【作者】 刘淑艳；

【导师】 郑国光；

【作者基本信息】 中国协和医科大学 ， 细胞生物学， 2010， 硕士

【摘要】 干细胞因子(stem cell factor, SCF)是一种多功能细胞因子,参与机体发育中多种细胞的增殖调控,是多种哺乳动物干细胞增殖分化的关键因子。它可与细胞表面c-kit受体结合,作用于早期造血干／祖细胞,在维持其存活、促进增殖、及调控其向各系造血分化中起重要作用。人SCF由外显子6蛋白酶解位点选择性剪接形成可溶型(sSCF)与膜结合型(mSCF)两种形式,二者在造血中的作用有所不同。sSCF参与干／祖细胞的动员,而mSCF则被认为是造血微环境"niche"的重要组成分子,参与造血干细胞与"niche"之间的相互作用,造血微环境缺乏mSCF导致造血干细胞重建造血障碍。在恶性实体肿瘤中,sSCF/c-kit通过自分泌作用促进肿瘤生长和转移,而c-kit表达阳性的肿瘤细胞被认为具有肿瘤干细胞样性质。在血液系统恶性疾病中,63%的急性髓系白血病(AML)患者c-kit表达阳性,其中2／3同时伴有异常CD34+指标的患者不易缓解、易复发、预后不良。以往的研究主要阐明了sSCF通过自分泌机制促进肿瘤细胞的恶性表型,我们已有的结果提示mSCF在部分白血病中有高表达,在AML患者和正常人骨髓单个核细胞中均有sSCF、mSCF、c-kit三种基因产物的表达,其中AML患者与正常人骨髓细胞c-kit mRNA、mSCF mRNA水平差异有统计学意义,而sSCF mRNA水平差异无统计学意义。本论文在此基础之上,用DNA重组技术成功构建了人可溶型及膜结合型干细胞因子的重组逆转录病毒表达载体MSCV-PGK-GFP-sSCF、MSCV-PGK-GFP-mSCF,二者与空载体MSCV-PGK-GFP分别转染Phoenix细胞,包装出含有目的基因的假病毒颗粒并成功感染了NIH3T3细胞,流式细胞术筛选后的阳性克隆经合适剂量射线照射后,各自作为滋养层细胞与c-kit受体阳性的K562细胞共培养,CCK8法分别检测共培养体系中K562细胞的增殖情况,结果发现,10%血清浓度条件下共培养48、72小时,NIH3T3-S、NIH3T3-M孔中的K562细胞增殖能力均显著高于NIH3T3-V孔(P<0.01),而前两者K562细胞的增殖没有显著差异(P>0.05)；1%血清浓度条件下共培养24、48和72小时,NIH3T3-S、NIH3T3-M孔中的K562细胞增殖能力均显著高于NIH3T3-V(P<0.01),且NIH3T3-M孔高于NIH3T3-S孔(P<0.05)。上述结果提示导入的两种形式人干细胞因子基因可以在细胞中稳定有效的表达,并发挥作用,感染的NIH3T3细胞通过表达sSCF、mSCF促进K562细胞的增殖；在低血清条件下mSCF的作用更强；同时,我们建立了一种可行的共培养方法,可以提供稳定的SCF信号,为积极探索有效的重建造血的方法奠定宝贵的实验基础。本室前期的研究初步表明了本身有c-kit受体表达的K562细胞分别转染两种形式人干细胞因子后,mSCF与c-kit之间的自身并置性作用显著促进K562细胞的增殖,为了进一步证明这种作用是二者通过自激环路实现的,我们将已经构建好的sSCF前体的真核表达质粒pTARGET-s和mSCF的真核表达质粒pTARGET-m以及空载体pTARGET-v通过脂质体介导转染Ramos细胞(c-kit阴性),通过G418筛选,并用RT-PCR、Western blotting鉴定,成功建立了两种人干细胞因子的稳定表达细胞株,通过CCK-8细胞增殖实验、集落形成实验研究不同细胞株在白血病细胞中的作用。实验结果提示Ramos-S、Ramos-M与转染空载体对照的Ramos-V细胞增殖差异没有统计学意义(P>0.05)；集落形成实验中三者所形成的集落在大小及数量上也均没有什么差别(P>0.05)。上述结果说明,缺乏sSCF介导的自分泌作用及mSCF介导的自身并置性作用,表达任何一种SCF均不能促进白血病细胞的增殖。因c-kit高表达率低,故两类SCF在淋巴细胞白血病中不发挥重要作用。综上所述,本文成功构建了两种形式人干细胞因子的逆转录病毒表达载体,经Phoenix包装的重组及对照逆转录病毒成功感染NIH3T3细胞,并在细胞中得到功能性的稳定表达,同时我们首次建立了一种可行的共培养方法,可以提供稳定的干细胞因子信号,为深入研究mSCF在细胞间通讯及造血系统中的作用提供重要的手段和有益的实验基础；同时实验证实了,mSCF通过并置性作用机制促进白血病细胞的增殖,为研究白血病发生、发展及治疗提供有益的线索。更多还原

【Abstract】 Stem cell factor is a multifunctional cytokine that regulates cell proliferation during development. It is an important regulator in proliferation and differentiation of stem cells in mammals. Binding to its surface receptor c-kit, it plays essential roles on maintaining the survival, promoting the proliferation and regulating the differentiation of hematopoietic stem/progenitor cells. Two forms of human SCF, the soluble and transmembrane ones are generated by alternative splicing that includes or excludes a cleavage site at exon 6. Both of them are biologically active with distinct roles in hemocytogenesis. sSCF participates in the mobilization of stem/progenitor cells, while mSCF is a component of hematopoietic "niche" that mediate the interaction of hematopoietic stem cells and microenvironments. Absence of mSCF in hematopoietic microenvironment would lead to the disability of Hematopoietic system reconstruction. sSCF/c-kit promotes tumor growth and metastasis by paracrine in malignant solid tumors. Meanwhile the tumor cells expressing c-kit (SCF receptor) is thought to show a feature of tumor stem cells. In the hematopoietic malignancies, expression of the c-kit receptor was detected in 63% of AML samples investigated, Among which approximately two thirds of patients accompanying abnormal CD34+ phenotype are not able to remission, easy to relapse and may get unfavorable prognosis.Previous studies mainly explained that sSCF promoted the malignant phenotype of tumor cells by autocrine mechnism. Researches in our lab indicated that sSCF, mSCF and c-kit were detected in the marrow mononuclear cells of AML patients and healthy controls with a different mRNA level. mSCF was highly expressed in AML patients compared with normal controls, while no different expression of sSCF was found between AML patients and normal ones.Based on the research background above, the retroviral vectors MSCV-PGK-GFP-sSCF and MSCV-PGK-GFP-mSCF were constructed by DNA recombinant technologies. Recombinant and blank vectors were packed in Phoenix cells. NIH3T3 cells were infected respectively by viral supernatant which contains the corresponding genes. After sorted by flow cytometry, three positive cell lines were obtained. Then proper irradiation was given to them.As trophoblastic cells, they were cocultured with K562 cells respectively. And the proliferation of K562 cells in coculture system was detected by means of CCK8 methods. Results showed that the proliferation potential of K562 cells cocultured with NIH3T3-S and NIH3T3-M were significantly higher than that cocultured with NIH3T3-V under 10% serum levels culture conditions at the time of 48h and 72h (P<0.01), however, no difference was detected in the former two cells(P>0.05). In addition, under 1% serum levels culture conditions at the time of 24h, 48h and 72h, growth promotion effects on K562 cells could be observed in NIH3T3-S and NIH3T3-M coculture systems (P<0.01). Furthermore, NIH3T3-M showed higher effects than NIH3T3-S (P<0.05). These results suggested that the imported two forms of human SCF could express in target cells stably and effectively. Infected NIH3T3 cells promoted the proliferation of K562 cells by expressing SCF. Moreover, under low serum culture conditions, NIH3T3-M showed higher effects than NIH3T3-S. At the same time, we established a feasible method for coculture system, which could give a stabile signal of SCF and provide solid platform for further working towards the reconstruction of hemopoiesis.Our previous research showed that after transfection of the two forms of SCF, the K562 cells (c-kit+) could be promoted proliferation, and the juxtacrine of mSCF/c-kit is more powerful in the process. To further explore that this effect was accomplished by self-excitation, another leukemic cell line Ramos, which is negative in c-kit expression, were transfected with the constructed vectors (pTARGET-s, pTARGET-m) or blank vector pTARGET via Lipofectamine 2000. Stable transfectant clones were obtained by G418 selecting, and verified by RT-PCR and Western blot. In vitro characteristics of the stable transfectants were studied by liquid culture and colony-formation assays. Studies showed that the proliferation potential of Ramos-S, Ramos-M and Ramos-V had no difference (P>0.05); Furthermore, the colony either showed no difference in size and numbers (P>0.05). The above results suggested that expression of either mSCF or sSCF could not promote the proliferation of leukemia cells in absence of autocrine and juxtacrine mechanism. Since the high expression rate of c-kit was low, two forms of SCF were not important in Lymphocytic leukemias.In conclusions, the recombinant retroviral vectors expressing sSCF and mSCF were successfully constructed. High titer recombinant retroviruses were obtained after packed in Phoenix cells. NIH3T3 cells were successfully infected by retroviruses. And in NIH3T3 cells, they could express stably and effectively. Furthermore, we first established a feasible coculture method, which could give a stabile signal of SCF and provide the useful basis for better understanding of SCF-mediated intercellular communication in leukemias. At the same time, our studies showed that the juxtacrine mechanism of m-SCF and c-kit contributed to the proliferation of leukemia cells. The knowledge will help us for further study to explore potential target for the treatment of leukemias.更多还原