原免疫血清。结果 得到了特异性IgG抗体。结论 利用这种纯化方法在实验室简易可行。     

食管分层应力-应变研究

目的　探讨食管壁各层环向应力 应变分布特征。方法　将 8只Wister大鼠的食管标记、测量在体长度后剪下置于去钙离子Krebs液中。剪下标记的中段食管用做充压试验 ,将其在显微镜下分离成内层食管 (粘膜和粘膜下层 )和外层食管 (固有肌层 )。充压时全层和分层食管均被拉长至在体长度 ,以每分钟升高 2cmH2 O速度匀速充压。根据所测图像的几何数据计算全层和分层食管的Kirchhoff应力和Green应变。零应力状态作为计算应力和应变的基本参照状态。结果　 (1 )食管离体后较在体长度缩短 2 4%。 (2 )全层食管和分层食管的环向应力 应变曲线均符合指数方程τ =(τ +β)eα(ε-ε ) -β(判定系数 >0 .96) ,为非线性关系。 (3 )与全层食管相比 ,分层后内层食管应力 应变曲线较陡直 ,且移向左侧 ,差别有统计意义 (P <0 .0 5 ) ,外层食管的曲线移向右侧 ,差别无统计意义 (P >0 .0 5 ) ,内外两层食管的应力 -应变曲线亦有统计差别 (P <0 .0 5 )。结论　食管壁各层环向应力 -应变均符合非线性关系 ,食管内层不如外层易变形     

心肌肽素对豚鼠心室肌细胞钠通道的影响

目的： 研究心肌肽素对豚鼠心室肌细胞钠通道的影响，探讨心肌肽素在离子通道水平的作用机制。 方法： 用急性酶解分离法获得豚鼠心室肌细胞，标准全细胞膜片钳技术记录钠电流(I_Na)。 结果： 心肌肽素1、5、10、50、100、500 mg/L使豚鼠心室肌细胞I_Na分别减少(0±1)%、(6±2)%、(10±2)%、(15±1)%、(22±9)%、(30±6)%，呈浓度依赖性抑制I_Na。心肌肽素50 mg/L使I_Na激活时间(TTP)从(2.8±0.7) ms延长至(3.0±0.8) ms (P<0.05)；使I_Na电流密度-电压曲线上移，但不改变激活电位、峰电位、反转电位和I-V曲线的形状；不影响稳态激活曲线、稳态失活曲线和稳态失活后恢复曲线。 结论： 心肌肽素浓度依赖性抑制豚鼠心室肌细胞I_Na，可能是其抗心律失常作用的机制之一。     

结核分枝杆菌Rv2450蛋白诱导小鼠免疫应答的实验研究

目的:以原核表达的结核分枝杆菌Rv2450蛋白在小鼠体内诱导体液和细胞免疫应答。方法:采用皮下包埋的方法,以预先转移到硝酸纤维素膜上的原核表达的Rv2450蛋白免疫小鼠(10只)3次,每次间隔2周。用间接ELISA法检测免疫小鼠血清特异性抗体的滴度。末次免疫完成后4周,处死3只免疫小鼠并分离脾淋巴细胞,体外经PPD(2μg/孔)刺激后,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖指数。用ELISA法检测脾淋巴细胞悬液中IFN-γ、IL-10及IL-12的水平。结果:Rv2450蛋白免疫小鼠血清特异性抗体的滴度为1∶3200,淋巴细胞增殖指数为3.76±0.19。免疫小鼠脾淋巴细胞培养液中IFN-γ、IL-10及IL-12的含量,分别为(1740±19)ng/L、(678±15)ng/L、(469±13)ng/L,均高于各组的生理盐水对照组(P<0.05)。结论:Rv2450有可能作为新型结核疫苗的候选组分。     

慢性乙型肝炎患者血清维生素E水平与肝组织病理的关系研究

目的 探讨慢性乙型肝炎(CHB)患者血清维生素E水平与肝组织病理的关系.方法 选择66例慢性乙型肝炎患者进行肝穿刺病理学检查,健康对照组10例.化学比色法检测血清维生素E表达水平,同时检测HBV DNA、HBeAg及肝功能.结果 与正常对照组相比,慢性乙型肝炎患者血清维生素E水平明显降低(P<0.01);HBeAg阳性组与HBeAg阴性组比较,血清维生素E水平无明显差别(P0.05).CHB患者血清维牛素E水平与肝脏组织炎症呈负相关关系(r=-0.451,P<0.05),而与肝脏组织纤维化程度无明显相关(r=0.02,P0.05).结论 维生素E作为体内重要的抗氧化剂,参与了慢性乙型肝炎的炎症过程.在肝脏炎症过程中,适当补充维生素E,可能有助于缓解病情.     

大鼠急性肺栓塞后SMP-30表达下降及其对细胞凋亡的影响

目的：研究大鼠急性肺栓塞模型肺组织中衰老标记蛋白质30(SMP-30)的表达变化及其对Fas诱导的细胞凋亡的影响。方法:建立大鼠急性肺栓塞模型，分别在急性肺栓塞后1、8、24和48 h进行支气管肺泡灌洗，然后开胸取出肺组织。常规提取肺组织的总RNA和总蛋白，以正常组为对照，采取半定量RT-PCR的方法研究SMP-30在mRNA水平表达的变化；采用Western blotting方法进一步验证SMP-30在蛋白水平表达的变化；采用免疫组织化学方法检测大鼠肺组织中SMP-30以及肺泡巨噬细胞中IL-8在肺栓塞前后表达的变化及其组织分布情况；采用TUNEL法研究急性肺栓塞后组织细胞的凋亡情况；最后采用ELISA法检测急性肺栓塞后肺泡灌洗液中sFasL的浓度变化。结果:在大鼠急性肺栓塞后的不同时点，SMP-30的mRNA水平和蛋白水平均逐渐降低，在24和48 h下降最为明显。免疫组化研究表明SMP-30主要分布在支气管黏膜上皮细胞和肺泡上皮细胞，急性肺栓塞后SMP-30在上述细胞内的表达均明显降低。TUNEL染色发现随着SMP-30表达的降低，肺组织内出现明显的细胞凋亡现象，同时肺泡灌洗液中sFasL的浓度升高，肺泡巨噬细胞内IL-8的表达也明显升高。结论:大鼠急性肺栓塞后肺组织内SMP-30的表达明显降低，可能促进Fas－FasL细胞凋亡系统的活化。     

针对肺结节检测的肺实质CT图像分割

目的：针对CT图像上肺结节的自动检测，开发并评价对全肺螺旋CT扫描中的肺实质进行自动分割的一种综合方法。方法：首先利用全局阈值对CT图像进行二值化，然后消除由于支气管、细支气管等低密度影和由于结节、血管等高密度影以及由检查床造成的条状伪影等噪声，最后对包含胸膜连接结节的图像利用数学形态学运算和图像凸包运算进行完善形成肺实质掩膜。结果：利用该方法对从LIDC数据库中所有包含结节的505张CT扫描片（来自69个病例）进行肺实质分割，正确率为95．4％。其中，包含胸膜连接结节的139张CT扫描片的正确分割率为94．2％。结论：本文提出的方法较好地完成了肺实质分割任务，为利用CT图像进行计算机辅助肺结节的检测打下了基础。