低渗介质对影泡载体的影响

本文提供了利用低渗透析技术将生物大分子物质牛血清白蛋白、L-天门冬酰胺酶包封入人红细胞影泡制备载体的技术方法,并且比较了PBS、HEPES-EDTA两种缓冲系统制备的影泡载体的性能。发现以后一种缓冲系统制备的载体H-Gs无论在形态、包封率还是在稳定性等方面均比P-Gs有明显优点。为载体应用于实验研究提供了必要的参数。     

甘油包埋绿豆SOD的稳定性研究

用不同浓度的甘油包埋绿豆(Phczeolus rodiatus L.)超氧化物岐化酶(Superoxide dismutase,SOD),对不同条件下制备的甘油SOD的稳定性进行评估.结果表明:甘油包埋绿豆SOD的稳定性受甘油浓度影响明显,12.5%～25%为最适甘油浓度.在10℃、pH 6.8、搅拌速度1000 r min-1.条件下包埋的甘油SOD在55℃的平均半衰期为25.1 d,为非包埋SOD的5.1倍.甘油浓度为25%,温度分别为5℃、10℃、25℃、45℃条件下,pH 3.8中包埋的绿豆SOD耐酸性较好,pH 8.9下包埋的绿豆SOD的抗碱能力较强.在包埋过程中添加一定浓度的Zn2 、Cu2 、Fe2 有利于其热稳定性的提高.甘油包埋SOD对一些常见的化妆品添加剂也有一定的抗性.由此可见,甘油包埋技术可望作为一项有应用前景的SOD活性保持新技术,有利于极端温度和极端pH条件下的SOD的应用.     

海藻糖载入红细胞及其冷冻干燥的实验研究

冷冻干燥保存是长期保存人体红细胞的理想方案之一。冻干保护剂海藻糖渗入细胞内后,对细胞膜和细胞内物质有保护作用,其中的一个作用是增加细胞质的浓度,使冻干过程容易形成稳定的玻璃态。应用高渗法处理红细胞,通过考察胞内海藻糖含量、红细胞冻干后的存活率、腺苷三磷酸酶(ATPase)、超氧化物歧化酶(SOD)活力以及细胞形态变化,研究胞内海藻糖含量对红细胞冻干后活性的影响。结果显示:海藻糖对红细胞冻干具有明显的保护作用,随胞内海藻糖浓度升高,其保护性能逐渐增强;43.8mmol/L的胞内海藻糖浓度对红细胞保护最好,细胞存活率达到53.6%,形态保持良好,ATP和SOD活力均在正常的范围内。     

过表达miR-153非小细胞肺癌细胞稳转株的构建及鉴定

目的:构建过表达miR-153的非小细胞肺癌的稳转细胞株,为进一步探讨miR-153在肺癌发生发展中的作用奠定基础。方法:构建具有嘌呤霉素抗性的miR-153慢病毒载体;体外培养非小细胞肺癌细胞系SPC-A-1和A549,加入浓度梯度的嘌呤霉素溶液,筛选出最佳浓度;使用慢病毒转染细胞株,并在转染72 h后用荧光显微镜检测绿色荧光蛋白(EGFP)的表达,评价转染效率;体外实验使用PCR检测miR-153在稳转株的表达;稳转株移植裸鼠体内成瘤,取出瘤体后检测瘤体内EGFP和miR-153的表达。结果:完成过表达miR-153慢病毒载体及阴性对照载体的构建;确定嘌呤霉素最佳筛选浓度:在SPC-A-1细胞中的浓度为2.0 g/mL,A549细胞中的浓度为3.0 g/mL;目的基因miR-153被慢病毒成功导入SPC-A-1细胞和A549细胞中,荧光显微镜下能直接观察到EGFP。转染miR-153组和阴性对照组的PCR实验结果显示:在SPC-A-1细胞中,miR-153的表达量为92.9±20.6,明显高于阴性对照组(P=0.016);在A549细胞中,miR-153的表达量为2624.6±153.4,显著高于阴性对照组(P0.001)。稳转株能在裸鼠体内形成移植瘤,瘤体内明显有EGFP的表达;与阴性对照组的瘤体相比,移植瘤组miR-153的表达量为显著上调(P=0.048)。结论:成功构建过表达miR-153的非小细胞肺癌的稳转细胞株。     

蛋白激酶C活化对L-6TG大鼠肌母细胞缺血/再灌注损伤中细胞凋亡的影响

目的:蛋白激酶C(PKC)活化对L-6TG大鼠肌母细胞缺血/再灌注损伤过程中细胞凋亡的影响.方法:将培养的L-6TG大鼠肌母细胞随机分为3组:①正常对照组(C组);②缺血/再灌注组(I/R组);③PMA 缺血/再灌注组(PMA组).观测了细胞内SOD、XOD、Ca2 含量的变化;采用MTT法检测线粒体的功能;利用流式细胞仪和细胞DNA电泳结果检测细胞凋亡情况;采用免疫组织化学的方法检测caspase-3的蛋白表达情况,结合自动图像分析系统对其结果进行定量分析.结果:蛋白激酶C活化可显著降低L-6TG大鼠肌母细胞I/R 4 h后细胞内XOD、Ca2 含量及凋亡细胞百分率,增加细胞内SOD活性及线粒体呼吸功能,DNA电泳无梯状条带出现,caspase-3的表达明显下调.结论:蛋白激酶C活化可明显减轻L-6TG大鼠肌母细胞缺血再灌注损伤后的细胞凋亡的发生,其机制可能与减轻氧化损伤、调节细胞内钙稳态、减轻线粒体损伤、减少caspase-3表达有关.     

淋巴细胞单抗致敏的红细胞花环试验和荧光染色法检测人外周血液的淋巴细胞

本文应用T、B淋巴细胞和T细胞亚群单抗致敏的红细胞花环试验结合荧光染色法,检测了50例健康人外周血液内的淋巴细胞。人外周血液中与T_3单抗致敏红细胞形成花环的T_~+细胞平均百分率为61.29±11.686,与T_4单抗致敏红细胞形成花环的T_4~+细胞平均百分率为32.76±8.974,与T_8单抗致敏红细胞形成花环的T_8~+细胞平均百分率为21.66±7.443,B细胞以单抗致敏红细胞识别的B细胞平均百分率为19.55±10.018,但标准差过高,个体间淋巴细胞的百分率波动范围相当大。用同一方法和试剂连续检测同一个体的淋巴细胞和T细胞亚群的百分率亦很难得到重复性结果。我们对检测人外周血液中的淋巴细胞的方法判断人体的免疫状态,和作为各种疾病的诊断指标进行了讨论。     

中枢氧化应激抑制高血压大鼠压力感受性反射功能

目的:观察中枢氧化应激对压力反射功能的影响,探讨自发性高血压大鼠(SHR)压力反射敏感性降低的中枢机制。方法:24周龄雄性SHR和正常大鼠在乌拉坦和α-氯醛糖混合麻醉下,静脉注射苯肾上腺素(PE)和硝普钠(NP)诱发动脉压力感受性反射,以心率变化与血压变化的比值代表压力反射敏感性(BRS)。侧脑室给予超氧化物歧化酶(SOD)拟似剂tempol和SOD抑制剂DETC,检测给药前后BRS变化。结果:高血压大鼠BRS明显低于正常大鼠(P<0.01),侧脑室应用Tempol明显改善高血压大鼠BRS(P<0.05),但不影响正常大鼠BRS;而DETC则衰减两组大鼠BRS(P<0.05),对正常大鼠BRS抑制作用更明显。高血压大鼠下丘脑丙二醛(MDA)含量明显高于正常大鼠(P<0.01),但总抗氧化能力、总SOD、CuZn-SOD、过氧化物酶(CAT)等活性均明显低于正常大鼠(P<0.05)。结论:高血压大鼠压力反射功能减弱与中枢氧化应激有关,脑内抗氧化酶活性降低和抗氧化能力下降可能导致中枢氧化应激。     

EGFP—pBABE逆转录病毒载体的构建及鉴定

目的构建表达增强绿色荧光蛋白（enhancedgreenfluorescentprotein,EGFP）的EGFP—pBABE逆转录病毒载体并对其表达进行鉴定。方法从pEGFP—N3质粒上切下EGFP片段,连接到pBABE载体,构建EGFP—pBABE重组质粒;将该重组质粒转染到Fr67细胞后,荧光显微镜下观察EGFP的表达;收集逆转录病毒感染宫颈癌SiHa细胞后荧光显微镜下观察EGFP的表达。结果成功构建EGFP—pBABE重组质粒。该质粒转染PT67细胞24h后,荧光显微镜下可观察到EGFP的表达;用该重组质粒包装的逆转录病毒感染宫颈癌SiHa细胞36h后,在荧光显微镜下可观察到明显的EGFP表达。结论成功构建表达EGFP的EGFP—pBABE逆转录病毒载体,为进一步利用该载体制备逆转录病毒并观测逆转录病毒感染细胞的效率奠定了良好的实验基础。     

棉铃虫核型多角体病毒sod基因在大肠杆菌中的表达

用PCR方法从棉铃虫(Helicoverpa armigera)单粒包埋型核型多角体病毒(HaSNPV) C1株基因组中扩增sod基因编码区,克隆到pGEM—T—easy vector,测定了核苷酸序列。将基因编码区克隆到原核表达载体pETblue2,构建了含表达质粒pETblue2／HaSNPV SOD,转化大肠杆菌DE3 (BL21)进行IPTG诱导表达。SDS—PAGE分析表明SOD的表达量约为细胞总蛋白的37％。邻苯三酚法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物校正酶活力单位为694U／mg。     

In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure

Glutamate dehydrogenase (GDH) has been encapsulated into mouse erythrocytes by a hypotonic dialysis/isotonic resealing method. Although a low GDH entrapment yield was achieved (3.8%), this percentage appeared sufficient enough to metabolize high quantities of ammonia. Carrier cell recovery yield was 56%. Due to the decrease in cell volume and haemoglobin content, constant mean cell haemoglobin concentration (MCHC) values were obtained. The osmotic fragility curves (OFC) indicated that dialyzed/resealed-RBCs are more resistant to hypotonic haemolysis than native-RBCs. The successful in vitro ammonia degradation by GDH-RBCs was reflected in its total disappearance from the incubation medium at around 48 h. In contrast, initial ammonia levels were not affected during the incubation in the presence of native-RBCs and remained constant. Two different methods were used for the preparation of hyperammonaemic mice model. Since the intraperitoneal (i.p.) administration of ammonium acetate produced high ammonia levels that lasted only a few minutes, the i.p. administration of urease was chosen, given that it generated elevated ammonia levels for longer periods of time. Hyperammonaemic mice quickly removed high levels of circulating ammonia in the presence of GDH-RBCs, whereas in the presence of native-RBCs ammonia was slowly metabolized. These results suggest that loaded GDH-erythrocytes can be used as a potential carrier systems for the in vivo removal of high levels of ammonia from blood.     

Intraperitoneal administration of carrier erythrocytes in dogs: an improved method for delivery of L-asparaginase

Carrier erythrocytes were prepared to encapsulate L-asparaginase by a hypotonic dialysis process. Dogs received either intravenous or intraperitoneal injections of cells containing both L-asparaginase and the marker [3H]inulin. The route of administration had no effect on the circulating survival of carrier erythrocytes. For enzyme therapy, intraperitoneal injection of a large volume of cells is technically feasible. Cells circulate with a maximum of 30% of the cells reaching circulation 24 h after injection. Those cells reaching circulation have a 7-day half-life for the encapsulated enzyme L-asparaginase.     

Human red blood cells as bioreactors for the inactivation of harmful xenobiotics

Human red blood cells are able to inactivate lipophilic electrophiles by conjugation with reduced glutathione. This metabolic ability was found to be limited by the rate of permeation of the xenobiotic into erythrocytes and by the amount of available reduced glutathione. By a procedure of hypotonic dialysis, isotonic resealing and reannealing human red blood cells were overloaded with increasing amounts of reduced glutathione up to three- to fourfold the normal level without modification of their metabolic functions or of their energetic state. These overloaded erythrocytes were able to conjugate increasing amounts of xenobiotics and to export the resulting conjugates from the cells. These properties of glutathione overloaded erythrocytes are significant for the use of carrier erythrocytes in cases of acute intoxication by lipophilic electrophiles.     

Encapsulation of proteins into human erythrocytes: a kinetic investigation

Moderate osmotic shocks of human erythrocytes by hypotonic dialysis (0.06 mosmol/kg) induce cell swelling and formation of pores, without causing apparent lysis. Using 125I-labeled macromolecules of different molecular weight and net charge, we followed the kinetics and efficiency of their encapsulation into erythrocytes. After a 20-30 min period of cell dialysis, macromolecules of up to 50 kDa begin diffusing into the swollen cells by a process which can be described by a first-order two-compartment kinetics. Adsorption to the external cell surface was insignificant, while adsorption to the inner membrane surface was substantial (15-20%) only for positively charged proteins, at physiological pH. After resealing, pores of a 12-14 kDa cut-off might remain open allowing some release of entrapped material (20-30%), depending on the final cytocrit, while the remaining might be associated with inner membrane or cytosolic components. Although the method of hypotonic dialysis is known to affect minimally the biophysical and immunological properties of red blood cell membranes, the interaction of encapsulated material with cell constituents would need to be further assessed when considering red cells as macromolecular carriers.     

Optimized recirculation survival of mouse carrier erythrocytes.

Carrier mouse erythrocytes prepared by a hypotonic dialysis technique and reinjected into mice have a 24 hour survival of approximately 50%. Twenty-four hour survival can be improved substantially to 74% by removing the more fragile erythrocytes by a hypotonic wash treatment. The mean cell volume of the carriers prepared by this modification is significantly (p less than 0.01) different from cells prepared by the standard method with a isotonic wash treatment. Carriers prepared by the hypotonic treatment wash modification exhibit a different 50% hemolytic value (15% difference) from isotonically prepared carriers, and normal erythrocytes. Carrier-erythrocytes removed from mice 24 hour post-injection exhibit an osmotic profile that is independent of the treatment. Carriers were also prepared by another modification of the encapsulation procedure and held in a permeable state overnight before resealing and annealing. Carriers prepared in this manner showed a much lower 24 hour survival (13%).     

Entrapment of protein protease inhibitors in red blood cells.

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.     

Encapsulation by hypotonic dialysis in human erythrocytes: a diffusion or endocytosis process

Human erythrocytes subjected to controlled hypotonic dialysis are capable of encapsulating and retaining drugs. Under selected conditions encapsulation has been reported to occur by an endocytosis process. The mechanism by which encapsulation occurs under conditions which are conducive for endocytosis to occur was studied. An analysis of the percentage of cells with endocytic vacuoles was made for cells dialyzed to optimal and suboptimal osmotic pressures for encapsulation. No differences were found with approximately 20% of cells from all preparations containing vacuoles. Transmission electron micrographs of cells in different stages of carrier cell preparation reveal endocytic vacuoles both with and without hemoglobin. However, based on the percentage of exogenous substance encapsulated, encapsulation appears to occur primarily by diffusion and secondarily by endocytosis.     

Interaction of carboplatin with carrier human erythrocytes.

The antineoplastic drug Carboplatin (CBDCA) was encapsulated in human erythrocytes by means of transient hypotonic hemolysis, followed by isotonic resealing. Up to 5 mg/ml of packed cells could be entrapped, with about 70% cell recovery. In vitro incubation of the CBDCA-loaded erythrocytes in autologous plasma caused a very slow release of the drug from the cells (12% approximately in 3 h). The encapsulation conditions, performed at a low hematocrit, in order to obtain high amounts of the drug inside the carriers, impaired the metabolic properties of the loaded erythrocytes significantly. In particular, an almost complete disappearance of GSH was observed. Analysis of the intraerythrocytic metabolism of CBDCA showed that, in spite of its relatively high stability in aqueous solutions, in hemolysates and in the loaded erythrocytes a significant percentage of CBDCA is rapidly converted to other species that still retain an antiproliferative activity in vitro. This fast conversion could be extensively inhibited by previous conversion of oxyhemoglobin to methemoglobin or carbomonoxyhemoglobin, suggesting an important role of heme iron in this process. Encapsulation of CBDCA in selectively targeted human erythrocytes may represent a therapeutic strategy for increasing the drug concentration in specific organs, notably liver.     

Intraerythrocytic administration of a synthetic Plasmodium antigen elicits antibody response in mice, without carrier molecules or adjuvants

The synthetic peptide (NANP)₂₀, reproducing the tandem-repeated epitope of the circumsporozoite protein of Plasmodium (Laverania) falciparum, was entrapped into murine, autologous erythrocytes by a hypotonic dialysis method. Mice immunized intravenously with minute amounts of encapsulated peptide produced considerable antibody titres. This result indicates that intraerythrocytic antigen administration may have a potential as an immunization system for humans, since it dispenses with adjuvants and carrier molecules.     

Delivery to macrophages and toxic action of etoposide carried in mouse red blood cells

Erythrocytes could be used as physiological carriers of active compounds. Several substances can be loaded into erythrocytes by hypotonic dialysis methods. Furthermore, carrier erythrocyte membrane can be chemically modified in order to promote increased arrival of the loaded compound to macrophages. In this work, we have prepared erythrocytes loaded with etoposide. We found conditions to obtain high etoposide encapsulation yields with minor alteration of some cell parameters of these carrier erythrocytes. Etoposide loaded into erythrocytes is mainly localised in the cytoplasmic compartment. Membrane modification of etoposide-loaded erythrocytes with band 3 crosslinkers produces an increased incorporation of the drug into macrophages mainly by phagocytosis process. The toxic effect of etoposide conveyed in these carrier erythrocytes determined as DNA fragmentation in macrophages was higher than that shown by free etoposide added at the same concentration in the culture medium to macrophages. These results seem to indicate the usefulness of this model to deliver this anti-tumour compound to macrophages, which might be useful in therapy.