体外培养淋巴细胞基因转移的实验探讨

以ＰＨＡ、ＩＬ－２体外诱导培养２～３ｗｋ的淋巴细胞作为受体细胞，在脂质体介导下进行腺苷脱氨酶及β－半乳糖苷酶基因转移。结果表明：外周血淋巴细胞在体外经ＰＨＡ、ＩＬ－２诱导可大量增殖，其中ＣＤ３＋细胞占９０％以上。脂质体介导外源性ＤＮＡ阳性转染率可高达３０％～４０％以上。转移后细胞表达有活性的基因表达产物。提示应用脂质体对体外扩增的淋巴细胞实施基因转移，其方法简便，转移率高，转移后基因在受体细胞中能较好地表达     

吸入糖皮质激素对哮喘儿童细胞免疫功能的影响

为了解糖皮质激素对免疫功能的作用，检测了哮喘发作期患儿。对经丙酸倍氯松（ＢＤＰ）吸入治疗的患儿及正常儿童外周血淋巴细胞膜ＩＬ－２受体（ｍＩＬ－２Ｒ）、超微结构、血浆游离ＩＬ－２受体（ｓＩＬ－２Ｒ）、淋巴细胞亚群及ＢＤＰ对体外培养淋巴细胞ｍＩＬ－２Ｒ的表达进行比较。结果显示，ＢＤＰ可以明显抑制ＰＨＡ刺激引起的体外培养淋巴细胞ｍＩＬ－２Ｒ的表达，引起淋巴细胞的凋亡，而吸入ＢＤＰ（２００μｇ／ｄ）的哮喘患儿（缓解期）除ｓＩＬ－２Ｒ比哮喘发作期显著降低（Ｐ＜０．０５）外，外周血淋巴细胞ｍＩＬ－２Ｒ及淋巴细胞亚群并未出现显著变化．结果提示，抑制淋巴细胞ｍＩＬ－２Ｒ表达和淋巴细胞活化可能是ＢＤＰ治疗哮喘等变态反应的重要机制之一。吸入ＢＤＰ主要是呼吸道局部作用，对全身细胞免疫指标影响不大     

ADA活性紫外吸收测定法的改良及复感儿淋巴细胞该酶活性的…

检测了４２例健康儿童和１７例反复上呼吸道感染患儿（复感儿）的血淋巴细胞腺苷脱氨酶（ＡＤＡ）活性，结果表明，复感儿的血淋巴细胞中ＡＤＡ活性较健康儿童低下，且大多同样伴有不同程度的免疫功能低下，从复感儿组中筛选了两例ＡＤＡ活性和免疫功能量显低下的患儿，拟采用这两例患儿的血淋巴细胞进行ＡＤＡ－ＳＣＩＤ基因治疗的实验研究。     

可溶性人干细胞因子基因导入真核细胞及其表达

本文构建了编码可溶性人ＳＣＦ基因的逆转录病毒载体ｐＬＸＳＮ－ＳＣＦ，应用Ｌｉｐｏｆｅｃｔｉｎ基因转移法将重组质粒导入Ψ２和ＰＡ３１７病毒包装细胞，经Ｇ４１８选择性培养基筛选，获得产重组病毒的包装细胞ＰＡ３１７－ＳＣＦ，其病毒效价为２．４×１０５～８．５×１０５ＣＦＵ／ｍｌ，继而感染人造血干祖细胞和ＮＩＨ３Ｔ３细胞。应用ＰＣＲ、ＡＰＡＡＰ免疫组化染色和化学发光一直接酶标法检测上述细胞中人ＳＣＦ基因的转染和表达，结果表明逆转录病毒载体介导转染的ｒｈ～ＳＣＦ基因在真核细胞中获得有效的表达。并将转有外源基因的人骨髓细胞进行体外液体长期培养（ＬＴＣ），观察造血干祖细胞增殖分化期间基因的表达情况。     

神经肽Y基因在COS－7细胞中的表达

构建大鼠ＮＰＹ基因的瞬时表达载体ｐＳＶＬ－ＮＰＹ，以Ｌｉｐｏｆｅｃｔｉｎ~（ＴＭ）介导将其导入ＣＯＳ－７细胞中，Ｎｏｒｔｈｅｒｎｂｌｏｔ和ＨＰＬＣ检测结果显示：ｐＳＶＬ－ＮＰＹ质粒转染的ＣＯＳ－７细胞可表达ＮＰＹｍＲＮＡ及成熟肽ＮＰＹ。     

IL—11 cDNA—逆转录病毒载体的构建及亲本细胞系的转染

本文采用Ｌｉｐｏｆｅｃｔｉｎ介导法，将构建的带有白细胞介素１１（ＩＬ－１１）ｃＤＮＡ的逆转录病毒载体导入制备人Ｂ细胞杂交瘤的亲本细胞系ＨＦ２、Ａｇ８．６５３和Ｓｐ２／０中，Ｇ４１８筛选稳定表达的抗性细胞，经克隆化后获得稳定分泌ＩＬ－１１的新型亲本细胞系。     

IL—1受体相关激酶1和2协同调控IL—1诱导的NF—？…

目的 研究细胞介素１受体相关激酶１（ＩＲＡＫ－１）和ＩＲＡＫ－２在白细胞介素１（ＩＬ－１）诱导核因子－кＢ（ＮＦ－кＢ）活化中的作用。方法 Ｌｉｐｏｆｅｃｔｉｎ介导反义和ＩＲＡＫ－１寡核苷酸和反义ＩＲＡＫ－２寡核苷酸转染ＨｅｐＧ２细胞。用逆转录ＰＣＲ法检测ＩＲＡＫ－１ ｍＲＮＡｔ ＩＲＡＫ－２ ｍＲＮＡ表达水平,以夹心ＥＬＩＳＡ法检测ＮＦ－кＢ的活化。结果 （１）反义ＩＲＡＫ－１寡核苷酸和反义ＩＲ     

白介素—1受体相关激酶—2调控白介素—1诱导的NK—kB活化

目的 研究白介素－１（ＩＬ－１）受体相关激酶－２（ＩＲＡＫ－２）在ＩＬ－１诱导ＮＦ－ｋＢ活化中的作用。方法 分别以逆转录ＰＣＲ法和Ｗｅｓｔｅｒｎ杂交法,检测Ｌｉｐｏｆｅｃｔｉｎ介导的反义Ｉ－ＲＡＫ－２寡核苷酸转染的人脐静脉内皮细胞中,ＩＲＡＫ－２ｍＲＡＫ－２寡核苷酸转染的人脐静脉内皮细胞中,ＩＲＡＫ－２ｍＲＮＡ和蛋白表达水平。用夹心ＥＬＩＳＡ法检测ＮＦ－ｋＢ的活化。结果 反义ＩＲＡＫ－２寡核苷酸可     

胰岛素样生长因子1的免疫调节作用的体外实验研究

目的：探讨胰岛素样生长因子１（ＩＧＦ１） 的免疫调节作用。方法：随机抽取健康个体８２ 名和肿瘤个体１２６ 名,体外提取外周血淋巴细胞后将ＩＬ２ 和或ＩＧＦ１ 共同培养,用３ ＨＴｄＲ 掺入法、３ ＨＴｄＲ 释放法及流式细胞仪检测淋巴细胞增殖活性、杀伤活性和ＣＤ４＋ ＣＤ８＋ 比率。结果：适当浓度的ＩＧＦ１（５０ ｎｇｍｌ）在体外能协同ＩＬ２ 促进ＬＡＫ细胞的增殖,增强其杀伤Ｒａｊｉ细胞的活性,提高淋巴细胞亚群ＣＤ４＋ ＣＤ８＋ 的比率。结论：ＩＧＦ１ 在体外对人体外周血免疫细胞起正向调节作用。     

IL—11 cDNA－逆转录病毒载体的构建及亲本细胞细胞系的转染

本文采用Ｌｉｐｏｆｅｃｔｉｎ介导法，将构建的带有白细胞介－素１１（ＩＬ—１１）ｃＤＮＡ的逆转录病毒载体导入制备人Ｂ细胞杂交瘤的亲本细胞系ＨＦ２、ＡＰ８．６５３和ＳＰ２／０中，Ｇ４１８筛选稳定表达的抗性细胞，经克隆化后获得稳定分泌ＩＬ－１１的新型亲本细胞系。     

ADA同工酶醋纤薄膜电泳法的建立及其在基因转移实验中的应用

本文建立了一种ADA同工酶醋纤电沐法．本电泳方法选用醋酸纤维薄膜为电泳主持物，可将人、鼠的红细胞及淋巴细胞中的ADA同工酶有效分离开来；以进行了人ADA基因转移后的鼠T淋巴细胞株Yac-I作为检测样品，用本电泳法进行了检测，结果显示：经基因转移并筛选后的细胞较基因转移前的细胞额外出现了一条泳动速率与人ADA同工酶相同的电泳带。这表明本法对ADA基因转移后的表达产物的分析检测具有应用价值。     

Activities of purine metabolising enzymes in lymphocytes of neonates and young children: correlates with immune function

Depressed activities of the following purine enzymes have been shown to result in immunodeficiencies: adenosine deaminase (ADA), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP). These enzymes and adenosine kinase (AK) were measured in cord blood lymphocytes of premature and small-for-gestational age infants since they have partial immunodeficiencies of unknown biochemical etiology which can persist for many years. We also measured these enzymes in 3 infants with various immunodeficiencies. Activities were compared with appropriate matched control groups. The results indicated normal ADA and PNP but significantly depressed AK (P less than 0.05) and HGPRT (P less than 0.001) activities in 10 premature/SGA infants when compared to 35 full-term normal infants. In the 3 immunodeficient children the results were as follows: Child 1 had a 2- to 3-fold decrease in ADA with normal PNP and AK activities; Child 2 had a 2- to 3-fold decrease in AK, 4-fold decrease in HGPRT with normal PNP and ADA activities; Child 3 had confirmed AIDS and a 4-fold decrease in ADA, 6-fold decrease in HGPRT with normal PNP activity. The possible role of these depressed purine enzyme activities found in lymphocytes is discussed in relation to the imparied immunity seen in these infants.     

滇西八个少数民族人群六种红细胞酶的遗传多态性

采用ＥｓＤ－ＰＧＭ１－ＧＬＯＩ同步电泳分型法和ＥＡＰ－ＡＤＡ－ＡＫ，同步电泳分型法，对滇西８个少数民族人群的红细胞酶ＥｓＤ、ＰＧＭ１、ＧＬＯＩ、ＥＡＰ、ＡＤＡ和ＡＫ１的表型分布进行了调查。计算出上述６种红细胞酶在僳僳族、纳西族、景颇族、普米族、怒族、阿昌族、德昂族和独龙族中的基因频率范围分别为：ＥｓＤ１０．６１２７～０．７２８２，ＰＧＭ１０．６６６７～０．８１００，ＧＬＯ１０．０８３３～０．２２１２，ＥＡＰＡ０．０９２２～０．３５２４，ＡＤＡ１０．９１２６～０．９９００，ＡＫ１１０．９９０５～１．００００．本次调查在６个民族中检出ＰＧＭ１６基因，其基因频率范围为０．００３３～０．０２９４．未发现其它酶型的变异型，亦未检出ＥＡＰｃ基因。对被调查人群中存在着的各酶型分布差异进行了分析。     

Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene

Adenosine deaminase (ADA) is expressed ubiquitously by diverse mammalian cells and tissues but at levels that vary according to tissue and species. In humans, the thymus exhibits levels of the enzyme up to 100-fold higher than most other tissues. Using transgenic mice, we identified human ADA gene regulatory domains. Up to 3.7 kb of 5'-flanking and first exon DNA from the human ADA gene failed to promote the expression of a chloramphenicol acetyl transferase (CAT) reporter gene in an efficient, reproducible, or tissue-appropriate manner in transgenic mice. However, when 12.8 kb of DNA from the first intron of the human ADA gene was placed 3' of CAT-coding and -processing sequences, transgenic mice reproducibly expressed CAT activity in most tissues, with profoundly high levels in the thymus. DNase I hypersensitivity studies demonstrated that among transgenic mouse tissues, human thymus, and a variety of human cell lines, a region of the intron 4-10 kb downstream of the first exon exhibited an array of hypersensitive sites that varied according to tissue and cell type. Deletion of this region from the gene construction eliminated high-level expression in transgenic mice. In transfection-transient expression assays, the 12.8-kb intron fragment exhibited enhancer activity in several cell types. A 1.3-kb fragment encompassing two of the hypersensitive sites exhibited some of these activities. The results of these studies suggest that the diverse pattern of human ADA gene expression is determined, in part, by a cluster of cis-regulatory elements contained within its large first intron.     

Prognostic significance of adenosine deaminase determinations in subjects with the lymphoadenopathy syndrome

The association between human immunodeficiency virus type I (HIV-I) infection and high levels of erythrocyte adenosine deaminase (ADA) has been suggested by Cowan et al [1986]. We have analyzed the specific activities of the same enzyme during different stages of acquired immunodeficiency syndrome (AIDS), including asymptomatic subjects at high risk and patients with lymphoadenopathy syndrome (LAS), AIDS-related complex (ARC), full-blown AIDS, and AIDS encephalopathy (AIDS enc). The ADA activities were significantly higher (P less than .05) in asymptomatic HIV-I serum-positive individuals (13.1 U +/- 1.1) and in different groups of patients (LAS = 23.6 U +/- 10.2; ARC = 23.7 +/- 4.1) than those found in controls (9.5 U +/- 1.8) and in HIV-I serum-negative subjects (10.4 +/- 1.5). In patients with AIDS the mean ADA activity was of 32.3 U +/- 7.1, whereas in two cases with AIDS enc it was of 10 U. A tendency to increase in median ADA values with the progression of the disease was observed. In LAS patients the ADA values presented two distinct subsets falling below and above the cut-off line of 15 U/10(9) erythrocytes, respectively. A specific correlation to drug addition and its duration was observed: LAS subjects who discontinued drug abuse (median addiction time: 3 years) presented ADA values (median = 13 U) that are lower than for addicts (median = 27.2 U; median addiction time = 7 years) and are close to those observed for asymptomatic HIV-I serum-positive group. Evidence was also obtained for a progressive increase of ADA values of LAS patients with disappearance of the product of gag gene. These results suggest that LAS subjects with elevated ADA activities present a longer history of HIV-I infection and a higher probability of developing AIDS.     

Activities of purine nucleotide metabolizing enzymes in human thymocytes and peripheral blood T lymphocytes: in vitro effect of thymic hormones

Adenosine deaminase (ADA, E.C. 3.5.4.4) and purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) activities are essential for the normal development and function of T lymphocytes. A comparison of the specific activity of these two enzymes and of ecto-5'-nucleotidase (5'-N, E.C. 3.1.3.5) of human thymocytes with that of peripheral T lymphocytes of children and young adults shows that significant differences exist between the activities of ADA and 5'-N in thymocytes and peripheral T cells. ADA activity is six-fold higher in thymocytes than in peripheral T cells whereas 5'-N activity is approximately one-fourth lower in thymocytes than in T lymphocytes. PNP activity is two-fold higher in T cells. The apparent Km and Vmax values for the three enzymes in thymocytes and peripheral T lymphocytes have been determined. The observed differences in enzyme activity are probably not entirely due to differences in the affinity of the enzymes for their substrates. The enzyme activities of a thymoma removed from a patient with myasthenia gravis had intermediate enzyme levels for ADA and PNP, but 5'-N was similar to peripheral T cells. Exposure of thymocytes in short-term culture to the thymic hormones thymopoietin (the pentapeptide TP5), thymosin fraction V or serum thymic factor (FTS) had no significant stimulatory or inhibitory effect on the activities of the three enzymes under our experimental conditions.     

Transfer of the adenosine deaminase (ADA) gene of a B-lymphoblastoid cell line (LCL) to an ADA-deficient LCL by a microcell-mediated chromosome transfer technique

As a model of somatic cell gene therapy, a normal adenosine deaminase (ADA) gene was introduced into a B-lymphoblastoid cell line (LCL) established from a patient with ADA deficiency by microcell-mediated chromosome transfer (MMCT). A LCL derived from his mother was used as a gene donor. Seven fusion experiments were performed and hybrid cells were pipetted into 123 wells. After selection in the presence of deoxyadenosine, cells grew in 12 wells at Week 9 after fusion. Among these wells, ADA activity of hybrids was low in 4 wells, 130-280% of that of the donor LCL in 7 wells and very high in one well. Hybrid cells in 4 wells with ADA-positive cells were investigated for the time-course of expression of ADA activity. In one well, ADA activity was expressed until Week 36, while, in 3 wells, ADA activity decreased or was lost between 21-36 weeks after fusion. These findings indicate the transfer of chromosome 20 containing a normal ADA gene into recipient cells and the deletion of this chromosome from some part of the hybrid cells. Karyotyping at Week 35 or 37 revealed 47 chromosomes in about 30% of the cells in 2 wells, which suggests that these hybrids were relatively stable in culture.     

Full genetic rescue of adenosine deaminase-deficient mice through introduction of the human gene

We have shown recently that adenosine deaminase (ADA)-deficient mice die perinatally with severe liver cell degeneration. In addition to enzyme substitution, we report the restoration of viability through introduction of the human ADA gene. The ADA gene is subject to complex developmental and tissue-specific regulation. To include the cis- regulatory elements necessary for correct regulation of the human ADA gene, a large transgenic locus constituting the human ADA gene with 10 kb of 5' and 4 kb of 3' flanking sequences was generated by co- injection of two overlapping DNA fragments into murine zygotes. Probably as a result of extrachromosomal (homologous) recombination between the fragments, one of the two transgenic lines contained a reconstituted, functional human ADA gene. As in man, human ADA expression generally was low in these transgenic mice, but high in the thymus, spleen and gastro-duodenal part of the gut. Apparently, all cis- regulatory elements essential for a human expression pattern were incorporated in the transgene and were functional in the murine background. Similarly to man, the upper alimentary tract of the transgenic mice revealed low human ADA activity in contrast to extremely high levels of murine ADA. The human gene probably lacks the cis-regulatory elements that target high level murine ADA expression to the murine upper alimentary tract. ADA-deficient mice rescued by introduction of the human ADA transgene appeared histologically and immunologically normal. Apparently, human ADA can complement murine ADA in all tissues, even in the epithelium of the upper alimentary tract where human ADA activity is as much as 70-fold lower than murine ADA activity in wild-type mice. Clearly, the lethal phenotype of ADA- deficient mice is due to the absence of ADA.      

A comparison of the kinetic properties of the common and rare variants of adenosine deaminase

Adenosine deaminase activity has been measured in red cells from individuals of known ADA phenotype (ADA 1, ADA 2-1, ADA 3-1, ADA 3-2) using adenosine and 2'-deoxyadenosine as substrates. No significant differences were observed among the phenotypes in their relative deaminase activity with the two substrates. However, evidence suggests the occurrence of an uncommon allele designated ADA ^1wdetermining low levels of ADA activity.
The deaminase activities of the phenotypes were in the order ADA 1 > ADA 2-1 > ADA 3-1 > ADA 3-2 with both substrates. The relative activities of the alleles were estimated to be: ADA ¹ 100%, ADA ² 89%, ADA ³ 28% and ADA ¹ 67% with adenosine, and ADA ¹ 100%, ADA ² 87%, ADA ³ 39% and ADA ¹ 66% with 2'-deoxyadenosine.
The Michaelis constants for adenosine and 2'-deoxyadenosine were determined for the different phenotypes. There were no significant differences in these values among the phenotypes.