组织切片肾小球微分离技术在肾脏病研究中的应用

MICRODISSECTION OF SINGLE GLOMERULUS FROM ARCHIVAL SECTION OF RENAL BIOPSY AND ITS APPLICATION IN MOLECULAR NEPHROLOGY

目的 :建立组织切片肾小球微分离技术 ,并将其应用于肾脏病分子病因学的研究。 方法 :选取肾脏病患者的肾活检组织石蜡切片。选取结构正常的肾小球、节段硬化肾小球和球性硬化肾小球分别进行肾小球微分离。一步法提取微分离肾小球DNA。PCR 微型凝胶电泳检验DNA质量。质量合格DNA应用于高保真PCR扩增TGF βⅡ型受体基因片段和聚丙烯酰胺凝胶电泳。 结果 :在成功获取肾小球丝球体的同时 ,相邻组织肾小囊壁保留完好 ;微分离小球一步法提取的DNA合格率 87 5 % ;各样本经高保真PCR扩增后 ,杂带少 ,目的条带清晰。密度随肾小球硬化程度递减。 结论 :本研究利用常规肾活检组织石蜡切片建立了肾小球微分离技术 ,进而用微分离肾小球提取DNA成功地进行了DNA扩增 。

To establish the technique of glomerular microdissection from archival renal biopsy sections and to apply it in molecular nephrology. METHODOLOGY ]Formalin fixed,paraffin embedded 5μm thick renal tissue sections were used for microdissection under phase microscope.A thin layer of distill water was applied onto the sections before the aimed glomeruli were microdissected.After microdissection,DNA was extracted from the procured glomeruli by a single step assay.In the pressent study,non sclerotic glomeruli,segmental sclerotic glomeruli and global sclerotic glomeruli were respectively microdissected from renal biopsy sections of thirty two patients with focal segmental glomerulosclerosis(FSGS).The DNA quality was examined by PCR amplification and minigel electrophoresis.Qualified DNA samples were further applied in high fidelity amplification of TGF β type Ⅱ receptor gene and denatured polyacrylamide gel electrophoresis(PAGE). RESULTS All glomeruli were successfully procured by manual microdissection with Bowmans capsule remaining intact.The DNA extracted by the single step assay was of high quality and 87 5% samples were qualified for further molecular studies.Most of the samples that failed in PCR amplification derived from sclerotic glomeruli.High fidelity amplification of TGF β type Ⅱ receptor gene using the qualified DNA samples and subsequent denatured PAGE revealed clear cut specific bands.The density of the bands were decreased accordingly with the development of glomerulosclerosis. CONCLUSION Manual microdissection of glomeruli is a promising technique of high efficiency in molecular nephrology.