Mee A P, Dixon J A, Hoyland J A, Davies M, Selby P L, Mawer E B
Bone Disease Research Centre, University Department of Medicine, Manchester Royal Infirmary, UK.
Bone. 1998 Aug;23(2):171-5. doi: 10.1016/s8756-3282(98)00079-9.
Previous evidence implicating paramyxoviruses in the aetiopathology of Paget's disease of bone has been controversial. While several groups have demonstrated the presence of paramyxoviruses using electron microscopy, immunohistochemistry, and molecular biological techniques, others have found no evidence of viruses using reverse transcriptase-polymerase chain reaction (RT-PCR). We have previously provided evidence that canine distemper virus (CDV) is present in approximately 65% of samples of pagetic bone, using in situ hybridization and RT-PCR; however, these results have been criticized. To further investigate the possible Role of CDV, we have now developed the technique of in situ-RT-PCR (IS-RT-PCR) to examine for the presence of CDV-nucleocapsid (CDV-N) ribonucleic acid (RNA) in pagetic bone. Control samples consisted of uninvolved sites from patients with the disease, normal bone, and several active remodeling states. IS-RT-PCR was optimized to detect CDV-N using distemper-infected vero cells. The specificity of the technique was confirmed using vero cells infected with CDV, which showed amplified signal following IS-RT-PCR, and cells infected with measles virus (MV), in which no positive signal for CDV was detected by IS-RT-PCR. Following conventional in situ hybridization, CDV-N was detectable in 10 of 15 pagetic bone samples. However, after five, and particularly 10, cycles of IS-RT-PCR, CDV-N was found in all 15 samples. There was no evidence of CDV in four samples from uninvolved sites from pagetic patients, or in any of the other control samples. In this study, using the novel technique of IS-RT-PCR, CDV was found to be present in 100% of pagetic samples examined. There was no evidence of the virus in any of the control samples, including samples of bone from uninvolved sites from patients with Paget's disease. These results provide additional proof that CDV is present within pagetic bone and further support the hypothesis that paramyxoviruses are involved in the etiopathology of Paget's disease.
先前有关副粘病毒与骨Paget病病因病理相关的证据一直存在争议。虽然有几个研究小组利用电子显微镜、免疫组织化学和分子生物学技术证实了副粘病毒的存在，但其他研究小组通过逆转录聚合酶链反应（RT-PCR）却未发现病毒存在的证据。我们之前利用原位杂交和RT-PCR技术提供证据表明，约65%的骨Paget病样本中存在犬瘟热病毒（CDV）；然而，这些结果受到了批评。为了进一步研究CDV可能的作用，我们现在开发了原位RT-PCR（IS-RT-PCR）技术，以检测骨Paget病样本中CDV核衣壳（CDV-N）核糖核酸（RNA）的存在。对照样本包括该病患者的未受累部位、正常骨以及几种活跃的重塑状态。通过感染犬瘟热的非洲绿猴肾细胞（Vero细胞）对IS-RT-PCR进行优化以检测CDV-N。利用感染CDV的Vero细胞证实了该技术的特异性，这些细胞在IS-RT-PCR后显示出扩增信号，而感染麻疹病毒（MV）的细胞在IS-RT-PCR中未检测到CDV的阳性信号。经过传统的原位杂交后，在15份骨Paget病样本中的10份中可检测到CDV-N。然而，经过5轮，尤其是10轮IS-RT-PCR后，在所有15份样本中均发现了CDV-N。在骨Paget病患者未受累部位的4份样本或任何其他对照样本中均未发现CDV的证据。在本研究中，使用新的IS-RT-PCR技术，在所检测的100%骨Paget病样本中均发现了CDV。在任何对照样本中均未发现该病毒的证据，包括骨Paget病患者未受累部位的骨样本。这些结果进一步证明CDV存在于骨Paget病骨组织中，并进一步支持了副粘病毒参与骨Paget病病因病理的假说。
