New approaches for successful islet transplantation

The long-term control of glucose metabolism is expected to be achieved with insulin-secreting pancreatic islets transplanted as the whole pancreas or isolated islet tissue. The current clinical outcomes of such procedures are unsatisfactory compared with that of whole organ transplantation or experimental islet transplantation, although the safety of the transplantation procedure itself is assured. In this review, we first describe the immunological characteristics of purified islets in an attempt to explain the differences between the results for human and experimental islet transplantation. We then introduce new approaches currently undertaken in our laboratory that could, we hope, make islet transplantation more successful.     

薏苡仁酯诱导人宫颈癌HeLa细胞凋亡的实验研究

目的　探讨薏苡仁酯诱导人宫颈癌HeLa细胞凋亡的作用机制。方法　应用形态学方法 ,流式细胞术 (FACS) ,DNA凝胶电泳等方法检测细胞凋亡的发生 ,应用RT PCR检测凋亡相关基因Fas与FasL的变化。结果　薏苡仁酯对人宫颈癌HeLa细胞的生长有明显的抑制作用 ,并诱导肿瘤细胞发生凋亡。凋亡细胞表现为细胞固缩 ,核染色质碎裂 ,DNA凝胶电泳显示清晰的DNA梯形条带 ,FACS检测到凋亡率最高为 13%。AnnexinV标记的方法检测凋亡时发现 ,坏死与凋亡共存。在薏苡仁酯诱导人宫颈癌HeLa细胞凋亡过程中 ,凋亡相关基因Fas转录水平比用药前增强 ,而FasL转录水平减低。结论　除了坏死 ,凋亡也为薏苡仁酯抑癌的机制之一。薏苡仁酯诱导人宫颈癌HeLa细胞凋亡可能与Fas基因与FasL基因表达有关。     

精胺预处理对离体灌流大鼠心肌缺血/再灌注损伤及细胞凋亡的影响

目的探讨精胺预处理对离体灌流大鼠心肌缺血/再灌注损伤的影响及其可能的抗凋亡作用。方法应用Langehdorff离体灌流大鼠心脏复制模拟心肌缺血/再灌注损伤模型。24只大鼠随机分为3组,每组8只:对照组(Con-trol)、缺血/再灌注组(IR)、精胺干预组(Spermine)。比色法测定冠脉流出液中乳酸脱氢酶(LDH)活力,心肌组织中超氧化物歧化酶(SOD)活性及心肌丙二醛(MDA)含量;多导生理记录系统记录心脏功能;HE染色光镜下观察心肌形态学变化;三苯基氯化四氮唑(TTC)染色检测心肌梗死面积;透射电镜和TUNEL方法检测细胞凋亡;免疫组化检测心肌Fas与Bcl-2蛋白的表达。结果 (1)与对照组比,IR组冠脉LDH漏出明显增多、心肌组织中MDA水平增加、SOD活性降低(P<0.01);心功能明显下降(LVDP,HR,CF均低于对照组,P<0.05);光镜下可见心肌细胞呈凝固性或带状坏死。与Control组比,IR组心肌梗死面积及心肌细胞凋亡率明显增加(P<0.01);心肌Fas蛋白表达上调,Bcl-2蛋白表达下调。透射电镜下心肌细胞核染色质浓缩、凝聚且边集,染色质在核膜周边聚集形成新月形,线粒体嵴排列紊乱。(2)与IR组比,Spermine组冠脉LDH漏出减少,心肌组织中MDA减少,SOD增加(P<0.01);心功能有明显改善(LVDP,HR,CF均明显高于IR组,P<0.05);光镜下心肌细胞结构清晰。Spermine组与IR组比心肌梗死面积及心肌细胞凋亡率均明显降低(P<0.01);心肌Fas蛋白表达下调,Bcl-2蛋白表达上调;电镜下可见心肌肌节结构清晰,线粒体嵴完整、基质致密,未见核染色质凝聚。结论外源性精胺能明显减轻离体灌流大鼠心肌缺血/再灌注损伤,其机制可能与精胺抑制细胞凋亡有关。     

Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta-cells by T lymphocytes. Beta-cell death that is induced by autoreactive CTL in diabetes involves both Fas/Fas ligand (FasL)- and perforin/granzyme-mediated pathways, although their relative contributions during the progression of the disease remain unknown. We demonstrate here that despite the preferential use of the Fas/FasL pathway for cytolysis of beta-cell targets, transgenic beta-cell-specific CTL were able to kill targets via the perforin pathway when triggered by a higher affinity stimulus. In addition, we show that the killing mechanism used by islet-associated CD8(+) T cells from non-obese diabetic mice changed as the mice aged and correspondingly, with the stage of diabetes. These results provide direct evidence for age-related changes in the cytotoxic pathways used by diabetogenic T cells during the progression of autoimmune diabetes.     

Expression of Fas/APO-1 and apoptosis of keratinocytes in human cholesteatoma

OBJECTIVES: The Fas/APO-1 is a cell surface receptor protein known to trigger apoptosis when conjugated with Fas ligand or specific agonistic antibodies. The present study investigated the expression of Fas/APO-1 and apoptotic cell death in both normal and cholesteatoma epithelia. STUDY DESIGN: Ten cholesteatomas and retroauricular skins were taken during middle ear operations in the Ajou University Hospital for this study. METHODS: In situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining and agarose gel electrophoresis of genomic DNA were used for detection of apoptosis. The expression patterns of Fas/APO-1 protein were confirmed by immunoblot and immunohistochemical analysis. RESULTS: In situ TUNEL staining revealed many positively stained nuclei in 9 of 10 suprabasal layers of cholesteatoma epithelium, whereas few positively stained cells were found on the granular layer of retroauricular skins. Typical "ladder pattern" was seen on the gel electrophoresis of the genomic DNA of cholesteatoma. Immunoblot analysis and immunohistochemical studies revealed that Fas/APO-1 protein was expressed in 8 of 10 cholesteatoma epithelia, whereas retroauricular skin showed negative reaction. CONCLUSION: These results show the upregulated expression of Fas/APO-1 and increased apoptotic cell death in cholesteatoma epithelium. Because Fas/APO-1 is known as an apoptosis-triggering protein, it is suggested that the accumulation of keratin debris is due to increased apoptotic cell death related to Fas/APO-1 protein.     

急性胰腺炎肝巨噬细胞表达的FasL在肝损伤中的作用

目的:探讨肝巨噬细胞表达的Fas配体(FasL)在实验性急性胰腺炎(AP)肝损伤中的作用。方法:建立AP小鼠模型。雄性ICR小鼠30只,随机分为5组,每组6只,其中一组为对照组,腹腔注射0.1m l等渗盐水,4 h后心脏穿刺抽血,并处死小鼠留取标本;另4组均腹腔注射雨蛙素50μg/kg,每4 h注射1次至24 h,分别在4、8、16和24 h心脏穿刺抽血,并处死小鼠留取标本。用自动化生化仪检测血清ALT、AST、LDH和AMY的浓度,用ELISA试剂盒检测血清FasL浓度,用W estern b lot检测肝FasL蛋白的表达。结果:雨蛙素诱导的小鼠AP组与对照组相比,各个检测时间点血清AST、ALT、LDH、AMY水平明显增加。在4、8、16和24 h时AP组血清FasL浓度为(532.17±46.92)、(496.73±32.62)、(485.57±18.31)和(448.08±26.44)pg/m l,均显著高于对照组(80.34±6.81)pg/m l,AP组各检测时间点肝FasL蛋白表达也增加,与对照组相比,差异有显著统计学意义(P<0.01)。结论:AP通过上调肝巨噬细胞表达的FasL介导肝损伤。     

移植肾急性排斥时Fas配体和T细胞内抗原1的表达

目的:探讨移植肾发生急性排斥反应时肾组织Fas配体(Fas ligand,FasL)和T细胞内抗原1(T-cell intracellular antigen-1,TIA-1)的表达及意义.方法:应用免疫组织化学技术分别检测32例移植肾标本、2例供肾标本及8例肾癌周围肾组织标本FasL和TIA 1的表达,标本按Banff标准分为急性排斥(14例)、慢性排斥(15例)和无排斥改变(13例)3组,分析移植肾组织标本病理形态学改变与FasL和TIA 1表达的相关性.结果:FasL主要在肾小管上皮细胞胞质内表达,急性排斥组表达明显强于慢性排斥组和无排斥组(P＜0.05);TIA-1在肾小管上皮细胞和间质浸润淋巴细胞均有表达,小动脉内皮细胞亦可见阳性着色,急性排斥组阳性强度较高,与慢性排斥组和无排斥组间存在显著差异(P＜0.05);急性排斥标本FasL和TIA-1的表达强度与组织损害的严重程度呈正相关.结论:急性排斥时移植肾组织FasL和TIA-1表达增强是T细胞活化的反映,其检测可能有助于移植肾急性排斥的早期诊断.     

P38MAPK、caspase-8在Fas-放线菌素D诱导Bel-7402细胞凋亡中的作用

目的 确定P38MAPK、caspase-8的关系,以探讨Fas-actinomycin D(Fas-AD)诱导Bel-7402细胞凋亡的信号转导机制.方法 通过流式细胞仪检测Fas-AD、SB203580(P38MAPK特异性抑制剂)及Ac-IEFD-cho(caspase-8特异性抑制剂)对Bel-7402细胞凋亡的影响;通过Western-blot法检测Fas-AD、SB203580及Ac-IEFD-cho对培养的Bel-7402细胞的P38MAPK、caspase-8的激活及其表达的影响.结果 Fas-AD能明显诱导Bel-7402细胞凋亡,SB203580和Ac-IEFD-cho能明显抑制Fas-AD诱导的细胞凋亡.Fas-AD能诱导P38MAPK、caspase-8激活并表达,而SB203580和Ac-IEFD-cho能分别抑制它们的激活及表达.结论 在Bel-7402细胞凋亡中,P38MAPK与caspase-8参与Fas-AD凋亡途径,并且相互调节.     

丁酸钠对人绒毛膜癌JAR细胞生长抑制作用及其机制的实验研究

目的:探讨丁酸钠对人绒毛膜癌JAR细胞生长抑制作用及其可能机制。方法:通过流式细胞仪定量分析细胞周期分布及细胞凋亡;并采用酶联免疫吸附法(ELISA)检测丁酸钠对Fas蛋白表达水平的影响。结果:丁酸钠对JAR细胞生长有抑制作用,呈剂量依赖关系(P<0.05);丁酸钠组细胞凋亡率明显高于对照组(P<0.01);随着丁酸钠浓度升高,Fas蛋白的表达水平逐步升高(P<0.05)。结论:丁酸钠可以抑制人绒毛膜癌JAR细胞的生长和诱导其凋亡,其机制可能与Fas蛋白的高表达有关。     

泰素帝诱导人肺腺癌细胞凋亡的实验研究

目的 探讨泰素帝诱导人肺腺癌细胞凋亡的作用机制。方法 应用形态学方法、流式细胞术（ＦＡＣＳ）、ＤＮＡ凝胶电泳、ＡｎｎｅｘｉｎＶ标记等方法检测细胞凋亡的发生,应用ＲＴ－ＰＣＲ检测凋亡相关基因Ｆａｓ、ｂｃｌ－２、ｃ－ｍｙｃ表达的变化。结果 泰素帝对人肺腺癌细胞系Ａ５４９细胞的生长有明显的抑制作用,使细胞分裂阻滞于Ｇ２Ｍ期,并诱导肿瘤细胞发生凋亡。凋亡细胞表现为细胞固缩,核染色质聚集或断裂,ＤＮＡ凝胶电