羧乙基壳聚糖-纳米羟基磷灰石复合材料细胞相容性和生物力学性能的实验研究

制备羧乙基壳聚糖-纳米羟基磷灰石(NCECS/nHA)复合材料,研究其生物力学性能以及与气管软骨细胞的生物相容性。方法 气管软骨片段取自8周龄大耳白兔,Ⅱ型胶原酶消化,将所获得软骨细胞传代培养。将体外制备的NCECS/nHA复合材料分别进行干态标本和湿态标本的生物力学检测。将第3代软骨细胞种植到NCECS/nHA复合材料,分别计算材料表面软骨细胞在2h、6h、12h细胞贴壁率,并用噻唑蓝(MTT)法测定细胞增殖活性。结果 NCECS/nHA复合材料具有良好的生物力学性能。兔气管软骨细胞在NCECS/nHA复合材料表面上12h的贴壁率达(88.4±2.1)％,与其他组差异无统计学意义(P＞0.05)。同时MTT显示气管软骨细胞在NCECS/nHA复合材料表面生长状态良好。扫描电镜结果显示软骨细胞在NCECS/nHA薄膜上增殖和分化良好。结论 NCECS/nHA复合材料具备良好的细胞相容性和适宜的生物力学强度,作为一种具有开发潜力的生物材料,可用于组织工程气管的体外构建。     

人工听骨链重建材料生物相容性、特性及修复喉软骨缺损的效果

BACKGROUND: DefectedLaryngeal cartilage has many alternatives, including autologous cartilage, allograft cartilage and metal stents. Although these materials can achieve desired outcomes in laryngeal cartilage defect repair, certain limitations exist.     

MHC五聚体方法检测基因疫苗体外诱导的抗原特异性细胞毒T淋巴细胞

目的 探讨MHC五聚体方法检测在体外用抗B细胞淋巴瘤基因疫苗(简称基因疫苗)刺激PBMC生成抗原特异性CTL的效率.方法 用于细胞培养的4份血液标本来自2例B淋巴细胞肿瘤患者(1例为滤泡性淋巴瘤患者,另1例为毛细胞白血病患者)和2名健康对照者.分别检测上述标本中PBMC受基因疫苗刺激后CTL生成情况.PBMC贴壁培养获得DC前体,在重组细胞因子GM-CSF和IL4的支持下诱导培养DC,采用基因枪方法将基因疫苗质粒导入DC,RT-PCB方法检测转染后细胞IgHV1-GM-CSF mRNA的转录,ELISA方法检测细胞因子GM-CSF的表达,并验证转染是否获得有效表达.转染后的DC再与从PBMC中获得的淋巴细胞共培养,诱导抗原特异性的CTL;共进行2次DC刺激,共计培养24 d,分别在培养前、培养第7天、第17天及第24天收获培养细胞,培养分为转染基因疫苗组和转染对照质粒组,流式细胞术分析培养细胞中CD3+ CD8+细胞亚群的水平;用针对基因疫苗抗原肽抗原表位的MHC五聚体荧光抗体检测CTL产生水平.结果 基因枪颗粒轰击方法转染DC后,RT-PCR结果显示转染得到了表达;转染基因疫苗组的DC中GM-CSF含量为(28±6)ng/106个细胞,而转染对照质粒组的DC中GM-CSF含量为(10±3)ng/106个细胞,差异有统计学意义(t=5.191,P<0.01).分析培养后的T淋巴细胞亚群,显示随着培养时间的延长,CD3+CD8+细胞亚群比例明显增加,转染对照质粒组在培养前、培养第7天、第17天和第24天的比例分别为(34.24±2.72)%,(46.06±3.08)%,(65.34±4.26)%,(73.86±4.85)%,而在转染基因疫苗组,其比例分别为(32.28±2.08)%,(45.32±3.81)%,(63.37±4.21)%,(75.01±3.20)%.两因素方差分析显示培养不同时间点的CD3+CD8+细胞亚群比例差异有统计学意义(F培养时间=176.966,P<0.01),但转染因素本身对其产生的影响差异无统计学意义(F转染因素=0.657,P>0.05).MHC五聚体检测结果显示转染基因疫苗组的DC能够诱导针对该表位的抗原特异性CTL产生,CTL水平随着DC的刺激逐渐增高;在培养第24天,健康对照者最高获得2.03%的CTL,滤泡性淋巴瘤患者的CTL达4.36%,而毛细胞白血病患者为3.89%.结论 MHC五聚体方法能够有效检测基因疫苗诱导的抗原特异性CTL;该方法对于抗肿瘤细胞免疫的检测、肿瘤患者的细胞免疫状态及早期预后判定可能是一项有前景的临床检验方法.     

HLA半相合未去除T细胞骨髓移植治疗白血病的初步观察

目的探索半相合未去除T细胞骨髓移植治疗白血病的可行性.方法15例白血病患者接受HLA2～3个位点不匹配亲缘骨髓移植.用阿糖胞苷、环磷酰胺和γ射线全身照射进行预处理,供者应用G-CSF250μg/d,连用7d后采髓,移植物抗宿主病(GVHD)预防除用环孢菌素A(CsA)和甲氨蝶呤(MTX)外,在移植前第4天～第1天用抗胸腺细胞球蛋白ATG5mg@kg-1@d-1,移植后第7天开始加用霉酚酸酯1.0g/d.结果患者移植后均获得造血重建,中性粒细胞＞0.5×109/L和血小板＞20×109/L的中位时间分别是19d(13～23d)和21d(16～32d).5例(33.3%)发生急性Ⅱ～Ⅳ度GVHD,其中急性Ⅱ度肠道GVH2例,急性Ⅲ度肠道GVHD2例,急性肠道和肝脏Ⅳ度GVHD1例.可评价的9例患者中8例发生慢性GVHHD,无一例发生广泛性慢性GVHD.中位随访时间395d(110～690d),死亡6例,其中死于急性GVHD3例,死于感染2例,复发死亡1例.无病存活9例,其中6例存活在1年以上.结论供者应用G-CSF后采髓,多种免疫抑制剂联合应用的HLA半相合未去除T细胞骨髓移植,在治疗自血病过程中,有效地降低了急性重症GVHD发生,提高了无病生存,对拓宽供髓来源有重要实用价值.     

人类白细胞抗原DRB1和DQB1等位基因多态性与大肠癌的关联性

目的：探讨人类白细胞抗原HLA－DRB1,－DQB1等位基因与大肠癌遗传关联性。方法：运用序列特异性引物聚合酶链反应,结合等位基因序列分析,检测无亲缘关系的湖北籍汉族健康人136名、大肠癌组54例患者的HLA－DRB1、－DQB1基因。结果：大肠癌患者与正常人比较,HLA－DRB1＊0901等闪基因分布频率明显增高（0．2315、0．1397,P＝0．033）;而－DRB1＊080X等位基因频率明显降低（0．0093、0．0809,P＝0．0071）。两组间HLA－DRB1其余等位基因及HLA－DQB1等位基因分布频率,差异均无显著意义。结论：HLA－DRB1＊0901与人大肠癌呈正关联,而HLA－DRB1＊080X则与其负关联,上述峡谷等位基因结构还得到基因序列分析印证。     

人白细胞抗原G1及G5在重度子痫前期患者胎盘组织中的表达

目的 探讨重度子痫前期(sPE)患者胎盘组织中人白细胞抗原(HLA)G1及G5表达的变化.方法 选取早发型及晚发型sPE患者各10例、早产及正常足月妊娠产妇各10例,通过蛋白印迹法及免疫组化方法检测胎盘组织中HLA-G1及G5蛋白的表达.结果 与早产产妇相比,HLA-G1蛋白在早发型sPE患者的胎盘组织中表达量明显减少,分别为2.9±1.1、2.4±0.6,两者比较,差异有统计学意义(P＜0.05);与正常足月妊娠产妇相比,晚发型sPE患者的胎盘组织中HLA-G1蛋白的表 达量明显减少,分别为4.2±2.4、3.5±2.1,两者比较,差异有统计学意义(P＜0.05).与正常足月妊娠产妇相比,HLA-G5蛋白在晚发型sPE患者的胎盘组织中表达量增加,分别为1.1±0.9、1.8±1.1,两者比较,差异有统计学意义(P＜0.05);与早产产妇相比,HLA-G5蛋白在早发型sPE患者胎盘组织中的表达量无明显变化(分别为1.4±0.7、1.6±0.9,P＞0.05).HLA-G1蛋白在早产产妇胎盘组织中的表达量较正常足月妊娠者减少(P＜0.05);HLA-G5蛋白在早产及正常足月妊娠产妇胎盘组织中的表达量无明显差异(P＞0.05).HLA-G1及G5蛋白主要在胎盘的绒毛外细胞滋养细胞中有表达,在血管周围及胚外中胚层中也有高表达.结论(1)HLA-G1在早发型及晚发型sPE患者胎盘组织中的表达均减少.(2)HLA-G5在早发型及晚发型sPE患者胎盘组织中的表达均增加,这一增加趋势在晚发型sPE患者中更为明显.(3)在妊娠晚期,HLA-G1在早产产妇胎盘组织中的表达较少.(4)胎盘组织中能够检测到HLA-G1及G5的表达,其主要位于胎盘的绒毛外细胞滋养细胞中.     

Intrathecal synthesis of soluble class I antigens (sHLA) in patients with HIV infection and tuberculous meningitis

sHLA are soluble class I antigens produced by lymphocytes on early activation. We have studied the sHLA indexIH=(CSF sHLA/serum sHLA)/(CSF albumin/serum albumin), which reflects the intrathecal synthesis (ITS) of sHLA in 23 intravenous drug abusers with central nervous system (CNS) HIV infection. Their mean IH value was increased and directly correlated with ITS of IgG against HIV when the total group of patients was studied; however, 8 of them, who suffered from concomitant tuberculous meningitis, had a decreased IH. The relationship between this index, blood-brain barrier (BBB) function, and HIV and tuberculous infection was also studied. We consider IH an index of lymphocyte activation within the CNS. Its decrease in patients with CNS HIV infection may reflect the presence of a meningeal opportunistic infection due toMycobacterium tuberculosis.     

A novel HLA-DR13 allele (DRB11314) identified by single-strand conformation polymorphism analysis and confirmed by direct sequencing

A novel variant of the HLA-DR13 group is described. The new allele was found in a DR 10, 13 heterozygous patient of Turkish origin, two HLA genotypically identical children of the patient typed as DR11, 13, and one child typed as DR13, 13. DR13 subtyping of the patient was initially performed by SSCP analysis of PCR-amplified DNA, using the 11th IHWS primers DRBAMP-3 and DRBAMP-B. Due to an unusual SSCP banding pattern, the PCR product was subjected to solid-phase sequencing. The sequence of the new allele, DRB1^*1314, is different from that of DRB 1^*1307 by a single nucleotide substitution in codon 47, with T replacing consensus A. This results in a single amino acid change of tryptophan to phenylalanine in the first domain of the DRβ chain.     

New cell lines of gastric and pancreatic cancer: distinct morphology,growth characteristics,expression of epithelial and immunoregulatory antigens

Two new cell lines from stomach cancers and one from a pancreatic carcinoma are presented. MZ-GC-1 was established from a hepatic metastasis of a well differentiated gastric adenocarcinoma. MZ-GC-2 was derived from ascites induced by a poorly differentiated gastric adenocarcinoma. MZ-PC-1 originated from the pleural effusion of a moderately well differentiated pancreatic ductal adenocarcinoma. MZ-GC-1 cells were adherent and partially polarized, connected tightly via desmosomes. In contrast MZ-GC-2 cells consisted of slightly adherent or floating subpopulations and displayed no desmosomes. MZ-PC-1 cells were adherent and showed polarized growth, connected by apical junctional complexes. Cell doubling times were 7 days for MZ-GC-1 and 45 h for MZ-GC-2 and MZ-PC-1 cells. MZ-GC-2 and MZ-PC-1 gave rise to nude mouse tumours, resembling the original lesions. Chromosome analysis of the cell lines revealed a high range of numerical abnormalities. Each cell line had cytokeratin patterns fitting well to typical in vivo patterns. Furthermore the cell lines expressed a panel of antigens typical for gastrointestinal epithelia. Unique for MZ-PC-1 were high amounts of secreted Ca19-9. -Interferon enhanced HLA-class I antigens up to twofold and induced ICAM-1 expression on each cell line. HLA-class II antigens were differentially enhanced by -interferon. Due to their distinct characteristics the three tumour cell lines may be useful models in the investigation of the cell biology and immunogenicity of gastrointestinal tumours.This work has been supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (He 1752-2-1) and by an Investigator Award from the Cancer Research Institute, New York, USA (A. Knuth).     

A pilot study on Hla-G locus control region haplotypes and cervical intraepithelial neoplasias

Human papillomavirus (HPV) can induce cervical intraepithelial neoplasias (CIN) grades 1, 2 and 3. Untreated, these lesions may progress to cervical cancer (CC) which is the third most common cancer in women worldwide. HLA-G plays an immunotolerant role in the immune response. The aim of this study was to characterize the configuration of SNPs located at the distal promoter of HLA-G in patients with CIN2 and CIN3 and control women. The study sample was composed of 207 women as follows: 73 diagnosed with CIN2 lesions, 56 with CIN3 and 78 healthy control women. Genotyping was performed by sequence base typing. Eleven haplotype configurations subdivided in two main haplogroups (H1dist and H2dist), were characterized and compared between patients and controls. The haplotypes H1.1Dist (GAGAACGC) and H2.1Dist (AGGTACAC) were more frequent in Euro-Descendants as well as in Brazilian Mixed. Nevertheless, the haplotype H2.1Dist standed out as a susceptibility haplotype in Brazilian Mixed patients while the H1.1Dist presented a protector effect in this same ethnic group. Whether such LCR haplotype configurations can impact on HLA-G gene expression levels in women who developed cervical intraepithelial neoplasia is still unknown and it is of utmost importance that more investigation on this field be pursued.