目的：探究非甾体抗炎药不良反应发生的表现以及预防手段。方法：选取我院2010年4月～2013年6月收治的208例使用非甾体抗炎药后出现不良反应病例。归纳其病例的临床资料，并进行总结。结果：在使用药物后，其不良反应累及器官主要为胃肠道损伤以及肝功能损伤等，在使用非甾体抗炎药所产生的严重不良反应常见于皮疹和肝功能损伤。结论：非甾体抗炎药在临床应用时还具有相应的不良反应，因此在临床治疗中需要科学合理地用药，以此来降低不良反应的发生率。

目的 分析门诊处方用药差错情况并提出相应的干预对策.方法 对2014年1月~2014年6月间的12 076例门诊处方的用药差错情况进行分析,据此提出相应的干预对策,并于2014年7月起开始实施相应的干预对策,调查本院门诊在2014年7月~2014年12月间所开具的14 895例门诊处方的用药差错情况,比较实施干预前后门诊处方用药差错的发生几率.结果 干预前12 076例门诊处方中共有67例存在差错(0.55%),其中出错最多的项目为用药数量错误、其次依次为用药规格错误、用药品种错误以及用药剂量错误.干预后的14 895例门诊处方中共有24例存在差错(0.16%),差异具有统计学意义(P<0.05).结论 严格执行临床安全用药的相关制度、强化门诊药剂师的培训力度、强调医师、药师以及患者之间的有效沟通是降低门诊处方用药差错率的有效对策.

目的：分析研究抗菌药物不合理应用致药物不良反应的原因，并提出相应的解决对策。方法对本院在2014年1月～2014年12月间开具的26971张处方进行回顾式分析，调查不合理使用抗菌药物的情况以及药物不良反应的发生情况。结果26971张处方中抗菌药物处方3268张，占总处方比例为12.1%。其中存在不合理用药情况的处方共计310张，占抗菌药物处方的9.5%。其中因不合理使用抗菌药物而导致患者出现不良反应现象的处方287张，占总不合理用药处方的92.6%。抗菌药物不合理应用致药物不良反应的主要原因：用药时间不正确、无使用抗菌药物指征、不合理联合用药或重复用药。不良反应主要集中发生在消化系统、皮肤及软组织、心血管系统以及中枢神经系统。结论通过科学有效的干预对策来降低抗菌药物不合理使用的发生率和药物不良反应发生率，规范医务人员的用药行为对提高患者的用药安全性以及抗菌药物的使用效果具有十分重要的意义。

长期来国内发表的一些有关造血干细胞移植文章常常把单倍体相合(haploidentical)误称为"HLA单倍型相合"或"HLA半相合".有的文章则把haploidentical transplant错译为"单倍体移植"、"单倍型移植"等.

一、干细胞基础研究的新发展和有关干细胞的概念更新 1.成体干细胞研究:成体干细胞是21世纪初生物学的伟大发现,它改变了干细胞传统的理念.20世纪末,一些科学家报道,在许多成体组织中,有一些细胞可以在体外被某些细胞因子诱导分化,变为和原组织不同的组织细胞.例如肌肉匀浆中诱导发生造血细胞,脂肪组织中诱导发生神经原细胞等.

During the recent 5 years,an upsurge of cell biology research has appeared around the world with a huge number of papers published.A part of them,however,concluded with a hypothesis of so called or stem cell plasticity that cells could transform into any other tissues such as fats becoming liver or muscle changing into nervous cells and so on,that all lacks of strong evidence.The discovery of adult cells(ASC) in the adult tissues was actually a great scientific epoch-making towards the end of 1999 though the authors made a wrong conclusion of transdifferentiation at that time.More and more evidences showed that the ASC in the adult tissue are the remnants of cells from every stage of embryonic development.The tissue specific cells harbored in the adult tissues can only be induced to differentiate in the progeny cells of its own committed origin.ASC includes hematopoietic and non-hematopoietic cells and there are lots of cells in the latter,like nervous cells and mesenchymal cells(MSC).The progenies of MSC are the connective tissues,such as osteoblasts,chodroblasts,myocytes,angio-endothelio cells,etc,which are most important during the recovery and reconstitution of tissue injury,ischemic necrosis and hematopoietic damage.

Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFbeta, 78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD). It has been demonstrated that hSCGFbeta is strictly species-specific in regulating he-matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37 degrees C. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature (28 degrees C), the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFbeta, hSCGF had the granulocyte/macrophage promoting activity (GPA) for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFbeta, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFbeta does not directly bind to corresponding SCGF receptor, but may have certain biological function.

The article reviews concisely around the history of hematopoietic stem cell research, basic and clinical, since its very beginning after the nuclear explosions at the end of the Second World War, that explains why the stem cell study in the world began with the hematopoietic stem cells and the existence of non-hematopoietic stem cell in vivo had been left out of account for many decades till 21st century. During 50-60s of the last century, it was known from the animal experiments that there must be hematopoietic stem cells existing in vivo and believed that the effective bone marrow transplant is actually the stem cell transplantation. It is reviewed how the basic studies of stem cell interacted with the clinical stem cell transplantation and how great the contribution was given to strongly push forward the development of contemporary stem cell biology and modern hematology from the basic studies especially in the immunological and molecular biological fields, for instance, the applications of HLA technology and monoclonal antibody produced, flow cytometry, and genetic recombinant cytokines, the novel technique for gene cloning, genomics, proteomics, and iRNA as well as bioinformatics. It has lead to the pluralistic cell therapy as a novel trend in stem cell transplantation as to combine immunotherapy and mesenchymal stem cells with the conventional stem cell transplants. This paper looks back in the past several decades, however, on every achievement of stem cell study that were usually accompanied with some idealistic one-sidedness or even errors in design and conclusion of some experiments. Usually it took a period of 2 or even 4 decades to clarify some basic idea, that seemed normal in the science development, for examples, the dividing line between the hematopoietic stem cell and progenitor cells, possibility to expand or clone the real stem cell ex vivo, and whether the majority of leukemias are originated from stem cell level, etc. Towards the end of 20th century, the greatest discovery was the existence of adult stem cells in adult tissues, which are no doubt the remnants from the embryonic development including all stages of embryonic stem cells, very earlier and later, hematopoietic and nonhematopoietic, and could be induced to differentiate into all kinds of tissue cells by proper ways in vitro. No evidence has really provided for the hypothesis of so called trans-differentiation or de-differentiation, which brought about strong calls in questions in the past 5 years. The problems in developing the clinical gene therapy by using hematopoietic stem cell as carrier of interested gene still remained to be solved so far. Because of the relatively weak base of related basic studies, the clinical application of gene therapy resulted in the failure of clinical practice with lots of lessons towards the end of last century. The whole history of stem cell study in the world was an endless process of continuous redress for theoretical ideas in stem cell biology that had never been consummate.

目的研究间充质干细胞(mesenchymal stem cell,MSC)对T细胞的免疫调节作用.方法从骨髓中分离培养MSC,从外周血中分离单个核细胞,进一步用免疫磁珠纯化CD3+T细胞,观察MSC对T淋巴细胞转化的影响;且用ELISA 实验检测上清中IFN-γ和IL-10的表达.结果当CD3+T细胞量为2×105/孔时,SC对其增殖反应主要表现为负调控,且随MSC细胞量的增多,这种负调控越明显;而当体系中CD3+T细胞量为5×104/孔时,SC对其增殖反应表现为正调控,而且随MSC细胞量的增多这种促进作用越明显.ELISA结果表明,在没有MSC的存在下,×105/孔的T细胞分泌细胞因子IFN-γ和IL-10均高于5×104/孔的T细胞;但在有MSC存在情况下,×105/孔的T细胞分泌细胞因子IFN-γ和IL-10反而低于5×104/孔的T细胞.结论 MSC对T细胞的调控是复杂的,不仅与MSC的细胞量有关,还与T细胞的量有关.

白介素-11是体内不可缺少的调控正常细胞生理功能的一种细胞因子.人体发生疾病或创伤时,特别是肿瘤放化疗后体内的白介素-11的量远远不能满足抗御疾病或创伤的需要,所以必须使用与天然白介素-11分子结构完全相同的、基因重组表达的人白介素-11纯化制品,才能达到治疗的目的.本文综述了白介素-11的药效和药理,供临床医生在使用迈格尔(R)(注射用重组人白介素-11)治疗肿瘤放化疗引起或其他原因引起的血小板减少症时参考.

目的:克隆表达人CD40-Ig融合蛋白,并在小鼠移植物抗宿主病(GVHD)模型中研究利用其阻断CD40/CD40L相互作用的保护性效果.方法:利用RT-PCR技术自人Daudi细胞系中克隆CD40基因胞外区,并插入含有人IgG1 Fc段基因的pIG1载体中,构建携带CD40-Fc融合基因的瞬时表达载体转染COS-7细胞,间接夹心ELISA法检测CD40-Ig融合蛋白的表达.再将CD4-Fc融合基因连接入pcDNA3.1的相应位点构建稳定表达载体转染CHO细胞,筛选分泌CD40-Ig融合蛋白的阳性重组CHO细胞并进行无血清大规模培养,收获培养上清利用Protein A亲和层析法纯化融合蛋白.SDS-PAGE、Western blot和配基结合实验鉴定CD40-Ig的性质.将C57BL6/J(H-2b)小鼠的脾细胞经尾静脉注射入亚致死剂量照射的BALB/c(H-2d)小鼠体内建立急性GVHD模型,通过体内注射CD40-Ig融合蛋白评价其对急性GVHD小鼠的保护效果.结果:按上述方法分别构建了哺乳动物表达载体pIG/40Ig和p3.1/40Ig.ELISA 和Western blot确定在COS-7和CHO细胞中表达了CD40-Ig融合蛋白.SDS-PAGE结果显示该蛋白具有通过二硫键结合的二聚体结构并以同源二聚体的形式存在.纯化的CD40-Ig可与CD40L结合.体内应用CD40-Ig融合蛋白治疗可延缓小鼠GVHD病情发展并显著延长小鼠的平均存活时间.结论:CD40/CD40L相互作用在GVHD的病理过程中可能扮演了十分重要的角色,提示人CD40-Ig融合蛋白在临床预防和治疗GVHD方面的巨大应用潜力.

Immunoglobulins (Igs) are found thus far only to be produced by differentiated B lymphocytes. By immunohistochemistry analysis, in situ hybridization, and laser capture microdissection-assisted single-cell PCR, we demonstrate that human cancers of epithelial origin, including carcinomas of breast, colon, liver, lung, established epithelial cancer lines, as well as some normal lung tissues, also produce IgG in both cytoplasmic and secreted forms. Furthermore, blockade of tumor-derived IgG by either antisense DNA or antihuman IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro. More importantly, administration of antihuman IgG antibody also suppressed the growth of an IgG-secreting carcinoma line in immunodeficient nude mice. Our results support a role of tumor-derived IgG as growth factor for epithelial cancers. Prevalent expression of IgG in human carcinomas and its growth-promoting functions may have important implications in growth regulation and targeted therapy of human cancers.

The basic studies selected were mainly published since 1998 and related to stem cell biology and engineering and particularly the efforts for developing new sources of hematopoietic stem/progenitor cells ex vivo. Hematopoietic cells and lymphocytes can be developed by induced differentiation in a appropriate way of culture, originating in the embryo- or adult-derived stem cells or tissue-committed stem cells which still exist in the tissue of adults. The most primitive multipotential embryonic stem cell from embryo or adult tissue has the plasticity to differentiate into every kind of progenies, the committed tissue-specific stem cell, by different proper ways of induction in vitro. The committed tissue-specific stem cell, however, can only be induced to differentiate along the line of its committed origin of tissue. No studies in China strongly confirmed yet the existence of "transdifferentiation" among the tissue- or organ-specific stem cells.

一般而言,干细胞可根据来源的不同划分为胚胎干细胞(Embryonic Stem Cells)和成体组织干细胞或成体干细胞(Adult Stem Cells),按其分化能力的不同又可区分为全能干细胞(Totipotent Stem Cells)、三胚层多能干细胞(Pluripotent Stem Cells)、单胚层多能干细胞(Multipotent Stem Cells)和单能干细胞(Monopotent Stem Cells).

慢性粒细胞白血病(CML)是人造血干细胞发生的一种恶性肿瘤,临床上慢粒是以异常的、不成熟的恶性造血祖细胞的大量增生为特征.病理特征是因第九与第22位染色体易位{t(9:22)}形成的pH1染色体,该染色体易位产生的BCR/ABL融合基因表达的具有高酪氨酸激酶活性的p210BCR/ABL融合蛋白是慢粒发生的分子基础,它能结合或激活细胞内多种信号相关的分子,为造血细胞的恶性转化必要而充分的条件.该病的p210BCR/ABL是通过什么分子机制引起这些变化仍不清楚,目前临床上除干细胞移植外尚无治愈的方法.

Objective To obtain CD40 Ig fusion protein, and investigate the therapeutic potentials of blocking the CD40/CD40L signal pathway in the prevention and treatment of graft versus host disease (GVHD). Methods The CD40 Fc fusion gene, derived from mammalian transient expressing vector pIG/40Ig, was inserted into constitutive expressing vector pcDNA3.1. Then the recombinant expression vector was transfected into CHO cells with Lipofectamine reagent. The CHO cells, secreting CD40 Ig fusion protein in the culture supernatant, were obtained after selecting by G418. That was confirmed by Sandwich ELISA and Western bolt assay. The transfected CHO cells were cloned two times by limited dilution method. After big batch serum free culture using roller bottle, the concentrated supernatant was purified by rProtein A affinity chromatograph method. The purity of the purified recombinant was confirmed by SDS PAGE. The binding of the CD40 Ig fusion protein to Jurkat cell was detected by FCAS. Results Mammalian constitutive expression vector p3.1/40Ig was constructed as described above. Then the mixed CHO clones, expressing CD40 Ig fusion protein, were selected by G418 0.8g/L for two weeks after transfection. Through twice limited dilution cloning, five single clones were obtained, and the best one named as B2. The big batch culture supernatant of B2 was harvested, concentrated, and purified through rProtein A affinity chromatograph. The purity of recombinant CD40 Ig protein is above 95%. FACS results shown that CD40 Ig can bind to the CD40L expressing on the Jurkat cells, which were stimulated by PHA P. Conclusion The CD40 Ig could mimic the native CD40 molecule and bind to its ligand. It provided a useful tool to investigate the role of blockade CD40/CD40L costimulation signal pathway in the prevention and therapy of GVHD.

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34+ cells by 15.7-fold. Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.

为探讨转染FL和/或TPO基因骨髓基质细胞系对脐血CD34+细胞的体外扩增效应, 建立了转基因骨髓基质细胞系共培养体系. 采用免疫磁珠法分离人脐血CD34+细胞, 在CD34+细胞不同体外培养体系中取样测试细胞总数、CD34+细胞百分率和CFC(包括CFU-GM 和BFU-E). 结果表明, 在8种不同组合的培养体系中, 转基因基质细胞共培养体系较无基质液体培养体系对细胞总数, CFC, CD34+细胞均具有明显的扩增效应, 其中以SCF + IL-3 + HFT扩增效果最好, 分别扩增了(893.3±52.1), (74.5±5.2)和15.7倍. CFU-GM和BFU-E在第2周时达扩增高峰, 扩增倍数分别为(78.1±5.5)和(57.0±19.7). LTC-IC测定结果显示, 只有SCF + IL-3 + FL + TPO和SCF + IL-3 + HFT组有LTC-IC的存在, 统计学检验无显著性差异. 上述结果提示, 转基因骨髓基质细胞系可通过细胞间的接触协同其他细胞因子增强对脐血CD34+细胞的体外扩增作用.

Aim To explore antitumor responses induced by recombinant vaccinia viruses expressing a point mutant p53 (rVVp53 FL )and its antigenic peptide rVVp53 M). Methods P815 mastocytoma transduced with a 135 Cys to Tyr point mutation p53 was used as an experimental tumor (P815mp53) and the p53 protein as the model of tumor associated antigen. rVVp53 FL and rVVp53 M were used as tumor vaccine to investigate their induction of CTL and antitumor immunity. Results Immunizing Balb/c mice with rVVp53 FL and rVVp53 M could induce specific CTLs, which specifically lysed P815mp53 cells. After the immunized mice were challenged with a lethal dose of P815mp53, a part of the mice survived. The mice bearing tumor was treated with rVVp53 FL and rVVp53 M, which could significantly prolong the mice′ s survival time. Antitumor response induced by rVVp53 M was comparable to that by rVVp53 FL . Conclusion antigenic peptide derived from mutant p53 protein may replace the protein as vaccine for antitumor immunotherapy.