T-大颗粒淋巴细胞白血病临床特点及疗效IFN-γ在造血调节中的作用

【作者】 赵馨；

【导师】 王建祥； 张凤奎； 王敏； 饶青；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 目的提高对T-大颗粒淋巴细胞白血病(T-LGLL)的认识。方法回顾性分析我院2000年2月～2009年11月间确诊的59例T-LGLL患者病例资料。结果T-LGLL起病潜隐、进展缓慢,中位确诊年龄50岁。贫血相关症状最为突出,40.7%患者脾脏轻中度肿大,13.6%患者肝脏轻度肿大。1例患者合并类风湿性关节炎。外周血细胞计数中性粒细胞<1.5×109／L者占66.1%,<0.5×109／L者占13.6%。48例患者贫血,中位血红蛋白65g/L,合并纯红细胞再生障碍性贫血(PRCA)者31例,占53.4%。外周血大颗粒淋巴细胞(LGL)绝对值中位数1.64×109／L,大多数(62.0%)患者LGL数≤2.0×109／L。48例(81.3%)患者LGL免疫表型为CD3+CD8+CD57+CD56-。48例患者接受治疗,治疗总有效率87.5%,完全血液学缓解(HCR)者占60.4%。33例患者接受环孢素为主的治疗,有效率为81.8%,HCR率60.6%。复发率35.7%,主要由药物减量、停药或感染所致。中位随访时间20个月(3.5-120月)。结论T-LGLL起病及进展缓慢,主要表现血细胞减少和脾脏肿大,常合并PRCA。外周血LGL计数多≤2.0×109／L,免疫表型以CD3+CD8+CD57+为主。治疗有效率高,对以环孢素为主的免疫抑制治疗方案反应良好。目的：通过基因转导研究小鼠IFN-y在造血调节中的作用。方法：从小鼠脾脏细胞中提取mRNA, RT-PCR方法扩增mIFN-y片段,连接到pCDHl-MCS1-EF1-copGFP (pCDH-GFP)载体上,构建小鼠IFN-y (mIFN-y)慢病毒表达载体pCDH 1-mIFN-y-EF 1-copGFP (pCDH-mIFN-γ-GFP)通过磷酸钙沉淀法转染293T细胞,分别制备带有pCDH-mIFN-γ-GFP和pCDH-GFP空载体的慢病毒,感染C57小鼠的骨髓单个核细胞(BMMNC),48小时后进行甲基纤维素集落培养,观察BMMNC集落形成能力,并将转导后的BMMNC尾静脉注射至致死剂量照射的受体C57小鼠体内,定期尾静脉取血检查血象,并用流式细胞仪检测外周血细胞GFP阳性率。另外,取小鼠骨髓基质细胞培养传代,用慢病毒感染后经尾静脉注射至正常C57小鼠体内,定期检测血象。结果：用带有pCDH-mIFN-γ-GFP或空载体pCDH的病毒感染293T细胞,检测病毒滴度分别为4.5×105／ml和3.4×105／ml。经ELISA方法检测上清液中mIFN-y含量约为188pg/ml,证实感染的细胞中有mIFN-y的表达。慢病毒感染后的C57小鼠BMMNC集落培养结果显示：感染mIFN-y的BMMNC集落生成数量较空载体对照组显著减少,提示转导mIFN-y可能直接损伤造血前体细胞,导致其集落形成能力下降。接受骨髓移植的C57小鼠2周后尾静脉血象显示：转导mIFN-y组的小鼠血象恢复较对照组晚,提示mIFN-y可能通过损伤早期造血细胞导致造血恢复延迟。流式细胞仪检测结果显示：移植后8周外周血中仍可见GFP阳性细胞,表明转导的细胞在小鼠体内可存活8周以上。移植了经病毒感染的小鼠骨髓基质细胞后的小鼠,2周开始尾静脉取血查血象,至血象出现下降时处死小鼠,取骨髓组织送病理检查。该部分实验结果较少,有待完善。综上所述,本研究通过慢病毒转导mIFN-y至小鼠BMMNC中,发现不仅造血细胞体外集落形成受抑制,而且移植小鼠骨髓造血恢复延迟。以上研究结果表明mIFN-y可能损伤造血前体细胞,支持IFN-y在骨髓造血衰竭的发生中起重要作用。更多还原

【Abstract】 Objective: To improve the recognization of characteristics of T-LGLL.Methods: Retrospectively analyze the documents of 59 patients with T-LGLL diagnosed between 2000 and 2009 in our hospital.Results: The median age at T-LGLL diagnosis was 50 years without a male or female predilection. The patients mainly complained of fatigue. Of those 59 patients, 40.7% had splenomegaly, and 13.6% heptomegaly. Rheumatoid arthritis was present in 1 patient. The most frequent hematological abnormalities were anemia in 81.4% of patients with median Hgb level of 65g/L. Pure red cell aplasia (PRCA) was more common in this study. Neutropenia was present in 66.1% of patients and 13.6% of patients were agranulocytosis. The median count of LGL in peripheral blood was 1.64×109/L and most of them were less than 2.0×109/L. Fourty-eight patients (81.3%) had the CD3+CD8+CD57+CD56- LGL phenotype. Fourty-eight patients accepted treatment and 87.5% of patients responded. With CsA therapy in 33 patients, 60.6% achieved HCR. The total relapse rate was 35.7%. The median follow-up time was 20 months.Conclusion: T-LGLL was an indolent disease, mainly presented with anemic symptoms. Nearly half of the patients had splenomegaly. Pure red cell aplasia was commonly associated with T-LGLL in this group while rheumatoid arthritis was found only in 1 patient. LGL count in peripheral blood was mostly≤2.0×109/L. The patients had a good response to immunosuppressive therapy. Purpose:To explore the role of murine IFN-γ-(mIFN-γ) in the regulation of hemopoiesis through lentivirus transduction.Methods:The mRNA was extracted from the splenic cells of C57 mouse and mIFN-y ORF was amplificated using RT-PCR and then cloned into lentiviral vector pCDHl-MCS1-EF1-copGFP (pCDH) to construct vector pCDH1-mIFN-y-EF1-copGFP (pCDH-mIFN-y-GFP). The lentiviruses were prepared following transfecting 293T cells by calcium phosphate precipitation. The murine bone marrow mononuclear cells (BMMNCs) were transduced with those two kinds of lentiviruses, and cultured in methylcellulose semisolid medium for colony-forming assay 48 hours later. The transduced BMMNCs were also transplanted into lethally-irradiated mice by tail vein injection and the peripheral blood cell counts 2 weeks later and green fluorescent protein (GFP) were monitored regularly. Bone marrow stromal cells (BMSCs) were transduced with lentivirus and transplanted into normal C57 mice, and the peripheral blood cell counts were also monitored regularly.Result:The titer of lentivirus was measured to be about 4.5×105/ml and 3.4×105/ml for pCDH-mIFN-γ-GFP or pCDH-GFP respectively using a bioassay with the 293T cell line. The expression of mIFN-y in the supernant of 3T3 was confirmed with ELISA kit and the concentration was 188pg/ml. The colony-forming assay showed that the number of colonies in the mIFN-y group was significantly decreased compared to the control. It indicated that mIFN-y may injure the hematopoietic progenitors directly and make its colony-forming capacity decreased. The circulating white blood cell count, platelet count and hemoglobin in mIFN-y group was significantly lower than control recipient mice at 2 weeks, while no difference was observed at 8 weeks. This indicated that the hematopoiesis rebuilding in the mIFN-y group was delayed than control group. It may be caused by the injury of the hematopoietic progenitors which overexpressed mIFN-y. GFP positive cells were detectable in the peripheral blood 8 weeks after transplantation and this indicated that the transduced cells could survive in vivo for at least 8 weeks. BMSCs trandsduced with lentivirus were transplanted into normal C57 mice. When the peripheral blood cell count decreased, the myeloid tissue was examined for pathologic analysis. This part was just ongoing and there was little results for analysis.In conclusion, we constructed mIFN-y expressing vector pCDH-mIFN-γ-GFP. It showed that not only the colony-forming capacity of transduced BMMNCs was inhibited, but also the hematopoiesis rebuilding was delayed. This result indicates that mIFN-y may damage the hematologic progenitors, supporting the hypothesis that IFN-y plays an important role in the mechanism of bone marrow failure.更多还原