阳离子脂质体介导hGM-CSF真核表达载体在骨髓基质细胞系中的表达

目的 构建人粒－巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）真核表达载体ｐＩＲＥＳ１ｎｅｏ／ｈＧＭ－ＣＳＦ,并观察其在人骨髓基质细胞系ＨＦＣＬ中的表达,方法 用ＤＮＡ重组技术,将ｈＧＭ－ＣＳＦ ｃＤＮＡ插入到真核表达载体ｐＩＲＥＳ１ｎｅｏ的多砍隆位点中,获得阳性重组载体ｐＩＲＥＳ１ｎｅｏ／ｈＧＭ－ＣＳＦ。以脂质体介导法转染人骨髓基质细胞系ＨＦＣＬ细胞筛选获得的阳性细胞用Ｓｏｕｔｈｅｒｎ ｂｌｏｔ和Ｎｏｒｔｈｅｒｎ ｂｌｏｔ方法分别检测其基因组ＤＮＡ整合和ｍＲＮＡ转录,用ＥＬＩＳＡ及依赖株（ＴＦ－１）法检测其蛋白表达量和活性。结果 酶切鉴定表明成功地构建了重组真核表达载体ｐＩＲＥＳ１ｎｅｏ／ｈＧＭ－ＣＳＦ;ｈＧＭ－ＣＳＦ基因已整合到ＨＦＣＬ细胞基因组ＤＮＡ中,在ｍＲＮＡ和蛋白质水平上均可检测到其表达;ｈＧＭ－ＣＳＦ的表     

人粒系巨噬系克隆刺激因子基因导入真核细胞及其表达

目的：获得带糖链、近天然的重组人粒系巨噬系克隆刺激因子（ｈＧＭ－ＣＳＦ），以期为临床提供更安全、稳定的细胞因子。方法：采用基因重组技术，构建编码ｈＧＭ－ＣＳＦ的稳转载体ｐＭＥｎｅｏ－ｈＧＭ－ＣＳＦ，以酶切和猴肾细胞ＣＯＳ瞬时转染鉴定后，用磷酸钙－ＤＮＡ共沉淀法将重组质粒导入中国仓鼠卵细胞（ＣＨＯ）。结果：经Ｇ４１８加压筛选（８００μｇ／ｍｌ～１ｍｇ／ｍｌ），获得可表达ｈＧＭ－ＣＳＦ的细胞株。常规或载体培养ＣＨＯ－ｈＧＭ－ＣＳＦ细胞株上清可直接刺激ｈＧＭ－ＣＳＦ依赖细胞株的增殖，ＭＴＴ测定活性单位可达１×１０７ｕ／Ｌ原液。ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ分析显示ｒｈＧＭ－ＣＳＦ主带在２８ＫＤ位左右，为２Ｎ糖链型。结论：此ＣＨＯ－ｈＧＭ－ＣＳＦ细胞株表达稳定，活性较高，具有开发价值。     

CHO-rhGM-CSF克隆稳定性与不同浓度G418维持选择相关性分析

ＣＨＯｒｈＧＭＣＳＦ克隆稳定性与不同浓度Ｇ４１８维持选择相关性分析陈静娴宋宁陈强钙磷ＤＮＡ沉淀法转染我组构建的重组人粒巨噬细胞集落刺激因子（ｒｈＧＭＣＳＦ）ｐＭＥｎｅｏ质粒进入中国仓鼠卵细胞（ＣＨＯ），经Ｇ４１８选择后得到稳定表达ＧＭＣＳＦ...     

粒—巨噬细胞集落刺激因子对Vp16诱导白血病细胞凋亡的调控作用

目的：探讨粒－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）对Ｖｐ１６诱导白血病细胞凋亡的调控作用，指导白血病临床治疗中正确使用ＧＭ－ＣＳＦ。方法：在构建高效表达ＧＭ－ＣＳＦ的逆转录载体Ｎ２Ａ／ＣＭＶ／ＧＭ－ＣＳＦ表达系统的基础上，对转ＧＭ－ＣＳＦ基因和未转ＧＭ－ＣＳＦ基因的白血病ＨＬ－６０细胞进行研究。结果：未转ＧＭ－ＣＳＦ基因及转空载体Ｎ２Ａ的ＨＬ－６０细胞经Ｖｐ１６处理后均出现凋亡的特征性表现：ＤＮＡ电泳呈梯状；流式细胞仪ＤＮＡ直方图上呈现特征性的亚二倍体（亚Ｇ１）峰；透射电镜观察细胞形态可见凋亡的特征性改变。结论：Ｖｐ１６具有诱导白血病细胞发生凋亡的作用，而ＧＭ－ＣＳＦ能够抑制Ｖｐ１６诱导的细胞凋亡。以上结果表明，白血病临床治疗中为预防和改善化疗所致骨髓抑制而应用ＧＭ－ＣＳＦ时，应谨慎地选择时机，在化疗前和化疗期间不宜使用，以避免白血病细胞对抗癌药物诱导的凋亡产生抵抗而导致耐药。     

人FⅧ基因高效表达质粒的构建及其在Cos-7细胞中的表达

目的：构建人ＦⅧ真核表达质粒——ｐＡｄＣＭＶＬｉｎｋＦⅧＤＢ并观察其在Ｃｏｓ－７细胞中的表达活性。方法：采用缺失大部分Ｂ结构域的人凝血因子ⅧｃＤＮＡ（ＦⅧＤＢ），长度为４．６ｋｂ，将其插入含腺病毒序列表达质粒——ｐＡｄＣＭＶＬｉｎｋ１，构建了ＦⅧ真核表达质粒——ｐＡｄＣＭＶＬｉｎｋＦⅧＤＢ，用脂质体介导法将其转染Ｃｏｓ－７细胞，转染后２４，４８，７２小时分别用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）、ＥＬＩＳＡ法及一期法测定培养细胞中ＦⅧＤＢｍＲＮＡ及ＦⅧ的含量与活性。结果：ＲＴ－ＰＣＲ法可检测到ＦⅧＤＢｃＤＮＡ转录的ｍＲＮＡ，ＥＬＩＳＡ法测得７２小时后ＦⅧ的含量为每２４小时１８ｎｇ／１０６细胞，一期法测得活性为每２４小时０．６Ｕ／１０６细胞，相当于正常人血浆中１００μｇ／ＬＦⅧ所产生的凝血活性的６０％。结论：所构建的ＦⅧ真核表达质粒在Ｃｏｓ－７细胞中具有一定的表达活性。     

人凝血因子Ⅷ cDNA 体外真核表达的初步研究

目的：建立人凝血因子ⅧｃＤＮＡ体外真核高效表达的体系。方法：将人ＦⅧＢ区大部分缺失（Δａ７６０１６３９）的ＦⅧｃＤＮＡ插入ｐＣＩ真核表达载体、ｐＧＲＥ５．２／ＥＢＶ载体和逆转录病毒载体ｐＭＳＣＶ，构建了５种表达框架，在体外转染和感染了多种靶细胞。结果：ｐＣＩⅧ在ＮＩＨ３Ｔ３细胞产生了低水平表达，而ｐＧＲＥ５．２／ＥＢＶⅧ和ｐＭＳＣＶⅧ系列分别在Ｈｅｌａ细胞和Ｂｏｓｃ２３逆转录病毒包装细胞中呈较高水平的表达。Ｂｏｓｃ２３细胞培养上清感染的ＮＩＨ３Ｔ３和３２ＤＣ不能有效地产生ＦⅧ。结论：在ＦⅧ体外表达及基因治疗的方案设计中，靶细胞的选择是一重要因素。     

G418选择与稳转克隆表达分泌hGM-CSF的相关性考察

目的：进一步考察不同Ｇ４１８维持用量与ｒｈＧＭ－ＣＳＦ表达水平相关性。方法：第２期历时３个月（第４～６个月），本室构建的ＣＨＯ－ｒｈＧＭ－ＣＳＦ稳转克隆，在不同Ｇ４１８维持用量（０、３００μｇ／ｍｌ、６００μｇ／ｍｌ）下传２５代左右，各批次传代上清用依赖ＧＭ－ＣＳＦ生长的ＴＦ－１细胞作ＭＴＴ活性测定。结果：无Ｇ４１８选择维持，ＣＨＯ稳转细胞株表达和分泌ｈＧＭ－ＣＳＦ的水平从第４个月开始下降，再次加Ｇ４１８（６００μｇ／ｍｌ）后，ｈＧＭ－ＣＳＦ的表达水平迅速回升，２周后至正常。结论：Ｎｅｏ抗性基因作为遗传选择标志进入真核细胞，上述结果对于选择Ｇ４１８用量以维持宿主细胞表达分泌目标蛋白具有一定参考价值。     

IL—6和GM—CSF对人多发性骨髓瘤细胞与血管内皮细胞粘附效应…

用＾５１Ｃｒ标记细胞株的方法，研究了白细胞介素６（ＩＬ－６）及粒－巨噬系集落刺激因子（ＧＭ－ＣＳＦ）对人多发性骨髓瘤（ＭＭ）细胞株（Ｕ２６６、ＸＧ－７）、ＥＢ病毒阳性细胞株（Ｄａｕｄｉ）和内皮细胞间粘附的调控作用，观察了粘附作用对ＸＧ－７细胞表面粘附分子表达的影响。结果表明：①ＩＬ－６及其受体（ＩＬ－６Ｒ）ｇｐ１３０相关性生长因子ＧＭ－ＣＳＦ不仅是人ＭＭ细胞体内、外的生长因子，而且可以提高ＭＭ细胞     

caspase 3在细胞因子调节急性白血病细胞凋亡中的变化

目的 研究ｃａｓｐａｓｅ３在粒细胞集落刺激因子（Ｃ－ＣＳＦ）和粒巨洚细胞集落因子（ＧＭ－ＣＳＦ）调节白血病细胞系ＮＢ４细胞凋亡过程中的变化及其意义。方法 利用细胞这、流式细胞仪分析及ＤＮＡ片段化比率测定,检测Ｇ－ＣＳＦ及ＤＭ－ＣＳＦ对ＮＢ４细胞凋亡的影响,应用荧光分光光度法检测ｃａｓｐａｓｅ３活性变化,并利用ＡＥ－ＤＥＶＤ－ＣＲＯ进行ｃａｓｐａｓｅ３抑制试验。结果 Ｇ－ＣＳＦ能诱导ＮＢ４细胞凋亡,     

纤维连结蛋白,粘附抗原和造血祖细胞

纤维连结蛋白（ＦＮ）是广泛存在于多种细胞表面、基质及血浆中的一种糖蛋白，在细胞粘附、迁移、分化及成熟中的作用已有较多研究。但ＦＮ与髓系祖细胞分化成熟的关系研究尚少。除ＢＦＵ－Ｅ、ＣＦＵ－Ｅ和ＣＦＵ－ＧＭ皆能和ＦＮ分子中ＲＧＤ短肽相结合外，最近发现，原始造血祖细胞（ＬＴＢＭＣ－ＩＣ）多向造血祖细胞（ＣＦＵ－ＭＩＸ）、ＢＦＵ－Ｅ、ＣＦＵ－Ｅ及ＣＦＵ－ＧＭ还能和ＦＮ分子肝素结合片段中的“ＣＳ１”多肽结合     

Intercellular transfer of the virally derived precursor form of acid alpha-glucosidase corrects the enzyme deficiency in inherited cardioskeletal myopathy Pompe disease

Pompe disease is a lethal cardioskeletal myopathy in infants and results from genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Genetic replacement of the cDNA for human GAA (hGAA) is one potential therapeutic approach. Three months after a single intramuscular injection of 10(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level. Direct transduction of a target muscle, however, may not correct all deficient cells. Therefore, the amount of enzyme that can be transferred to deficient cells from virally transduced cells was studied. Fibroblasts from an affected patient were transduced with AdhGAA, washed, and plated on transwell culture dishes to serve as donors of recombinant enzyme. Deficient fibroblasts were plated as acceptor cells, and were separated from the donor monolayer by a 22-microm pore size filter. Enzymatic and Western analyses demonstrate secretion of the 110-kDa precursor form of hGAA from the donor cells into the culture medium. This recombinant, 110-kDa species reaches the acceptor cells, where it can be taken up by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks to lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme. Patient fibroblasts receiving recombinant hGAA by this transfer mechanism reach levels of enzyme activity that are comparable to normal human fibroblasts. Skeletal muscle cell cultures from an affected patient were also transduced with Ad-hGAA. Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunocytochemistry, and enzyme activity is transferred to deficient skeletal muscle cells grown in coculture. Transfer of the precursor protein between muscle cells again occurs via mannose 6-phosphate receptors, as evidenced by competitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA-knockout mice demonstrate that hepatic transduction with adenovirus encoding either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism. Taken together, these data show that the mannose 6-phosphate receptor pathway provides a useful strategy for cell-to-cell distribution of virally derived recombinant GAA.     

Cathepsin B and H activities and cystatin C concentrations in cerebrospinal fluid from patients with leptomeningeal metastasis

BACKGROUND: Cysteine proteases are involved in the extension of cancer into the subarachnoid space. The presence of cathepsins B and H along with their potent inhibitor cystatin C in the cerebrospinal fluid (CSF) was investigated in patients with leptomeningeal metastasis of cancer (LM). MATERIALS AND METHODS: CSF samples were obtained in 16 cases of LM (10 solid tumors and 6 leukemia or lymphoma) and compared with 11 cancer cases without involvement of the central nervous system, 12 multiple sclerosis cases and 34 healthy volunteers. The activity of the enzymes was measured, their molecular forms were analyzed by the Western blotting, and the concentration of cystatin C was measured by ELISA. Immunohistochemistry of the leptomeningeal tissues was also performed in six autopsy cases of LM. RESULTS: High activities of cathepsins B and H along with decreased cystatin C concentration were observed in CSF of LM as compared with three control groups. Western blot analysis revealed higher concentration of the enzyme protein as well as its active forms in samples with higher enzyme activity. Cells metastasizing leptomeningeal tissue were clearly positive in immunohistochemical staining of cathepsins, indicating active production by tumor cells. CONCLUSION: Production of cathepsins B and H by tumor cells and their high activity along with concomitant decrease of their potent inhibitor, cystatin C, in the CSF might contribute in the process of metastasis and spread of the cancer cells in the leptomeningeal tissues. A high enzyme activity/cystatin C concentration ratio in the CSF could be useful when diagnosing LM in combination with other parameters.     

带J链的α-防御素-1基因转染COS-7细胞后的表达检测

目的将α-防御索-1（HNP-1）基因与J链基因重组为一种新的杀菌肽分子J-HNP-1，观察J-HNP-1在细胞内的表达及分泌情况。方法采用聚合酶链反应（PCR）方法分别从pCH-J质粒和pBabeNco-HNP-1质粒中扩增出J链和HNP-1；进行重组PCR，获取J-HNP-1 DNA片段．并插入哺乳动物细胞表达载体pcDNA3．1（-）／Myc-HisC中；用脂质体转染法将此重组真核表达载体导入COS-7细胞。然后应用蛋白质免疫印迹法（Western blot）检测His-tag标签基因。结果用抗His抗体检测到细胞可溶性蛋白及培养上清在约24ku处均有强反应条带显示，其大小与预测相符；对照组未见相应条带显示。结论采用Western blot检测到新的杀菌肽分子J-HNP-1在细胞内得到表达并分泌到细胞外．为进一步研究J-HNP-1杀菌肽的抗菌活性奠定了基础。     

血管生成素在COS-7细胞中的表达及其生物活性研究

本研究旨在实现血管生成素（ANG）在真核细胞中的表达并探讨其生物学功能。通过RT—PCR获得ang基因，构建真核表达载体pcDNA3．1-ang，在转染COS-7细胞后进行瞬时表达，通过Western blot对表达产物进行鉴定。利用MTT法分析表达上清对ECV304细胞的促增殖作用，同时利用鸡胚分析表达上清的促血管生成作用。结果表明：瞬时转染后细胞培养上清中有重组ANG的表达，并能与抗-ANG单克隆抗体发生特异性反应。与转染空载体的对照相比．转染pcDNA3．1-ang的细胞培养上清具有促进ECV304细胞增殖的作用，并能显著促进鸡胚尿囊膜血管生长。结论：ang可在COS-7细胞中瞬时表达，其表达产物具有显著的促细胞增殖和促进血管生成的作用。     

Blood counts in healthy donors 1 year after the collection of granulocyte-colony-stimulating factor-mobilized progenitor cells and the results of a second mobilization and collection

BACKGROUND : Granulocyte–colony-stimulating factor (G–CSF)-mobilized blood cells are being used for allogeneic transplants, but the long-term effects of G–CSF on healthy individuals are not known. Furthermore, it is not certain how many CD34+ cells can be collected in a second mobilization and collection procedure. STUDY DESIGN AND METHODS : Nineteen people were given 2, 5, 7.5, or 10 μg of G–CSF per kg per day for 5 days, and blood progenitor cells were collected by apheresis on the sixth day; this was done on two occasions separated by at least 12 months. Blood counts obtained before and after each course of G–CSF and the quantity of cells collected were compared. RESULTS : There were no differences in white cell (WBC), platelet, red cell, and WBC differential counts measured before each course of G–CSF, and all the values were in the normal range. In a subset of 12 people who received 7.5 or 10 μg of G–CSF per kg per day for both courses, the numbers of neutrophils, mononuclear cells, and CD34+ cells in the blood after each course were similar (34.1 ± 7.31 × 10⁹/L vs. 36.4 ± 12.3 × 10⁹/L, p = 0.24; 6.59 ± 2.28 × 10⁹/L vs. 5.63 ± 2.11 × 10⁹/L, p = 0.24; and 92.0 ± 55.6 × 10⁶/L vs. 119.2 ± 104.6 × 10⁸/L; p = 0.48, respectively), as were the quantities of mononuclear cells (31.0 ± 8.4 × 10⁹ vs. 31.0 ± 6.1 × 10⁹; p = 0.64) and CD34+ cells (417 ± 353 × 10⁶ vs. 449 ± 286 × 10⁶; p = 0.53) collected in the two apheresis procedures. Furthermore, there was a positive correlation between the quantity of CD34+ cells collected from each of the 12 people per liter of whole blood processed in the two procedures (r² = 0.86, p<0.001). CONCLUSION : One year after the administration of G–CSF to healthy people, their blood counts were normal and unchanged from pretreatment counts. If healthy people donate blood progenitor cells after a second G–CSF course, the quantity of CD34+ cells collected will be similar to that obtained in the first collection.     

Adenovirus-Mediated MUC1 gene transduction into human blood-derived dendritic cells

MUC1 protein is widely expressed on various human cancer cells and has a specific highly glycosylated core structure with multiple tandem repeats, which may include an immunogenic peptide sequence. The potency of MUC1 protein to induce human histocompatibility leukocyte antigen-class I-restricted cytotoxic T-lymphocyte (CTL) induction remains to be fully clarified in human beings. In the current study, we made MUC1-expressing human dendritic cells (DCs) using recombinant adenovirus vector. Adenovirus vector plasmid containing human MUC1 cDNA, pAdHM4-MUC1 was constructed using in vitro ligation with a shuttle vector, pHMCMV5. Adenovirus vector expressing MUC1 was generated by the transfection of PacI-digested recombinant vector plasmid into 293 cells. Human blood DCs were obtained from 7-day culture of monocytes with recombinant human (rh) granulocyte-macrophage (GM) colony-stimulating factor (CSF) and (rh)interleukin (IL)-4. Then, 1 x 10(6) DCs were incubated with viral supernatant at a multiplicity of infection of 200 for 24 h in the presence of rhGM-CSF and rhIL-4. Flow cytometric analysis showed that 30% to 40% of the transduced DCs expressed MUC I protein; by contrast, nontransduced or transduced DCs with mock virus expressed only small amounts of MUC1 protein. Adenovirus-mediated MUC1 gene transduction into DCs had no significant effect on DC surface marker expressions or functions such as mixed leukocyte reaction. Furthermore, MUCI-specific CD8+ CTLs could be induced from healthy donor blood lymphocytes using MUC1-expressing DCs as stimulators. These results suggested that MUC1 gene-transduced DCs are a functional and potent tool for triggering a CTL response against MUC1 cancer cells.     

人干细胞因子与其它造血生长因子对造血集落形成的协同作用

本文观察了含有人造血干细胞因子(rh-SCF)的重组质粒转染的COS7上清液,在半固体培养中,单独或协同rhGM-CSF、G-CSF、IL-3、EPO刺激人粒单系和红系造血祖细胞形成集落的影响。结果表明:单独rh-SCF对造血祖细胞集落形成无明显作用,SCF主要是协同其它因子,明显增加集落形成数量和集落的大小。