Kirk A D, Bollinger R R, Finn O J
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
Hum Immunol. 1995 Jun;43(2):113-28. doi: 10.1016/0198-8859(94)00158-m.
Cytokine mRNA analysis was performed on human renal allograft needle core biopsies by a PCR-based assay. The assay was specifically developed to be capable of simultaneous analysis of multiple interleukin transcripts (IL-1-IL-12), as well as those of other relevant cytokines, by one person in less than 1 day from cultured cells or directly from tissue samples. It was initially used on preparations containing known amounts of plasmid DNA encoding individual cytokine cDNA sequences, confirming that the sensitivity of this technique was both well defined and comparable for all target sequences tested. Analysis of human PBLs prior to stimulation, after polyclonal stimulation with PHA and after simultaneous treatment with PHA and MP or CyA, was also performed to show a proportional relationship between mRNA levels measured by PCR and protein release measured by ELISA (R2 = 0.86). This correlation was not adversely altered by pharmacologic immunosuppression by MP or CyA. Thus, this method of PCR primer design and usage was appropriate for the clinical study of cytokine mRNA levels during allograft rejection. Direct study of cytokine mRNA in allograft biopsy tissue showed that IL-2 was specifically and significantly (p = 0.006) elevated during ACR when compared to other causes of graft dysfunction. Transcripts from the IFN-gamma and IL-6 genes were also increased in ACR (p = 0.001 and 0.017, respectively), whereas increased IL-8 mRNA was correlated with irreversible loss of graft function (p = 0.02). TNF-alpha, IL-1 beta, and IL-10 gene transcripts were also detected during ACR, but were not quantitatively increased compared to other forms of graft injury (p > 0.2). We conclude that acute cellular rejection is associated with intragraft mRNA from the IL-2 gene. Other transcripts, including those from the IFN-gamma, IL-6, and IL-8 genes, are detected in increased amounts during this process. Messenger RNA from the TNF-alpha, IL-1 beta and IL-10 genes is also detected during ACR, but the presence of these transcripts is not exclusive to this process.
通过基于聚合酶链反应（PCR）的检测方法，对人肾移植针芯活检组织进行细胞因子信使核糖核酸（mRNA）分析。该检测方法经过专门开发，能够由一人在不到1天的时间内，从培养细胞或直接从组织样本中同时分析多种白细胞介素转录本（IL-1 - IL-12）以及其他相关细胞因子的转录本。最初，该方法用于含有已知量编码单个细胞因子互补脱氧核糖核酸（cDNA）序列的质粒DNA的制剂，证实了该技术对所有测试靶序列的灵敏度均已明确界定且具有可比性。还对人外周血淋巴细胞（PBL）在刺激前、用植物血凝素（PHA）进行多克隆刺激后以及同时用PHA和霉酚酸酯（MP）或环孢素A（CyA）处理后进行了分析，以显示通过PCR测量的mRNA水平与通过酶联免疫吸附测定（ELISA）测量的蛋白质释放之间的比例关系（R2 = 0.86）。MP或CyA的药物免疫抑制并未对这种相关性产生不利影响。因此，这种PCR引物设计和使用方法适用于移植排斥反应期间细胞因子mRNA水平的临床研究。对移植活检组织中细胞因子mRNA的直接研究表明，与其他移植功能障碍原因相比，急性细胞排斥反应（ACR）期间白细胞介素 - 2（IL-2）特异性且显著升高（p = 0.006）。γ干扰素（IFN-γ）和白细胞介素 - 6（IL-6）基因的转录本在ACR中也增加（分别为p = 0.001和0.017），而白细胞介素 - 8（IL-8）mRNA增加与移植功能的不可逆丧失相关（p = 0.02）。肿瘤坏死因子 - α（TNF-α）、白细胞介素 - 1β（IL-1β）和白细胞介素 - 10（IL-10）基因转录本在ACR期间也被检测到，但与其他形式的移植损伤相比，其数量没有定量增加（p > 0.2）。我们得出结论，急性细胞排斥反应与IL-2基因的移植内mRNA相关。在此过程中还检测到其他转录本，包括IFN-γ、IL-6和IL-8基因的转录本，其数量增加。在ACR期间也检测到TNF-α、IL-1β和IL-10基因的信使核糖核酸，但这些转录本的存在并非此过程所特有。
