生物素标记HBVDNA探针原位杂交碱性磷酸酶过氧化物酶显色检测肝内HBVDNA

In Situ Hybridization for Detecting HBV—DNA in Liver:using Biotinylated Probe,Staining with Avidin—Alkaline Phosphatese Peroxydase Complex.

本研究应用国产生物素标记HBVDNA探针,于福尔马林固定,石蜡包埋肝组织切片上,建立了碱性磷酸酶过氧化物酶显色的原位杂交技术,在此过程中发现,蛋白酶的消化是杂交能否成功的关键因素,酸、三乙醇酐、洋地黄的预处理,能促进探针参入,降低非特异背景,减低蛋白酶作用浓度或作用时间,因而提高实验稳定性、特异性。并用这一技术检测了34例肝活检和4例尸检肝组织,结果:尸肝组织均检出了HBV—DNA,其中2例核型见于肝硬化,肝癌旁组织,而2例浆型HBV—DNA见于慢活肝,亚重肝。34例肝活检组织中,只检出12例浆型HBV—DNA,其中CAH—CPH无症状携带者中检出率分别为42.9％,38.8％,33.3％,均明显高于肝硬化组织(16.7％)(P<0.05)。由此可见浆型HBV—DNA分布同病变轻重程度的关系可能并不密切,不同分布型的HBV—DNA可能只反映HBV的复制状态。

A rapid and reproducible technique for in situ hybridization. Using national produced biotin labelled HBV-DNA probe,on formalin fixed,paraffin embedded liver tissue,was developed. It was found that protelytic digestion of tissue is critical element. while the pretreatment of hydrochloric acid,triethanolamine and digitonin can improve the penetration of probe, and decrease the background of non-specific staining. Using this method ,the HBV-DNA was detected in 34cases of biopsies and 4 cases of autopsies the results show that the 4 autopsies which were positive for HBsAg and HBcAg, were positive for HBV-DNA. Among them, two case of nucleous HBVDNA were found in tissues from a cirrhosis and a hepatocarcinoma patient,while tvo cases of cytoplasmic HBV-DNA were in CAH,subfulminat hepatitis tissue respectively. Among 34 cases biopsies ,only twelve Cytoplasmic HBV-DNA were detected,The positive rates in CAH,CPH,and non-symptom HBsAg carrier were 42.8%,38.9%and 33.3%respectively. Those are significantly high than that in cirrhosis (16.7%)P<0.05. This results suggests that: (1) the method discribed here is a rapid,stable and special technique (2) There may be not a closely relationship between the expression of cytoplasmic HBV-DNA and the damage of tissue,The different forms of HBVDNA perhaps only the replication of HBV.