银屑病患者骨髓CFU-HPP集落形成及集落细胞p16基因启动子甲基化的研究

CFU-HPP Colony Formation of Bone Marrow Hematopoietic Proginitor Cells in Psoriatic Patients and Methylation of p16 Gene Promotor in CFU-HPP Colony Cells

本研究检测银屑病患者骨髓高增殖潜能集落形成单位(CFU-HPP)的集落形成能力及集落细胞p16基因启动子甲基化状态,并探讨两者之间的关系。收集了24例银屑病患者及正常对照者骨髓,采用密度梯度离心法分离单个核细胞,并于含SCF/GM-CSF/IL-3/IL-6细胞因子组合的甲基纤维素半固体培养液中,培养14天计数CFU-HPP集落,然后收集集落。提取纯化集落细胞的DNA,经亚硫酸盐修饰后,采用甲基化特异PCR(MSP)检测CFU-HPP集落细胞的p16基因启动子甲基化状态。结果发现:在甲基纤维素半固体培养基中,银屑病患者骨髓CFU-HPP集落数显著低于正常对照(t=5.91,p<0.01),且集落形态较小;正常对照骨髓CFU-HPP集落细胞的p16基因启动子甲基化阳性率较高(66.7%),而银屑病患者骨髓CFU-HPP集落细胞p16基因启动子甲基化阳性率(37.5%)低于正常人。结论:银屑病患者骨髓CFU-HPP细胞集落形成能力降低;银屑病患者骨髓CFU-HPP集落细胞的p16基因甲基化降低可能与其相对较低的CFU-HPP集落形成能力密切相关。

This study was purposed to investigate the colony formation of high-proliferative potential colony-forming units（CFU-HPP） from bone marrow-derived hematopoietic cells of psoriatic patients and p16 gene promotor methylation in CFU-HPP cells, and to explore the relationship between the colony formation and the methylation status of p16 gene promoter. Bone marrow-derived mononuclear cells from psoriatic patients and normal controls were separated by density gradient centrifugation, and were cultured in methycellulose semi-solid culture medium with SCF, GM-CSF, IL-3 and IL- 6 for 14 days to measure the colonies of CFU-HPP. The CFU-HPP colony cells were collected and methylation status of p16 gene promoter of CFU-HPP cell DNA modified with sodium bisulfite was detected by the methylation-specific polymerase chain reaction （MSP）. The results showed that in methycellulose semi-solid culture system, the number and the size of CFU-HPP colonies of bone marrow of psoriatic patients were all significantly less than that of normal controls, the positive frequency of p16 gene promoter methylation in CFU-HPP cells was lower than that in CFU-HPP colony cells of normal controls. It is concluded that the colony formation capability of CFU-HPP from bone marrow hematopoietic progenitor cells in psoriatic patients is lower than that in normal controls, and the lower positive frequency of P16 gene promoter methylation in CFU-HPP cells perhaps closely correlated with lower CFU-HPP colony-forming capability.