体外细胞微核试验的制片新技术介绍

体外细胞微核试验,作为一种鉴定化学物致突变性的体外试验已广泛应用。常规采用的制片方法是在细胞收获后,先作低渗处理,然后涂片、固定和染色.由于细胞经受试化学物处理后,细胞膜对低渗的耐受性常常会发生变化,使得低渗处理不易掌握,易造成低渗不够或过头。我们采用美国制造的离心制片机(Cytaspin Shandon Inc.pittsb-urgh,PA15275),在细胞收获后,将细胞混悬液调整至适当的细胞密度,不经低渗直接制片,胞     

羊水细胞培养及染色体制备方法的探讨

目的:探讨羊水细胞培养及染色体制备方法。方法:孕15～29周具有产前指征的50例孕妇,抽取羊水细胞培养6～7天,换液第二天取出观察,见有许多大而亮圆的分裂期细胞时,加入秋水仙素继续培养1小时后收获、低渗、固定、制片、消化、染色。结果:50例羊水细胞培养全部成功,成功率100%。有49例获得满意的染色体分裂相。结论:该方法细胞培养成功率高、有效分裂相多、实验稳定、易于操作、成功率高。     

染色体畸变分析实验条件探讨

目的探讨如何确立外周血淋巴细胞培养染色体畸变分析实验条件,以获得良好的制片效果。方法在已消毒好备用的超净台内,用碘伏消毒采血管的头部,轻轻混匀后接种,加入秋水仙素工作液,经混匀、培养、收集细胞并离心后,弃上清液,加入低渗液混匀,给予预固定;重复固定后依细胞数量加入适量固定液,制片、染色、气干。于低倍镜下细致观察,计数200个分裂相。结果细胞生长良好,分裂指数高,能够满足分析要求,染色体分散均匀,着色良好,长短适中,两条姐妹染色体大致平行分离,着丝粒清晰。结论寻找适合自己实验室的实验条件能提高染色体畸变的检出率和阅片速度,及时准确地反映辐射损伤情况,对放射工作人员健康进行监护,为意外放射性损伤事故进行生物剂量估算,为放射患者诊断与治疗提供依据。     

六种抗肿瘤药物影响小鼠精子生成过程的定量组织学比较

目的通过对小鼠睾丸的定量组织学研究,观察丝裂霉素C等6种抗肿瘤药物对小鼠睾丸生精过程的影响。方法制作小鼠睾丸组织切片,显微镜下观察,记录第Ⅶ相细精管的精原细胞数及第Ⅳ相细精管的次级精母细胞和处于减数分裂中期、后期的细胞数。结果腹腔注射给药,丝裂霉素C和美法仑可使小鼠睾丸精原细胞接近完全消失,而多柔比星、长春新碱、甲氨蝶呤和5-氟尿嘧啶对精原细胞没有影响。此6种抗肿瘤药物对次级精母细胞以及处于减数分裂中期、后期的细胞均无明显影响。给小鼠腹腔注射丝裂霉素C2mg.kg-1,3d后第Ⅶ相细精管的精原细胞接近完全消失,14d后精母细胞接近完全消失,21d后精子细胞消失,35d后3种细胞数目基本恢复正常。结论不同抗肿瘤药物对小鼠睾丸精原细胞的影响有明显的差异。精原细胞对丝裂霉素C高度敏感。6种抗肿瘤药物对次级精母细胞和处于减数分裂中期、后期的细胞数量无明显影响。     

二甲苯致小鼠角膜损伤以及角膜染色体损伤的研究

将二甲苯分别滴入四个实验组的小鼠眼内，每只眼两滴，固定30min。加入60％的冰醋酸水溶液2—3滴软化角膜0．5—1min。离心，滴片，晾干，Giemsa染色，在显微镜下观察并照相。染色体制片上可见到染毒后角膜细胞分裂增殖活跃，分裂相明显增多。在50％二甲苯染毒组的染色体制片中，可观察到染色体粘连现象及染色单体裂隙。     

115例不育男子的细胞遗传学分析

目的对１１５例不孕男子进行染色体检查，以分析不育男性染色体异常检出率及异常类型和频率分布。方法 取外周血０．５ｍｌ，按常规制片法制片，每例标本均在油镜下计数个分裂相，发现嵌合体细胞或异常细胞时，分别再加数３０个分裂相，每例标本均选择５个染色体分散良好、显带清晰的分裂相镜下分析。结果 １１５例男性患者检出异常染色体１３例，检出率为１１．３％，染色体异常类型及频率分别为：４７，ＸＸＹ（Ｋｌｉｎｅｆｅｌ     

五加双参片对60Co辐射小鼠外周血细胞和骨髓三系细胞的影响

目的 探讨五加双参片对60Co辐射小鼠外周血细胞和骨髓造血细胞的作用，了解其作用途径。方法 采用6．0GY60Co辐射小鼠造成严重造血功能损伤，尾静脉采血计数外周血细胞及其分类，取一侧股骨骨髓计数骨髓有核细胞，取另一侧骨髓制片，观察计数骨髓粒系分裂池，成熟池，贮存池骨髓细胞绝对值的动态变化。结果 五加双参片对辐射损伤细胞与骨髓造血细胞有一定保护作用，使损伤脾细胞向正常逆转，加速粒细胞增殖与激活作用，促进成熟池粒细胞成熟进人贮存池、从而进人到外周血循环，对淋巴细胞有激活作用。结论 五加双参片对粒细胞和淋巴细胞有促进增殖作用。     

小鼠精细胞微核试验方法的初步研究

本文采用不同于L?hdetie和Tates的制片方法,通过低渗、固定、软化、离心、分离出精细胞、滴片、吉姆萨染色。比较3种离心方式对制片的影响,发现低于300转/分离心3分钟比较合适,膜片上细胞均匀分散。杂物较少。不同剂量(40、80、100mg/kg)的环磷酰胺诱发小鼠精细胞微核率不同,剂量越高,微核率也越高。给药后不同处     

胸腹水脱落细胞制片及影响因素的分析

目的分析胸腹水脱落细胞制片及影响因素。方法选择我院2016年2月至2017年2月收治的进行常规检查和脱落细胞检查的胸腹水标本56份,制片后分别进行水平湿固定和垂直湿固定,然后进行水平湿固定(HE染色)和干燥法(瑞姬氏染色),观察制片质量。结果水平湿固定对细胞成分的保留情况更理想,水平湿固定HE染色对细胞结构的影响更轻微。结论在对胸腹水脱落细胞制片时,选择水平湿固定不但能让细胞成分流失和细胞退变的人为因素减少,让交叉污染有效减少,而且还能让诊断符合率显著提高。     

改良法秋水仙素处理技术在染色体畸变分析实验中应用的比较性研究

目的在染色体畸变分析实验中,探讨与常规方法相比较改良法秋水仙素处理技术的应用价值。方法采取30份样本,每份标本分成两份,分别采用A方法(常规法)和B方法(改良法)进行细胞培养和秋水仙素处理。然后均给予细胞收集、离心、固定、制片和染色等步骤。镜检计算每种方法的有丝分裂指数。然后利用SPSS 17.0软件进行t检验分析(P<0.05时有统计学差异)。结果两种方法处理后的细胞均生长良好,分裂指数高,均能够满足分析要求。两种方法所得有丝分裂指数比较,无统计学差异(P>0.05)。结论改良法秋水仙素处理技术具有秋水仙素用量少,毒性低,可控性好等优势,且实验效果可靠,具有推广价值。     

Mucosal cytokine production during remission after resection for Crohn's disease and its relationship to future relapse

AIM: To examine whether mucosal cytokine production during remission after resection for Crohn's disease is a predictor of future relapse. METHODS: Thirty-six patients who remained in remission after resection for terminal ileal or ileo-caecal Crohn's disease were included. At enrollment, blood and mucosal (ileal and rectal biopsies) samples were collected. All patients were followed up regularly for 1 year after enrollment and the disease activity was assessed according to the Crohn's disease activity index. RESULTS: Twenty patients remained in remission and 16 patients relapsed during the 1-year follow-up. Interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha levels in the ileal mucosa were significantly higher in relapsed patients than in patients in remission. These cytokine levels in the rectal mucosa were not associated with relapse. Conventional blood markers and plasma cytokine levels did not correlate with relapse. Amongst the clinical parameters, a younger age, short disease duration before operation and fistulating disease were risk factors for relapse. In multivariate analysis, only the ileal interleukin-6 level was an independent significant predictor for relapse. CONCLUSIONS: The interleukin-6 level in the ileal mucosa during remission after resection for ileal or ileo-caecal Crohn's disease is an independent significant predictor for future relapse.     

唐古特大黄多糖对大鼠应激性胃溃疡的保护作用

目的：探讨唐古特大黄多糖(Rheum tanguticum polysaccharides，RTP)对大鼠应激性胃溃疡的保护作用。方法：用水浸束缚应激法(water immersion and restraint stress，WRS)复制大鼠应激性胃溃疡模型，提前灌胃给予大黄多糖，应激6h后处死动物，观察溃疡指数和胃粘膜损伤程度的变化，检测血清及胃粘膜组织中超氧物歧化酶(SOD)和丙二醛(MDA)的变化。结果：RTP能明显降低应激大鼠胃粘膜溃疡指数和胃粘膜MDA水平，升高血清和胃粘膜SOD活性。结论：RTP对水浸束缚应激引起的大鼠应激性胃溃疡有明显的保护作用，可能是大黄治疗应激性胃溃疡的有效成分之一。     

Gut mucosal uptake and retention characteristics contribute to the high intestinal selectivity of budesonide compared with prednisolone in the rat

BACKGROUND: Budesonide and prednisolone are both effective for the treatment of inflammatory bowel disease, but budesonide produces fewer adverse systemic effects. High first-pass hepatic inactivation of budesonide partially explains its favourable ratio between topical and systemic activity, but it is probable that its uptake and retention in intestinal target tissues are also contributory. AIM: To compare the uptake and retention of radio-labelled budesonide and prednisolone in rat ileal mucosa in vivo. METHODS: 3H-Budesonide and 3H-prednisolone were applied for 10 min directly to a perfused segment of rat ileum in vivo, followed by saline lavage every 10 min. Steroid uptake into the mucosa and submucosa was assessed at 20 min and 4 h. The uptake of budesonide was also measured in allergen-challenged animals vs. saline-challenged controls to assess whether inflammation of the mucosa with ongoing plasma exudation would impair its uptake. RESULTS: Budesonide was better absorbed into ileal tissue (15-fold at 20 min) than prednisolone and better retained (50-fold at 4 h) after topical administration. The uptake of budesonide was not impaired by exudation processes following allergen challenge. CONCLUSIONS: The higher uptake and retention characteristics of budesonide in gut mucosa should contribute to its greater intestinal selectivity compared with that of prednisolone.     

银杏叶提取物的胃粘膜保护作用(英文)

目的:研究银杏叶提取物的胃粘膜保护作用.方法:采用大鼠束缚-冷冻应激(RCS)模型和小鼠无水乙醇损伤模型观察GbE对胃粘膜损伤指数的影响;采用幽门结扎法收集胃液,观察GbE对胃液分泌量,胃液酸度和胃蛋白酶活性的影响;采用硫代巴比妥酸(TBA)法测定胃粘膜及血清中丙二醛(MDA)含量.结果:GbE(25,50,100 mg/kg,bid×5 d,ig)剂量依赖性地抑制RCS和无水乙醇引起的胃粘膜损伤.用药组应激后的胃粘膜损伤指数分别为对照组的58％,43％和31％;用药组乙醇诱发的胃粘膜损伤指数降至对照组的62％,36％和26％;GbE尚能增强西米替丁对胃粘膜的保护作用,但对大鼠胃液分泌量、胃液酸度及胃蛋白酶活性GbE并无明显影响.小鼠经无水乙醇ig后1 h,胃粘膜和血清中的MDA含量显著升高(P<0.01),而GbE(25,50,100 mg/kg,ig)预处理则可以明显抑制MDA的升高.结论:GbE具有胃粘膜保护作用,并且与西米替丁在治疗急性胃粘膜损伤方面具有协同作用.     

A protective effect of melatonin on intestinal permeability is induced by diclofenac via regulation of mitochondrial function in mice

Aim:

This study investigated the effect of intragastrically administered melatonin on intestinal mucosal permeability induced by diclofenac in mice.

Methods:

Intestinal mucosal permeability was induced in mice by intragastric administration of diclofenac (2.5 mg/kg). Melatonin was given intragastrically (10 mg/kg) once per day for 3 d after diclofenac administration. The small intestine was examined macroscopically and microscopically for pathologic injury to the intestinal mucosa. Intestinal mucosal permeability was evaluated by Evans blue and FITC-dextran methods. Mitochondrial functional parameters, including mitochondrial membrane potential, mitochondrial ATPase and succinate dehydrogenase (SDH) activity, were assessed. The malondialdehyde (MDA) and myeloperoxidase (MPO) levels were determined from small intestinal mucosal homogenates.

Results:

As compared with control mice, the permeability, pathologic score, MDA and MPO levels and ulceration of the intestinal mucosa were increased significantly by diclofenac treatment, and a broadened junctional complex and enlarged intercellular space were observed by transmission electron microscopy (TEM). Melatonin treatment significantly reduced the intestinal mucosal permeability, pathologic score, MDA, and MPO levels and ulceration of the intestinal mucosa. By TEM, the small intestine villus morphology and intercellular spaces were nearly normal in melatonin-treated mice. At the level of the mitochondria, melatonin treatment significantly restored the activities of ATPase and SDH.

Conclusion:

The intestinal damage and increased intestinal permeability induced by diclofenac in mice was limited by melatonin; moreover, melatonin preserved several aspects of mitochondrial function.     

Regulation of guinea pig ileal electrolyte transport by M3-muscarinic acetylcholine receptors in vitro

To determine the muscarinic receptor subtype mediating guinea pig ileal mucosal electrolyte secretion, we compared the potencies (Kb) of selective M1 (pirenzepine) (PZ), M2 (AF-DX 116, methoctramine), and M3 [4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladifenidol (HHSiD)] antagonists as inhibitors of carbachol-induced reductions in guinea pig atrial heart rate and ileal longitudinal muscle contractions, responses mediated by M2 and M3 receptors, respectively. Pretreatment with all five muscarinic antagonists shifted the carbachol concentration-response curve to the right, in a manner suggesting competitive antagonism. The following affinity profiles (Kb, nM) were obtained for: 1) ileal mucosa: 4-DAMP (2.7) greater than HHSiD (23.0) greater than PZ (110) greater than or equal to methoctramine (395) greater than AF-DX 116 (784); 2) atrial heart rate: 4-DAMP (9.5) congruent to methoctramine (11) greater than AF-DX 116 (63) greater than HHSiD (222) greater than PZ (256); and 3) ileal longitudinal muscle: 4-DAMP (3.1) greater than HHSiD (21) greater than PZ (143) greater than methoctramine (388) greater than or equal to AF-DX 116 (482). The selectivity profiles of these antagonists suggest that muscarinic receptors in the ileal mucosa more closely resemble those in the ileal muscle (M3) than those in atrial muscle (M2). Moreover, M1-muscarinic receptors appear to be relatively unimportant in mediating the effects of carbachol on short circuit current (ISC). Carbachol-induced increases in ISC were also unaffected by pretreatment with 0.5 microM tetrodotoxin, suggesting that electrolyte transport in the guinea pig ileal mucosa may be mediated, in part, by postsynaptic M3-muscarinic receptors on the enterocytes.     

Interleukin-8 increases acetylcholine response of rat intestinal segments

BACKGROUND: Interleukin-8 (IL-8) is a pro-inflammatory cytokine highly expressed in inflammatory bowel diseases, but whose effects on intestinal motility are unknown. AIM: To characterize the role of IL-8 in the contraction of rat intestinal segments. METHODS: Contractile response to acetylcholine (ACh 10-6 M) in terminal ileal segments (including mucosa) from Wistar rats was measured before and after incubation (15, 30, 60 or 90 min) with IL-8 (1 ng/mL), and after 60 min of incubation with different doses of IL-8 (0, 0.1, 0.5, 1, 10 and 100 ng/mL). The effects of blocking neural transmission with tetrodotoxin (TTX) and inhibiting protein synthesis (cycloheximide) were tested. The contractile response of longitudinal muscle-myenteric plexus preparations (i.e. without mucosa) was measured after 60 min of incubation with 0.1 and 1 ng/mL of IL-8. RESULTS: IL-8 increased ileal contraction induced by ACh 10(-6) M. This augmentation was significant after 60 min of incubation (58%, P=0.01) and persisted after 90 min (18%, P=0.04). A 60-min incubation period showed a dose-related effect, beginning at 0.5 ng/mL (30%, P=0.003) and reaching a peak at 1 ng/mL (58%, P=0.01). The same effect was also observed on colonic segments. TTX did not affect the IL-8 increase of ACh-induced contractions, which was completely abolished by cycloheximide. IL-8 had no significant effect on longitudinal muscle-myenteric plexus preparations. CONCLUSION: In vitro, IL-8 increases contractile response of the ileum to ACh in a dose-dependent manner. This effect is not neurally mediated, but seems to involve protein synthesis by intestinal mucosa.     

The effect of PARS inhibition on ileal histopathology, apoptosis and lipid peroxidation in LPS-induced obstructive jaundice.

In our experimental study, we investigated the protective effect of 3-aminobenzamide (3-AB), the poly (ADP-ribose) synthetase (PARS inhibitor), on the ileal histopathology and the apoptosis in lipopolysaccharide (LPS)-induced inflammation in rats with obstructive jaundice (OJ). We randomized 40 rats into five groups. Group 1: sham group; Group 2: OJ group; Group 3: OJ+LPS; Group 4: OJ+3-AB+LPS; Group 5: OJ+LPS+3-AB. At the fifth day; the rats were jaundiced. In Group 3; 10 mg kg(-1) LPS was injected intraperitoneally at the fifth day and then after 6h the rats were sacrificed. In Group 4; 10 mg kg(-1) 3-AB was administrated intraperitoneally at the fifth day and repeated daily for 3 days and at the eighth day, 10 mg kg(-1) LPS was injected intraperitoneally. In Group 5, 10 mg kg(-1) LPS was injected intraperitoneally at the fifth day and after 6h 10 mg kg(-1) 3-AB was administrated intraperitoneally and repeated daily for 3 days. At the eighth day, rats were sacrificed. Blood samples were taken for detection of serum MDA levels. Ileum samples were taken after relaparotomy for histopathological examination to evaluate the endotoxin-related intestinal injury and Caspase-3 apoptosis and for detection of tissue MDA and ATPase activities. There was marked destruction of villous and crypt epithelial cells and extensive apoptosis in Groups 3 and 5 in histopathological examination. In Group 4, the scores of intestinal mucosal damage and apoptotic cells were reduced significantly (P<0.05). On the other hand, the scores of intestinal mucosal damage and apoptotic cells were not improved in Group 5. After the administration of 3-AB (Group 4), serum and ileal MDA levels decreased, ileal ATPase increased as compared to Groups 1 and 2. Our study showed that 3-AB prevented the mucosal damage and apoptotic loss of intestinal epithelial cells significantly if it was administrated before LPS. However, 3-AB failed to prevent the mucosal damage and apoptotic loss of intestinal epithelial cells significantly if there was established endotoxemia in OJ.     

Analysis of a carcinogen, 4,4'-methylenedianiline, from thermosetting polyurethane during sterilization

Polyurethane (PU) is widely used in medical devices such as potting material in artificial dialysis devices, plasma separators, etc. Gamma-ray irradiation is frequently used for the sterilization of such devices. This paper reports that a carcinogen, 4,4'-methylenedianiline (MDA, p,p'-diaminodiphenylmethane), is produced from medical thermosetting PU by gamma-ray irradiation. Gamma-ray irradiated PU was immersed in methanol or equine serum. The serum was treated with a mixture of 5N HCIO4:acetonitrile (1:10) in order to deproteinate and recover MDA. It was found that MDA is formed from thermosetting PU at around a few ppm in the original sample. The production of MDA increased with increasing irradiation dose. The MDA amount formed was related to the irradiation dose by a second order equation. Results of methanol and serum extraction were similar. Pressurized steam (autoclave) sterilization in place of gamma-ray sterilization was also examined. MDA production was not found in autoclave sterilization procedures. Gel permeation chromatography (GPC) of methanol or N,N-dimethylformamide (DMF) extract of irradiated PU showed that the PU oligomers eluted. Time course of methanol extract of irradiated PU was detected at 245.5 nm. This showed an exponential decline regardless of doses of irradiation.     

超氧化物歧化酶对急性胃粘膜损伤的保护作用

制作不同的动物急性胃粘膜损伤模型，应用外源性超氧化物歧化酶（ＳＯＤ）观测对急性胃粘膜损伤的保护作用。发现ＳＯＤ抑制应激、结扎幽门和阿斯匹林性胃溃疡的形成，降低大鼠胃溃疡形成过程中胃粘膜丙二醛（ＭＤＡ）含量，保护胃粘膜上皮并促进其粘液的分泌，但对胃酸的分泌量无明显影响。提示：ＳＯＤ的胃粘膜保护作用，可能与提高胃粘膜的防御能力有关。