【作者】 洪海燕；

【导师】 戚中田； 沈倍奋；

【作者基本信息】 第二军医大学 ， 微生物学， 2002， 博士

【摘要】 干细胞因子（stem cell factor，SCF），是原癌基因c-kit的配体，是作用在造血过程中较早期的造血因子，其基因定位于人第12号染色体q^22～24区。SCF在体内有两种存在方式：膜结合型和可溶性，可溶性人SCF分子，由胞外区1～164个氨基酸组成，在体内有时通过非共价键形成二聚体存在，是一种多功能细胞因子，作用于多谱系造血细胞。自1990年SCF被发现以来，其功能也不断被发现，并被阐述清楚。SCF能够协同其他的细胞因子，如EPO、GM-CSF、G-CSF、IL-2、IL-3、IL-11和TPO等，SCF都有明显的协同效应。临床上应用SCF治疗贫血、提高放、化疗后白细胞数量、体外造血、基因治疗等，是一种前景看好的细胞因子药物。已经证实，在E.coli中表达的未糖基化的可溶性人SCF，与天然的可溶性人SCF具有同样的生物学效应。 重组人干细胞因子在E.coli中较难实现高表达，从1992年开始研究hSCF在E.coli中的表达，为了获得高效表达，我们曾经尝试过许多方法，虽然都没有达到满意的效果，但是积累了许多经验。本研究以pBV-220温度敏感表达载体为模板，借助计算机分析软件分析原来工作获得的结果，寻找影响rhSCF在E.coli中高效表达的因素。pBV-220表达载体，是国内科学家自己构建的原核高效表达载体，目前在国内被广泛应用于生产基因重组药物。普遍认为影响外源基因在该载体中表达效率主要有三个因素：SD序列长度、基因起始码ATG和终止码TAA处RNA二级结构和密码子偏性。通过分析认为：SD序列长度在hSCF的表达中不起关键性作用，SD序列长度的改变不能提高起表达效率；天然hSCF两端的RNA二级结构基本符合高表达判别函数，试验中没有得到高效表达，通常有目的的改造外源基因与载体连接处的RNA二级结构，就能够获得高效表达，修改hSCF两端的RNA二级结构后，也没有实现高表达，提示可能还有另外的原因存在。我们认为是由于密码子偏性影响了hSCF基因在E.coli中的高效表达。可溶性hSCF蛋白由164个氨基酸组成，其中有57个氨基酸的密码子表达系数低于0.1，占总氨基酸数量的34.8％。由于编码hSCF基因中的密码子不是E.coli所偏爱，并且低表达指数的氨基酸成组存在，造成该基因在E.coli中较难实现高＿第。军医大学博十学位论义”小义仰业g g 【 效表达。Z 找到影响hSCF基因在E。。h中高效表达的因素之后，尝试通过对hSCF ｛ 基因进行改造，以实现hSCF在大肠秆菌中的高效表达。对hSCF的基因进行了1 改造，修改编码hSCF氨基酸的部分密码子，使编码hSCF中每个氨基酸的表达8＿指数不低于0＊。分段合成改造后的基因，PCR一次性充成全基因的拼接，将丛Z－g 因克隆到叼爪220载体上。测序结果显示插入的基因序列与原设计一致，诱导g 表达后山沈F占菌体总蛋白的2＊％，与基因改造前10％的表达效率利I比，竹了￥ 近2倍的提高。Western七＊ 鉴定，表达的蛋白能够为hSCF的单克隆抗体所识f＿别。小量发酵，初步纯化的rhSCF蛋白，纯度大于80％，能够促进Mo7e细胞。－的增殖活性。‘ 1998年至今，红细胞分化相关的研究，不断取得进展，红细胞分化机制正g 在被逐渐阐述清楚。SCF及其配体仁七it在红系分化中，不仅仅是协同红细胞生j 成素＊rythrpoietin，EPO人而且参与其中，并且起着重要作用。新近的研究发 ＿现，S＊F和＊PO一样，在红系分化中起非常重要作用，它能够抑制红系造血扭＿细胞的凋亡，促进红系干、祖细胞增殖和分化，而且是红系祖细胞增州、分化－＿的必要条件。但是S＊F对非正常发育的细胞，是否也同样具有抑凋亡、促增则 的作用呢？在8＊＊L大鼠白血病造血叶。，＊＊F具有两利吓同的生物效应，山］既 能有效地刺激正常造血，又可抑制白血病细胞增殖。那么在人体中SCF足否也＿－具有这样两种不同的生物效应呢？一、以红白血病细胞系一K562细胞为研究模板，尝试探讨 SCF小kit在红系“l“／ 祖细胞增殖分化上的作用。已经有大量文献报道，hEpO、HCmill和 RA能够诱 －导K562细胞向红系分化。经过诱导后的K562细胞，血红蛋白表达增加，红系 ＿相关的转录因子激活，具有了红系祖细胞一些特征。本研究用rhEPO、Flemin ＿和RA分别诱导K562细胞向红系分化，检?更多还原

【Abstract】 Stem cell factor (SCF) and its ligand type III tyrosine kinase receptor are closely involved in the regulation of a wide range of tissue at different stages of life. They play an important role in various biologic phases,such as hematopoiesis, reproduction and regeneration. Human SCF is encoded by a gene located on chromosome 12q22-24,which including 8 exon. The gene is alternatively spliced at exon 6 encoding two isoform of SCF: soluble and transmembrane forms of SCF, and both of them have the same biological active. Soluble SCF is generated from SCF248 which is composed of 248 amino acids by spliced at a specific proteolytic cleavage site at Ala165. Soluble SCF sometimes exists as homedimer by noncovalent. It promotes viability and induced the proliferation of haemopoietic progenitor cells. SCF is potently synergistic when combined with growth factor such as: EPO> GM-CSF, G-CSF, IL-2, IL-3, IL-11, TPO. There are 3 clincal areas where SCF has been used today: (1)in vivo and ex vivo expension of stem cell, (2)stem cell mobilization, (3)therapetic of aplastic anaemia. It was claimed that rhSCF produced from E.coli has the same biological active as the natural glycoprotein of soluble hSCF.It is difficult to reach a high expression of rhSCF in E.coli. We have been studying the expression of rhSCF in E.coli since 1992. Though we have not got a high expressing bacteria, we have got lots of experience. In order to find the reason for rhSCF could not high express in E.coli, we analysed the result gained before with the help of software on computer.lt was pBV-220 vector that we selected to express rhSCF in DH5 a becteria. pBV-220 vector is constructed for high expressing gene in E.coli by the scientist in China. This vector is temperature sensitive. It has been used to express many genetic medicine and poly peptide, and often gave high expression. There are 3 factor that play an important role in high expression of pBV-220: (1) length of SD sequence, (2)RNA secondary structure of the sequence around ATG andTAA, (3) condon adaptive. We analysed the work done before, and draw the conclusion like this: both SD sequence and RNA secondary structure have little effect on high expression of rhSCF. Soluble SCF is composed of 164 amino acids. There are 57 amino acids whose condon adaptive is lower than 0.1, and it is 34.8 percent in totle amino acids. Also amino acids with lower condon adaptive often connected side by side existing here and there in the gene of rhSCF. So we concluded that condon adaptive maybe the reason for low expression of rhSCF.To highly express hSCF in bacterial strain,the gene of hSCF was corrected and primer was designed with the help of Goldkey software. The gene was synthesized, ligated by PCR in one tube, and cloned into a prokaryotic temperature sensitive vector pBV-220. Sequence analysed showed the insert gene has the same sequence as we designed After induction the recombinant bacteria could produce rhSCF about 27 percent of all bacterial protein. Western-blot showed a stained band at 19000 Da. The biologic assay showed that rhSCF could stimulate the proliferation of Mo7e cells. It is the amino acid codon adaptive that make SCF low expression.10 years before, researches on signal transduction of cytokine are developed in cell line for the limitation of the technology. With the development of CD34+ cell separating and culturing technology, it is possible to study the signal transduction of cytokine with the body cell in vitro. Erythropoiesis is clearly expatiated from 1998. Stem cell factor and its receptor have an important role in erythropoiesis. There are two main functions of stem cell factor in erythropoiesis. One is inhibiting apoptosis of CFU-E and BFU-E , the other is stimulating proliferation of CFU-E and BFU-E. So stem cell factor has its clinical usage in anaemia. We wonder if SCF has the same function on tumor cell as it is on the stem cell.The continuous cell line K562 is a human erythroleukemia line.lt could beinduced and show some specific marker of erythroid by so更多还原