红系分化发育过程中珠蛋白基因表达开关进程的分析及相关调控因子基因探寻

【作者】 张昕；

【导师】 张俊武；

【作者基本信息】 中国协和医科大学 ， 生化与分子生物学， 2005， 博士

【摘要】 在人类个体发育过程中存在ε(胚胎型)→γ(胎儿型)→β(成人型)珠蛋白基因表达开关过程。在成人骨髓造血干细胞向红系分化发育过程中也存在γ→β基因表达的“压缩性”开关过程。虽然对于珠蛋白基因表达调控及开关的研究已取得许多进展，但至今对于调节这些开关的决定性调节因子仍不明确。这些因子的确定对于揭示珠蛋白基因开关机制具有决定性意义，本课题的目的就是希望能向此目标迈进。 本研究首先利用密度梯度离心方法，从正常成人和异细胞型遗传性胎儿血红蛋白持续存在综合症(HPFH)家系患者的骨髓组织(Bone Marrow)、健康足月正常分娩胎儿脐带血(Umbilical Cord Blood)中分离得到了含有造血干／祖细胞的单个核细胞(Mono-Nuclear Cell，MNC)，进行初步纯化及体外(in vitro)扩增后，组合使用Epo、IL-3、GM-CSF等细胞因子，将造血干／祖细胞向红系方向进行诱导培养。鉴于在以往报告中使用的红细胞体外培养方法往往由于添加胎牛血清及诸如c-kit配体、Tpo等促生长添加剂而使得成人外周血或骨髓来源的红细胞内胎儿型γ-珠蛋白基因的表达水平升高，不能真正模拟体内成人红系分化发育的过程，无法满足研究珠蛋白表达及开关调节的需要，我们针对成人骨髓来源的红细胞尝试使用了无血清培养方法，针对人脐带血来源的红细胞则使用了含脐带血清的培养方法，并且尽量去除其他影响珠蛋白表达的添加剂，希望能够找到并建立尽可能模拟生理情况的培养条件与方法，并以此为基础，为研究珠蛋白基因表达调控及开关机制提供适当的实验材料。 在向红系诱导过程中，我们在不同的时间点收集了红细胞培养物，提取总RNA，进行逆转录合成cDNA，通过实时荧光定量PCR(Real time PCR)技术详细检测了细胞分化成熟不同时间点的红细胞内α-类与β-类珠蛋白基因的表达水平。结果显示：在使用无血清培养基诱导培养的成人骨髓红细胞内，其α-、β-及γ-珠蛋白基因的表达水平及变化趋势同正常生理情况下的表达过程即所谓的“压缩性开关”非常相似，其中的β-珠蛋白基因的表达同γ-珠蛋白基因相比占明显优势，胚胎型ε-珠蛋白基因表达水平极低，而胚胎型ζ-珠蛋白基因的表达则未能检测到；在使用含人脐带血清的培养基诱导培养的脐带血红细胞内检测到了包括α-、β-、γ-、ε-及ζ-等在内的所有珠蛋白基因的表达，其中的β-珠蛋白基因表达变化趋势同无血清诱导培养的正常成人骨髓红系细胞相似，而与前者不同的是在整个成熟过程中γ-珠蛋白基因同β-珠蛋白基因的表达相比占更多还原

【Abstract】 During human development, two switches of globin gene expression, ε (embryonic) →γ (fetal) →β (adult) occur. Adult erythropoiesis is also postulated to recapitulate γ- to β- globin switching, which is called "compressed switch". Although advance has been made in the study on control of globin gene expression and switching, key factors of globin gene switching is still unknown till now. As the validation of these factors is of significance for disclosing mechanisms of globin gene switching, we hope that our work could contribute to this target.In this study, Mono-nuclear cells (MNCs) containing hematopoietic stem/progenitor cells were isolated from umbilical cord blood of normal full-term deliveries, healthy adult and heterocellular HPFH patient bone marrow by density gradient centrifugation. After simple purification and expansion in vitro, MNCs were induced to erythroid cell differentiation using different culture systems and cytokines including Epo, IL-3 and GM-CSF. In the previous study, fetal bovine serum and the other components such as c-kit ligand and Tpo were often used when erythroid cells were cultured in vitro. However, the fetal (γ-) globin gene was reactivated in cultured erythroid cells from adult bone marrow or peripheral blood in these culture conditions. That is, these culture methods failed to mimic the erythropoiesis in physical condition and couldn’t meet the needs for study of globin gene regulation and switching. In this study, serum free medium were used for induction of erythroid cells from adult bone marrow and human cord serum-containing medium were used for erythroid cells from cord blood. Further, components that reactivated γ-globin gene were never used in our culture system. We hope that the culture condition and methods mimicking erythropoiesis in vivo could be established and thus, can provide us the materials for further study of globin gene regulation and switching.During induction, erythroid cells were collected from cultures at different time points and total RNA was extracted for reverse transcription reaction. Then the expression level of α-like and β-like globin genes in erythroid cells at different time points were determined by real time PCR. The results showed that the expression level and fluctuation of -α、β- and γ-globin gene in cultured erythroid cells of adult bone marrow is very similar to the so called "compressing switch" in vivo when cultured in serum free medium. Compared to y-globin gene, the expression of β-globin gene is predominant, whereas the expression of 8- (embryonic) and ζ,-(embryonic) globin genes is nondetectable. However, the expression of α-、β-、γ-、更多还原