Reproductive death of cancer cells induced by femtosecond laser pulses

Purpose: High intensity femtosecond (1 fs = 10^?15 s) laser pulses may, via multi-photon processes, cause reproductive cell death at wavelengths that otherwise are harmless. We study the efficacy of inducing reproductive death of cancer cells by ultraviolet (UV), visible (VIS) and near infrared (IR) femtosecond laser pulses.

Materials and methods: Human squamous carcinoma cervical cancer cells are irradiated by femtosecond laser pulses at 800 nanometers (nm), 400 nm, 266 nm and 200 nm. The reproductive death is assessed by colony forming assay. The contribution from multi-photon processes is evaluated by comparing the cell reproduction subsequent to irradiation by collimated (low intensity) and focused (high intensity), pulsed laser beams with identical fluences.

Results: Suitable femtosecond pulses are capable of arresting cell reproduction at all the tested wavelengths. Irradiation at 266 nm is far more efficient than the other wavelengths, both in terms of the fluence and the absorbed dose needed to induce reproductive cell death. The collimated 800 nm beam is unable to induce reproductive cell death even at a fluence of 230 Joule/square centimeters (J/cm²). However, focused 800 nm pulses with much higher intensities, but lower fluences efficiently arrest cell reproduction, thus highlighting the dramatic effect of multi-photon processes. At the intensities used in the present work focusing the 400 nm beam improves its efficacy by an order of magnitude, whereas focusing the 266 nm beam does not improve its efficacy.

Conclusion: Femtosecond pulses at 200, 266, 400 and 800 nm induce reproductive cell death if the intensity is sufficiently high. Multi-photon processes can improve the efficacy substantially and even result in reproductive cell death at wavelengths, where single-photon processes are harmless.     

树突状细胞诱导抗胃癌效应的实验研究

目的探讨树突状细胞（DC）抗胃癌效应的作用。方法分离正常人外周血单个核细胞（PBMC），加入GM－CSF和IL－4培养，于第5d加入TNF—α继续培养，诱导分离DC。将淋巴细胞与DC混合培养4d后，加入胃癌细胞MKN45、MKN28及SGC7901抗原培养至10d，诱导产生肿瘤特异性的CTL。ELISA检测IL－12和IFN－γ的水平，MTF法检测DC诱导的CTL对MKN45、MKN28、SGC7901和A549体外杀伤效应，流式细胞术检测CTL对肿瘤细胞周期、凋亡的影响。结果培养10d后，IL－12、IFN－γ的分泌量明显提高，DC对同种不同分化类型的胃癌细胞株MKN45、MNK28和SGC7901均有强烈的杀伤效应，杀伤活性显著高于A549（P〈0．01）。与A549比较，MKN45、MKN28和SGC7901的细胞周期显著抑制、凋亡率增加（P〈0．01）。结论DC体外可诱导出高效而特异的抗胃癌效应，显著抑制胃癌细胞分裂增殖，促进其凋亡。     

Analysis of death pattern in cancer cells by using different kinds of LET radiation

We investigated the death pattern of cancer cells by using different kinds of linear energy transfer (LET) radiation. We used two human squamous cell carcinoma cell lines with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with the mp53 gene to SAS cells having functional p53 (wtp53). As the control, a neovector was transfected to the SAS cells (SAS/neo cells). Both types of cells were exposed to X-rays (1.5 KeV/micron) or accelerated C-beams (30-100 KeV/micron). The frequency of cell death (apoptosis and necrosis) was measured by acridine orange/ethidium bromide(AO/EB) double staining for fluorescence microscopy. We found that (1) low-LET radiation induced apoptosis only in SAS/neo cells; (2) high-LET radiation at an iso-survival dose induced apoptosis not but necrosis in SAS/neo cells at a higher frequency; (3) high-LET radiation induced p53-independent apoptosis even in SAS/mp53 cells. Our findings suggest that high-LET radiotherapy is expected to (1) have validity in its application to patients carrying mutated p53 cancer cells and (2) reduce injury to adjacent normal tissue for high-frequency-induced apoptosis without inflammatory response. We propose that elucidation of p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.     

程序性细胞死亡4基因增强胰腺癌细胞放射敏感性的研究

目的 探讨程序性细胞死亡4（PDCD4）对胰腺癌细胞放疗敏感性的影响及机制。方法 收集胰腺癌组织及对应的癌旁组织,采用RT-PCR和Western blot检测PDCD4表达水平。以人胰腺癌细胞Sw1990为研究对象,细胞转染PDCD4过表达载体（pIRES2-PDCD4组）、空载体（pIRES2组）,同时以只加入转染试剂的细胞为未转染组,RT-PCR和Western blot检测PDCD4表达水平。细胞经放射处理后,流式细胞术检测细胞凋亡情况,细胞克隆实验检测细胞放射敏感性,Western blot检测细胞中β-连环蛋白、Wnt信号通路下游靶基因c-myc、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）表达水平。结果 胰腺癌组织中PDCD4 mRNA和蛋白表达水平均明显低于癌旁组织（t=4.869、9.208,P<0.05）。pIRES2-PDCD4组细胞中PDCD4 mRNA和蛋白表达水平均明显高于未转染组（t=9.074、18.927,P<0.05）。照射处理后,pIRES2-PDCD4组细胞凋亡率及细胞中Cleaved Caspase-3水平均明显高于未转染组（t=3.670、4.086,P<0.05）,而细胞中β-连环蛋白、c-myc表达水平均明显低于未转染组（t=9.242、17.644,P<0.05）。pIRES2-PDCD4组放射敏感性高于未转染组,增敏比为1.843。结论 PDCD4能够增加胰腺癌细胞放射敏感性,促进胰腺癌细胞凋亡,作用机制可能与Wnt信号通路有关。     

白藜芦醇诱导人宫颈癌Hela细胞凋亡及其机制

目的探讨白藜芦醇诱导人宫颈癌Hela细胞的凋亡作用及其分子机制。方法应用不同浓度的白藜芦醇作用于人宫颈癌Hela细胞,采用四甲基偶氮唑蓝（MTT）法检测药物对细胞的增殖抑制率;流式细胞术（FCM）检测细胞凋亡、细胞周期分布：免疫组化法检测Survivin及Caspase-3的表达。结果白藜芦醇能明显抑制Hela细胞的增殖,并呈剂量和时间依赖性。经白藜芦醇处理Hela细胞后,FCM分析发现各实验组S期细胞比例增高,G2／M期细胞比例减少,凋亡率明显高于对照组,并呈剂量依赖性（P〈0．01）;各实验组Heal细胞Survivin的表达均低于对照组,而Caspase-3的表达均高于对照组（P〈0．01）。结论白藜芦醇能明显抑制人宫颈癌Hela细胞的增殖,诱导其凋亡,其机制可能与抑制Survivin的表达、上调Caspase-3的表达有关。     

Cell death triggered by alpha-emitting 213Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death

Purpose Radioimmunotherapy with -particle-emitting nuclides, such as ²¹³Bi, is a promising concept for the elimination of small tumour nodules or single disseminated tumour cells. The aim of this study was to investigate cellular damage and the mode of cell death triggered by ²¹³Bi-immunoconjugates.Methods Human gastric cancer cells (HSC45-M2) expressing d9-E-cadherin were incubated with different levels of activity of ²¹³Bi-d9MAb targeting d9-E-cadherin and ²¹³Bi-d8MAb, which does not bind to d9-E-cadherin. Micronucleated (M) cells, abnormal (A) cells and apoptotic (A) [(MAA)] cells were scored microscopically in the MAA assay following fluorescent staining of nuclei and cytoplasm. Chromosomal aberrations were analysed microscopically following Giemsa staining. The effect of z-VAD-fmk, known to inhibit apoptosis, on the prevention of cell death was investigated following treatment of HSC45-M2 cells with sorbitol as well as ²¹³Bi-d9MAb. Activation of caspase 3 after incubation of HSC45-M2 cells with both sorbitol and ²¹³Bi-d9MAb was analysed via Western blotting.Results Following incubation of HSC45-M2 human gastric cancer cells expressing d9-E-cadherin with ²¹³Bi-d9MAb the number of cells killed increased proportional to the applied activity concentration. Microscopically visible effects of -irradiation of HSC45-M2 cells were formation of micronuclei and severe chromosomal aberrations. Preferential induction of these lesions with specific ²¹³Bi-d9MAb compared with unspecific ²¹³Bi-d8MAb (not targeting d9-E-cadherin) was not observed if the number of floating, i.e. unbound ²¹³Bi-immunoconjugates per cell exceeded 2×10⁴, most likely due to intense crossfire. In contrast to sorbitol-induced cell death, cell death triggered by ²¹³Bi-immunoconjugates was independent of caspase 3 activation and could not be inhibited by z-VAD-fmk, known to suppress the apoptotic pathway.Conclusion ²¹³Bi-immunoconjugates seem to induce a mode of cell death different from apoptosis in HSC45-M2 cells.     

雌激素受体诱导乳腺癌细胞辐射抗性的机制研究

目的 探讨雌激素受体(ESR1)对乳腺癌细胞辐射抗性的影响及分子机制。方法 在雌激素受体阴性乳腺癌细胞中转染ESR1过表达质粒(过表达对照vector组、过表达ESR1 OE组),在雌激素受体阳性乳腺癌细胞中通过慢病毒转染方法构建稳定敲低ESR1(敲低对照shNC组、敲低shESR1组)的细胞模型,采用实时荧光定量聚合酶链式反应(qPCR)和免疫印迹方法检测自噬相关基因mRNA(ATG5)和蛋白表达水平(LC3B-I、LC3B-II、P62、FIP200、ATG5、ATG7、ATG12、Beclin1、ULK1);在8 Gy X射线的电离辐射处理下,免疫印迹法检测自噬通量;采用流式细胞术检测细胞死亡;采用集落形成实验检测乳腺癌细胞辐射敏感性(未照射sham组、照射IR组、电离辐射联合铁死亡抑制剂处理Fer-1+IR组、电离辐射联合凋亡抑制剂处理Z-VAD+IR组、电离辐射联合自噬抑制剂处理CQ+IR组);用台盼蓝染色检测自噬基因敲低和过表达细胞模型以及雌激素受体抑制剂他莫昔芬(TAM)与电离辐射联合治疗下乳腺癌细胞的死亡率(他莫昔芬未处理TAM-组、他莫昔芬处理TAM+组)。结果 在8 Gy X射线照射后24 h处理条件下,雌激素受体阳性乳腺癌ZR75细胞ESR1的敲低促进细胞死亡(t=3.49、3.13, P < 0.05),而雌激素受体阴性乳腺癌MDA-MB-231细胞ESR1的过表达抑制细胞死亡(t=4.16、7.48, P < 0.05);与单纯照射相比,自噬抑制剂氯喹的处理后增加了敲低ESR1的ZR75细胞集落形成数量(t=8.49, P < 0.05), 抑制自噬可以减少沉默ESR1所致的ZR75细胞死亡。在电离辐射处理下,MDA-MB-231细胞过表达ESR1促进了细胞保护性自噬;ZR75细胞敲低ESR1后细胞保护性自噬降低。在ZR75细胞中敲低ATG5发现自噬降低,细胞死亡增加(t=4.19、6.39, P < 0.05),而在ZR75中过表达ATG5可以逆转因敲低ESR1导致的细胞死亡增加(t=1.70、4.65, P < 0.05)。化疗药物他莫昔芬处理雌激素受体阳性乳腺癌细胞ZR75后发现自噬基因ATG5、ATG12的表达下降,自噬抑制,细胞死亡增加(t=18.70, P < 0.05),电离辐射处理促进了这一进程(t=16.82, P < 0.05)。结论 雌激素受体ESR1通过增加ATG5自噬相关蛋白,促进雌激素受体阳性乳腺癌细胞的保护性自噬,从而导致雌激素受体阳性乳腺癌细胞的辐射抵抗,化疗药物他莫昔芬联合电离辐射治疗增加了雌激素受体阳性乳腺癌细胞的辐射敏感性。     

外源性p53对不同LET辐射诱导人黑色素瘤细胞系A375死亡途径的影响

目的 观察外源性P53蛋白对不同传能线密度(LET)射线辐照诱导肿瘤细胞凋亡和坏死的影响,并探讨其可能的机制。方法 人黑色素瘤细胞系A375(wild-type p53)经携带人野生型p53基因的腺病毒载体(AdCMV-p53)感染后分别给予X射线和碳离子束照射,采用克隆形成法测定细胞辐射敏感性,Hoechst 33258和吖啶橙-溴化乙锭双染荧光显微镜下观察细胞凋亡和坏死。结果1高LET辐照时,A375细胞和转导人野生型p53基因的A375细胞(A375/p53)的辐射敏感性没有明显差异;2虽然辐射诱导细胞凋亡比例的增加依赖于LET升高,但是无论高LET或低LET,外源性P53蛋白均可有效诱导细胞凋亡。3高LET辐照时,A375细胞的坏死细胞明显高于A375/p53细胞。结论 尽管高LET辐射对A375和A375/p53细胞的存活无明显影响,但是对细胞凋亡的诱导却部分依赖于P53蛋白的功能,P53蛋白可能在调节细胞死亡类型中发挥重要作用。这对临床应用高LET辐射联合p53基因治疗恶性黑色素瘤有一定参考意义。     

SKP2表达对受照食管癌细胞诱导辐射旁效应的影响

目的：探究SKP2表达水平对受照食管癌细胞所诱导辐射旁效应的影响。方法：Western blot法筛选出SKP2高表达和低表达的食管癌细胞系,微核检测法探讨SKP2对受照食管癌细胞所诱导辐射旁效应的影响。建立SKP2高表达和低表达的转染细胞系,Western blot法验证转染效率,微核检测法进一步验证SKP2对受照食管癌细胞所诱导辐射旁效应的影响。通过γ-H2AX聚集点检测法探究SKP2影响受照食管癌细胞所诱导辐射旁效应的作用机制。结果：微核检测结果表明,SKP2高表达的受照细胞诱导的辐射旁效应显著低于SKP2低表达的受照细胞诱导的辐射旁效应（t=8.06,P<0.01）。SKP2转染细胞系的微核检测结果进一步验证了SKP2的表达对受照食管癌细胞诱导的辐射旁效应的抑制作用,SKP2表达升高,辐射旁效应减弱（t=11.12、10.16,P<0.01）;SKP2表达降低,辐射旁效应增强（t=8.39、8.83,P<0.01）。γ-H2AX聚集点检测结果表明,受照细胞SKP2表达升高可提高旁效应细胞的DNA损伤修复能力（t=6.85、7.10,P<0.01）。反之,会抑制旁效应细胞的DNA损伤修复能力（t=7.66、8.47,P<0.01）。结论：SKP2的表达抑制受照食管癌细胞诱导的辐射旁效应,并且这一机制至少部分是通过SKP2对DNA损伤修复能力的调节来实现的。     

脱氧胞苷激酶Ser-74位点在辐射诱导的乳腺癌细胞死亡中的作用

目的 研究脱氧胞苷激酶(dCK) S74位点在辐射诱导乳腺癌细胞MCF-7死亡中的作用.方法 采用脂质体转染方法在MCF-7中分别构建dCK-Vector(空载体)、dCK-WT(野生型)、dCK-S74A(非磷酸化型)及dCK-S74E(过磷酸化型)4种细胞模型,均分别予以0,2,4,6和8 GyX射线照射.实时定量PCR(QPCR)及Western blot验证细胞模型,CCK-8法和集落形成实验检测细胞存活率,单丹磺酰尸胺(MDC)检测自噬发生率,流式细胞术检测细胞凋亡率.结果 成功构建4种细胞模型;CCK-8结果显示,与0 Gy组相比,MCF-7和dCK-Vector经8 Gy照射后存活率下降(t=14.469和9.357,P＜0.05),dCK-WT、dCK-S74E和dCK-S74A没有改变(P＞0.05);与dCK-Vector比较,dCK-WT和dCK-S74E照射后的总死亡率降低(x2=3.857 ～3.971和3.857～3.859,P＜0.05),dCK-S74A则没有变化(P＞0.05);与各自0 Gy组比较,MCF7、dCK-Vector和dCK-S74A照射后凋亡发生率升高(t=-4.531、-3.688和-7.076,P＜0.05),而dCK-WT和dCK-S74E使照射诱导的凋亡率逆转了66％和68％;dCK-WT和dCK-S74E照射后自噬率分别增加22％和26％(t=-9.051和-8.411,P＜0.01),dCK-S74A、MCF-7和dCK-Vector照射后的自噬率没有明显变化(P＞0.05).结论 dCK-WT和dCK-S74E使电离辐射诱导的凋亡率增加发生逆转,同时使自噬率增加,总死亡率降低,提示dCK在Ser-74的磷酸化与乳腺癌细胞MCF-7的辐射敏感性有关.     

顺铂诱导人宫颈癌Hela细胞凋亡后氧自由基水平的变化

 目的 探讨顺铂诱导人宫颈癌Hela细胞凋亡后氧自由基水平的变化特点.方法 应用MTT比色法、流式细胞术、电子显微镜观察化疗前后Hela细胞活性、细胞周期,以及凋亡形态的改变;用化学比色法检测细胞凋亡前后细胞内丙二醛(MDA)、超氧化歧化酶(SOD)和谷胱甘肽(GSH)的含量.结果 顺铂处理Hela细胞48 h的IC50值为3 mg/L,当DDP浓度在3 mg/L以下时,对Hela细胞无明显毒性作用,超过此浓度时,其毒性呈剂量效应关系(P<0.001);与亲本细胞相比较,Hela+DDP细胞的G2期细胞数增多,为(90.80±8.37)%,而G1、S期的细胞数明显减少,分别为(30.84±8.02,5.30±0.60)%.扫描及透射电镜下可观察到典型的凋亡早期细胞形态学改变;当DDP作用48 h后,细胞内SOD的活性比对照组显著降低(P<0.05),细胞内的脂质过氧化产物MDA的含量与对照组相比显著升高(P<0.01),抗氧化剂GSH的含量较对照组明显降低(P<0.05).结论 经DDP可诱导Hela细胞的凋亡,其机制可能与细胞的氧化损伤有关.     

Biological characteristics of gene expression features in pancreatic cancer cells induced by proton and X-ray irradiation

Abstract

Background: Radiation therapy is an important alternative treatment for advanced cancer. The aim of the current study was to disclose distinct alterations of the biological characteristics of gene expression features in pancreatic cancer cells, MIAPaCa-2, following proton and X-ray irradiation.

Materials and methods: Using cDNA microarray, we examined the gene expression alterations of MIAPaCa-2 cells following proton or X-ray irradiation. We also isolated the surviving MIAPaCa-2 cells after irradiation and analyzed their gene expression profiles.

Results: Although the cytocidal effects of both types of irradiation were similar at sufficient doses in vitro and in vivo, the affected gene expression profile alterations of MIAPaCa-2 cells irradiated with protons were distinct from those irradiated with X-ray. Interestingly, clustering analysis of gene expression of the surviving MIAPaCa-2 cells was also completely discernible between the two types of irradiation. However, a similar cytocidal effect was still observed in the proton- and X-ray-irradiated surviving cells after re-irradiation, commonly showing biological effects related to apoptosis and cell cycle processes.

Conclusions: Proton irradiation treatment for pancreatic cancer provides the distinct biological effect of steady gene expression alterations compared to X-ray irradiation; however, surviving cells from both types of irradiation were still susceptible to the cytocidal effects induced by proton re-irradiation treatment.     

铜蒸气激光照射下光敏剂血卟啉衍生物和血卟啉单甲醚杀伤特性研究

目的 测定血卟啉衍生物(HpD)和血卟啉单甲醚(HMME)在铜蒸气激光照射下的半数杀伤浓度(IC50)和半数杀伤光剂量(IED50),了解HpD与HMME杀伤特性的差异.方法 将小鼠肺血管内皮细胞接种于96孔培养板,然后加入用DMEM(低糖)培养基配置的光敏剂中,4h后应用铜蒸气激光(510.6nm和578.2nm)单色光分别照射,24h后用MTT法测定细胞存活率.结果 在510.6nm和578.2nm波长下,饱和光剂量条件下HMME的IC50分别是HpD的1.31和1.24倍,在饱和光敏剂剂量条件下,HpD的IED50分别是HMME的1.18和1.17倍.结论 HpD在510.6nm和578.2nm波长的杀伤效应均较HMME强并和光剂量、波长密切相关.     

IntraLase FS60飞秒激光与Moria M2微型角膜刀制作LASIK角膜瓣的效果比较

目的 探讨应用IntraLase FS60飞秒激光与Moria M2微型角膜刀制作准分子激光原位角膜磨镶术（laser in situ keratomileusis,L ASIK）角膜瓣的特点.方法 选取IntraLase FS60组（激光组）患者100例200眼及Moria M2微型角膜刀组（角膜刀组）99例198眼,应用RTVueOCT傅立叶光学相干断层扫描仪分别测量LASIK术后1周每个角膜上0°和90°两条子午线所在截面上特定7个点的角膜瓣厚度.两组角膜瓣预计值均为110 μm.结果 术后1周激光组制作的角膜瓣形态均一、规整,形状近似规则的平面,两条子午线间角膜瓣厚度无明显差异（f=1.582,P=0.114）.角膜刀制作的角膜瓣呈周边厚,中央薄的新月形,两条子午线间角膜瓣厚度无明显差异（t=1.010,P=0.313）.激光组与角膜刀组相比,仅中央点两者厚度无明显差别,其余各点两者间均存在明显差异（P＜0.05）.激光组角膜瓣实际厚度与预计值的差值（7.15 ±4.89） μm明显小于角膜刀组（18.97±1 1.56）μm,差异有统计学意义（P＜0.01）.结论 飞秒激光组制作的LASIK角膜瓣比微型角膜刀制作的LASIK角膜瓣更精确;重现性更好;形态更均一、更规整.