用于电穿孔效应实验的亚微秒脉冲电源

针对肿瘤细胞电穿孔效应的实验需求,用幅值可调的高压直流电源为电容充电,具有一定时间延时的两路TTL信号分别控制串联的两个IGBT模块的通与断状态,以便从电容放电曲线中截取适当脉宽电脉冲的方法,研制了一台输出脉冲电压0.5～3.5kV连续可调,脉宽300～800ns准连续可调,重频1～400Hz连续可调的亚微秒脉冲电源。实验测试结果表明,这种脉冲电源性能稳定可靠,可用于恶性肿瘤电穿孔实验。     

心肌细胞电脉冲作用下电场分布仿真结果与分析

目的 细胞电脉冲刺激仿真是研究心脏电脉冲消融的一种仿真方式，本文建立椭球形细胞电脉冲刺激仿真模型，模拟心肌细胞受到电脉冲刺激下的情况，研究电场入射方向和细胞长度对其电场分布和跨膜电位的影响。方法 通过COMSOL5.5软件进行仿真，以球形细胞模型为基础，在边长60μm的立方体空间中建立椭球形细胞模型。于垂直于Z轴的两面施加2.4 V电压，用以模拟心肌细胞在外加匀强电场作用下的电压分布情况。改变脉冲电场与细胞长轴的夹角，研究0°、30°、60°、90°的不同电场入射角度对心肌细胞电压分布和跨膜电位的影响。保持入射角为0，研究跨膜电位最大值与细胞长轴直径的关系。结果 对于椭球形的心肌细胞，电场的入射角从0°增大到90°时，跨膜电位从1.068 V减小至0.373 V，同时最大跨膜电位的位置也发生改变。入射角为0°时，跨膜电位最大值V与细胞长轴直径D的线性回归方程为V=81.191 6+38.607 9D,r²=0.998 1。结论 电场入射角越大，细胞跨膜电位越低；细胞长轴直径越长，跨膜电位最大值越大。该研究对后续心肌细胞电脉冲刺激实验及心脏电脉冲消融的临床试验具有参...     

电穿孔技术的研究及应用进展

脉冲电场的电穿孔效应在医学上的应用是一门多学科的生物医学工程学技术,已证实其主要的生物学效应是使靶细胞膜发生可逆及不可逆性电击穿。近年电穿孔效应所介导的电化学治疗应用广泛。对电脉冲的量—效关系、电场分布、膜穿标记物及生物电阻成像等基础研究及临床应用中的进展及尚存问题作一综述。     

电穿孔技术的研究及应用进展

脉冲电场的电穿孔效应在医学上的应用是一门多学科的生物医学工程学技术，已证实其主要的生物学效应是使靶细胞膜发生可逆及不可逆性电击穿。近年电穿孔效应所介导的电化学治疗应用广泛。对电脉冲的量—效关系、电场分布、膜穿标记物及生物电阻成像等基础研究及临床应用中的进展及尚存问题作一综述。     

不同中心频率下双极电脉冲的生物医学效应分析

采用多层球对称细胞模型,针对恶性Tonsillar B细胞和Jurkat T淋巴细胞,利用电路分析方法,计算了不同中心频率的一阶高斯脉冲在细胞各部分的电压分布;给出了中心频率与细胞膜跨膜电压间的定量关系和细胞核膜电穿孔的最佳频率;研究了细胞膜电穿孔和纳秒脉冲致DNA降解的原因。本文工作为双极瞬态脉冲应用于恶性肿瘤治疗提供了参考。     

利用电磁脉冲提高抗癌药物治疗肿瘤细胞疗效的实验研究

本文采用一种新的方法--电穿孔结合化学药物进行肿瘤治疗的电化学疗法.电穿孔由于能使细胞膜出现瞬时微孔,从而能大大提高癌细胞对药物的吸收率、促进了药物等大分子进入细胞.这里利用电穿孔现象结合抗癌药物治疗昆明小鼠身上的S-180肉瘤,从实验数据可看出,这种方法取得了很好的效果.这种癌症治疗新技术具有易于控制、便于操作等优点,特别适用浅表肿瘤的治疗,为临床治疗肿瘤提供了一条新的途径.     

利用电磁脉冲提高抗癌药物治疗肿瘤细胞疗效的实验研究

采用一种新的方法——电穿孔结合化学药物进行肿瘤治疗的电化学疗法。电穿孔由于能使细胞膜出现瞬时微孔，从而能大大提高癌细胞对药物的吸收率、促进了药物等大分子进入细胞。这里利用电穿孔现象结合抗癌药物治疗昆明小鼠身上的S-180肉瘤，从实验数据可看出，这种方法取得了很好的效果。这种癌症治疗新技术具有易于控制、便于操作等优点，特别适用浅表肿瘤的治疗，为临床治疗肿瘤提供了一条新的途径。     

利用电磁脉冲提高抗癌药物治疗肿瘤细胞疗效的实验研究

采用一种新的方法——电穿孔结合化学药物进行肿瘤治疗的电化学疗法。电穿孔由于能使细胞膜出现瞬时微孔，从而能大大提高癌细胞对药物的吸收率、促进了药物等大分子进入细胞。这里利用电穿孔现象结合抗癌药物治疗昆明小鼠身上的S-180肉瘤，从实验数据可看出，这种方法取得了很好的效果。这种癌症治疗新技术具有易于控制、便于操作等优点，特别适用浅表肿瘤的治疗，为临床治疗肿瘤提供了一条新的途径。     

小鼠大脑皮质神经元电穿孔转染条件的优化

目的:以原代培养的小鼠大脑皮质神经元为研究工具,优化电穿孔转染条件。方法:通过设置不同的电压和脉冲时间组合等电转染条件,将表达绿色荧光蛋白(GFP)的shNC质粒导入小鼠大脑皮质神经元,24 h后观察并比较神经元的转染效率,确定最佳电转染条件。结果:200 v电压,25 ms脉冲时间或250 v电压,15 ms脉冲时间的转染条件下,小鼠大脑皮质神经元可获得较好的转染效率,分别为(31.15±1.89)%和(29.10±1.93)%。结论:电穿孔转染是一种高效的基因转染方法,通过条件的优化,可以提高小鼠大脑皮质神经元的电转染效率。     

瞬态电磁脉冲加速肿瘤消亡的实验研究

当一定强度的瞬态电磁脉冲作用于细胞时 ,在细胞膜上将形成瞬时微孔 ,可促进药物等大分子进入细胞内。本研究利用电穿孔现象结合抗癌药物治疗在昆明小鼠上接种和生长的 S- 180肉瘤 ,发现这种方法可以明显地抑制肿瘤细胞的生长。这种癌症治疗技术具有操作简单、容易控制、毒副作用小等优点 ,特别适用于浅表肿瘤的治疗。它为临床治疗肿瘤提供了一种新途径     

Induction of secretory and tumoricidal activities in peritoneal macrophages activated by an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco)

The immunomodulatory and anti-tumoral effects of an acidic heteropolysaccharide containing mainly galactose and arabinose (ARAGAL), isolated from the gum of the leguminous tree Anadenanthera colubrina (Angico branco) native to Brazil, were studied. It has been demonstrated that activation of mice peritoneal macrophages both in vivo and in vitro, increases phagocytic ability and anion superoxide production. In order to obtain further insights on the biological effects of ARAGAL, the capacity of eliciting peritoneal macrophages and tumor necrosis factor-alpha (TNF-alpha) production, and anti-tumoral effect against Sarcoma 180 (S-180), are now evaluated. Cell eliciting activity was observed in ARAGAL-treated animals in a dose dependent manner. Treatment of animals with 50, 100 or 200 mg/kg of ARAGAL increased peritoneal exudate cell (PEC) numbers by approximately 18, approximately 44 and approximately 88%, respectively. ARAGAL also increased 26-fold TNF-alpha production by peritoneal macrophages. Macrophages, treated in vitro for 18 h with ARAGAL, were able to kill Sarcoma 180 cells, as observed by their structures inside the macrophage cytoplasm. ARAGAL (100 mg/kg) showed anti-tumoral activity against S-180 in ascites or solid tumors, the tumoral inhibition being 63 and 38%, respectively. The results suggest a possible role as a BRM for ARAGAL.     

电化学治疗昆明小鼠肉瘤及其机理分析

研究了电化学治疗昆明小鼠肉瘤的疗效,并分析其机理。在体外将S-180细胞用不同参数的电场处理,研究适合电化学治疗的电场条件。通过复制肉瘤模型,将肉瘤小鼠随机分成4组:对照组、电疗组、化疗组、电化疗组。研究了不同处理组的肿瘤抑瘤率、治愈率以及小鼠的自由基代谢水平。结果电化疗组的抑瘤率、治愈率都显著高于化疗组和电疗组(P<0.05),电化疗组小鼠受到氧自由基的攻击显著降低,免疫力提高。分析机理发现,电化学治疗肿瘤的机理可能至少涉及细胞膜通透性提高、细胞的耐药性降低、机体的免疫力提高三个方面。     

Evidence of dual function of macrophage migration inhibitory factor relevant to tumor progression and regression

Macrophage migration inhibitory factor (MIF) is known to play an important role in broad-spectrum inflammation and immune responses. To evaluate the role of MIF in tumor growth, we established transgenic (Tg) mice (ICR strain) driven by cytomegalovirus (CMV) enhancer and beta-actin promoter. We inoculated Tg mice in the back with murine sarcoma cell line S-180 cells. The tumor growth rate was more enhanced in Tg mice than in littermate non-Tg mice up to day 9 after tumor inoculation. Surprisingly, most tumors embedded on the back of Tg mice regressed at day 10 after inoculation and eventually disappeared. Tumor volumes of non-Tg mice incessantly increased until death. We reinoculated the Tg mice with S-180 cells, which had been recovered from the first challenge, and found that the tumor cells were completely rejected in all cases. To identify the effector cells that eradicated the tumor cells, we prepared spleen cells from tumor-bearing Tg mice and carried out cell lysis assay. The magnitude of cytolytic activity of spleen cells obtained from Tg mice was significantly higher against S-180 cells, as well as natural killer cell-sensitive YAC-1 cells, than was the activity of cells from non-Tg mice. Furthermore, we observed that CTL activity of Tg mice against S-180 cells was significantly decreased by the deletion of CD8+ T cells or NK cells. On the other hand, the deletion of CD4+ cells minimally affected the cytolytic activity. Taken together, these results suggest that MIF has the potential to promote tumor growth and angiogenesis in the early phase and, by contrast, this protein could activate CD8+ cytotoxic T cells and NK cells, leading to tumor regression.     

Stimulation of tumor cell growthin vitro by a monoclonal antibody to a tumor specific protein (TSP-180) present on the cell surface of 3LL cells

Proliferation capacity and MHC class I antigen expression of two Lewis lung carcinoma (3LL) metastatic variants (C87, BC215) grown under defined experimental conditions (serum-free defined medium or 10 per cent serum) have been studied following exposure to Mo Ab 135-13C which recognizes on these cells a tumor surface protein of 180000 daltons (TSP-180).The results of this study indicate that the high metastatic clone (C87) binds higher amounts of MoAb to TSP-180 and Db antigens than does the low metastatic one (BC215), while both clones express very low amounts of Kb antigens. 3LL clones grown in 10 per cent serum or adapted in serum-free, defined medium show the same metastatic phenotype and MHC class I antigen expression, but when grown in defined medium exhibit increased capacity to bind MoAb 135-13C. However, the relative binding rate of 3LL clones grown in 10 per cent serum or in defined medium is unchanged: the high metastatic clone always showing higher capacity to bind MoAb to TSP-180. Furthermore, comparison of EGF binding sites on the cell surface of 3LL clones, grown in different culture conditions, demonstrates that the C87 clone binds higher amounts of labelled EGF and that this amount increases in serum-free defined medium, exactly as reported for TSP-180. In addition, competition experiments demonstrated that MoAb 135-13C does not compete for EGF binding sites on 3LL cell surface. Studies on cell proliferation following exposure to MoAb 135-13C, revealed that the low metastatic clone (BC215) is more actively stimulated than the high metastatic one. Moreover, similar data were obtained after exposure of 3LL clones to physiological amounts of different growth factors (i.e. EGF, MSA, insulin). Analysis of MHC class I antigen expression following exposure to MoAb 13S-13C indicated that MoAb 135-13C induces on the cell surface of the C87 clone a transient low modulation of Db antigens.These results suggest that 3LL cells endowed with lower metastatic potential are more dependent on the microenvironmental conditions than the high metastasizing ones, and that MoAb 135-13C binding to 3LL cell surface stimulates proliferation as reported for several known growth factors.     

Increased translocation of bacteria from the gastrointestinal tracts of tumor-bearing mice.

Aerobic gram-negative bacilli and other indigenous gastrointestinal (GI) bacteria are important opportunistic pathogens in immunosuppressed cancer patients. These same bacteria frequently translocate from the GI tracts of mice immunosuppressed by single injections of certain anticancer drugs or by T-lymphocyte impairments. Since similar cellular and humoral immune deficiencies may be present in the tumor-bearing host, we sought to determine if progressive growth of a tumor alone would be sufficient to enhance the translocation of indigenous bacteria from the murine GI tract. Pathogen-free DBA/2 mice were injected intraperitoneally with 10(6) viable sarcoma 180 (S-180) cells or 0.5 ml of sterile buffer. Mesenteric lymph nodes, livers, spleens, and kidneys were tested for the presence of translocated aerobic GI bacteria on various days after tumor injection. Immunity was assessed by measuring footpad delayed-type hypersensitivity and serum hemagglutinins to sheep erythrocytes. Overall, translocated aerobic GI bacteria infected 33 of 92 S-180-bearing mice (36%) and only 9 of 99 control mice (9%) (P less than 10(-6)). Cumulatively, 50 of 460 sites (10.9%) in S-180-bearing mice were infected with translocated GI bacteria as opposed to only 9 of 485 sites (1.9%) in control animals (P less than 10(-7)). GI bacteria often translocated to infect more than one site in tumor-bearing mice, but not in controls. Aerobic gram-negative bacilli translocated 11 times in tumor-bearing mice, but only once in controls, even though the mean cecal population levels of these bacteria were relatively low (range, 4.33 to 5.28 log10 bacteria per g). The population levels of cecal aerobic bacteria were similar in S-180 and control mice throughout the period of observation. S-180 mice had significantly suppressed (P less than 0.04) delayed-type hypersensitivity and serum hemagglutinin responses when sensitized 4 or 8 days after S-180 injection. S-180 growth was associated with a neutrophilic leukocytosis and a slight drop in platelet counts; no bleeding was detected. Thus, the translocation of gram-negative bacilli and other indigenous aerobic bacteria from the GI tract to the mesenteric lymph nodes and other organs was increased in immunosuppressed S-180-bearing mice, and this increase was not caused by bacterial overgrowth in the intestines or by neutropenia.     

Correlation of dendritic cell infiltration with active crypt inflammation in ulcerative colitis

The aim of this study was to evaluate the histological localization and phenotypic characteristics of infiltrating dendritic cells (DCs) and to examine the relationship between the degree of DC infiltration and the severity of inflammation in the colonic mucosa in ulcerative colitis (UC). Also explored was the expression of macrophage inflammatory protein-3alpha (MIP-3alpha), and its receptor CC-chemokine receptor 6 (CCR6), to evaluate the significance of immature DCs in the crypt inflammation evident in UC. There was a significant positive correlation between the number of infiltrating DCs and the degree of crypt inflammation, mononuclear cell infiltration, crypt atrophy, and comprehensive active inflammation. No significant correlation between the number of S-100 protein(+) cells and the severity of crypt atrophy was found. S-100 protein(+), MIP-3alpha(+), and CCR6(+) cells were frequently localized in or around the crypt with inflammation. MIP-3alpha(+) neutrophils and S-100 protein(+) CCR6(+) cells with dendritic morphology were detected in or around the crypt inflammation. Both S-100 protein(+) DCs and CCR6(+) cells were frequently clustered in the surface mucosa beneath the surface epithelium when the crypt was not inflamed. CD1a(+) Langerhans-cell-type DCs were not found in any of the tissues examined. These data indicate that DCs participate not only in chronic inflammation but also in active crypt inflammation.     

ADP-ribosyltransferase mutations in the catalytic S-1 subunit of pertussis toxin.

The ADP-ribosyltransferase activity of pertussis toxin resides within the S-1 subunit of the toxin. Deletion mapping of a recombinant S-1 subunit produced in Escherichia coli showed that amino acids 2 through 180 are required for ADP-ribosylation of Gi protein. Mutants of the S-1 subunit which lacked either amino acids 2 through 22 or amino acids 153 through 180 failed to express enzyme activity, implicating a functional or structural role for these residues in catalysis. The catalytic carboxy-terminal S-1 deletion, C-180, was found to be more soluble than the recombinant S-1 subunit, making it a useful construct for future structure-function studies on enzyme catalysis. Four independent single-amino-acid substitutions which decreased ADP-ribosyltransferase activity were constructed in the recombinant S-1 subunit. Substitution of Asp-11 by Ser, Arg-13 by Leu, or Trp-26 by Ile decreased enzyme activity to below detectable levels (less than 1.0% of that of the recombinant S-1 subunit). The Glu-139-to-Ser substitution reduced ADP-ribosyltransferase activity to 15% of that of the recombinant S-1 subunit. Both the oxidized and reduced forms of the recombinant S-1 subunit and recombinant S-1 subunits containing single-amino-acid substitutions were degraded through identical immunoreactive tryptic peptides, suggesting that the conformations of the mutants are similar to that of the recombinant S-1 subunit. Identification of noncatalytic forms of the S-1 subunit of pertussis toxin which have conserved protein structure is an initial step in the generation of a recombinant noncatalytic form of pertussis toxin which may be tested as a candidate for an acellular vaccine against Bordetella pertussis.     

Surface properties of Chlamydia psittaci.

The S-1 clone of dengue type 2 virus was used for the preparation of a live-attenuated vaccine after passage in DBS-FRhL-2 cell culture. The vaccine virus had a relatively higher replicative capacity at superoptimal temperatures than its precursor virus, S-1, passaged in primary green monkey kidney cells (S-1 PGMK). There was also a tendency for the S-1 vaccine virus to exhibit leakiness at increased temperatures. Another in vitro marker, replication in monkey peripheral blood leukocytes, indicated less host restriction for the S-1 vaccine in comparative assays with S-1 PGMK virus. Mouse virulence appeared to remain stable on passage in DBS-FRhL-2 cells, whereas monkey immunogenicity decreased. Cautious trials of the dengue type 2 S-1 vaccine in humans are indicated.     

IMMUNOHISTOCHEMICAL STUDY OF MALIGNANT MELANOMA

Fifty-two malignant melanoma cases were divided morphologically into round, spindle and mixed-cell types and were studied immunohistochemically on the localization and stainig intensity of S-100 protein and neuron specific enolase (NSE). Most of malignant melanomas were positively stained for S-100 protein and NSE. There were no correlation between the localization of S-100 protein and three cell types. However S-100 protein and degree of melanin production seemed to have an inverse relationship. On the other hand, for NSE, there were some differences on immunostaining intensity among the three cell types. Especially, deeply invasive melanomas showed strong reactivities for NSE and it may clinicopathologically indicate their poorer prognosis.     

Immunohistochemical study of malignant melanoma

Fifty-two malignant melanoma cases were divided morphologically into round, spindle and mixed-cell types and were studied immunohistochemically on the localization and staining intensity of S-100 protein and neuron specific enolase (NSE). Most of malignant melanomas were positively stained for S-100 protein and NSE. There were no correlation between the localization of S-100 protein and three cell types. However S-100 protein and degree of melanin production seemed to have an inverse relationship. On the other hand, for NSE, there were some differences on immunostaining intensity among the three cell types. Especially, deeply invasive melanomas showed strong reactivities for NSE and it may clinicopathologically indicate their poorer prognosis.