免疫组织化学染色防脱片几种方法的比较及PCNA、MMP-7、VEGF、Cath-D和HSP70在食管癌中的表达及临床意义

发布时间：2018-01-05 00:23

本文关键词：免疫组织化学染色防脱片几种方法的比较及PCNA、MMP-7、VEGF、Cath-D和HSP70在食管癌中的表达及临床意义　出处：《河北医科大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的: 在今天的免疫学实验研究及病理诊断中,免疫组织化学(免疫组化)是一种应用较广的标记免疫技术。免疫组化具有操作简便,结果易于观察,特异性较高等优点,但免疫组化染色结果并不十分不稳定,影响因素较多。研究发现,玻片的清洁程度,不同的抗原修复方法(微波、高压锅、水浴煮沸和酶消化),缓冲液的pH 值,是否经高压处理切片以及不同公司的试剂类型对免疫组化染色都有影响。有些学者致力于探讨免疫组化染色的影响因素,制定出统一的操作规范和评定标准,以保证免疫组化的正确使用。石蜡切片在免疫组化染色中的脱片问题由来已久。切片经抗原修复和缓冲液的长时间浸泡后,往往会部分或全部从载玻片上脱落,影响染色结果的判断。不同的粘附剂和粘附剂不同的涂胶方法防脱片效果不尽相同。我们在工作中也经常遇到石蜡切片脱落问题,为此本实验对三种粘附剂即APES(氨醛三已氧基硅烷),多聚赖氨酸和白乳胶的染色结果和防脱片效果作了比较;又进一步对多聚赖氨酸的涂胶方法作了比较和改进,成功地总结出一种重复性好的多聚赖氨酸涂胶法。方法: 本实验用3 种粘附剂APES、多聚赖氨酸、白乳胶涂胶,APES 涂胶按说明书,白乳胶涂胶参照文献。多聚赖氨酸涂胶除了按说明书用浸泡法以外,还对其涂胶条件做了探讨,0.01%多聚赖氨酸不同体积(40μL, 80μL, 120μL,160μL)单次涂胶;同量多聚赖氨酸不同浓度单次涂胶(0.02%多聚赖氨酸40μL、0.0133%多聚赖氨酸60μL、0.01%多聚赖氨酸80μL);0.01%多聚赖氨酸80μL 单次涂胶和双次涂胶;0.01%多聚赖氨酸80μL 单次涂胶后37℃保存的影响(0.01%多聚赖氨酸80μL 单次涂胶后当日贴片或在37℃放置4 周后再贴片)。柠檬酸加热做抗原修复以后进行免疫组化染色,HPIAS-1000 高清晰度彩色病理系统测量切片染色强度;肉眼观察脱片情况。结果: 1 不同粘附剂染色强度和防脱片效果的比较特异性显色OD 均值,APES 组为0.1980±0.0120,多聚赖氨酸浸泡法组为0.2020±0.0082,白乳胶组为0.2580±0.0164。非特异性显色OD 均值,APES 组为0.0105±0.0010,多聚赖氨酸浸泡法组为0.0106±0.0009,白乳胶组为0.0298±0.0027。APES 组和多聚赖氨酸浸泡法组特异性显色、非特异性显色的差别均没有显著性(P0.05);白乳胶组特异性显色、非特异性显色均显著强于APES 组(P0.01)和多聚赖氨酸浸泡法组(P0.01)。特异性显色OD 均值除以非特异性显色OD 均值,APES 组为18.81,多聚赖氨酸浸泡法组为19.08,白乳胶组为8.66。三种粘附剂的完整贴片百分率分别为APES 组55%,多聚赖氨酸浸泡法组57.5%,白乳胶组50%,三组均不能获得高比例的完整贴片。 2 多聚赖氨酸不同涂胶方法防脱片效果的比较 1)相同浓度不同体积多聚赖氨酸的比较0.01%多聚赖氨酸40μL, 80μL, 120μL, 160μL 四种不同体积单次涂胶的完整贴
[Abstract]:Objective: in today's study on immunological function and pathological diagnosis, immunohistochemical (IHC) staining technique is a widely used. Immunohistochemistry has the advantages of simple operation, easy observation results, the specificity is relatively high, but the results of immunohistochemical staining and not very unstable factors research found that the clean degree of slide, different methods of antigen retrieval (microwave, pressure cooker, water boiling and enzyme digestion), buffer pH value, whether by high pressure treatment section and different company type reagents on immunohistochemical staining are affected. Some scholars committed to the study of immunohistochemical staining effect factors, formulate uniform operating standards and evaluation standards, to ensure the correct use of immunohistochemistry in paraffin. Immunohistochemical staining in the unfalling sections of the long-standing problem. The antigen retrieval and buffer time Between after soaking, often partly or wholly from the slide off, affect the judgement of the staining results. Gluing method of adhesion agent and adhesion agent of different anti stripping effect is not the same. In our work we often encounter paraffin shedding problem, this experiment of three kinds of adhesive APES (ammonia three aldehyde silane), poly lysine staining and white latex and anti off effect were compared; and further to the polylysine coating method of acid ammonia were compared and improved, successfully concluded a reproducible polylysine coating method.
Methods: 3 adhesives APES, polylysine and white latex were applied to the experiment, and APES coating was applied according to the instructions. The white latex was coated with reference literature.
On 0.01% poly-L-lysine of different volume (40 L, 80 L, 120 L, 160 L) gelatinizedonce; the same amount of poly-L-lysine of different concentrations of gelatinizedonce (0.02% poly lysine 40 L 0.0133% poly lysine 60 L 0.01% poly lysine 80 L); 0.01% poly lysine 80 L single coating and double coating; 0.01% poly lysine save 37 C 80 L gelatinizedonce after 0.01% (poly lysine 80 L gelatinizedonce after the patch or in 37 C placed 4 weeks before the patch). After antigen repairing withcitric acid by immunohistochemical staining, HPIAS-1000 high-resolution color measurement system pathological staining intensity; the unfalling observed with naked eye.
Result:
1 different adhesive strength and anti staining effect and comparison of specific color OD mean, APES group is 0.1980 + 0.0120, poly-L-lysine dipping group is 0.2020 + 0.0082, white latex color group mean OD was 0.2580 + 0.0164. nonspecific, APES group is 0.0105 + 0.0010, poly lysine ammonia acid immersion group is 0.0106 + 0.0009, white latex group is 0.0298 + 0.0027.APES group and poly-L-lysine dipping group specific color, non-specific color differences were not significant (P0.05); white latex specific color, non-specific staining was significantly stronger than that of APES group (P0.01) and poly-L-lysine dipping group (P0.01). The color OD value divided by the non-specific staining of OD mean specificity, APES group was 18.81, poly-L-lysine dipping group was 19.08, white latex group complete patch percentage of 8.66. of three kinds of adhesion agent were 55% in group APES, poly-L-lysine dipping Group 57.5%, white latex group 50%, three groups were not complete patch to obtain a high proportion.
Comparison of the effect of more than 2 polylysine with different coating methods
1) the comparison of the same concentration of polylysine with different volumes of polylysine 0.01% polylysine 40 mu L, 80 mu L, 120 micron L, 160 L four different volume single coating adhesive

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R735.1;R392

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