实时定量PCR法检测骨髓间充质干细胞移植到梗死心肌后的存活数量

Detection of the survival number of the bone marrow derived from mesenchymal stem cells implanted into myocardial infarction with Real-time PCR

目的:建立一种干细胞移植到梗死心肌后检测其存活数量的有效方法。方法:选取10只雌性SD大鼠结扎前降支,心肌梗死3周后,随机分成移植组与对照组,同时选取雄性大鼠培养出骨髓间充质干细胞(MSCs),分别将MSCs(3×106个,50μL)及等体积的PBS液移植到梗死心肌中,24h后通过实时定量PCR(Real-timePCR)检测雄性大鼠Y染色体上特有的基因SRY,从而得知移植MSCs的存活数量。结果:MSCs移植24h后,移植组梗死心肌中检测到SRY基因数占移植总数的14.9%±8.3%,存活细胞的绝对数量为447195±248090,而对照组均未检测到SRY基因的存在。结论:用Real-timePCR的方法能有效地检测到具有SRY基因的干细胞,从而能准确地得知移植干细胞的存活数量,Real-timePCR技术在心梗后干细胞移植的基础研究及临床应用中具有重要的价值。

Objective： To establish an effective method to detect surviving number of the stem cells in vivo. Methods： Ten adult female SD rats were randomly assigned into two groups： the bone marrow derived from mesenchymal stem cells （MSCs） transplantion group and the control group. Anterior myocardial infarction was induced by ligation of the proximal left anterior descending artery. Using an insulin syringe, PBS（50μL） and MSC （3×10^6/50μL） of male rat sources were injected into infarcted area in transplantation group and control group, respectively. To evaluate the viability of MSCs injected into infarcted area, Real-time PCR was used to detect the SRY gene which is a specific marker of Y chromosome in 24 hours after MSC transplantation, Results： Twenty four hours after MSC transplantation, the cells were detected in the infarcted myocardium in six rats of transplantation group. The number of surviving cells was 447195±248090; the number of detected SRY to the number of transplanted cells was 14.9%±8.3%. In contrast, no sry gene were detected in control group. Conclusion： Real-time PCR is a sensitive and accurate method to detect the viability of transplanted stem cells in infarcted myocardium, and this method is valuable for both the laboratory study and clinical practice in the field of stem cell therapy for myocardial infarction.