多重定量PCR检测PDX模型小鼠细胞浸润比例的构建

Detection of the Proportion of Mouse Cell Infiltration in PDX Model by Multiplex Quantitative PCR

目的:建立一种快速、灵敏的方法检测人源异种移植模型(Patient-Derived tumor Xenograft,PDX)中小鼠细胞的浸润比例,以确保PDX模型的保真性。方法:选择人和小鼠的特异性基因PSMB2、Ren 2的部分区段设计引物和探针,利用多重荧光定量PCR(TaqMan探针法)在单管中检测PDX模型中小鼠-人细胞的相对量。另外,构建了不同浸润比例的标准品作为阳性对照,根据标准曲线进行样本比例计算。结果:建立了一种多重荧光定量PCR检测PDX模型小鼠细胞浸润比例的检测方案,该方案显示,PDX模型中不同比例的人鼠混合样本与△Ct值(Ct(小鼠)-Ct(人))之间线性关系良好,相关系数r~2为0.9998。利用该方法对14个样本进行检测,结果表明在绝大部分样本中小鼠细胞的比例低于30.00%,平均小鼠细胞比例为13.38%。结论:本研究建立的多重定量PCR检测PDX模型中小鼠细胞浸润比例的方法能够根据△Ct值快速、准确推断PDX模型中人、小鼠细胞的比例。该方法操作简单、耗时短、结果可靠,是一种快速、灵敏的PDX模型的质控方案。

Objective: A rapid and sensitive method to detect the proportion of mouse cell infiltration in PDX model was established in order to ensure the fidelity of PDX model. Methods: Partial segments of human and mouse specific genes PSMB2 and Ren2 were selected to design primers and probes, and the relative amount of mouse-human cells in PDX model was detected by multiplex fluorescence quantitative PCR(TaqMan probe method) in a single tube. In addition, we also constructed standards with different infiltration ratios as positive controls, and calculated the sample proportion according to the standard curve. Results: We established a method to detect the proportion of mouse cell infiltration in PDX model by multiplex fluorescence quantitative PCR. The scheme showed that there was a good linear relationship between different proportions of human and mouse mixed samples and △Ct value(Ct mouse-Ct human) in PDX model, and the correlation coefficient r~2 was 0.9998. This method was used to detect 14 samples, and the results showed that in most of the samples, the proportion of mouse cell was less than 30%, and the average proportion of mouse cell was 13.38%. Conclusion:The multiplex quantitative PCR method established in this study to detect the proportion of mouse cell infiltration in PDX model can quickly and accurately infer the proportion of human and mouse cells in PDX model according to △Ct value. This method has the advantages of simple operation, short time-consuming and reliable results, and provides a fast and sensitive quality control scheme for PDX model.