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目的 建立一种适用于检测人巨细胞病毒（ Ｈ Ｃ Ｍ Ｖ）感染新生乳鼠大脑皮质神经元原代培养物中病毒 Ｄ Ｎ Ａ 的原位杂交法。方法 制备和培养新生乳鼠大脑皮质神经元并进行病毒感染；在感染后的第３ ，５ 天及第１ ～４ 周连续取样用地高辛标记的 Ｈ Ｃ Ｍ Ｖ 即刻早期抗原（ Ｍ Ｉ Ｅ） 和晚期抗原（ Ｌ Ａ） 寡核苷酸特异性探针分别进行核酸原位杂交，检测受染神经元内相应病毒核酸及分布。结果 Ｍ Ｉ Ｅ 寡核苷酸探针可在 Ｈ Ｃ Ｍ Ｖ 感染后第４ ～７ 天检出神经元核内 Ｈ Ｃ Ｍ Ｖ Ｄ Ｎ Ａ；而 Ｌ Ａ 寡核苷酸探针则可稳定地检出感染后第７ ～２８天细胞培养物中的相应病毒核酸；并显示： Ｈ Ｃ Ｍ Ｖ Ｄ Ｎ Ａ 杂交阳性信号在感染的早期主要分布在核内，呈灶性；而在感染的晚期，杂交阳性信号均匀分布于受染神经元的核内及胞浆内。结论 地高辛标记的 Ｈ Ｃ Ｍ Ｖ Ｄ Ｎ Ａ Ｍ Ｉ Ｅ 和 Ｌ Ａ 寡核苷酸探针在病毒感染机制研究以及临床诊断等方面，具有广泛的应用前景。
Objective To establish an in situ hybridization assay for the detection of virus D N A in primary cultures of neonatal rat cortical neurons infected with human cytomegalovirus (H C M V). Methods Neonatal rat cerebral cortical neurons were prepared and cultured for virus infection. Digoxin-labeled H C M V immediate early antigen (M I E) was serially sampled on days 3, 5 and 1 to 4 after infection. And late antigen (L A) oligonucleotide-specific probes were used for nucleic acid in situ hybridization to detect the corresponding virus nucleic acid within the infected neurons and distribution. Results M I E oligonucleotide probes were able to detect H C M V D N A in the nucleus of neurons from day 4 to day 7 after H C M V infection, whereas L A oligonucleotide probes could stably The corresponding viral nucleic acids in the cell cultures were detected on the 7th to 28th day after infection and showed that the positive signals of the hybridization of the H C M V D N A were mainly located in the nucleus and focal in the early stage of infection and focal in the late stage of infection , Hybrid positive signal evenly distributed in the nucleus and cytoplasm of infected neurons. Conclusion Digoxin-labeled H C M V D N A M I E and L A oligonucleotide probes have broad application prospects in the study of viral infection mechanism and clinical diagnosis.