摘要
目的 探讨固定剂及固定时间对石蜡包埋RNA病毒感染组织中RNA降解及标本病理形态学的影响。方法 取汉坦病毒 76～ 118株接种的乳鼠感染组织 ,以沉淀脱水类固定剂 10 0 %甲醇、75 %乙醇、10 0 %丙酮以及醛类固定剂 4 %的多聚甲醛磷酸缓冲液 (PFA)、10 %福尔马林共 5种固定剂 ,在室温下固定病毒感染组织 6h、12h、2 4h和 4 8h后 ,将各种方式固定后的病毒感染组织标本进行石蜡包埋 ,组织包埋块在室温下保存 1年～ 1 5年 ,然后进行切片HE染色 ,镜下观察各种固定剂对组织病理形态学的影响。各种方式固定后石蜡包埋组织中以RT_PCR扩增出不同片段长度的病毒RNA及 β_actinRNA序列 ,以判断各种固定方式对病毒RNA和细胞总RNA降解的影响。每种固定方法对RNA的影响是通过获得产物最大长度或对长片段扩增的可重复性及假阴性结果的多少来反映。结果 病理切片结果显示 ,经 10 %福尔马林和 4 %PFA固定的石蜡切片 ,组织结构完整清晰 ,组织经其他三种沉淀脱水类固定剂固定后有不同程度收缩 ,但不影响组织病理形态学检测 ,其中甲醇固定的收缩程度最小。从固定后石蜡包埋组织中提取RNA进行RT_PCR扩增时 ,扩增的效果受固定剂类型和固定时间的影响 ,同时也受扩增的基因片段长度的影响。在可达到固定作用的前提下 ,同一固定?
Objective To study the effect of fixatives and fixed_time on the morphology and detectability of viral RNA from paraffine_embedded tissues by RT_PCR. Methods The infected tissues removed from milky mice inoculated with Hantavirus (HTV) strain 76～118 were fixed with dehydration fixatives including 100% methanol,70% ethanol and 100% acetone,and other two fixatives including 4% PFA 10% formalin at room temperature for 6h,12h,24h and 48h respectively.The infected tissues after fixed with different fixatives and fixed_time were embedded paraffin and preserved for 1～1.5 years at room temperature.Then paraffin_embedded tissues were sectioned for morphological observation.RT_PCR amplification of RNA extracted from paraffin_embedded tissues.The degrading effect of fixatives and fixed_time on total RNA and viral RNA was analyzed by the ability to amplified the longest sequences,the repetition of longer sequence amplification and the false_negative results. Results There are no morphological differences between tissues fixed with 4%PFA and 10% formalin.The sections were fragile,and histological examination revealed regional tissue shrinkage with other three dehydration fixatives.Which did not cause examination on morphology.And the morphology of sections prepared by methanol fixation was preserved better than that of acetone_ or ethanol_fixed sections.The degrading effect of fixatives and fixed_time on total RNA and viral RNA.Smaller than 403bp fragment from virus and β_actin RNA sequences of all fixed paraffin_embedded tissues were detected by nest RT_PCR.All three dehydration fixatives were able to preserve relatively higher_molecular_weight RNA,compared with formalin and 4% PFA. Conclusions Detecting viral RNA by RT_PCR using is feasible.In retrospective studies on virus_infected diseases with paraffin_embedded tissues,knowledge of the effect of tissue fixation on fragile RNA molecules is necessary for interpreting negative PCR results.
