激光、血卟啉对肿瘤细胞DNA单链断裂及其重接的影响

用简化的Kohn氏碱洗脱法,观察了光敏剂血卟啉衍生物(HPD)对小鼠S-180肿瘤细胞DNA单链断裂及其重接修复的影响。激光HPD能导致S-180细胞DNA单链断裂明显增加,而且这种断裂随着保温时间的延长,继续增多。在本实验条件下没有观察到HPD对X线的增敏作用,HPD不能增加X线所致的DNA单链断裂,也不能影响其重接。单链断裂重接动力学的实验进一步证明了这个论点。     

血卟啉衍生物的生物学作用与制备

一、血卟啉衍生物是一种诊治癌瘤的光敏物质早在本世纪初,人们在研究血卟啉衍生物(Hematoporphyrin derjrative,简写 HPD)的光动力学过程时就发现其对人和哺乳动物癌瘤的光敏治疗作用。近年来,随着激光技术的发展,利用血卟啉加激光诊治癌瘤,在世界各国得到了广泛的应用。我国学者也在进行大量研究,还组织过攻关组来协调这方面的工作。HPD 光敏诊治癌瘤具有安全、简便、诊断率高,治疗效果好,对正常组织损伤少等优点,是一种很有前途的诊治癌瘤方法。     

血卟啉衍生物(BHPD)光敏反应的原初过程Ⅰ.活性氧的产生

为了阐明血卟啉衍生物(HPD)的光敏作用机理,本文以我国自行研制、并已应用的第一代产品——北京血卟啉衍生物(简称BHPD)为对象,采用电子自旋共振(ESR)和自旋捕捉(Sping-Traping)等技术,从光生物物理角度,探讨了BHPD光敏反应的原初过程.结果表明,不仅观察到~1O_2的产生,而且还观测到O_2~(?)和·OH的产生.这提示,在BHPD对生物系统的光敏损伤过程中,不只是~1O_2,O_2~(?)和·OH等活性氧,很可能也起重要作用.     

血卟啉衍生物加光对人红血球膜蛋白构象的影响

大量资料表明,细胞质膜可能是光敏引起细胞死亡的临界靶结构之一。有的作者认为临界靶分子是膜上的蛋白质,有的则认为膜上的靶事件是膜脂的过氧化。由此可见,为了揭示光敏的靶分子机理,必需进一步探讨膜脂和膜蛋白在光敏损伤中的地位。血卟啉衍生物(HPD)除了能较多地潴留于癌细胞外,也能潴留在正常细胞膜中,它杀死正常细胞和癌细胞的靶分子机理是相似的。基于这一考虑,我们采用血影细胞为材料,观察了HPD对血影细胞的圆二色性的影响。     

血卟啉衍生物(BHPD)光敏反应的原初过程 Ⅱ.由产生活性氧转变为生成非氧自由基

为了阐明血卟啉衍生物(HPD)的光敏作用机制,本文仍以北京血卟啉衍生物(BHPD)为对象,继前文(Ⅰ)观测到其光敏反应原初过程产生活性氧(~1O_2,O~(?)_2和OH)之后,又利用ESR技术,在一定条件下,观测到由活性氧向生成非氧自由基的转变过程,并且通过几种方法,都证实了非氧自由基BHPD~-阴离子的生成.这表明,BHPD的光敏作用,不仅归因于活性氧,而且还包含着非氧自由基.     

NaN_3血清等对血卟啉衍生物光敏致溶血作用的影响

含有血卟啉衍生物(HPD)的细胞在红光照射后,其光动力学过程的主要产物是一些HPD的自由基和单态氧等。这些极为活泼的物质使膜或其它细胞器上的重要大分子因过氧化而损伤,并进而可引起癌细胞或正常细胞死亡。人血红细胞材料容易得到,其死亡指标(溶血)容易测量,是一个研究光敏作用的良好模型。已有人用红细胞研究原卟啉等光敏机理。本文用人红血球为材料,探讨HPD的光动力学过程,以及某些~1O_2的淬灭剂对这一过程的影响及血清等的保护效应。     

铝酞菁:人癌细胞的吸收及光敏杀伤的研究

本文比较了光敏剂铝酞菁(AISPC)及血卟啉衍生物(HPD)在人癌细胞的吸收及光敏杀伤中的差异.探讨了两光敏剂在细胞中的分布、摄入量,血清的影响、及细胞对两光敏剂的吸收动力学过程,结果显示,除了类似的特征外,细胞对AISPC的吸收速度慢于HPD.光敏杀伤的结果表明,在红光照射下,两光敏剂对细胞有相近的杀伤效率;在室内自然光下,AISPC对细胞的损伤明显低于HPD,显示出其较低的光毒付作用,具有适合临床应用的优点.     

HPD对人工膜的光敏作用

本文用ESB、荧光和冰冻断裂电子显微镜技术在分子水平上研究了血卟啉衍生物(HPD)作用于人工膜的光敏作用过程,损伤细胞膜的途径以及各种因素的影响.结果表明,HPD光敏作用与羟基·OH有关的动力学过程和靶分子密切相关.光照HPD的主要靶物质是不饱和分子,双键是其重要的进攻部位,由此导致脂质过氧化和膜的破坏.但这并不一定是引起膜损伤的唯一途径.HPD光敏作用也可破坏靶分子的空间排列或构象,从而导致膜结构的无序和损伤.牛血清白蛋白可以加强HPD的光敏作用.此外,HPD的光敏作用还受温度,光照时间、HPD和靶物质的浓度等因素的影响.     

血卟啉衍生物对红细胞膜的光辐射损伤

Kessel曾观察到血卟啉衍生物(HPD)对癌细胞膜的转运功能有深刻影响,我们的工作也证实了这一点,由此推测,细胞膜可能是HPD的初始作用部位。细胞膜的磷脂内含有大量的不饱和脂肪酸,很容易受到自由基的攻击,红细胞由于直接与分子氧接触对脂质过氧化作用很敏感。我们用小鼠红细胞和提纯的红细胞膜,观察两种HPD对它们的光辐射效应,以便探讨HPD杀伤癌细胞的作用机理及其引起的光溶血效应。     

血卟啉光敏化作用在细胞生物学中的研究进展

本文主要总结了近年来有关血卟啉对细胞的光敏化作用的研究进展。对HPD在细胞内的动态分布机理及卟啉-脂蛋白亲和机理进行了讨论;阐述了细胞生物膜的光反应中心学说及HPD对各种细胞器的光敏作用;探讨了HPD对细胞周期的光动力学作用,认为不同周期时相的细胞其光动力学作用的敏感性是不同的。     

HPD在胃癌细胞各时相中的转运分布和损伤部位的关系

本文探讨了HPD衍生物加红光对人胃低分化腺癌MGC 80-3细胞不同周期的生物学效应。我们观察到HPD的转运与分布决定于细胞周期。G_1期在30分至60分钟内HPD从膜转运至胞质;S、G_2期则直接进入胞质的不同部位;而M期在核部位弥散分布。同步化细胞经HPD加红光处理后,引起细胞大量光敏杀伤,S与G_1期较明显,而M期光敏性最小。我们还观察到:不同周期细胞HPD的分布和HPD的光敏损伤部位密切相关。核仁对HPD的选择性结合也很明显。     

Influence of hematoporphyrin derivative concentration, incubation time, temperature during incubation and laser dose fractionation on photosensitivity of normal hemopoietic progenitors or leukemic cells

Photodynamic therapy represents a new approach for the local control of cancers. It has recently been claimed that photodynamic therapy mediated by hematoporphyrin derivative (HPD) is selectively more efficient for killing leukemic cells than normal progenitors. To improve this effect, we studied the influence of hematoporphyrin dose, temperature during incubation and/or treatment, hematoporphyrin derivative incubation time, and fractionation of the argon laser light (488-514 nm) used for hematoporphyrin stimulation. Plating efficiency calculated after a 7-day period of growth on collagen gel medium showed a dose-dependent phototoxicity of HPD reaching 0.01% for normal hemopoietic progenitors and 0.001% for leukemic cells (dose = 12.5 micrograms/ml). The 10:1 ratio of normal hemopoietic progenitors to leukemic cells was also found to be the same or increased when temperature was 37 degrees C during incubation and 4 degrees C during laser irradiation. Similar results were also found when incubation time was varied from 75-120 min, or when laser irradiation dose was fractionated into 2 or 3 periods. The ratio of normal progenitors to leukemic cells reached 100:1 when 75 J/cm2 were fractionated into 3 periods after an incubation time of 120 min with 10 micrograms/ml HPD. Selectivity in photodynamic treatment seems to occur between normal hemopoietic progenitors and leukemic cells. The mechanism of this selectivity remains unclear, but experiments with the fractionated irradiation dose suggest that as in radiotherapy, better potentially lethal damage repair in normal cells could be a factor for selectivity in photodynamic therapy. Our results obtained with leukemic cells are fully in agreement with data in the literature concerning similar experimental models.     

Preparation of suspensions of pancreatic islet cells: a comparison of methods

A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method. The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods. Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.     

Thymidine phosphorylase and uridine phosphorylase of Lactobacillus casei

Abstract Comparison of the whole cell protein profiles of Staphylococcus epidermidis grown in pooled human peritoneal dialysate (HPD) with those of cells grown in nutrient broth (NB) revealed proteins of 27, 39, 45, 54 and 98 kDa which were absent or poorly expressed in NB-grown cells. IgG, but not transferrin, was detected bound to the surface of bacteria grown in HPD. Immunoblotting experiments revealed that IgG antibodies present in pooled HPD recognised staphylococcal protein antigens of 16, 27, 35, 39, 45, 54 and 98 kDa. The 16-, 35- and 39-kDa antigens which were associated with the cytoplasmic membrane were repressed following growth in NB or in HPD supplemented with excess iron.     

The effect of hematoporphyrin derivative and human erythrocyte ghost encapsulated hematoporphyrin derivative on a mouse myeloma cell line

The effect of hematoporphyrin derivative (HPD) in combination with HeNe laser radiation on a mouse myeloma cell line, P3X63Ag8U1, has been examined. The effects of varying doses of radiation and photosensitizer were assessed using both dye exclusion and clonogenic assay systems. Development of photosensitivity by those cells in the presence of hematoporphyrin (HP) and HPD has been examined. In addition, with a view towards assessing erythrocyte encapsulation as a vehicle for an immunotargeting system for HPD, the effect of HPD encapsulated in such a manner, on the mouse myeloma cell line has been examined. From the results obtained from these in vitro experiments the authors believe that this may be a convenient means of packaging HPD for antibody-mediated delivery to tumour cells.     

Experimental aspects of in vitro and in vivo photochemotherapy

The selectivity of in vitro photodynamic reactions and the in vivo effects induced by PRT, whether the irradiation is applied interstitially or externally, still remains unclear. In vitro studies were performed using leukemic cell lines and syngeneic normal hemopoietic progenitors. For these, cells incubated with hematoporphyrin derivative (HPD) and non-incubated cells were irradiated with an argon laser. Data were obtained as the count of cell colonies found after a 7-day incubation period on semi-solid collagen gel medium. In vivo studies employed the HT 29 tumor model grafted into nude mice. Both animals injected with HPD and non-infected controls were irradiated with a dye laser pumped by an argon laser (Coherent) using a 400 micron optic fiber located either at a distance of 65 mm from the skin or inserted into the tumor. The temperature increase occurring during PRT was measured using non-absorbing thermocouples. In vitro, after HPD treatment and argon irradiation leukemic cells showed a greater phototoxicity (greater than 2 log10) than did the normal cells (0.25 log10). In vivo, when the heat rise is very similar (less than 4 degrees C) in both the tissues irradiated externally and those irradiated interstitially after HPD injection, histological examination of these did not reveal any quantitative differences (90% of tumor mass). These results are discussed.     

A test for adequacy of chromium nutrition in humans--relation to type 2 diabetes mellitus

When Na(2)51CrO(4) is added to blood the 51CrO(4)(2-) ions enter the erythrocytes readily, and nearly exclusively, and are reduced to 51Cr(III) ions. We have observed that a fraction of these ions becomes bound to the cell membrane in a concentration which seemingly depends on that of the dietary derived intracellular Cr(III) ions. Thus, when constant amounts of 51CrO(4)(2-) ions enter constant amounts of erythrocytes, the resulting 51Cr(III) ions become bound to the cell membrane in a concentration that varies inversely as the initial, intracellular concentration of Cr(III) ions which, in turn, depends directly on the adequacy of chromium nutrition. Therefore, we have determined an arbitrary set of conditions under which the concentration of 51Cr(III) ions bound to the erythrocyte membrane becomes an indicator of the adequacy of chromium nutrition. The application of this test to 25 Type 2 diabetes mellitus subjects and 35 normal controls, both randomly selected, indicates that the concentrations of membrane bound 51Cr(III) ions in the two groups were not significantly different. Consequently, it is concluded that the level of chromium nutrition which is normally adequate in humans has only a minor role, if any, in the genesis of Type 2 diabetes mellitus.     

Preferential uptake of cytotoxic porphyrins from hematoporphyrin derivative in murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells

Photodynamically induced loss of clonogenicity of murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells was determined with two different assays. It appeared that the loss of clonogenicity was much higher when 20 cells/cm2 were incubated with hematoporphyrin derivative (HPD) and illuminated, than when confluent cell layers were incubated with the same amount of HPD and illuminated prior to plating out. This dependency of cell killing on the experimental protocol was also observed when protoporphyrin (90-95% pure) was used as photosensitizer, but not when the cells were photodynamically treated with rose bengal or exposed to mitomycin C. Further, when cell layers were incubated with the residual solution that remained after the previous incubation of a confluent cell layer with HPD, illumination of these layers appeared to be almost non-toxic, although the overall porphyrin concentration in the residual solution was only slightly lower than in HPD. These results indicate that the porphyrins, responsible for loss of clonogenicity, are present in relatively small amounts in HPD and unpurified protoporphyrin and are preferentially taken up by the cells. Although 2-aminoisobutyric acid transport and DNA synthesis are among the most photosensitive targets with HPD, photodynamic treatment of L929 cells with the residual solution did not result in inhibition of the transport system and DNA synthesis. In contrast, the K+ content of the cells still decreased considerably, when utilizing the porphyrins, remaining in the residual solution as sensitizer. This indicates that under the present experimental conditions the disturbance of the membrane barrier function does not contribute to loss of clonogenicity of these cells and, moreover, that the photodynamically induced K+ leakage is caused by a component of HPD other than inhibition of 2-aminoisobutyric acid transport and DNA synthesis.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.