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目的探讨应用于乙肝携带者卵巢颗粒细胞涂片原位杂交技术。方法选择6例乙肝携带者颗粒细胞涂片原位杂交,每例涂片12张,分别进行蛋白酶K浓度(1μg、2μg、3μg)、消化时间(1min、3min、5min)、探针浓度(1∶10、1∶20、1∶30)及显色时间(10min、20min、30min)优化。结果 3例颗粒细胞核均检测到HBVDNA阳性信号。最佳反应条件为,蛋白酶K浓度为2μg,消化时间为3min,探针∶预杂交液比为1∶20,显色时间为20min。结论蛋白酶K浓度、消化时间、探针浓度及显色时间是原位杂交中关键性步骤,该技术可用于颗粒细胞涂片的研究。
Objective To investigate the in situ hybridization technique of ovarian granulosa cell smear applied to hepatitis B carriers. Methods Six cases of hepatitis B carriers granulosa cell smear were selected and in situ hybridization was performed. Twelve smears were used in this study. Proteinase K concentration (1μg, 2μg, 3μg), digestion time (1min, 3min, 5min) 1:10, 1:20, 1:30) and color development time (10min, 20min, 30min). Results HBVDNA positive signals were detected in all three granulosa cells. The optimal reaction conditions were as follows: the concentration of proteinase K was 2μg, the digestion time was 3min, the ratio of probe to prehybridization was 1:20 and the color time was 20min. Conclusions Proteinase K concentration, digestion time, probe concentration and color time are the key steps of in situ hybridization. This technique can be used for the study of granular cell smears.