Analysis of the HLA-A,-B allele polymorphism in 5844 umbilical cord blood samples taken from Han population of Shandong province

To investigate the HLA-A, -B allele polymorphism in Han population of Shandong province and to explore the possibility to find out the HLA-A, -B-matched cord blood donors for stem cell transplantation to be used in other area in China, 5844 umbilical cord blood samples were taken from Han population donors of Shandong province, and assayed with PCR-sequence-oligonucleotide (PCR-SSO) assay. In Shandong Han donors, 20 alleles at HLA-A locus and 46 alleles at HLA-B locus could be detected as revealed in the present study. Among the 20 alleles at HLA-A locus, the most prevalent five alleles included A * 02(0.3041), A * 11(0.1443), A * 24(0.1434), A * 30(0.0975) and A * 33(0.0859), while, the alleles with lower gene frequencies included A * 34(0.0006), A * 25 (0.0005), A*66(0.0005), A* 74(0.0004) and A* (0.0001). Of the 46 HLA-B alleles detected, the most prevalent five alleles were B * 13(0.1348), B * 51(0.0713), B * 62(0.0712), B * 61 (0.0676) and B * 60(0.0642); while alleles with lower gene frequencies included B * 77(0.0001), B * 76(0.0002), B * 47(0.0003), B * 42(0.0003) and B * 72(0.0004). In comparison with those of the other Han population in China, the HLA-A, -B gene frequencies in the umbilical cord blood of Shandong province possess unique distribution features among the investigated populations from various regions of the same race origin, and the differences in various regions of the same race were less than those among the different race. It is evident that the HLA-A,-B alleles of the umbilical cord blood taken in Shangdong province show high degree of polymorphism, and it might be part of those of Northern Han population in China. So, it is reasonable for patients of Northern Chinese to receive HLA class I -match transplant of cord blood stem cells for tissue and organ transplantation from Shangdong umbilical cord blood bank.     

汉滩病毒G1蛋白胞质区ITAM样基序与Syk细胞内相互作用

目的：研究汉滩病毒（HTNV）G1蛋白胞质区ITAM样基序与Syk的细胞内相互作用。方法：构建用于研究Syk和G1ITAM样基序之间相互作用的哺乳动物细胞双杂交系统，验证G1ITAM样基序与Syk之间是否存在细胞内相互作用。结果：哺乳动物细胞双杂交分析证实G1ITAM样基序在哺乳动物细胞内可以与Syk相互作用；而突变体分析表明，这种相互作用依赖于该基序中两个高度保守的酪氨酸残基的存在。结论：HTNVG1蛋白胞质区高度保守ITAM样基序在体外和哺乳动物细胞内可以与Syk相互作用，为进一步探讨G1蛋白ITAM样基序在HFRS免疫信号传递中的作用及其与血管内皮损伤的关系奠定了基础。     

抗幽门螺杆菌单克隆抗体与人血小板交叉识别的实验研究

目的：研究抗幽门螺杆菌（Hp）尿素酶B（ureB）单克隆抗体与血小板膜糖蛋白（GP）的结合情况。方法：运用Western blot、流式细胞术（FCM）和免疫放射法（IRMA）分析血小板膜GP成分与幽门螺杆菌ttreB成分的相关性。结果：抗幽门螺杆菌ureB单抗1F11与血小板成分GPⅢa、P-选择素有一定程度结合，但与GPIb、GPⅡb及GPⅥ无结合。结论：血小板成分GPⅢa、P-选择素与幽门螺杆菌ureB存在交叉识别的共同抗原表位，提示Hp感染与部分特发性血小板减少性紫癜（ITP）的发病机理有关。     

抗汉坦病毒单克隆抗体F（ab‘）2的制备及其纯度和活性鉴定

利用木瓜蛋白酶分别制备了抗汉坦病毒鼠源性单克隆抗体（ｍＡｂ）３Ｇ１和３Ｄ８Ｆ（ａｂ′）２片段。通过ＷａｔｅｒＤＥＡＥ４０ＨＲ阴离子交换色谱柱纯化后，非还原型ＳＤＳＰＡＧＥ呈现单一条带，相对分子质量（Ｍｒ）约１０００００。两种Ｆ（ａｂ′）２片段均具有良好的血凝抑制活性、病毒中和活性及对汉坦病毒感染乳鼠的保护作用     

p38MAPK参与高糖刺激大鼠肾小管上皮细胞产生细胞外基质胶原Ⅲ

目的：探讨p38MAPK信号通路在高糖刺激大鼠肾小管上皮细胞产生细胞外基质胶原Ⅲ中的作用。 方法： 采用体外培养和Western blotting等方法，以不同浓度D-葡萄糖、p38MAPK信号通路特异性阻断剂SB203580以及用不同时间刺激正常大鼠肾小管上皮细胞NRK52E，分别检NRK52E细胞p38MAPK磷酸化水平和细胞外基质胶原Ⅲ的表达。 结果： 随D-葡萄糖浓度增加，p38MAPK磷酸化水平、胶原Ⅲ的产生也增加，SB203580可有效阻断高糖引起p38MAPK磷酸化水平的升高和细胞外基质胶原Ⅲ的表达的增高。 结论： 高糖引起p38MAPK磷酸化水平的升高可能在糖尿病肾病的肾间质纤维化中发挥重要作用。SB203580有潜在的糖尿病肾病防治的临床应用价值。     

人CGT52TGT MBL突变体在CHO细胞中的表达及其产物分析

目的: 初步探索MBL基因CGT52TGT点突变引起调理吞噬缺损的机制。方法: 采用PCR技术, 从质粒pMBLm52中获取含CGT52TGT点突变的MBL基因, 将其插入真核表达载体pcDNA4 /HisMaxC中构建重组表达载体。经测序验证后, 电转染入CHO细胞。以800mg/LZeocin筛选转染后的CHO细胞30d; 随后的30d中, 维持Zeocin的浓度在200mg/L, 以获取稳定转染的细胞。以RT PCR分析其mRNA的表达情况。表达产物经Ni NTAagarose纯化后, 以非还原SDS PAGE和Westernblot对表达产物进行初步鉴定。结果:以PCR扩增的MBLm52基因片段长约750bp, 将其插入表达载体构建重组真核表达载体pcDNA4 /HisMaxC MBLm52, 测序验证序列正确后将其电转染入CHO细胞。从细胞培养上清中获得的纯化的表达产物, 主要为相对分子质量(Mr)约60 000的分子, 寡聚化程度明显低于重组人野生型MBL和从人血浆中分离的MBL。结论: MBL基因CGT52TGT点突变可能并不影响其表达产物向胞外分泌的过程, 但突变后产生的Cys可能形成新的二硫键, 影响MBL结构单位和/或寡聚分子的形成, 推测该突变MBL蛋白不能发挥正常的功能。     

Raji 细胞系特异性Fab噬菌体抗体库的构建及筛选

目的构建针对B细胞淋巴瘤Raji细胞系噬菌体抗体库并从中筛选出特异性的抗体。方法以Raji细胞免疫BALB/c小鼠,RT-PCR法从脾淋巴细胞中扩增抗体轻链к和重链Fd基因。经SacI/XbaI和XhoI/SpeI双酶切,依次克隆入噬菌体载体pComb3H-SS中,并电转化大肠杆菌XL1-Blue,以辅助噬菌体VCSM13进行超感染,构建B细胞淋巴瘤Raji细胞系特异性Fab噬菌体抗体库。以Raji细胞为抗原进行筛选,获得抗Raji细胞的抗体,通过ELISA法进行抗原结合活性的测定,并对所得阳性克隆进行基因测序分析。结果构建了容量为2.18×107的抗人B细胞淋巴瘤Raji细胞系的Fab噬菌体抗体库,并筛选获得了与Raji细胞系特异性识别结合的8株阳性克隆。对其中两个阳性克隆进行基因序列分析,结果显示其重链可变区、轻链可变区分别与免疫球蛋白基因库中已注册的鼠源性免疫球蛋白重链可变区和轻链可变区序列具有高度同源性。结论成功地构建Fab噬菌体抗体库并筛选出针对Raji细胞系膜抗原的抗体,为进一步研究B细胞淋巴瘤的免疫治疗提供了实验基础。     

慢性乙型肝炎患者外周血T细胞KIR表达研究

目的：检测慢性乙型肝炎患者外周血T细胞表面KIR的表达情况。方法：采用三色流式细胞术检测慢性乙型肝炎患者外周血CD4^＋T细胞和CD8^high T细胞表面KIR分子表达，并与正常外周血比较。结果：慢性乙型肝炎患者外周血中CD8^high T细胞KIR表达明显高于对照组CD8^high T细胞。正常外周血CD4^＋T细胞几乎不表达KIR，慢性乙型肝炎患者外周血中CD4^＋T细胞表达KIR。结论：慢性乙型肝炎患者T细胞表面KIR表达明显增加。     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

胫骨外侧髁骨折塌陷对关节轴线及接触压力的影响

目的:研究在胫骨外侧髁骨折中关节面塌陷和外侧半月板切除对膝关节轴线、接触面积及压力的影响。方法:6个新鲜尸体膝关节标本制成胫骨外侧髁劈裂骨折模型,通过使用支撑垫片制成关节面塌陷0、1、2、4和6mm。膝关节在屈膝0°,负荷500N和屈膝30°,负荷350N。通过数码相机记录关节轴线的变化;而关节内外间隙的压力和压力扩散则由F-Scan感应器记录。每个标本在保留和切除外侧半月板下进行测试。结果:随着关节面塌陷高度的增加,膝关节外翻角度、外侧间隙的平均和最大的接触压力逐渐增加,而接触面积则逐渐减少。在屈膝0°,关节面塌陷6mm时,外翻角度平均增加7.6°,平均接触压力和最大接触压力分别增加208%和97%;而接触面积则减少33%。在同一关节面塌陷高度,切除半月板平均增加38%的外翻角度和外侧间隙45%的接触压力;而接触面积则减少26%。结论:研究结果表明在治疗胫骨外髁劈裂骨折中,减少关节面的塌陷十分重要,特别是在需切除半月板的时候。