凋亡或程序化死亡是基因控制的细胞自我毁灭的过程，它在几乎所有动物的发展和内稳定的维持中起着非常重要的作用。凋亡是严格控制的过程，涉及到许多基因,这些基因在种属之间往往非常保守。在细胞凋亡研究中，利用去除细胞因子的途径诱导白血病细胞凋亡已成为广泛应用的模型，通过这种凋亡模型，许多凋亡相关基因已被克隆，并对其编码蛋白之间的相互关系进行了深入的研究。为了在分子水平上研究细胞因子撤除导致凋亡所参与的基因及其分子机理，本文利用近年来发展起来的代表差异分析(cDNA Representational Differences Analysis, cDNA-RDA)技术,研究了在人红白血病细胞株TF-1细胞撤除细胞因子导致凋亡过程中诱导表达的基因。经基因克隆和测序，发现了6个新基因片段，其中有三个经查询GenBank nr和dbEST数据库均未发现高度同源的序列，已向GenBank 登记，登记号分别为U83208、U83279和U83397。此外还发现一批在凋亡细胞中高表达的已知基因，其中包括编码Nmi和人硫氧还原蛋白的基因，提示它们在凋亡中可能起作用。这项工作为进一步研究凋亡相关基因奠定了基础。

在上述工作基础上，我们对其中的一个新基因cDNA进行了深入的研究。经mRNA斑点杂交和Northern 杂交证实此基因在TF-1的凋亡过程中表达明显增高。利用RACE的方法，克隆出了它的全长cDNA序列(已在GenBank 登记，登记号为AF014955)，并命名为TFAR19(TF-1细胞凋亡相关基因19)。TFAR19 cDNA全长为559个碱基，与Northern blot 分析的大小一致，包括polyA和“AATAA”加尾信号。第25-399位碱基读码框架编码125个氨基酸的蛋白质,其ATG起始密码周边序列(GCCATGG)符合Kozak 规律。根据蛋白质序列检索GenBank数据库发现， TFAR19蛋白质在原核和真核生物不同种属间具有保守性，它与小鼠、线虫(Caenorhabditis elegans)、真菌(Archaeoglobus fulgidus)、Methanococcus jannaschii、酵母菌等类似分子量的一些未知功能蛋白有较高同源性，说明它在进化上相当保守，可能具有重要的功能。运用Prosite软件分析推测的蛋白质序列，没有发现信号肽、穿膜区序列、没有DNA结合位点及N-糖基化位点，Ser¹⁰⁰为可能的cAMP和cGMP依赖的蛋白激酶磷酸化位点，另有4个可能的PKC蛋白激酶磷酸化位点。计算分子量为14,285道尔顿，等电点为pH 5.65。50种正常和胚胎人类组织的mRNA斑点杂交分析表明，TFAR19的表达不局限于造血细胞，它在所有组织中均有不同程度的表达，其中在成年心、睾丸、肾脏、垂体腺、肾上腺及胎盘中的表达相对高于其它组织。这些结果表明TFAR19不仅在进化中具有保守性，而且对机体各个组织均有功能上的重要性。令人感兴趣的是，TFAR19在胚胎期的表达水平远低于成年组织中的表达，考虑到多数胚胎组织的细胞凋亡过程弱于成年组织，并结合本文发现的TFAR19促进凋亡效应和抑制细胞生长的功能实验结果，提示TFAR19在调节成熟组织细胞的增殖分化和内稳定中可能发挥重要的负调节作用。为研究TFAR19的细胞定位，构建了绿色荧光蛋白与TFAR19的融合蛋白表达载体，，转染细胞后，发现转染细胞的核区呈明显绿色着色，这提示TFAR19可能在核内发挥作用。

为在蛋白质水平上研究TFAR19的功能，构建成功原核表达载体，在大肠杆菌中表达并经变性、复性、柱层析等程序纯化了重组TFAR19蛋白质，免疫小鼠制备了抗TFAR19的多克隆抗体，Western Blot发现TFAR19蛋白质在凋亡的TF-1细胞中高表达，表达蛋白质分子量与预期分子量一致。高剂量的TFAR19重组蛋白质(2-10? g/ml)能诱导人滑膜细胞的凋亡，促进TF-1细胞去细胞因子所致的凋亡。为研究TFAR19的生物活性，构建了TFAR19正义和反义真核表达载体，瞬时转染TFAR19正义表达载体后，TF-1细胞去细胞因子后凋亡程度增加，胃癌细胞株MGC-803细胞和HeLa细胞的生长受到抑制。稳定转染TFAR19正义表达载体的MGC-803细胞生长速度明显减慢。稳定转染TFAR19正义表达载体的MGC-803及HeLa细胞去血清后凋亡速率明显快于对照，而稳定转染TFAR19反义表达载体后的Hela细胞凋亡慢于对照。以上结果显示TFAR19实际上可能是一种凋亡的增强剂，在某些条件下(如去除细胞因子、去除血清)对某些肿瘤(TF-1、MGC-803、HeLa)发挥凋亡促进作用，它与其他已知凋亡相关蛋白的关系及其作用机制有待进一步的研究。

综上所述，本文在国际上首次克隆了一个具有功能的凋亡相关基因的全长cDNA，证明TFAR19是一个进化保守的具有促进细胞凋亡作用的人类新基因编码蛋白质，很可能在细胞核内发挥重要的信号传导作用，从而调节细胞增殖、分化和凋亡。本文工作为进一步了解凋亡的分子机理提供了新的基础。

关键词： TF-1细胞 cDNA-RDA技术 凋亡 基因克隆 细胞因子

Apoptosis or programmed cell death (PCD) is a genetically controlled program of cellular self-destruction, which is of central importance for the development and homeostasis of virtually all animals. It is a highly regulated process involving an increasing number of genes that are conserved in all metazoans that can be induced by a variety of stimuli, which include deprivation of growth factors, signaling via certain surface receptors, treatment with DNA-damaging agents or inhibitors of macromolecular synthesis. In order to study the molecular mechanism of the apoptosis, we studied the genes involved in the process of the apoptosis induced by cytokine deprivation. Up withdrawal of GM-CSF from TF-1, a human cytokine-dependent leukemia cell line, most of them will go into the process of apoptosis within 8 hours. Therefore, the TF-1 cells after cytokine deprivation for 8 h were used as the tester and cytokine supplement-cultured cells were used as the driver. With Representational Differences Analysis (RDA)，we found seven gene fragments which include some known genes such as Nmi and thioredoxin that formerly suggested to play a role in the process of apoptosis uniquely expressed or highly expressed in the process of apoptosis of TF-1 cells. Three genes were verified novel after searching the Nr and EST data bases in GenBank and were submitted to GenBank. The accession numbers for them are U83208, U83279 and U83397, respectively.

In the course of the study above, a novel gene fragment was found highly expressed during the process by slot blot and Northern blot. Base on the novel gene fragment, RACE method was used to clone the full- length cDNA (AF014955) and it was named TFAR19 (TF-1 apoptosis-related clone 19). The full-length cDNA of TFAR19 is 559bp including poly(A) tail and “AATAA” tailing signal. The ORF from 25bp to 399bp encodes 125 amino acids. The predicted amino acid sequence was analyzed by Prosite program and found that Ser¹⁰⁰ is a possible site for cAMP-and cGMP-dependent protein kinase phosphorylation site. TFAR19 is not hemapoietic restricted and expresses in almost all human tissues, which shows that it may play an important role in the development of tissues. Localization of TFAR19 in the nucleus by GFP (Green Fluorescent Protein) staining suggests that TFAR19 may function at the transcription level. Recombinant TFAR19 protein was expressed and purified in E.coli and used as immunogene to produce polyclonal antibody. It was found that TFAR19 protein expressed highly in the apoptotic TF-1 cells detected by Western blot. High dosage of the purified recombinant protein (2-10? g/ml) could induce the apoptosis of human synoviocytes and enhance the apoptosis of TF-1 cell induced by withdrawal of cytokine. After transient transfection of TFAR19, the apoptosis of TF-1 cell was accelerated and the growth of two tumor cells (MGC-803 cell from human stomach tumor and Hela cell) was slowed down. The stable MGC-803 cell transfectant of TFAR19 sense gene grew obviously slowly than that of control and the stable MGC-803 and Hela cell transfectants of TFAR19 sense gene died faster than that of control when deprivation of serum in the culture medium. All the results showed that TFAR19 might be an enhancer of apoptosis for some of tumor cells. The relationship of TFAR19 with other apoptosis proteins is needed to study before we further understand its function in the apoptosis.

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