Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization.
Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization.
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DOI：
10.1038/ki.1991.234
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发表时间：
1991-09
影响因子：
19.6
通讯作者：
A. Yoshimura;K. Gordon;C. Alpers;J. Floege;P. Pritzl;R. Ross;W. Couser;D. Bowen-Pope;Richard J. Johnson
中科院分区：
文献类型：
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作者：
A. Yoshimura;K. Gordon;C. Alpers;J. Floege;P. Pritzl;R. Ross;W. Couser;D. Bowen-Pope;Richard J. Johnson
原位杂交检测系膜增生性肾炎肾小球PDGF B链mRNA表达我们使用原位杂交技术，以确定是否可以检测到表达PDGF B链mRNA的细胞在系膜增生性肾炎模型中的大鼠诱导的抗体针对Thy 1抗原存在于系膜细胞膜上。该方法涉及与地高辛标记的鼠PDGF B链cRNA探针杂交，然后用抗地高辛-碱性磷酸酶缀合物检测，随后进行比色反应。在正常大鼠（N = 4）中，大部分肾小球（74%）的PDGF B链mRNA阴性，而65%的肾小球系膜增生性肾炎大鼠（N = 4）的肾小球中有节段性或弥漫性PDGF B链mRNA染色的系膜模式。使用半定量量表测量的差异具有显著性（平均评分0.4 ± 0.2 vs. 1.9 ± 0.2;量表0至3+ ; P < 0.001）。PDGF B-链mRNA阳性细胞的增加定位于细胞过多的区域，并且与用特异性单克隆抗体免疫染色的PDGF B-链阳性细胞的显著增加相关（0.8 ± 0.1 vs. 1.7 ± 0.4，等级0至3+，正常大鼠vs.患病大鼠，P < 0.005）。补体耗竭，这阻止了系膜细胞增殖，也阻止了表达PDGF B链mRNA和蛋白的细胞的增加。因此，这种原位杂交的方法可以成功地检测细胞表达PDGF mRNA的活动性肾小球肾炎，并可能是有用的检测细胞表达基因的其他生长因子和细胞因子在人类和实验模型的肾小球损伤。
Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization. We used the technique of in situ hybridization to determine if cells expressing PDGF B-chain mRNA can be detected in a model of mesangial proliferative nephritis in the rat induced with antibody directed against the Thy 1 antigen present on the mesangial cell membrane. The method involved hybridization with a digoxigenin-labeled cRNA probe for the murine PDGF B-chain followed by detection with an anti-digoxigenin-alkaline phosphatase conjugate and subsequent colorimetric reaction. In normal rats (N = 4), the majority of glomeruli (74%) were negative for PDGF B-chain mRNA, whereas 65% of glomeruli from rats with mesangial proliferative nephritis (N = 4) had segmental or diffuse staining for PDGF B-chain mRNA in a mesangial pattern. The difference, as measured using a semiquantitative scale, was significant (mean scores 0.4 ± 0.2 vs. 1.9 ± 0.2; scale 0 to 3+ ; P < 0.001). The increase in PDGF B-chain mRNA positive cells localized to areas of hypercellularity and was associated with a significant increase in cells positive for PDGF B-chain by immunostaining with a specific monoclonal antibody (0.8 ± 0.1 vs. 1.7 ± 0.4, scale 0 to 3+ , normal vs. diseased rats, P < 0.005). Complement depletion, which prevents the mesangial cell proliferation, also prevented the increase in cells expressing PDGF B-chain mRNA and protein. Thus, this method of in situ hybridization can successfully detect cells expressing PDGF mRNA in active glomerulonephritis, and may be useful for detecting cells expressing genes for other growth factors and cytokines in both human and experimental models of glomerular injury.