Hart D A, Reno C
McCaig Centre for Joint Injury and Arthritis Research, Faculty of Medicine, University of Calgary HSC, 3330 Hospital Drive NW, Calgary, Alba T2N 4N1, Canada.
Biochim Biophys Acta. 1998 Oct 22;1408(1):35-43. doi: 10.1016/s0925-4439(98)00051-9.
Explants of tissue derived from the medial collateral ligament (MCL) of normal and pregnant NZW rabbits cultured in the presence of substance P (SP), calcitonin gene-related peptide (CGRP), or both neuropeptides were found to have altered mRNA levels for a number of relevant molecules. Using a very efficient RNA isolation method, semi-quantitative RT-PCR and rabbit-specific primers, mRNA for growth factors (TGFbeta, bFGF, IGF-2, ET-1), cytokines (IL-1, TNF), enzymes (COX-2, iNOS), metalloproteinases (collagenase, stromelysin) and metalloproteinase inhibitors (TIMP-1, TIMP-2) were assessed after culture with or without neuropeptide. The results indicate that SP was effective in lowering mRNA levels for all of the molecules assessed in RNA from normal ligaments except IL-1beta, IGF-2 and TIMP-1, for which there was no significant effect. Similarly, CGRP was effective in lowering mRNA levels for all molecules except TNF, ET-1 and the TIMPs. The extent of the lowering of mRNA levels was both molecule-specific and neuropeptide-specific. When the experiments were repeated with ligament tissue from pregnant animals, a very different pattern of responsiveness to the neuropeptides was observed. While mRNA levels for 9/12 genes assessed were significantly affected by SP when normal MCL tissue was investigated, pregnancy abolished all significant responsiveness to this neuropeptide except for iNOS mRNA levels. In the case of iNOS mRNA, SP induced an increase in the steady-state levels, the opposite to what was observed with tissue from non-pregnant animals. For CGRP and SP+CGRP, tissue from pregnant animals was still responsive, but the pattern of responsiveness was changed from strictly a lowering of steady-state mRNA levels to elevations in mRNA levels for a number of genes. These findings indicate that mRNA levels for a number of genes can be influenced by neuropeptides known to be in ligaments. Thus, neuropeptides likely are important regulators of ligament cell metabolism. As the responsiveness to SP was nearly completely abolished during pregnancy, neuroregulatory influences mediated by this peptide are altered in the pregnant female. This loss of responsiveness to SP may also be one aspect of the analgesia associated with pregnancy.
在存在P物质（SP）、降钙素基因相关肽（CGRP）或两种神经肽的情况下，对正常和怀孕的新西兰白兔内侧副韧带（MCL）来源的组织外植体进行培养，发现许多相关分子的mRNA水平发生了改变。使用一种非常有效的RNA分离方法、半定量逆转录聚合酶链反应（RT-PCR）和兔特异性引物，在用或不用神经肽培养后，对生长因子（转化生长因子β、碱性成纤维细胞生长因子、胰岛素样生长因子-2、内皮素-1）、细胞因子（白细胞介素-1、肿瘤坏死因子）、酶（环氧化酶-2、诱导型一氧化氮合酶）、金属蛋白酶（胶原酶、基质金属蛋白酶）和金属蛋白酶抑制剂（金属蛋白酶组织抑制剂-1、金属蛋白酶组织抑制剂-2）的mRNA进行了评估。结果表明，SP能有效降低正常韧带RNA中除白细胞介素-1β、胰岛素样生长因子-2和金属蛋白酶组织抑制剂-1外所有评估分子的mRNA水平，而对这三种分子无显著影响。同样，CGRP能有效降低除肿瘤坏死因子、内皮素-1和金属蛋白酶组织抑制剂外所有分子的mRNA水平。mRNA水平降低的程度既有分子特异性，也有神经肽特异性。当用怀孕动物的韧带组织重复实验时，观察到对神经肽的反应模式非常不同。在研究正常MCL组织时，当评估的12个基因中有9个基因的mRNA水平受到SP的显著影响，但怀孕后除诱导型一氧化氮合酶mRNA水平外，对该神经肽的所有显著反应都消失了。就诱导型一氧化氮合酶mRNA而言，SP诱导其稳态水平升高，这与非怀孕动物组织的情况相反。对于CGRP和SP + CGRP，怀孕动物的组织仍然有反应，但反应模式从严格降低稳态mRNA水平转变为许多基因的mRNA水平升高。这些发现表明，许多基因的mRNA水平可受到已知存在于韧带中的神经肽的影响。因此，神经肽可能是韧带细胞代谢的重要调节因子。由于怀孕期间对SP的反应几乎完全消失，该肽介导的神经调节作用在怀孕雌性动物中发生了改变。对SP反应性的丧失也可能是与怀孕相关的镇痛作用的一个方面。
