强激光作用下生物组织光致破裂效应的研究

基于激光与生物组织作用时产生的光致破裂效应,利用脉冲激光直接聚焦于孕囊内的胚胎内部,激光产生的光致破裂效应对胚胎心脏造成损伤,造成胚胎的快速死亡,初步证明利用激光进行减胎手术有望成为一种新方法。采用输出波长1 064 nm、脉宽8 ns、单脉冲能量60～96 mJ可调的脉冲激光作用于牛肌肉组织样品,并用光学相干层析成像(OCT)系统测量实验样品的损伤直径和损伤深度,建立了不同的激光参数与被作用样品的损伤直径及损伤深度之间的关系。得出结论:样品的损伤深度和损伤直径与激光的脉冲能量成正相关关系,而与激光的脉冲频率成负相关关系。这对于激光应用于减胎手术时参数的优化和精确控制起到一定的指导作用。     

血卟啉光动力作用对小鼠腹水肝癌细胞膜损伤的ESR波谱研究

已知血卟啉衍生物(以下简称HPD)能较长时间潴留在癌组织内,在可见光或低能量激光照射下受激,可以破坏生物分子和杀伤癌细胞。细胞膜在此过程中有肿胀和通透性发生改变等现象。然而,HPD对细胞膜类脂分子的物理性质有何影响未见报道。为此,我们用脂肪酸噁唑氮氧自由基自旋标记小鼠     

激光辐照微藻的显微图像观察及荧光定量分析

利用激光共聚焦扫描显微镜观察激光辐照前后微藻细胞叶绿体自体荧光图像,并对荧光变化进行定量分析。用Nd:YAP激光辐照扁藻、金藻及三角褐指藻。实验结果表明:Nd:YAP激光辐照后,藻细胞荧光光谱峰位不变,但荧光峰值发生较大变化,在激光促长剂量辐照下,几种微藻细胞的荧光强度均比对照组强。激光辐照微藻产生的生理刺激效应可以反映在细胞的荧光特性与强度变化上。激光共聚焦扫描显微镜可以作为微藻激光生物效应研究的一种有效方法。     

激光、血卟啉对小鼠S-180V肿瘤细胞膜通透性的作用及其影响因素

通过Na_2~(51)CrO_4在肿瘤细胞膜内外的分布比值的测定,观察了激光血卟啉衍生物(简称HPD)对小鼠S-180V肿瘤细胞膜通透性的作用及其影响因素:(1)通过紫外吸收光谱的测定对肿瘤细胞摄取HPD的动态过程作了观察。选择了实验所需的合适HPD浓度和作用时间,并观察到细胞悬液中血清蛋白能阻抑细胞对HPD的摄取。(2)在氦氖激光照射后即可观察到含有HPD的肿瘤细胞膜外~(51)Cr/膜内~(51)Cr的比值明显增加,而单照激光或单加HPD两组的~(51)Cr比值与正常对照组相比无明显变化。(3)上述的~(51)Cr比值变化随着照后保温时间的延长而逐渐加大。与此同时细胞形态也发生相应的变化,细胞死亡率也逐渐增加。说明除了原初的光敏反应外,还有继发的细胞损伤。(4)细胞悬液中血清蛋白的存在虽然对激光血卟啉对肿瘤细胞的杀伤作用有所减弱,但在这样条件下的光敏反应比较接近临床上治疗肿瘤的实际情况。     

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。     

电压敏感染料DiBAC4(3)用于检测胚胎细胞膜电位变化的研究

目的:探讨电压敏感染料DiBAC4(3)用于检测胚胎细胞膜电位变化的可行性。方法:分别取单细胞期胚胎、二细胞期胚胎、囊胚期胚胎,用M16孵育0.5 h后加入终浓度5μmol/L的DiBAC4(3)溶液,每隔10min在荧光显微镜下或激光共聚焦显微镜下观察胚胎细胞荧光强度变化,0.5 h后实验组加入终浓度100mmol/L的KCL溶液,对照组加入与KCL溶液等量的M16,每隔5 min观察胚胎细胞荧光强度变化。结果:每个时期的胚胎细胞都可以被DiBAC4(3)染色,没加KCL前半小时荧光强度随时间缓慢增强,加KCL后即刻荧光强度显著增强,且在15 min内荧光强度随时间增强。结论:电压敏感染料DiBAC4(3)可以用于胚胎细胞膜电位变化的检测。     

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。     

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。     

不同长度茎部shRNA表达载体构建与鉴定

本研究针对同一目的基因设计构建不同茎部长度的shRNA表达载体,并对其在细胞及胚胎水平的干扰效应做一比较。以绿色荧光蛋白基因为沉默效应的靶基因,设计茎部长度分别为21bp、27bp、29bp的干扰片段,退火后连入带有H6启动子的真核表达载体psiSTRIKE中(分别命名为EGFP-21siRNA、EGFP-27siRNA和EGFP-29siRNA),将构建成功的载体以脂质体法转染小鼠胚胎成纤维细胞,利用荧光定量PCR对其荧光表达进行精确定量。不同茎部长度的shRNA载体均使绿色荧光蛋白基因表达降低,茎部为29bp时比21bp、27bp表现出更明显的沉默效应。细胞水平沉默效应的初步验证,为筛选适合小鼠个体水平的最佳发夹结构奠定了基础。     

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.     

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.     

Photoradiation therapy of cutaneous angiosarcomas in mice

Cutaneous angiosarcoma is a relatively rare but devastating malignant vascular tumor. It has a high incidence of recurrence following conventional therapeutic modalities applied either singly or in combination. The increased vascularity of cutaneous angiosarcomas, facilitating selective uptake and retention of a photosensitizing agent, such as hematoporphyrin derivative (HPD), suggests that these tumors would respond well to photoradiation therapy. To study the feasibility of this treatment modality, transplantable hemangiosarcomas were implanted in B6C3F1 female mice. Within 2.5 to 3.5 hours after intraperitoneal administration of HPD, fluorescence was recorded in the tumor as compared with surrounding normal skin. When these photosensitized tumors were exposed to 70 J/cm2 of laser energy from an argon-pumped dye laser at 630 nm, the tumors showed marked necrosis within 24 hours. In another series, the tumors were initially photosensitized with HPD for 3 hours and then treated with laser energy ranging from 0 to 96 J/cm2. A dual labeling procedure demonstrated a dose-related decrease in DNA synthesis rate in tumors that were exposed to 0 to 30 J/cm2 at 24 hours after treatment. Furthermore, tumor tissue exposed to laser energy in excess of 30 J/cm2 showed no significant cellular DNA synthesis. These data, supported by histologic evidence of tissue destruction, suggest that photoradiation therapy has a great potential as a therapeutic modality for cutaneous angiosarcomas.     

The two-photon induced fluorescence of the tumor localizing photosensitizer hematoporphyrin derivative via 1064 nm photons from a 20 ns Q-switched Nd-YAG laser

We demonstrate the direct 1064 nm two-photon excitation of hematoporphyrin derivative (HPD), a complex mixture of photosensitizing porphyrins which is selectively retained in tumor tissue and used in cancer photochemotherapy. Although 1064 nm is outside of the one-photon HPD absorption spectrum, two-photon induced fluorescence from HPD was observed following excitation by the 20 ns output of an amplified, Q-switched Nd-YAG laser at peak power levels of 0.1 to 3 GW/cm2. Evidence for the successful two-photon excitation to vibrational levels of the S1 state consists of the observation of the known HPD fluorescence spectrum exhibiting peaks at approximately 615 and 675 nm, with the observed two-photon induced fluorescence intensity exhibiting a quadratic dependence on the excitation laser intensity as required for a direct two-photon process. More generally, these results suggest the possibility for the achievement of photosensitized oxidations utilizing photons of lower energy than that required for single photon excitation, offering the potential for both greater selectivity and a reduction in competing photochemical processes.     

The two-photon laser-induced fluorescence of the tumor-localizing photosensitizer hematoporphyrin derivative. Resonance-enhanced 750 nm two-photon excitation into the near-UV Soret band

The tumor-localizing photosensitizer hematoporphyrin derivative (HPD) is shown to undergo a simultaneous two-photon excitation into the near-ultraviolet Soret band system upon intense laser irradiation at 750 nm, a spectral region where there is no significant HPD one-photon absorbance in aqueous solution. Subsequent to this excitation, internal conversion and vibrational relaxation occur, resulting in the population of the vibrationless level of the first electronically excited singlet state. This state relaxes by two channels, the emission of fluorescence in the spectral region 600-700 nm and intersystem crossing into the triplet manifold, followed by near-resonant electronic energy transfer with surrounding oxygen to result in the generation of highly reactive singlet molecular oxygen (1 delta g). Evidence for the two-photon excitation consists in the observation both of the HPD fluorescence spectrum in the region of 615 nm as a result of 750 nm excitation and the quadratic dependence of this fluorescence emission intensity upon the excitation laser intensity. Since, in general, the penetration depth of ultraviolet and visible light into tissue varies directly with wavelength (red penetrating more deeply than blue), these studies suggest the possibility that two-photon-induced localization of tumor-bound HPD might facilitate the detection of deeper lying tumors than allowed by the current one-photon photolocalization method.     

Tissue distribution,intracellular localization and proteolytic processing of rat 4-hydroxyphenylpyruvate dioxygenase

4-hydroxyphenylpyruvate dioxygenase (HPD) is an important enzyme involved in tyrosine catabolism. HPD was shown to be identical to a protein named the F-antigen, exploited by immunologists because of its unique immunological properties. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. Decreased expression of HPD is often observed in association with the severe type I tyrosinemia, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. In this study we present a comprehensive analysis of tissue specific expression and intracellular localization of HPD in the rat. By combined use of in situ hybridization and immunohistochemistry we confirm previously known sites of expression in liver and kidney. In addition, we show that HPD is abundantly expressed in neurons in the cortex, cerebellum and hippocampus. By using immunoelectron microscopy and confocal laser scanning microscopy, we provide evidence that HPD contrary to earlier assumptions specifically localizes to membranes of the endoplasmic reticulum and the Golgi apparatus. Detailed mass spectrometric analyses of HPD purified from rat liver revealed N-terminal and C-terminal processing of HPD, and expression of recombinant HPD suggested that C-terminal processing enhances the enzymatic activity.     

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.