rIL-2工程菌诱导阶段细胞再循环培养的研究

为了减少rIL-2工程菌高密度培养时乙酸的积累,在诱导阶段对该工程菌进行细胞再循环培养的研究,比较了细胞再循环补料液、pH、细胞循环培养时间段对工程菌的生长及rIL-2表达的影响。结果表明在菌密度D_(600)为50时,细胞再循环补料液中酵母抽提物与胰蛋白胨浓度为发酵培养基的5倍就能满足rIL-2表达的需求,同时选择诱导后4～6h之间的细胞再循环培养能有效地防止乙酸的过高积累并减少营养物质的损失,有利于rIL-2的表达。根据以上研究结果得到了rIL-2工程菌诱导阶段细胞再循环培养方法,使得在诱导前菌密度D_(600)为50左右时rIL-2的表达水平约为40％。     

补料速度对工程菌生长和产物表达的影响研究

通过研究各培养阶段补料速度对温敏启动子控制的rIL-2工程菌E.coliK802(pLY-4)培养密度和rIL-2表达的影响,发现在各培养阶段控制不同的补料速度有利于提高菌密度和rIL-2表达,缩短培养周期。确定了rIL-2工程菌高密度培养方案,三批重复实验,平均菌密度为58OD_(600);菌干重18.0g/L,rIL-2表达水平为42.4％。     

白细胞介素-6在工程菌分批培养中的高效表达及其纯化

在确定了培养基及pH值的基础上,进一步观察了升温诱导过程中有机酸的产生及其对工程菌E.coli DH5α(pHV-hIL-6)生长和rIL-6表达的影响。当有机酸浓度低于70mmol/L以下时,菌密度达到干重2～3.5g/L之间收菌,rIL-6的表达水平为25％～32％;当有机酸浓度达到70mmol/L以上时,工程菌的生长不受影响,而rIL-6的表达明显受抑制。产生的有机酸以乙酸为主。收集菌体后,经过破菌,分离提纯的包涵体,其rIL-6的纯度可达到70％。用GuHCl缓冲液溶解包涵体,样品稀释后经过Q Sepharose F F柱纯化,可得到纯度达95％以上的rIL-6。采用依赖IL-6的小鼠杂交瘤细胞系7TD1及MTT比色法测定生物活性,rIL-6的比活性为2×10~8U/mg。     

基因工程菌培养过程中乙酸的积累及其对工程菌生长和产物表达的影响

在rIL-2工程菌K802(pLY-4)高密度培养中发现培养液中有大量代谢副产物乙酸积累,乙酸的存在对工程菌的生长和产物的表达均有明显的抑制作用,这种抑制作用是制约工程菌高密度培养的重要因素,为了减小这种抑制作用,研究了培养基pH与乙酸抑制作用的关系,发现适当提高培养基pH值,能有效地减小乙酸的抑制作用;高密度培养时,提高培养基的pH后,虽然仍有大量乙酸积累,但产物的表达水平和菌密度都有一定的提高     

乙酸积累对基因工程菌培养的影响及与培养基pH的关系

在rIL-2工程菌K_(802)(pLY—4)高密度培养中,发现培养液中有大量代谢副产物乙酸积累,乙酸的存在对工程菌的生长和产物的表达均有明显的抑制作用,这种抑制作用是制约工程菌高密度培养的重要因素。为了减小这种抑制作用,初步研究了培养基pH与乙酸抑制作用的关系,发现适当提高培养基pH值,能减小乙酸的抑制作用;高密度培养时,提高培养基的pH后,虽然仍有大量乙酸积累,但产物的表达水平和菌密度都有提高。     

大鼠白介素10真核表达质粒在大鼠体内外肝细胞中的表达及影响

目的探讨体外重组的大鼠白介素10（rIL-10）真核表达质粒能否在大鼠体内外肝细胞中表达及表达产物对肝细胞的影响。方法通过受体介导的脂介体转染法及尾静脉大容量注射法将rIL-10真核表达质粒分别转入大鼠BRL细胞及体内大鼠肝细胞中,采用RT—PCR法、ELISA法和免疫组织化学法检测体内外肝细胞rIL-10的表达情况,MTT法及流式细胞术检测rIL-10真核表达质粒转染对BRL细胞增殖与凋亡的影响。结果转染rIL-10真核表达质粒的BRL细胞及大鼠肝组织高表达rlL-10基因,BRL细胞培养上清与大鼠血清中rIL-10浓度分别为（12．78±O．94）ng／ml,（61．68±3．60）．g／ml。MTT法及流式细胞术显示rIL-10的表达对肝细胞有-定的保护作用。结论rIL-10真核表达质粒可在大鼠体内外肝细胞中表达并对肝细胞有-定的保护作用。     

半乳糖配体介导大鼠白介素10基因在大鼠肝脏内的靶向表达

目的 探讨大鼠白介素10（rIL-10）基因是否可通过半乳糖配体介导的脂质体转染法在大鼠肝脏内靶向表达。方法将已构建好的rIL-10基因真核表达质粒与半乳糖配体转染试剂按jetPEI．Gal／DNA（N／P=10）比例混合,通过尾静脉注射转移至大鼠体内。RT—PCR法和ELISA法检测rIL-10基因转移至体内0h、24h、7d和16d后大鼠肝、肾、脾和肺组织及血清中rIL-10的表达情况。结果rIL-10基因转移前大鼠肝、肾、脾和肺组织末扩增出明显rIL-10mRNA表达,转移7d后rIL-10表达主要分布在肝组织。肝组织中rIL-10mRNA表达在基因转移24h和7d时显著升高。血清中的rIL-10浓度在转移后24h和7d浓度分别为（107．92±12．26）pg／ml和（33．2±13．15）pg／ml。结论rIL-10基因通过半乳糖配体介导的脂质体转染法可有效的转移至大鼠体内,并可在肝脏靶向表达一周左右时间。     

大肠杆菌表达的重组白细胞介素-2的初步纯化

本文对大肠杆菌表达产生的重组白细胞介素-2进行了纯化研究。通过比较两种方法制备的rIL-2包含体的纯度,发现用4mol/L脲溶解可溶性细菌蛋白后可使rIL-2包含体纯度达70％;在高浓度变性剂条件下进行凝胶过滤,解决了rIL-2易聚合的问题;结合透析,利用空气氧化形成高活性氧化型rIL-2;经SephadexG-100凝胶过滤,DEAE离子交换等步骤纯化,得到了均一性rIL-2,纯度达98％,比活达4.3×10~6u/mg蛋白,得率为30.8％。     

乙酸对重组大肠杆菌生长及外源基因表达的影响

在重组基因工程菌ＤＨ５α（ＰＧ－ＦＧＦ）的高密度培养过程中,发现培养液中有大量代谢副产物－乙酸的产生和积累,乙酸的存在抑制了工程菌的生长及外源基因的表达。研究了乙酸在Ｍｇ培养基中对工程菌ＤＨ５α（ＰＧ－ＦＧＦ）生长及外源基因表达的影响。结果表明,乙酸的存在不仅导致重组菌生长速率的降低及延迟期的增长,而且对外源基因产物的表达具有强烈的抑制作用,这为该工程菌的高密度培养及外源基因产物的高表达打下了基础。     

乙酸对重组大肠杆菌生长及个源基因表达的影响

在重组基因工程菌ＤＨ５α（ＰＧ－ＦＧＦ）的高密度培养过程中,发现培养液中有大量代谢副产物－乙酸的产生和积累,乙酸的存在抑制了工程菌的生长及外源的表达。研究民乙酸在Ｍ９培养基中对工程菌ＤＨ５α（ＰＧ－ＦＧＦ）及生长外源基因表达的影响。结果表明,乙酸的存在不仅导致重组菌生长速率的降低及延迟期的增长,而且对外源基因产物的表达具有强烈的抑制作用,这为该工程菌的高密度培养及外源基因产物的高表达打下了基础。     

Characteristics of the low molecular antigens of pertussis bacteria]

The authors elaborated a method of obtaining pertussis soluble antigenic complex by dialysis through the cellophane membrane against the physiological saline at a temperature of 4 degrees C. An antigen which was active in the passive hemagglutination and neutralization of antibodies tests was revealed in the dialyzate. The amount of this antigen in the dialyzate increased gradually up to the 7th day and then became stabilized. The serological activity of the antigen after evaportation increased 4-16 times. The results of the antibody neutralization test pointed to the presence in the dialysate of substances common to those contained in the 1a and 1Da fractions isolated from the pertussis bacteria with the aid of ammounium sulfate.     

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.     

Estimation of nitric oxide level in vivo by microdialysis with water-soluble iron-N-methyl-d-dithiocarbamate complexes as NO traps: A novel approach to nitric oxide spin trapping in animal tissues

It was found that microdialysis, i.e., passage of aqueous solutions of iron-N-methyl-d-glucamine dithiocarbamate complexes through dialysis fibers implanted into heart, kidney and liver tissues of narcotized rats, was accompanied by effective binding of the complexes to nitric oxide from interstitial fluid. The walls of dialysis fibers used in this study were permeable for compounds with molecular weight not exceeding 5 kDa. The dialyzate samples collected every 20 min and containing diamagnetic nitrosyl Fe³⁺-MGD adducts were reduced to the paramagnetic state with sodium dithionite; their concentration was measured by the EPR method. The basic level of the adducts, which represented mononitrosyl iron complexes with MGD (MNIC–MGD), in the dialyzate samples of all tested organs were similar (1 μМ). Treatment of animals with the water-soluble nitroglycerine analog Isoket or a low-molecular dinitrosyl iron thiosulfate complex as a NO donor increased the concentration of MNIC–MGD with going out into a plateau. The novel approach allows determination of nitric oxide levels in tissue interstitial fluid from concentration of MNIC–MGD formed during microdialysis.     

Cytomodulation of interleukin-2 effect by l-2-oxothiazolidine-4-carboxylate on human malignant melanoma

Glutathione (GSH), the most prevalent intracellular non-protein thiol, plays an important role in the interleukin-2 (IL-2)-induced proliferative activity of normal and tumour cells expressing IL-2 receptor (IL-2R). In the present study, we investigate the effect of IL-2 on proliferation of the human melanoma A375 cell line, and the possible selective cytomodulation effect of this cytokine by l-2-oxothiazolidine-4-carboxylate (OTZ) on these melanoma cells and on human peripheral blood mononuclear cells (PBMCs). We found that recombinant IL-2 (rIL-2) significantly increased the proliferation rate of A375 melanoma cells, which was associated with an increase in GSH levels, the enhancement of IL-2Rα expression and the endogenous production of IL-2 in these tumour cells. In contrast, OTZ decreased GSH content and the proliferation rate of A375 cells, and abrogated the growth-promoting effects of rIL-2. Thus, compared to cells treated with rIL-2, pre-treatment with OTZ reduced IL-2Rα expression, and also decreased the consumption of rIL-2 and the endogenous secretion of IL-2 by these tumour cells. With regard to PBMCs, the combination of OTZ plus rIL-2 resulted in a more rapid and greater increase of IL-2Rα expression than rIL-2 alone, with the proliferation rate being similar in the first 24 h, but with a lower PBMC′ count found thereafter compared to rIL-2 treatment alone. These results suggest that OTZ plays a crucial role in obtaining a selective cytomodulation of rIL-2, enabling it to exert its growth-promoting effect on normal cells, but not on melanoma cells, thereby possibly improving biochemotherapy with rIL-2.     

Inhibition by fatty acids of the biodegradation of petroleum

The extent of biodegradation of petroleum by two marine bacterial isolates was found to increase when the organisms were grown in dialysis culture. This suggests that inhibitory products are formed during growth on petroleum. Fatty acids were produced by both organisms and were present in the dialyzate (dialyzable material). Fatty acids and crude oil were found to have a synergistic toxic effect. Short-chain acids were more toxic than longer-chain ones.     

Frequency analysis of CD4+CD8+ T cells cloned with IL-4

The coexpression of both CD4 and CD8 molecules on T cells occurs in the peripheral blood at a low frequency and can be generated transiently on CD4+ peripheral blood T cells by treatment with lectin which induces CD8 biosynthesis and cell surface expression. We have cloned T cells in a nonselective fashion from normal subjects in the presence of either IL-2, rIL-4 and IL-2, or rIL-4 and have examined the phenotypic expression of CD4 and CD8. The addition of excess rIL-4 increased the expression of CD8 on the surface of CD4+ T cell clones but did not increase CD4 expression on CD8+ T cell clones. There were three patterns of CD4 and CD8 expression observed: high density CD8 with no CD4 expression; high density CD4 with low CD8 expression; or high density CD4 with higher cell surface CD8 expression which was regulated by the presence of rIL-4. CD4+ T cell clones originally cultured in IL-2 and rIL-4 and subsequently grown in IL-2 alone exhibited decreased expression of the CD8 molecule. The increased expression of CD8 did not correlate with NK activity or lectin-dependent cytotoxicity in an antigen independent system. In addition, rIL-4 alone or in combination with IL-2 appeared to accelerate the growth curve of T cell clones as compared to IL-2 alone. These results show that IL-4 can upregulate CD8 expression on CD4+ T cell clones while not effecting CD4 expression on CD8+ T cell clones. As class I MHC is the ligand for the CD8 molecule, expression of CD8 induced by IL-4 on CD4+ T cells may allow for increased nonspecific cell to cell contact during the course of an inflammatory response.     

Both B151-T cell replacing factor 1 and IL-5 regulate Ig secretion and IL-2 receptor expression on a cloned B lymphoma line

In the present study, we have demonstrated that both B151-T cell-replacing factor 1 and rIL-5 are responsible for the activity to partially induce CL-3 cells into IgM-synthesizing cells and also to synergize with IL-2 to augment IL-2R expression on and IgM synthesis in CL-3 cells. These actions of rIL-5 on a homogeneous cloned line (BCL1-CL-3 cells) allow us to identify and characterize the two alternated B cell developmental pathways. One is an IL-2-independent, IL-5-driven differentiation pathway without preceding up-regulated IL-2R expression, and the other is an IL-5 plus IL-2-dependent augmented differentiation pathway with preceding up-regulated IL-2R expression. We have also demonstrated the functional difference of two distinct B cell growth-promoting factors, B cell-stimulating factor 1 (rIL-4) and rIL-5. CL-3 cells are equally stimulated to grow by rIL-4 and rIL-5, whereas only rIL-5 can render CL-3 cells responsive to rIL-2, indicating that these two lymphokines affect B cells in a strikingly different manner.