虎纹蛙消化道肥大细胞类胰蛋白酶免疫组化研究

研究采用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体AA1,应用ElivisionTM plus免疫组化染色法对虎纹蛙(Rana tigrina rugulosa)消化道组织中类胰蛋白酶阳性肥大细胞存在的可能性进行研究。研究发现单克隆抗体AAl可与中性缓冲福马林液固定的虎纹蛙组织的肥大细胞获得良好的交叉反应,类胰蛋白酶阳性细胞胞浆染成棕黄色,证实虎纹蛙肥大细胞胞浆颗粒中也存在类胰蛋白酶。虎纹蛙组织中AA1免疫染色阳性细胞的分布,与AB/SO和改良甲苯胺兰染色阳性细胞的分布存在较大的差异:虎纹蛙类胰蛋白酶阳性细胞数量很少,且阳性反应比人胃癌间质肥大细胞弱,主要见于黏膜型肥大细胞(MMC)分布区域,如消化道黏膜上皮下方和固有层,少量分布于肠绒毛基底部及食管腺和胃腺周围。而在结缔组织型肥大细胞(CTMC)分布区域,如消化道黏膜下层结缔组织中却未见类胰蛋白酶阳性细胞。AB/SO和改良甲苯胺兰染色阳性细胞数量多,广泛分布于消化道黏膜固有层、黏膜下层、腺体之间、肌间及外膜结缔组织,说明并不是所有的虎纹蛙肥大细胞都含有类胰蛋白酶。很有可能是虎纹蛙MMC中含有类胰蛋白酶,而CTMC中不含类胰蛋白酶。虎纹蛙类胰蛋白酶阳性细胞数量很少,且阳性反应比人胃癌间质肥大细胞弱,说明虎纹蛙肥大细胞胞浆颗粒类胰蛋白酶含量较少,虎纹蛙属于低等脊椎动物,可能与生物进化水平较低有关,有待进一步研究。       

天花粉注射后不同时间小鼠子宫肥大细胞的变化

雌性中国１号小鼠３６只,分为实验组和对照组。实验组分别腹腔注射天花粉后不同时间处死,取子宫组织,作甲苯胺蓝及Ａｌｃｉａｎ蓝,藏红染色,对肥大细胞进行计数后结果表明：给小鼠天花粉,可引起其子宫肥大细胞数量明显增多,而且随天花粉作用时间延长,肥大细胞数量逐渐增多。本文对肥大细胞的这一数量变化与子宫功能的关系也作了进一步探讨。     

天花粉对小鼠子宫肥大细胞超微结构的影响

本研究用雌性中国１号小鼠１２只,分实验组和对照组,实验组分别腹腔注射天花粉后处死,取子宫组织作超薄切片,电镜观察。在天花粉作用下,常见子宫肥大细胞脱颗粒,呈功能活化状态,并与子宫平滑肌细胞紧密相邻。提示肥大细胞可能通过脱颗粒释放组胺促进子宫平滑肌的收缩。同时,也见肥大细胞与子宫结缔组织中的成纤维细胞,淋巴细胞等密切接触,提示成纤维细胞和淋巴细胞与天花粉作用下子宫肥大细胞的数量增多有关。     

肥大细胞及类胰蛋白酶在人甲状腺肿瘤组织中的表达 及其与微血管密度的关系

目的:探讨肥大细胞(mast cell,MC)及类胰蛋白酶(tryptase)与甲状腺肿瘤微血管密度(microvessel density,MVD)的相关性及其对甲状腺癌发生发展的影响。方法:采用甲苯胺蓝组织化学染色和PV免疫组织化学染色对116例甲状腺癌、56例甲状腺腺瘤和10例正常甲状腺组织中MC和tryptase及其CD31的表达进行了检测;对MC和tryptase与MVD的关系进行了统计学分析。结果:甲状腺肿瘤中MC的数量和tryptase阳性表达高于正常甲状腺,而且与肿瘤的类型有关(P<0.01);Spearman等级相关分析显示各组甲状腺组织MC数量和tryptase表达与MVD呈正相关(r=0.900,r=0.636,P<0.05)。结论:MC及其分泌的tryptase有促进血管新生的作用,因而可促进甲状腺肿瘤的浸润和转移。     

实电解质钙可诱发人肥大细胞组胺和类胰蛋白酶分泌

目的: 利用人大肠组织的肥大细胞和肥大细胞激活的体外研究系统,评价实电解质钙(calcium ionophore A23187, CI)诱导肥大细胞释放类胰蛋白酶和组胺的能力和机制.方法: 经酶悬浮的人大肠肥大细胞与CI共同培养后收集上清液,并用酶联免疫吸附试验(ELISA)的方法检测类胰蛋白酶分泌量,用以玻璃纤维为基础的荧光比色法检测组胺释放量.结果: 经过15 min的培养,CI可引起浓度相关性的组胺和类胰蛋白酶释放.其中组胺的最大分泌量比基础分泌量超出了5.3倍以上,而类胰蛋白酶的最大分泌量则比基础分泌量超出了2.8倍以上.CI在浓度高于1.0 μmol/L时引起的组胺释放量明显多于类胰蛋白酶释放量.时间关系曲线显示,CI的作用从加样后10 s开始,6 min后达高峰并至少持续15 min.百日咳毒素和代谢抑制剂均能抑制CI引起的组胺和类胰蛋白酶释放.结论: 人大肠肥大细胞在受到CI刺激时具有释放类胰蛋白酶和组胺的能力,这个过程与肥大细胞膜G蛋白偶联受体的激活有关,并消耗能量.     

二甲基亚砜诱导人食管癌细胞分化的实验研究

The fact that treatment of leukemia (Acute Promyelocytic Leukemia) with ATRA (All-Trans Retinoic Acid) was so succeeded that it was considered as a good example for tumor therapy. In the treatment of solid tumors by means of induced differentiation, however, has not been yet so broken-through. DMSO (Dimethylsulfoxide) was a common and simple organic compound, which comprised a variety of biological activities. For example, DMSO induced differentiation of leukemia in many reports. However, the effect of DMSO on solid tumors was to be explored further. In the present study, DMSO was used to human esophageal cancer cell lines in vitro in comparison with the classical inducer ATRA. From the view of morphology, cell cycle, growth inhibition, cytokeratin 4 expression, dye transfer and tumorigenecity, the results demonstrated that DMSO as well as ATRA could induce differentiation of human esophageal cancer cells. Interestingly, DMSO was confirmed to be more effective in inducing differentiation of esophageal cancer cells than ATRA. It suggests that DMSO showed some good prospects for the treatment of solid tumors.     

蛋白酶激活受体2(PAR-2)激动剂对肥大细胞释放类胰蛋白酶的影响

研究反肉桂酰-亮-异亮-甘-精-亮-鸟-[酰胺]（tc-LIGRLO),一种PAR-2激动剂,对肥大细胞类胰蛋白酶释放的影响。结果显示,经过15min的培养,tc-LIGRLO可引起比基础分泌量增加1倍以上的类胰蛋白酶释放,作用强度超过抗IgE抗体和钙离子导入剂（calcium ionophore A23187,CI),而反PAR-2激动剂-反肉桂酰-鸟-亮-精-甘-异亮-亮-[酰胺]（tc-OLRGIL)无此作用,培养时间延长到30min时对tc-LIGRLO的作用无明显影响,其时间关系曲线表明,tc-LIGRLO的作用从1min开始,3min后达高峰,结果表明,PAR-2激动剂tc-LIGRLO是一种高效类胰蛋白酶释放刺激剂,在肥大细胞上可能有PAR-2存在。     

上皮组织增生与肥大细胞变化的形态学研究

Ｃ－１号小鼠４０只,随机分为４组,每组１０只,用食醋抹小鼠右耳皮肤,每日一次,以左耳作对照。在实验后第１０、３０、９０、１４０天各处死一组,取耳廓组织,常规石蜡制片,进行ＨＥ染色及肥大细胞染色,光镜下观察。结果发现：实验组１４０天皮肤上皮组织明显的增生,结缔组织内肥大细胞（ＭＣ）的数量比对照组显著增多（Ｐ＜０．０１）。在ＡＢ－Ｓ染色时,实验组ＭＣ由藏红阳性为主逐渐转变成以Ａｌｃｉａｎ蓝阳性为主,而对照组各时期ＭＣ均呈藏红阳性。实验组与对照组皮肤ＭＣ的临界电解质浓度分别为０．９５Ｍ,０．９７Ｍ。硫酸小蘖碱荧光染色均呈强黄色萤光反应。结果提示：①上皮组织增生可致结缔组织内ＭＣ数量增多,②增多的ＭＣ的组化性质发生了变化,③ＭＣ可参与上皮组织的增殖,ＭＣ与上皮细胞间具有相互促进作用     

小鼠骨髓来源肥大细胞的培养及鉴定

目的:探讨小鼠骨髓来源肥大细胞体外大量培养及鉴定方法。方法:采用IL-3(10 ng/mL)和不同浓度的SCF联合刺激小鼠骨髓细胞,培养四周,采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,甲苯胺蓝染色观察其胞浆颗粒物质,流式细胞仪检测细胞表面CD117和FcεRI的表达情况,β-己糖苷酶释放试验评估肥大细胞活化能力。结果:培养条件为10ng/mL IL-3和20 ng/mL SCF时,悬浮细胞数目能达到1×10~8细胞,其中CD117和FcεRI双阳性细胞比例达90%以上;甲苯胺蓝染色可见细胞胞浆含有大量紫红色颗粒;细胞经IgE-DNP致敏及DNP-HSA激发后,β-己糖苷酶释放率明显增加,且SCF能促进肥大细胞释放β-己糖苷酶。结论:采用10 ng/mL IL-3和20 ng/mL SCF诱导骨髓细胞能够获得大量高纯度的小鼠骨髓来源肥大细胞,为肥大细胞特性及功能学研究提供了基础。     

食管癌胃癌间质中肥大细胞淋巴细胞光镜与电镜研究

对３５例食管癌与３０例胃癌间质中的肥大细胞与淋巴细胞进行光镜与电镜观察。结果发现,两种癌间质中的肥大细胞数量在恶性程度低组均比恶性程度高组与对照组显著增加。在各组食管癌或胃癌中,有淋巴细胞高度浸润并形成淋巴小结者,肥大细胞也增多。用Ａｌｃｉａｎ蓝—藏红染色,对照组Ａｌｃｉａｎ蓝阳性和藏红阳性的肥大细胞都可以见到;而在食管癌胃癌间质中,大部分肥大细胞均呈Ａｌｃｉａｎ蓝阳性。电镜观察,对照组肥大细胞颗粒多具有涡卷状结构,而癌组者则显示多样化形态。一些肥大细胞和淋巴细胞与癌细胞接触,肥大细胞可见活跃脱颗粒现象。结果提示,在抗肿瘤生长的防御机制中,肥大细胞与淋巴细胞有协同作用。     

三氧化二砷对食管癌细胞增殖和热休克蛋白70表达的影响

目的：研究三氧化二砷（As2O3）对食管癌细胞增殖和热休克蛋白70（HSP70）表达的影响。方法：通过相差显微镜、流式细胞术、免疫细胞化学染色和免疫印迹分析等方法观察As2O3对人食管癌细胞株EC1的作用效果和作用机制。结果：与对照组相比,经2μmol/L和5μmol/Las2O3作用的细胞出现明显的生长抑制,G2/M期细胞比例增加;2μmol/Las2O3作用48h后经Ecl细胞HSP70（heat shock protein70）及HSC70(heat shock cognate protein70)表达均增加。结论：As2O3诱导食管癌细胞G2/M期阻滞抑制细胞增殖和生长;HSP70的升高是细胞对As2O3作用后出现的应激反应,并与细胞周期阻滞相关。     

双歧杆菌对食管癌细胞增殖的抑制作用及细胞周期的影响

目的探讨青春双歧杆菌对食管癌EC109细胞的增殖抑制作用及对细胞周期的影响。方法用MTT比色法测定EC109细胞活性,用流式细胞仪测定EC109细胞周期。结果青春双歧杆菌对EC109细胞具有显著的增殖抑制作用,并呈剂量和时间依赖性;经青春双歧杆菌处理后,EC109细胞周期发生变化：细胞分裂阻滞于G1期。结论青春双歧杆菌可通过影响细胞周期抑制食管癌EC109细胞的生长。     

Mastin is a gelatinolytic mast cell peptidase resembling a mini-proteasome

Mastin is a tryptic peptidase secreted by canine mast cells. This work reveals that mastin is composed of catalytic domain singlets and disulfide-linked dimers. Monomers unite non-covalently to form tryptase-like tetramers, whereas dimers aggregate with monomers into larger clusters stabilized by hydrophobic contacts. Unlike tryptases, mastin resists inactivation by leech-derived tryptase inhibitor, indicating a smaller central cavity, as confirmed by structural models. Nonetheless, mastin is strongly gelatinolytic while not cleaving native collagen or casein, suggesting a preference for denatured proteins threaded into its central cavity. Phylogenetic analysis suggests that mammalian mastins shared more recent ancestors with soluble alpha/beta/delta tryptases than with membrane-anchored gamma-tryptases, and diverged more rapidly. We hypothesize that gelatinase activity and formation of inhibitor-resistant oligomers are ancestral characteristics shared by soluble tryptases and mastins, and that secreted mastin is a mini-proteasome-like complex that breaks down partially degraded proteins without causing bystander damage to intact, native proteins.     

Arsenic trioxide inhibits the growth of A498 renal cell carcinoma cells via cell cycle arrest or apoptosis

Previously, we showed that arsenic trioxide potently inhibited the growth of myeloma cells and head and neck cancer cells. Here, we demonstrate that arsenic trioxide inhibited the proliferation of all the renal cell carcinoma cell lines (ACHN, A498, Caki-2, Cos-7, and Renca) except only one cell line (Caki-1) with IC(50) of about 2.5-10 microM. Arsenic trioxide induced a G(1) or a G(2)-M phase arrest in these cells. When we examined the effects of this drug on A498 cells, arsenic trioxide (2.5 microM) decreased the levels of CDK2, CDK6, cyclin D1, cyclin E, and cyclin A proteins. Although p21 protein was not increased by arsenic trioxide, this drug markedly enhanced the binding of p21 with CDK2. In addition, the activities of CDK2- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Arsenic trioxide (10 microM) also induced apoptosis in A498 cells. Apoptotic process of A498 cells was associated with the changes of Bcl-(XL), caspase-9, caspase-3, and caspase-7 proteins as well as mitochondria transmembrane potential (Deltapsi(m)) loss. Taken together, these results demonstrate that arsenic trioxide inhibits the growth of renal cell carcinoma cells via cell cycle arrest or apoptosis.     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.     

Enolase 1 and calreticulin regulate the differentiation and function of mouse mast cells

It has become widely accepted that the role of mast cells is not restricted to allergic processes. Thus, mast cells play an important role in innate and adaptive immune responses, but study of proteins related to differentiation of mast cells has not been done yet. Enolase 1 is a glycolytic enzyme expressed in most tissues and calreticulin, known as endoplasmic reticulum (ER) resident chaperon, has multifunctional responses. This study aimed to investigate the effects of these proteins on the differentiation and functions of mouse bone marrow-derived mast cells (BMMCs). To identify the target proteins related to the differentiation of BMMCs, we examined the protein expression pattern of BMMCs using 2-dimensional electrophoresis (2-DE) and MALDI-TOF analysis. Expressions of FcεRIα, surface molecules (c-kit, CD40, CD40L, VCAM-1), tryptase, and cytokines were examined in BMMCs using FACS analysis, Western blot, and RT-PCR respectively. Enolase 1 and calreticulin were transfected into BMMCs, and [Ca²⁺]i levels were determined by confocal microscope, while amounts of TNF-α and LTs were measured by ELISA. Eight proteins were identified by proteomic analysis. Enolase and calreticulin siRNA transfection inhibited the expressions of FcεRIα, surface molecules, tryptase, and cytokine mRNA, which are gradually enhanced during culture periods of BMMCs. Enolase 1 and calreticulin siRNA reduced the [Ca²⁺]i levels, amounts of total TNF-α, and the release of TNF-α and leukotrienes, all of which are increased in the BMMCs activated with antigen/antibody reaction. The data suggest that enolase 1 and calreticulin are important proteins in regulating the differentiation and functions of BMMCs.     

高转移潜力食管癌细胞株的建立及其生物学特征

目的建立具有高转移潜力食管癌细胞株并研究其生物学特征。方法将食管癌细胞系EC109细胞悬液异位移植到SCID小鼠胃壁,约3个月后或动物濒临死亡时处死,行病理学解剖,将肉眼可见的纵隔淋巴结转移瘤块接种于SCID鼠皮下扩增,然后取小鼠皮下瘤组织块进行细胞培养,得到性状稳定的细胞株NMC109后,用MTT法分析细胞生长曲线,Western bloting法检测与细胞分裂增殖能力密切相关的TopoⅡα表达,酶谱法检测MMP-2和MMP-9的活性,划痕实验和Transwell体外移动实验检测细胞的移动能力。结果与母本细胞EC109相比,所获得的细胞株NMC109其增殖能力和TopoⅡα表达明显增强,MMP-9的活性明显升高,移动能力明显增强。结论获得了具有高转移潜力的食管癌细胞株。     

A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.