Semiquantification and localization of brain cytokine by in situ RT-PCR

为了了解实验性自身免疫性脑脊髓炎（EAE）过程中星形胶质细胞与小胶质细胞之间的相互作用，我们在组织上扩增细胞因子mRNA后，采用原位RT-PCR方法定位中枢神经系统（CNS）细胞因子mRNA。冷冻或石蜡包埋切片用蛋白酶K预处理，用SuperScript预扩增系统将切片上的mRNA逆转录为cDNA。然后利用tgf - β - 1特异性引物对在热循环器中通过直接或间接方法扩增cDNA。直接法在DIG- dudp存在下扩增cDNA，用免疫染色法检测整合的DIG。在间接方法中，使用未标记的核苷酸进行扩增，然后使用dig标记的tgf - β - 1特异性探针进行原位杂交。结果表明，尽管经过多次预处理，DIG-dUDP整合到基因组DNA缺口中的所谓“DNA修复伪影”仍无法消除。结果，切片上的细胞核均呈阳性染色。另一方面，在间接方法中没有观察到这种伪影。在EAE的高峰期，许多浸润炎性细胞和小胶质细胞染色呈阳性。然而，难以获得稳定的染色，部分原因是扩增的PCR产物在组织上的固定较差。总之，最佳的预处理程序必须进行，以获得稳定和特异性染色。

In order to know the interaction between astrocytes and microglia during the course of experimental autoimmune encephalomyelitis (EAE), we tried to localize the cytokine mRNA in the central nervous system (CNS) by in situ RT-PCR after amplification of cytokine mRNA on tissue. Frozen or paraffin-embedded sections were pretreated with proteinase K and mRNA on sections was reverse transcribed into cDNA with the SuperScript Preamplification System. cDNA was then amplified using a TGF-beta1-specific primer pair in a thermal cycler by either the direct or indirect method. In the direct method, cDNA was amplified in the presence of DIG-dUDP and integrated DIG was detected by immunostaining for DIG.In the indirect method, amplification was performed using unlabeled nucleotides followed by in situ hybridization with DIG-labeled TGF-beta1-specific probe.It was revealed that so-called " DNA repair artifact " in which DIG-dUDP was integrated into nicks of genomic DNA could not be eliminated in spite of several pretreatment procedures. As a result, all the nuclei on section were stained positively. On the other hand, such artifact was not observed in the indirect method. At the peak stage of EAE,many infiltrating inflammatory cells and microglia were stained positively. However, it was difficult to obtain stable staining partly because of poor fixation of amplified PCR products on tissue. In conclusion, optimal pretreatment procedures must be undertaken to obtain stable and specfic staining.