聚焦超声结合血卟啉对S180肿瘤细胞的杀伤作用

初步研究聚焦超声激活血卟啉对S180肿瘤细胞的杀伤作用,并探讨其作用的生物学机制。通过台盼蓝拒染法检测频率为2.2MHz的聚焦超声激活血卟啉作用于S180肿瘤细胞的存活率,筛选出最佳的超声处理参数,采用自由基清除剂检测超声激活血卟啉产生的起主要作用的活性氧成分,通过丙二醛含量测定法检测细胞内脂质过氧化含量变化。单纯血卟啉组表现出无明显的细胞毒作用;同等条件下,超声结合血卟啉组对细胞的损伤显著高于单纯超声组,且这种协同杀伤作用可以被组氨酸和甘露醇所抑制,而不被SOD抑制;同时,超声激活血卟啉后细胞内的脂质过氧化含量显著增加。超声结合血卟啉对S180肿瘤细胞有协同杀伤作用,可能由于声动力学处理过程中产生的活性氧自由基作用于细胞膜,增加膜脂质过氧化水平,最终导致细胞死亡。     

MTT检测超声激活血卟啉对SW-480细胞的杀伤作用

本实验采用1.1、1.6、2.3MHz频率聚焦超声结合血卟啉的方式对体外培养的人结肠癌细胞SW-480进行照射,其介质分别为NaCl溶液、PRMI1640、完全培养基,并采用MTT法测试SDT对肿瘤细胞的杀伤效果。结果表明:SW-480细胞在声照强度为1.5W/cm2 ,频率2.3 MHz条件下,介质为NaCl溶液、PRMI1640和完全培养基中都存在超声激活血卟啉增强杀伤肿瘤细胞的效果,且在PRMI1640和完全培养基中这种效果和处理后细胞孵育的时间有一定的相关性。     

复频聚焦超声激活血卟啉杀伤离体肿瘤细胞的实验研究

在优选的超声辐照参数下 ,利用复频聚焦超声激活血卟啉 (Hp)杀伤肿瘤细胞K5 6 2 ,SW - 4 80并用MTT法检测分析了杀伤细胞的效率其初步结果为 :单频超声杀伤结果时低频优于高频 ;复频和单频比较 ,复频优于单频。而且杀伤效率不是单频的代数和 ,而是二者和的 2 - 3倍 ;由超声辐照后的孵育时间可以看出 :孵育 16h超声 Hp的杀伤效率高于 4h。     

聚焦超声结合原卟啉Ⅸ对S180肿瘤细胞杀伤作用的研究

采用频率为2.2MHz及不同强度的聚焦超声激活原卟啉对小鼠腹水型S180肿瘤细胞的杀伤作用进行研究。台盼蓝拒染法检测了不同处理后细胞的相对存活率,通过扫描电镜和透射电镜观察细胞超微结构的变化。实验结果表明:单纯原卟啉对S180肿瘤细胞几乎没有影响,单纯超声和超声结合原卟啉对细胞均有一定的损伤,肿瘤细胞受损程度随着声照强度的增加而加剧,并且在一定范围内随着原卟啉剂量浓度的加大,细胞受损也逐渐增强。当原卟啉浓度为120μM时,超声激活原卟啉对腹水型S180肿瘤细胞的协同杀伤效应表现最为明显。     

聚焦超声结合原卟啉Ⅸ对S180肿瘤细胞杀伤作用的研究

采用频率为2.2 MHz及不同强度的聚焦超声激活原卟啉Ⅸ对小鼠腹水型S180肿瘤细胞的杀伤作用进行研究.台盼蓝拒染法检测了不同处理后细胞的相对存活率,通过扫描电镜和透射电镜观察细胞超微结构的变化.实验结果表明:单纯原卟啉Ⅸ对S180肿瘤细胞几乎没有影响,单纯超声和超声结合原卟啉Ⅸ对细胞均有一定的损伤,肿瘤细胞受损程度随着声照强度的增加而加剧,并且在一定范围内随着原卟啉剂量浓度的加大,细胞受损也逐渐增强.当原卟啉Ⅸ浓度为120 μM时,超声激活原卟啉Ⅸ对腹水型S180肿瘤细胞的协同杀伤效应表现最为明显.     

聚焦超声激活原卟啉Ⅸ对S180肿瘤细胞的杀伤作用

目的初步研究超声激活原卟啉IX(PPIX)对S180肿瘤细胞的杀伤作用,并探讨其作用的生物学机制。方法采用频率为2·2MHz,强度为3W/cm2的聚焦超声,结合120μmol/L的PPIX作用于S180肿瘤细胞,通过台盼蓝拒染法检测细胞的存活率;扫描电镜观察细胞膜表面超微结构变化;活性氧试剂盒检测细胞悬液中活性氧的生成量;酶化学方法检测细胞内几种抗氧化物酶活性的变化。结果同等条件下,超声结合PPⅨ对细胞的损伤显著高于单纯超声组,而单纯PPⅨ表现出无明显的细胞毒作用;酶化学方法显示,超声激活PPⅨ后细胞内的丙二醛(MDA)含量显著增加,而细胞内的抗氧化物酶类均有不同水平的下降,并且与处理后细胞悬液中活性氧的生成量相关。结论超声激活原卟啉Ⅸ产生的活性氧自由基作用于细胞膜,增加膜脂质过氧化水平,降低细胞内的抗氧化物酶的含量,可能是其损伤S180肿瘤细胞的主要因素之一。     

聚焦超声激活原卟啉IX对S180 肿瘤细胞的杀伤作用

目的 初步研究超声激活原卟啉IX(PPIX)对S180肿瘤细胞的杀伤作用,并探讨其作用的生物学机制.方法 采用频率为2.2 MHz,强度为3 W/cm2的聚焦超声,结合120 μmol/L的PPIX作用于S180肿瘤细胞,通过台盼蓝拒染法检测细胞的存活率;扫描电镜观察细胞膜表面超微结构变化;活性氧试剂盒检测细胞悬液中活性氧的生成量;酶化学方法检测细胞内几种抗氧化物酶活性的变化.结果 同等条件下,超声结合PPIX对细胞的损伤显著高于单纯超声组,而单纯PPIX表现出无明显的细胞毒作用;酶化学方法显示,超声激活PPIX后细胞内的丙二醛(MDA)含量显著增加,而细胞内的抗氧化物酶类均有不同水平的下降,并且与处理后细胞悬液中活性氧的生成量相关.结论 超声激活原卟啉IX产生的活性氧自由基作用于细胞膜,增加膜脂质过氧化水平,降低细胞内的抗氧化物酶的含量,可能是其损伤S180 肿瘤细胞的主要因素之一.     

A-NK细胞培养和活化方法的改进

目的:比较A-NK细胞在完全培养基(CM)和无血清培养基(AIMV)中体外扩增及杀伤肿瘤细胞作用,同时探讨IL-12对于A-NK/IL-2治疗的辅助作用及其杀伤的形态学观察。方法:用MTT法比较不同培养条件下的A-NK细胞体外增殖能力,并测定细胞体外杀伤肿瘤细胞的活性。通过做扫描和透射电镜,对A-NK细胞杀伤的肿瘤细胞进行形态学观察。结果:不同培养条件下的A-NK细胞均可在短期内大量扩增(P<0.05),且均有很强的杀伤肿瘤的活性(P<0.05)。被A-NK细胞杀伤的肿瘤细胞的死亡形式是溶解坏死(necrosis)和凋亡(apoptosis)。结论:AIMV可替代CM用于A-NK细胞的培养。联合应用IL-12和IL-2激活A-NK细胞优于单独使用IL-2,可减轻高剂量IL-2带来的副作用。此研究为A-NK细胞的实验室研究和将来临床应用推广奠定了新的实验基础。     

超声结合原卟啉IX对腹水型S180肿瘤细胞的损伤作用

目的:根据原卟啉IX(PpIX)在S180肿瘤细胞内的含量变化及亚细胞定位分析,研究聚焦超声结合PpIX对S180肿瘤细胞的损伤作用.方法:应用荧光分光光度法测定PpIX在S180肿瘤细胞内的富集;激光共聚焦扫描显微镜观察PpIX的亚细胞定位;台盼蓝拒染法检测超声结合不同浓度的PpIX作用后细胞的存活率;环境扫描电镜观察细胞膜表面超微结构变化.结果:PpIX在S180肿瘤细胞内的富集是一个动态变化过程,45 min达到最高点,此时为最佳声照处理时间点,且此时PpIX主要定位于细胞膜;超声激活PpIX对S180肿瘤细胞的杀伤作用明显大于单纯超声组,其膜损伤程度也更为严重.结论:细胞膜可能是超声激活PpIX作用的一个主要靶点.     

超声结合原卟啉Ⅸ对腹水型S180肿瘤细胞的损伤作用

目的：根据原卟啉Ⅸ（PpⅨ）在S180肿瘤细胞内的含量变化及亚细胞定位分析,研究聚焦超声结合PpⅨ对S180肿瘤细胞的损伤作用。方法：应用荧光分光光度法测定PpⅨ存S180肿瘤细胞内的富集;激光共聚焦扫描显微镜观察PpⅨ的亚细胞定位;台盼蓝拒染法检测超声结合不同浓度的PpⅨ作用后细胞的存活率;环境扫描电镜观察细胞膜表面超微结构变化。结果：PpⅨ在S180肿瘤细胞内的富集是一个动态变化过程．45 min达到最高点,此时为最佳声照处理时间点,且此时PpⅨ主要定位于细胞膜;超声激活PpⅨ对S180肿瘤细胞的杀伤作用明显大于单纯超声组,其膜损伤程度也更为严重。结论：细胞膜可能是超声激活PpⅨ作用的一个主要靶点。     

顺铂诱导人胃癌细胞SGC7901和人肝癌细胞HepG2凋亡的细胞形态学变化的比较

目的观察顺铂诱导人胃腺癌细胞SGC7901和人肝癌细胞HepG2细胞凋亡的细胞形态学的异同并探讨其意义。方法用倒置显微镜、荧光显微镜、透射电镜观察细胞经顺铂处理后的形态学改变。采用流式细胞术研究CDDP对两株肿瘤细胞诱导凋亡中DNA含量的影响。结果经顺铂处理后，两株肿瘤细胞均表现出一些共同的凋亡形态学特征，如细胞及细胞核皱缩，微绒毛消失，细胞膜皱缩但完整，细胞内荧光强度增强。细胞超微结构变化如线粒体肿胀，胞质空泡化。但两者在细胞核形态方面表现出较大的差异；SGC7901表现为核染色质呈团块状或环状，聚集于核膜下；而HepG2主要表现为核碎裂、核膜崩解，核染色质呈梅花状散落在细胞中和一些细胞器通过细胞出芽裂解成由细胞膜包绕的凋亡小体并被周围细胞吞噬。在两者的流式细胞DNA的直方图上均检测到了亚二倍体峰。结论顺铂可诱导SGC7901和HepG2产生不同形态的细胞凋亡，其中SGC7901主要表现为核固缩，HepG2表现为核碎裂。这种差异具有重要的生理意义。     

Mechanism of radiosensitization by porphyrins.

According to our previous data, hematoporphyrin dimethyl ether (HPde) at concentrations useful for photodynamic therapy can radiosensitize aggressive Ehrlich ascite carcinoma (EAT) to 2Gy irradiation inducing total tumour growth inhibition. The aim of this study was to further investigate the possible mechanism of radiosensitization of EAT by dicarboxylic porphyrin-HPde. Our results reveal that HPde is inducing several rearrangements in the EAT cells: 1.2 x 10-6 M of the photosensitizer diminishes the number of cells in mitosis by a factor of 3, increases the number of cells in the S phase of the cell cycle, modifies the activities of antioxidant enzymes glutation S-transferase (GST) and DT-diaphorase (DTD), and eventually induces slight apoptosis. Moreover, it was shown that HPde is a ligand of peripheral benzodiazepine receptor (PBR). Named "house keeper," PBR is usually responsible for all these perturbations, which, in our case, act in concert with the following ionizing radiation, producing the interaction of two antiproliferative/destructive factors.     

IL-10 is necessary for FasL-induced protection from experimental autoimmune thyroiditis but not for FasL-induced immune deviation

Ectopic expression of FasL on thyrocytes confers immune privilege status to the thyroid by inducing apoptosis of Fas-expressing autoimmune effector T cells and anti-thyroglobulin (Tg) immune deviation away from the T1 type. Fas-mediated apoptosis of lymphoid cells leads to rapid production of anti-inflammatory cytokines such as IL-10. On the other hand, cytokines play a crucial role in the immunoregulation and pathology of experimental autoimmune thyroiditis (EAT), and systemic and local administration of IL-10 has a curative effect on EAT. To test the effect of endogenous IL-10 production in EAT, and to find out whether IL-10 production could be involved in FasL-induced protection, EAT was induced in IL-10(-/-) and in IL-10(-/-)xFasL-transgenic CBA/J mice.The results demonstrated that wild-type and IL-10 knockout (KO) animals developed similar EAT. In contrast, lack of endogenous IL-10 abolished the protective effect of FasL. Polymorphonuclear cells were observed significantly more frequently in the inflammatory cell infiltrates from IL-10(-/-)xFasL animals compared to IL-10(-/-) animals, but they were never detected in wild-type or IL-10(+/+)/FasL-transgenic mice. A shift away from T1 response was observed in FasL-transgenic mice irrespective of their IL-10 status, demonstrating that in our model, endogenous IL-10 plays no part in the T1-towards-T2 anti-Tg immune balance induced by FasL. In summary, endogenous IL-10 is not essential in EAT, or for the immune deviation induced by thyroid FasL expression, whereas it is necessary for the immune privilege status of the thyroid conferred by FasL expression on thyrocytes.     

Studies on inflammatory response induced by Ehrlich tumor in mice peritoneal cavity

In the present study we investigated the inflammatory response induced by the inoculation of Ehrlich tumor cells (EAT) into the peritoneal cavity of mice. It was found that after inoculation of 10³ EAT cells, the number of peritoneal leukocytes remained unchanged till the sixth day. Subsequently, the number of cells increased as a consequence of tumor growth. EAT cells did not induce influx of PMN leukocytes till six days after tumor implantation, but a significant influx was observed on the tenth day. Inoculation of the tumor cells did not induce production of H₂O₂ by peritoneal cells at any time examined and induced low levels of macrophage spreading only until the third day after tumor implantation but not later on. The levels of thromboxane in the peritoneal cavity were not affected by the presence of the tumor, whereas prostaglandin E₂ levels were significantly increased at all times examined. The biological significance of these results on the evolution and escape of the tumor from host defense mechanisms is under investigation.     

非聚焦超声对溶液中GLC-82肺腺癌细胞损伤的实验研究

目的:探讨不同功率及不同作用时间的非聚焦超声(none focused ultrasound,NFU)对溶液中肺癌细胞的作用,为杀灭弥漫在胸水及腹水中或浸润在组织中的癌细胞提供实验依据。方法:台盼蓝拒染法测NFU辐照GLC-82肺腺癌细胞后细胞的即刻存活率,MTT法检测其作用后24 h细胞的增殖率,流式细胞仪检测细胞周期和凋亡率。结果:(1)GLC-82细胞的即刻存活率随着辐照功率和作用时间的不断增加明显下降;(2)超声辐照24 h后,随着超声辐照时间和剂量的不断增加,细胞的增殖率明显下降,时间-效应各组之间、剂量-效应各组之间细胞的增殖率差异均有显著性(P<0.05);(3)超声辐照细胞的凋亡率均高于阴性对照,但其周期即刻未发生明显变化。结论:NFU能杀伤溶液中GLC-82肺癌细胞并抑制其增殖,较低功率的超声可能以诱导细胞凋亡为主,较高功率时则可能以杀伤细胞为主。超声对细胞周期的影响为迟发性效应。     

LAK 细胞培养上清及裂解液诱导 Raji细胞凋亡的研究

用流式细胞仪、 Ｈｏｅｃｈｓｔ３３２５８ 荧光素核染后显微镜下计数和电镜三种方法观察 Ｌ Ａ Ｋ 细胞培养上清和裂解液诱导 Ｒａｊｉ肿瘤细胞的凋亡。发现该上清和裂解液与靶细胞共同作用３ｈ 后瘤细胞凋亡率显著增高， 到９ｈ 达最高点， 亚二倍体 Ｄ Ｎ Ａ峰积分面积可达４８ ％ 。在电镜下观察到胞体缩小、表面平滑化、染色质靠边浓集、不连续凋亡小体形成等典型的细胞凋亡现象。提示诱导肿瘤细胞的凋亡是 Ｌ Ａ Ｋ 细胞杀伤机制之一， 并可能通过可溶性因子发挥作用。     

光量子加血卟啉疗法对动物移植瘤生长的影响及机制探讨

目的：探讨自血光动力学疗法对动物移植瘤生长的影响及机制。方法:本实验选用经腹股沟皮下接种W256瘤细胞株形成实验性荷瘤大鼠40只，采用光动力学疗法对患有移植瘤大鼠进行治疗，并同时检测血浆、肝脏及瘤组织中超氧化物歧化酶的活力及氧自由基代谢产物丙二醛含量。结果:大鼠输入经紫外光照射之血后，其肿瘤生长速度明显减慢（P<0.05），大鼠输入注有血卟啉的血后，其肿瘤生长亦显著减慢（P<0.01）；大鼠输入经紫外光照射并含有血卟啉的血后，其肿瘤生长速度比其它组均显著减慢（P<0.01）；光照射组大鼠在肿瘤生长减慢同时，其血浆和瘤组织中SOD活力明显升高；MDA含量显著降低（P<0.05，P＜0.01）；而血卟啉组大鼠其血浆和瘤组织中SOD活力明显降低，MDA含量显著升高（P<0.01）；光照射加血卟啉组大鼠，在肿瘤生长减慢同时，其血浆、肝脏及瘤组织中SOD活力较其它组均显著降低，MDA含量显著升高（P<0.01） 。结论：经紫外光照射的血或含有血卟啉的血均对肿瘤生长有抑制作用，而且紫外光照射与注入血卟啉相结合对抑制肿瘤生长效果最佳，该光动力学疗法对肿瘤治疗作用机制之一可能是通过产生单态氧而杀伤瘤细胞的。     

The effect of aspirin on human's gammadeltaT cells killing digestive system tumor cell lines

AIM: To explore the effect of aspirin on human's gammadeltaT cells killing digestive system tumor cell lines. METHODS: Use the isopentenyl pyrophosphate method to amplify human peripheral blood gammadeltaT cells in vitro. Various concentrations of aspirin were used to induce gammadeltaT cells and digestive system tumor cells SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines, LDH assays was used to measure the cytotoxic activity of gammadeltaT cells, flow cytometry analysis the apoptosis percentage of before and after induced gammadeltaT cells and SGC-7901, SW-1990, SW-480, SW-1116, LOVO cell lines. RESULTS: gammadeltaT cells were cultivated for ten days which proliferation ratio increase from 4.21% to 70.35%. When gammadeltaT cells induced via aspirin concentrations rang from 0.4 mmol/L to 0.8 mmol/L which cytotoxic activity on the five kinds of tumor cell lines was the highest, if aspirin's concentration surpassing 3.2 mmol/L, cytotoxic activity of gammadeltaT cells present decrease tendency. SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines were induced by different concentrations of aspirin for 24 hours, just SW-480, SW-1116 and LOVO were enhanced as far as the cytotoxic activity of gammadeltaT cells on these cell lines was concerned, other groups and control group have no notable changed. The apoptosis percentage of gammadeltaT cells(52.71%) were induced by aspirin which concentration was 3.2 mmol/L, which was strikingly higher than SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines, (respectively 7.88%, 8.89%, 6.21%, 4.47% and 3.67%). CONCLUSION: When aspirin's concentration for clinical routine used can enhance the effect of gammadeltaT cells killing the tumor cells, if surpassing this concentration can obvious inhibited the proliferation capacity and cytotoxic activity of gammadeltaT cells and augment apoptosis ratio, however, it is not obvious for digestive tract tumor lines.